# Patent

## Title
TRAF-3 deletion isoforms and uses thereof

## Abstract

The present invention provides an isolated TRAF-3 deletion isoform encoded by the nucleic acid sequence (SEQ ID NO:1) shown in FIG. 15 (deletion isoform Δ130 nucleid acid). One embodiment of the present invention is an isolated TRAF-3 protein which comprised the sequence in FIG. 16 (SEQ ID NO: 2) (Δ130 protein).

## Background
This application is a continuation of PCT International Application No. PCT/US00/06503, filed 10 Mar. 2000, designating the United States of America, which is a continuation-in-part and claims priority of U.S. Ser. No. 09/268,544, filed Mar. 11, 1999, now U.S. Pat. No. 6,410,710, the contents of which are hereby incorporated by reference into the present application.


BACKGROUND OF THE INVENTION
TRAF-3 gene products are signaling molecules that interact with the cytoplasmic tails of CD40 (Cheng et al., 1995; Hu et al., 1994; Sato et al., 1995), other Tumor Necrosis Factor-Receptor (TNF-R) family members (e.g. LTβ-R, CD30, CD27, Ox40) (Mosialos et al., 1995; Gedrich et al., 1996; Boucher et al., 1997; Yamamoto et al., 1998; Vanarsdale et al., 1997; Arch and Thompson, 1998; Kawamata et al., 1998) and the Epstein-Barr virus latent membrane protein, LMP1 (Mosialos et al., 1995). The finding that TRAF-3(−/−) lymphocytes are specifically defective in T-B lymphocyte collaboration, indiates that TRAF-3 gene products are required for signaling events that underlie this function (Xu et al., 1996). Full-length TRAF-3 alone is unlikely to account for such signaling, since over-expression of full-length TRAF-3 fails to induce NF-κB activation (Rothe et al., 1995; Takeuchi et al., 1996; Dadgostar and Cheng, 1998). However, alternative splicing of TRAF-3 transcripts generates mRNA species that encode at least 3 putative isoforms with altered Zn finger domains that may participate in transmitting receptor signals to the nucleus (Sato et al., 1995; Krajewski et al., 1997; van Eyndhoven et al., 1998). Therefore, the present study addressed whether TRAF-3 mRNA splice-deletion variants encode TRAF-3 protein isoforms that are able to induce NF-κB activation.
TRAF-3 is a member of the TRAF (TNF Receptor-associated factor) family of proteins, of which six have been identified (TRAF-1 through 6) (Rothe et al., 1994; Regnier et al., 1995; Ishida et al., 1996; Ishida et al., 1996; Kashiwada et al., 1998; Cheng et al., 1995; Hu et al., 1994; Sato et al., 1995; Mosialos et al., 1995). TRAF-3, like other TRAFs, appears to lack intrinsic catalytic activity, which suggests that TRAF-3 functions as a docking or adaptor molecule for other proteins that mediate signaling events. TRAF family members are related by significant homology in their carboxy-terminal TRAF-C domains (Rothe et al., 1994; Cheng et al., 1995). The TRAF-3 TRAF-C domain is known to be important for the interaction of TRAF-3 with the cytoplasmic tails of TNF-R family receptors (Cheng et al., 1995; Force et al., 1997; Vanarsdale et al., 1997), homo-oligomerization (Cheng et al., 1995; Force et al., 1997; Sato et al., 1995; Pullen et al., 1998) and binding to cytoplasmic proteins such as I-TRAF/TANK (Rothe et al., 1996; Cheng and Baltimore, 1996) and NIK (Song et al., 1997; Malinin et al., 1997). In addition to the TRAF-C domain, TRAF-3 contains an amino-terminal RING finger domain, five atypical Zn finger motifs, an iso-leucine zipper domain and a TRAF-N domain (Cheng et al., 1995; Hu et al., 1994; Sato et al., 1995; Mosialos et al., 1995). In TRAF-2 and TRAF-5, the RING finger and Zn finger domains have been shown to play important roles in mediating NF-κB activation (Takeuchi et al., 1996; Dadgostar and Cheng, 1998). The functional potentials of the TRAF-3 RING finger and Zn finger domains remain enigmatic, since TRAF-3 itself fails to induce NF-κB activation (Rothe et al., 1995; Takeuchi et al., 1996; Dadgostar and Cheng, 1998). However, the TRAF-3 RING finger domain is capable of supporting NF-κB activation in chimeric TRAF-3/5 molecules (Dadgostar and Cheng, 1998).
Alteration of the TRAF-3 Zn finger domain by alternative mRNA splicing was suggested by analysis of the sequences of the initial TRAF-3 cDNA clones isolated. One TRAF-3 cDNA clone (CAP1) contains a 75 bp deletion, relative to 3 other TRAF-3 cDNA clones (termed CRAF1, CD40 bp and LAP1) (Cheng et al., 1995; Hu et al., 1994; Sato et al., 1995; Mosialos et al., 1995). In addition, 2 other TRAF-3 mRNA variants containing 156 bp and 168 bp deletions were recently identified (van Eyndhoven et al., 1998). Each of the 3 mRNA species with deleted elements, encodes a putative TRAF-3 protein isoform with an altered number and composition of the Zn fingers. These putative isoforms have been termed, TRAF-3b (Δ25aa), TRAF-3c (Δ52aa) and TRAF-3d (Δ56aa) (Krajewski et al., 1997; van Eyndhoven et al., 1998). Characterization of the human TRAF-3 genomic structure indicated that these 3 TRAF-3 mRNA species result from alternative mRNA splicing (van Eyndhoven et al., 1998). Further analysis of the TRAF-3 gene suggested that a large number of additional splice variants may be generated, since each of the splice junctions of exons 4 through 10 are class “0”. Therefore, splice-deletion variants involving any or all of exons 5-10 would maintain open reading frames (ORFs) (van Eyndhoven et al., 1998). However, additional splice-deletion variants have not been characterized and the functions of the identified or predicted splice-deletion variants have not been studied.
SUMMARY OF THE INVENTION
The present invention provides an isolated TRAF-3 deletion isoform encoded by the nucleic acid sequence (SEQ ID NO: 1) shown in FIG. 15 (deletion isoform Δ130 nucleic acid). One embodiment of the present invention is an isolated TRAF-3 protein which comprises the sequence in FIG. 16 (SEQ ID NO: 2) (Δ130 protein). Another embodiment of the present invention is an isolated TRAF-3 deletion isoform encoded by the nucleic acid sequence (SEQ ID NO: 3) shown in FIG. 17 (deletion isoform Δ221 nucleic acid). A further embodiment of the present invention is an isolated TRAF-3 deletion isoform protein which comprises the sequence in FIG. 18 (SEQ ID NO: 4) (deletion isoform Δ221 protein)
The present invention also provides for a method of inhibiting activation by CD40 ligand of cells expressing CD40 on the cell surface, comprising contacting the cells with a TRAF-3 deletion isoform comprising the sequence shown in FIG. 18, the isoform protein being present in an amount effective to inhibit activation of the cells.
The present invention also provides for a method for inhibiting an autoimmune response in a subject which comprises administering to the subject an effective amount of a TRAF-3 deletion isoform protein comprising the sequence shown in FIG. 18 in an effective amount to inhibit an autoimmune response in a subject.
The invention also provides a method for identifying an agent that inhibits CD40-mediated cellular signaling in a cell which comprises: (a) contacting the cell with an agent under conditions wherein CD40-mediated cell activation occurs; and (b) determining whether CD40-mediated signaling is inhibited in the cell in the presence of the agent so as to identify whether the agent inhibits CD40-mediated cellular signaling.



DESCRIPTION OF THE FIGURES
FIG. 1: TRAF-3 mRNA species in Jurkat D1.1 cells. On top is a representation of the TRAF-3 genomic organization of the coding exons (3 through 12). Immediately below is a schematic representation of full-length TRAF-3 cDNA and the 5 predicted protein domains it encodes. Shown below are the 8 distinct mRNA splice-deletion species that encode putative TRAF-3 protein isoforms. All 8 cDNA clones lack mRNA segments from one or more exons that code for the Zn finger domain, thereby altering the composition and number of Zn fingers. TRAF-3 Δ25, Δ52 and Δ56 were previously identified and termed TRAF-3b, TRAF-3c and TRAF-3d, respectively (Krajewski et al., 1997; van Eyndhoven et al., 1998). Above TRAF-3 Δ25, two arrows depict the location of the primers used for PCR that allowed the isolation of the distinct TRAF-3 splice-deletion variant cDNAs from Jurkat T cell line D1.1 mRNA. The hatched bar depicts the location of the end-labeled probe (FF7GEN) used for detection of TRAF-3 specific PCR products.
FIG. 2: Expression of alternatively spliced TRAF-3 mRNA species in lymphoid cells. TRAF-3 specific RT-PCR was performed on poly (A)+ RNA from tonsilar B cells, and a panel of B cell (Ramos 2G6, Daudi, Raji and BA) and T cell tumor lines (D1.1, B2.7, H9 and CEM). PCR products were separated by gel electrophoresis and transferred onto membranes for Southern blot analysis using a TRAF-3 specific probe (FF7GEN). On the left are shown PCR products generated using cloned TRAF-3 cDNAs (full-length and isoforms) as templates, as indicated. On the right are shown the relative levels of expression of TRAF-3 mRNA species in the tonsilar B cells and in each of the B and T cell lines, as indicated.
FIG. 3: Expression of TRAF-3 protein isoforms in lymphoid cells. Cell lysates from tonsilar B cells, a panel of B cell (Ramos 2G6, Raji, BA and BJAB) and T cell tumors (D1.1, B2.7, H9, CEM) (50 μg of total protein) or transiently transfected 293T cells (15 μg of total protein) were separated by SDS-PAGE, transferred to a PVDF membrane and subjected to immunoblotting using anti-TRAF-3 (N-terminus) polyclonal antiserum (H-20). On the left are shown the overexpressed TRAF-3 protein isoforms in 293T cells as positive controls and on the right are shown the expression patterns of anti-TRAF-3 reactive protein species in lymphoid cells. The band migrating at 50 kD is non-specific (marked by an asterisk (*)) as are the bands at 85, 66 and 62 kD.
FIG. 4: Effect of over-expressing TRAF-3 splice-deletion variants and/or full-length TRAF-3 on NF-κB activation. 293T cells were transfected with 3 μg of expression vectors encoding TRAF-3 isoforms alone (open bars) or co-transfected with 3 μg of full-length TRAF-3 (black bars) As controls, 3 μg of TRAF-2 or 0.5 μg of CD40 expression vector was transfected alone or with 3 μg of full-length TRAF-3 expression vector. All samples were co-transfected with 0.3 μg of pRDII×4LUC (Firefly Luciferase) and 0.3 μg of pRL-TK (Renilla Luciferase). The amount of DNA transfected was equalized by the addition of empty pCEP4 vector. Luciferase activity was measured 40 h after transfection. All values were normalized by the individual Renilla Luciferase activity and are shown as the means of duplicate samples with error bars representing standard deviation. Results shown are representative of three independent experiments.
FIG. 5: Effect of full length TRAF-3 on TRAF-2 and CD40 mediated NF-κB activation. 293T cells were transfected with the indicated amounts of TRAF-2 or CD40 with 3 μg of TRAF-3 (full length) and 0.3 μg of pRDII×4LUC (Firefly luciferase) and 0.3 μg of pRL-TK (Renilla luciferase). The amount of DNA transfected was normalized for by adding empty pCEP4 vector. Cell lysates were prepared after 40 hours and used for determination of Luciferase activity. The open bars represent samples in which the expression vectors (as indicated) are transfected alone, whereas solid bars represent samples in which they are co-transfected with full length TRAF-3. All values were normalized by the individual Renilla luciferase activity and are shown as the means of duplicate samples with +/−S.D.
FIG. 6: Expression of co-expressed full length TRAF-3 and TRAF-3 splice-deletion isoforms in transfected 293T cells 293 T cells were transfected with 3 μg of full length TRAF-3 (full length or isoforms) or co-transfected with 3 μg of full length TRAF-3 and 3 μg of each of the TRAF-3 isoforms. 15 μg of total protein was separated by SDS-PAGE and subjected to immunoblotting using anti-TRAF-3 polyclonal antibody H-20. On the left are shown the cells over-expressing individual TRAF-3 isoforms. On the right are shown the cells over-expressing full length TRAF-3 in addition to TRAF-3 isoforms (as indicated on top). Asterixes indicate areas in which strong chenoluminescence signal resulted in “white-out” of the exposed film.
FIG. 7. Characterization of human TRAF-3 mRNA species in human tissues. Human tissue northern blots representing 15 tissues were hybridized with a 32P labeled cDNA probe containing the complete ORF of TRAF-3 (“P55”) or β-actin. Tissue origin and RNA molecular size markers are indicated on top and to the left, respectively.
FIGS. 8A-C. Expression of human TRAF-3 mRNA in B and T cell lines. (FIG. 8A) Poly (A)+ RNA from several B cell lines (BA, Ramos 2G6, Daudi, Raji) and several T cell lines (B2.7, D1.1, CEM, H9) were hybridized with the “P55” probe or β-actin probe. (FIG. 8B) The same blot hybridized with a 32P labeled cDNA probe representing the 3′-terminal 1.8 kb of exon 12 (“3′UTR”). (FIG. 8C) Shown is a schematic representation of the human TRAF-3 cDNA and the nucleotide homology to murine TRAF-3 (TRAFamn). The percentage homolgy between human and murine TRAF-3 is depicted by the color or hatching of discrete regions: solid black regions share 85% or more homology, gray regions share approximately 75% and the hatched areas shares 50% or less homology. The relationship of the “P55” and the “3′UTR” probes to the structure of the 8 kb mRNA is depicted below. Asterixes represent the polyadenylation signals at nt positions 2323, 2401 and 7617. Closed circles represent the AUUUA elements at nt positions 1944, 2133, 2335, 2529, 2774 and 7502.
FIG. 9. Alternative splicing in the Zn finger domain of human TRAF-3. Shown on top is a representation of the genomic organization of the coding exons (3 through 12). Shown below is the TRAF-3 cDNA and the 5 protein domains it encodes. Shown on the bottom are three different cDNA clones that code for putative isoforms of TRAF-3 by alternatively splicing of exons 7, 8 and 9, resulting in variation in the composition of the individual Zn finger domains. Referring to the Zn finger number of full length TRAF-3, the Zn finger “4-5” in TRAF-3b results from the fusion of the C-terminal half of finger 4 and the N-terminal half of finger 5. Similarly, the Zn finger “3-5” in TRAF-3c results from the fusion of the C-terminal half of finger 3 and the N-terminal half of finger 5.
FIG. 10. Alternative splicing in the 5′UTR of human TRAF-3. Shown on top is a representation of the genomic organization of the 5′UTR exons (exons 1a, 1b and 2) and the first coding exon (exon 3). Shown below are three different cDNA clones. cDNA clone “IIIb” and “Ib” start in exon 1b, while cDNA clone “2-1” starts in exon 1a. The IIIb cDNA clone does not contain exon 2. All cDNA clones identified contain the translational start site in exon 3 and the ORF coding for TRAF-3, represented by the ATG and arrow after exon 3, respectively.
FIGS. 11A-B. FISH analysis of human TRAF-3. (FIG. 11A) Metaphase spread showing hybridization of the TRAF-3 genomic probe (arrows) to the telomeric region of chromosome 14. (FIG. 11B) Metaphase spread from B cell line Ramos 2G6, carrying the Burkitt's lymphoma translocation t(8; 14)(q24.1; q32.3). The TRAF-3 probe is labeled with biotin and detected with streptavidin-rhodamine (red) while a telomeric 14qter probe is labeled with digoxigenin and detected with anti-digoxigenin-FITC (green). On the normal chromosome 14 the signals of the two probes are in close proximity and the level of resolution is not sufficient to establish order. However, the 14qter probe is translocated to derivative chromosome 8, whereas the TRAF-3 probe remains on the derivative chromosome 14. Therefore, the gene for TRAF-3 is located centromeric to the Burkitt's breakpoint in 14q32.3.
FIG. 12. Location of human TRAF-3 on chromosome 14q32.3. Shown is a representation of the telomeric region of chromosome 14 and the location of the gene for TRAF-3 in context of the Whitehead radiation hybrid map and the physical map of 4 overlapping PAC clones. Genomic markers are shown with their assigned position in parentheses. Genes that are represented by or linked to the genomic markers are listed below each marker.
FIG. 13. Physical map of the human TRAF-3 gene. Shown on top is a representation of the exon distribution of the TRAF-3 gene with positions of the restriction sites of NotI and SalI and two other genomic markers, KIAA0071 and SGC30775. Below are 4 overlapping PAC clones that span the complete TRAF-3 gene. Exons are indicated by bold lines for TRAF-3, KIAA0071 and SGC30775.
FIG. 14. Exon-intron structure of the human TRAF-3 gene. Shown are the exon numbers and their respective position in the TRAF-3 (CRAP-1) cDNA clone (as reported under GenBank accession number U21092). The exon sequences are in upper case, while intron sequences are in lower case. Splice donor (GT) and splice acceptor (AG) are in bold. Splice donors for exon 1a (SEQ ID NO:15); exon 1b (SEQ ID NO:16); exon 2 (SEQ ID NO:18); exon 3 (SEQ ID NO:20); exon 4 (SEQ ID NO:22); exon 5 (SEQ ID NO:24); exon 6 (SEQ ID NO:26); exon 7 (SEQ ID NO:28); exon 8 (SEQ ID NO:30); exon 9 (SEQ ID NO:32); exon 10 (SEQ ID NO:34); and exon 11 (SEQ ID NO:36) are shown. Splice acceptors for exon 2 (SEQ ID NO:17); exon 3 (SEQ ID NO:19); exon 4 (SEQ ID NO:21); exon 5 (SEQ ID NO:23); exon 6 (SEQ ID NO:25); exon 7 (SEQ ID NO:27); exon 8 (SEQ ID NO:29); exon 9 (SEQ ID NO:31); exon 10 (SEQ ID NO:33); exon 11 (SEQ ID NO:35); and exon 12 (SEQ ID NO:37) are shown. Splice junction class 0: exon junction falls between two codons; class 1: exon junction falls after first nucleotide of a codon; class 2: exon junction falls after second nucleotide of a codon. The IIIb cDNA clone ends at nt 2433 while exon 12 extends an additional 3855 bp in the 8 kb transcripts.
FIG. 15. Nucleotide sequence of Δ130 TRAF-3 deletion isoform (SEQ ID NO.: 1).
FIG. 16. Amino acid sequence of Δ130 TRAF-3 deletion isoform (SEQ ID NO.: 2).
FIG. 17. Nucleotide sequence of Δ221 TRAF-3 deletion isoform (SEQ ID NO.: 3).
FIG. 18. Amino acid sequence of Δ221 TRAF-3 deletion isoform (SEQ ID NO.: 4).
FIG. 19. RNAse protection analysis of TRAF-3 mRNA splice variant expression. (A) A schematic representation of the TRAF-3 genomic sequence, with exons represented as wide black bars, is mapped onto the protein domains encoded by full-length TRAP-3 cDNA. Below are depicted the protected probe fragments resulting from hybridization of an anti-sense probe corresponding to the full-length TRAF-3 mRNA with all previously identified alternative splice forms of TRAF-3 mRNA. As indicated, most “secondary” protected fragments, may have contributions from multiple splice-variants. (B), RNAse protection of the indicated samples was performed using an antisense human GAPDH probe as described in Materials and Methods. The indicated band (absent from the yeast mRNA sample) corresponds to the expected 316 nt protected fragment resulting from hybridization of the probe with GAPDH mRNA. (C), TRAP-3 splice variant RNAse protection assay for Yeast tRNA (negative control), Ramos CC total RNA (negative control), and Jurkat D1.1 total RNA (positive control) using indicated probes. (Full-length TRAF-3 probe is indicated by “FL”.) (D), TRAF-3 splice variant RNAse protection assay for BJAB, Daudi, and Raji total RNA using the indicated probes. In (C) and (D), diluted probes were run as size markers at the left and right of each gel. Displayed below each gel is the signal from each sample resulting from hybridization of probes to the internal control sense TRAF-3 RNA. Primary fragments corresponding to hybridization of a given probe with its complementary mRNA splice-variant are indicated by an asterisk (*). The asterisk in each Δ130 lane marks the expected position for the Δ130 primary fragment. An unexpected TRAF-3 specific band with an approximate size of 165 nt was protected by the probes FL, Δ25, Δ27, Δ52, Δ56, and Δ83 and is indicated by a cross (). Data shown are representative of three independent experiments.
FIG. 20. Relative expression of TRAF-3 splice isoforms. Phosphorimaging analysis of the RNAse protection assay shown in FIG. 19 as described in Materials and Methods. Data are presented as arbitrary units of TRAF-3 expression, scaled to the expression of full length TRAF-3 in Jurkat D1.1 as 100.
FIG. 21. Effect of TRAF-3 splice-variants on NF-κB signaling in BJAB cells. BJAB cells were transiently transfected with the indicated expression vectors by electroporation. Thirty-six hours after transfection, cells were harvested and NF-κB dependent luciferase activity was measured. Data are the mean of triplicate samples, scaled to signals from control, LacZ transfected cells. Error bars represent standard deviation. This experiment is representative of three independent experiments.



DETAILED DESCRIPTION
This invention provides for an isolated TRAF-3 deletion isoform protein encoded by the nucleic acid sequence shown in of FIG. 15 (SEQ ID NO.: 1)(Δ130 TRAF-3 isoform) or FIG. 17 (Δ221 TRAF-3 isoform) (SEQ ID NO.: 3).
The TRAF-3 deletion isoform protein may comprise the sequence shown in either FIG. 16 (Δ130 TRAF-3 isoform) (SEQ ID NO.: 2) or FIG. 18 (Δ221 TRAF-3 isoform) (Seq I.D. No. 4) capable of inhibiting CD40-mediated cell activation.
The present invention provides new TRAF-3 deletion isoforms which were not known to exist previously, although there has been a description of a few splice deletion isoforms of TRAF-3 in PCT International Application No. PCT/US97/05076, PCT International Publication No. WO 97/34473. The present invention provides several new isoforms which have been shown to actually be expressed in cells (i.e., not a theoretical isoform, but one that actually is produced by living cells) and which have been shown to modulate the CD40-mediated cell activation pathway.
The nucleic acid may be an isolated nucleic acid or a purified nucleic acid which nucleic acid is separated from cellular particles substantially. Such isolation or purification would be known to one of skill in the art, see Sambrook, et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989).
This invention provides for alleles of the TRAF-3 deletion isoform nucleic acids. This invention provides for human TRAF-3 deletion isoform nucleic acid (with a nucleotide sequence as shown in FIG. 15 or 17) and also provides for the homologous murine TRAF-3 nucleic acid, as well as the homologous TRAF-3 nucleic acids in other species of animals.
The present invention also provides for a TRAF-3 deletion isoform nucleic acid molecule linked to a vector. The vector may be a self-replicating vector or a replicative incompetent vector. The vector may be a pharmaceutically acceptable vector for methods of gene therapy. An example of replication incompetent vector is LNL6 (Miller, A. D. et al. (1989) BioTechniques 7:980-990).
The present invention provides an isolated protein comprising a TRAF-3 deletion isoform which comprises the amino acid sequence shown in FIG. 16 (SEQ ID NO.: 2). The present invention also provides an isolated protein comprising a TRAF-3 deletion isoform which comprises the amino acid shown in FIG. 18 (SEQ ID NO.: 4).
One embodiment of the present invention is an isolated nucleic acid molecule encoding the TRAF-3 deletion isoform protein. In one embodiment, the nucleic acid molecule is DNA or cDNA. The present invention also provides a vector comprising the nucleic acid molecule operably linked to a transcriptional control sequence recognized by a host cell transformed with the vector. In one embodiment, the vector is a plasmid.
The present invention also provides a method for identifying an agent that inhibits CD40-mediated cellular signaling in a cell which comprises: (a) contacting the cell with an agent under conditions wherein CD40-mediated cell activation occurs; and (b) determining whether CD40-mediated signaling is inhibited in the cell in the presence of the agent so as to identify whether the agent inhibits CD40-mediated cellular signaling. In one embodiment, the agent is a small molecule capable of inactivating a TRAF-3 deletion isoform. In one embodiment, the agent is a competitive inhibitor of a TRAF-3 deletion isoform. In one embodiment, the TRAF-3 deletion isoform is a protein having the amino acid sequence shown in FIG. 16 (SEQ ID NO.: 2) or FIG. 18 (SEQ ID NO.: 4). In one embodiment, the agent is a peptide or a nucleic acid. In one embodiment, the agent is an organic molecule. In one embodiment, the agent is a chiral compound or a racemic mixture of enantiomers. In one embodiment, the cell is a cell which overexpresses the TRAF-3 deletion isoform Δ130 protein having the amino acid sequence shown in FIG. 16 (SEQ ID. NO.: 2). In one embodiment, the cell is a 293T cell.
The present invention also provides a compound identified as an inhibitor of CD40-mediated cell activation.
The present invention also provides a method of inhibiting activation by CD40 ligand of cells expressing CD40 on the cell surface, comprising providing the cells with a TRAF-3 deletion isoform protein or an agent identified by the above method, the protein or agent being present in an amount effective to inhibit activation of the cells.
In one embodiment, the cells are provided with the protein by introducing into the cells a nucleic acid sequence encoding the protein under conditions such that the cells express an amount of the protein effective to inhibit activation of the cells.
In one embodiment, the nucleic acid sequence is operably linked to a transcriptional control sequence recognized by the cell. In one embodiment, the nucleic acid sequence is a plasmid. In one embodiment, the CD40-bearing cells are selected from the group consisting of B cells, fibroblasts, endothelial cells, epithelial cells, T cells, basophils, macrophages, Reed-Steinberg cells, dendritic cells, renal cells, and smooth muscle cells.
In one embodiment, the B cells are resting B cells, primed B cells, myeloma cells, lymphocytic leukemia B cells, or B lymphoma cells.
In one embodiment, the epithelial cells are keratinocytes. In one embodiment, the fibroblasts are synovial membrane fibroblasts, dermal fibroblasts, pulmonary fibroblasts, or liver fibroblasts.
In one embodiment, the renal cells are selected from the group consisting of glomerular endothelial cells, mesangial cells, distal tubule cells, proximal tubule cells, parietal epithelial cells, visceral epithelial cells, cells of a Henle limb, and interstitial inflammatory cells. In one embodiment, the parietal epithelial cells are crescent parietal epithelial cells.
In one embodiment, the smooth muscle cells are smooth muscle cells of the bladder, vascular smooth muscle cells, aortic smooth muscle cells, coronary smooth muscle cells, pulmonary smooth muscle cells, or gastrointestinal smooth muscle cells. In one embodiment, the gastrointestinal smooth muscle cells are esophageal smooth muscle cells, stomachic smooth muscle cells, smooth muscle cells of the small intestine, or smooth muscle cells of the large intestine.
The present invention provides a method of providing a subject with an amount of a TRAF-3 deletion isoform protein or an agent identified by the screening method hereinabove, effective to inhibit activation by CD40 ligand of cells bearing CD40 on the cell surface in the subject, comprising: introducing into CD40-bearing cells of the subject, a nucleic acid sequence encoding the protein or an agent identified by the screening method, under conditions such that the cells express in the subject an activation inhibiting effective amount of the protein.
In one embodiment, the introducing of the nucleic acid into cells of the subject comprises: a) treating cells of the subject ex vivo to insert the nucleic acid sequence into the cells; and b) introducing the cells from step a) into the subject. In one embodiment, the subject is a mammal. In one embodiment, the mammalian subject is a human.
The present invention provides a method of treating a condition characterized by an aberrant or unwanted level of CD40-mediated intracellular signaling, in a subject, comprising providing the subject with a therapeutically effective amount of a TRAF-3 deletion isoform protein or an agent identified by the screening method hereinabove, capable of inhibiting CD40-mediated intracellular signaling in cells bearing CD40 on the cell surface.
In one embodiment, the protein is provided by introducing into CD40-bearing cells of the subject, a nucleic acid sequence encoding the protein under conditions such that the cells express the protein. In one embodiment, the condition is organ rejection in a subject receiving transplant organs, or an immune response in a subject receiving gene therapy. In one embodiment, the transplant organ is a kidney, heart or liver. In one embodiment, the condition is a CD40-dependent immune response. In one embodiment, the CD40-dependent immune response is an autoimmune response in a subject suffering from an autoimmune disease. In one embodiment, the autoimmune disease comprises rheumatoid arthritis, Myasthenia gravis, systemic lupus erythematosus, Graves' disease, idiopathic thrombocytopenia purpura, hemolytic anemia, diabetes mellitus, a drug-induced autoimmune disease, psoriasis, or hyper IgE syndrome. In one embodiment, the drug-induced autoimmune disease is drug-induced lupus. In one embodiment, the immune response comprises induction of CD23, CD80 upregulation, rescue from CD95-mediated apoptosis, rescue from apoptosis in a subject undergoing chemotherapy against a tumor, or autoimmune manifestations of an infectious disease. In one embodiment, the autoimmune manifestations are derived from Reiter's syndrome, spondyloarthritis, Lyme disease, HIV infections, syphilis or tuberculosis. In one embodiment, the condition is an allergic response. In one embodiment, the condition is selected from the group consisting of atherosclerosis, reperfusion injury, allograft rejection, organ rejection, and chronic inflammatory autoimmune diseases. In one embodiment, the atherosclerosis is accelerated atherosclerosis associated with organ transplantation. In one embodiment, the chronic inflammatory autoimmune disease is vasculitis, rheumatoid arthritis, scleroderma, or multiple sclerosis. In one embodiment, the condition is dependent on CD40 ligand-induced activation of epithelial cells. In one embodiment, the epithelial cells are keratinocytes, and the condition is psoriasis. In one embodiment, the condition is an inflammatory kidney disease.
The present invention provides for TRAF-3 deletion isoform nucleic acid which is produced by polymerase chain reaction (PCR). An isolated TRAF-3 deletion isoform nucleic acid may be isolated by using PCR. Such reactions are well known to one of skill in the art. [U.S. Pat. Nos. 4,754,065; 4,800,159;; 4,683,195 and 4,683,202 provide PCR techniques and methods and these U.S. patents are hereby incorporated by reference in their entirties.]
In another embodiment of the present invention TRAF-3 deletion isoform nucleic acid may also be a synthetic nucleic acid or a mimetic of a nucleic acid which may have increased bioavailability, stability, potency or decreased toxicity. Such synthetic nucleic acids may have alterations of the basic A, T, C or G or U bases or sugars which make up the nucleotide polymer to as to alter the effect of the nucleic acid.
As used herein, “variants” encompass the following: Variants can differ from naturally occurring TRAF-3 peptide in amino acid sequence or in ways that do not involve sequence, or both. Variants in amino acid sequence are produced when one or more amino acids are substituted with a different natural amino acid, an amino acid derivative or non-native amino acid. When a nucleic acid molecule encoding the protein is expressed in a cell, one naturally occurring amino acid will generally be substituted for another. Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics such as substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. The non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
Other conservative substitutions can be taken from Table A, and yet others are described by Dayhoff in the Atlas of Protein Sequence and Structure (1988).







TABLE A







Conservative Amino Acid Replacements











For Amino Acid
Code
Replace with any of







Alanine
A
D-Ala, Gly, beta-ALa,





L-Cys, D-Cys



Arginine
R
D-Arg, Lys, homo-Arg,





D-homo-Arg, Met, D-Met,





Ile, D-Ile,





Orn, D-Orn



Asparagine
N
D-Asn, Asp, D-Asp, Glu,





D-Glu, Gln, D-Gln



Aspartic Acid
D
D-Asp, D-Asn, Asn, Glu,





D-Glu, Gln, D-Gln



Cysteine
C
D-Cys, S-Me-Cys, Met,





D-Met, Thr, D-Thr



Glutamine
Q
D-Gln, Asn, D-Asn, Glu,





D-Glu, Asp, D-Asp



Glutamic Acid
E
D-Glu, D-Asp, Asp, Asn,





D-Asn, Gln, D-Gln



Glycine
G
Ala, D-Ala, Pro, D-Pro,





Beta-Ala, Acp



Isoleucine
I
D-Ile, Val, D-Val, Leu,





D-Leu, Met, D-Met



Leucine
L
D-Leu, Val, D-Val, Met,





D-Met



Lysine
K
D-Lys, Arg, D-Arg, homo-





Arg, D-homo-Arg, Met,





D-Met, Ile, D-





Ile, Orn, D-Orn



Methionine
M
D-Met, S-Me-Cys, Ile,





D-Ile, Leu, D-Leu, Val,





D-Val, Norleu



Phenylalanine
F
D-Phe, Tyr, D-Thr,





L-Dopa, His, D-





His, Trp, D-Trp, Trans





3,4 or 5-phenylproline,





cis 3,4 or 5





phenylproline



Proline
P
D-Pro, L-I-thioazolidine-4-





carboxylic acid, D- or L-1-





oxazolidine-4-





carboxylic acid



Serine
S
D-Ser, Thr, D-Thr,





allo-Thr, Met, D-Met,





Met(O), D-Met(O),





Val, D-Val



Threonine
T
D-Thr, Ser, D-Ser,





allo-Thr, Met, D-Met,





Met(O) D-Met(O),





Val, D-Val



Tyrosine
Y
D-Tyr, Phe, D-Phe,





L-Dopa, His, D-His



Valine
V
D-Val, Leu, D-Leu, Ile,





D-Ile, Met, D-Met










Other variants within the invention are those with modifications which increase peptide stability. Such variants is may contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the peptide sequence. Also included are: variants that include residues other than naturally occurring L-amino acids, such as D-amino acids or non-naturally occurring or synthetic amino acids such as beta or gamma amino acids and cyclic variants. Incorporation of D- instead of L-amino acids into the polypeptide may increase its resistance to proteases. See, e.g., U.S. Pat. No. 5,219,990.
The protein of this invention may also be modified by various changes such as insertions, deletions and substitutions, either conservative or nonconservative where such changes might provide for certain advantages in their use.
In other embodiments, variants with amino acid substitutions which are less conservative may also result in desired derivatives, e.g., by causing changes in charge, conformation and other biological properties. Such substitutions would include for example, substitution of hydrophilic residue for a hydrophobic residue, substitution of a cysteine or proline for another residue, substitution of a residue having a small side chain for a residue having a bulky side chain or substitution of a residue having a net positive charge for a residue having a net negative charge. When the result of a given substitution cannot be predicted with certainty, the derivatives may be readily assayed according to the methods disclosed herein to determine the presence or absence of the desired characteristics.
Just as it is possible to replace substituents of the scaffold, it is also possible to substitute functional groups which decorate the scaffold with groups characterized by similar features. These substitutions will initially be conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. Non-sequence modifications may include, for example, in vivo or in vitro chemical derivatization of portions of the protein of this invention, as well as changes in acetylation, methylation, phosphorylation, carboxylation or glycosylation.
In a further embodiment the protein is modified by chemical modifications in which activity is preserved. For example, the proteins may be amidated, sulfated, singly or multiply halogenated, alkylated, carboxylated, or phosphorylated. The protein may also be singly or multiply acylated, such as with an acetyl group, with a farnesyl moiety, or with a fatty acid, which may be saturated, monounsaturated or polyunsaturated. The fatty acid may also be singly or multiply fluorinated. The invention also includes methionine analogs of the protein, for example the methionine sulfone and methionine sulfoxide analogs. The invention also includes salts of the proteins, such as ammonium salts, including alkyl or aryl ammonium salts, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate, thiosulfate, carbonate, bicarbonate, benzoate, sulfonate, thiosulfonate, mesylate, ethyl sulfonate and benzensulfonate salts.
Use of TRAP-3 Deletion Isoforms in Developing Immunomodulatory Agents
TRAF-3 is a cytoplasmic signaling molecule that interacts with the CD40 cytoplasmic tail (and cytoplasmic tails of other TNF-R family members). Full length TRAF-3 inhibits CD40 signaling, but the present invention shows that a number of splice deletion isoforms that are involved in transmitting the CD40 cellular signal. In looking at the nucleotide full length sequence of TRAF-3, one could predict about 65 splice isoforms based on splice donor and acceptor sequences. However, the present invention provides for several different splice isoforms of TRAF-3 which play a role in the activation of a cell based on CD40 signaling. For example, the Experimental Details section hereinbelow describes more fully the following active deletion isoforms: Δ56, Δ83, Δ103, Δ130 and Δ221. The present invention provides for use of these isoforms in identifying inhibitors of CD40 signaling. The present invention provides a method for identifying an agent that inhibits CD40 cellular signaling which comprises (a) contacting a cell with an agent which inhibits the acivity of a TRAF-3 deletion isoform; (b) determining whether CD40 signaling occurs in the cell in the presence of the agent so as to identify whether the agent inhibits CD40 cellular signaling. This application includes evidence that certain TRAF-3 isoforms are expressed in cells and are active in CD40 cell activation assays. Therefore, it is preferable that an agent is capable of inhibiting either the TRAF-3 deletion isoform termed Δ130 or the isoform termed Δ221.
Currently, administration of anti-CD40 ligand antibody (e.g., CD154) to a subject is one therapy to inhibit CD40 mediated cellular activation. Relatively large doses of such an antibody are necessary to achieve a blockade of the CD40 pathway. There is a lack of specific inhibitors of the CD40 signaling pathway for use in treating autoimmune diseases or transplant rejection in subjects. This invention provides a new assay useful for identifying agents which would be useful as specific inhibitors of CD40 signaling in a cell. In one embodiment, the assay utilizes a cell which is contacted by an agent and a determination is then made as to whether the agent is capable of inhibiting or modulating the CD40-mediated cell actiation. One can measure upregulation of NF-κB or CD23, for example, to measure the level of cell activation. In another embodiment, the assay utlizes a cell line which overexpresses the TRAF-3 deletion isoform, Δ130 (which hereinbelow is shown to activate cellular activation). The assay comprises contacting the protein isoform with a library of compounds and determining which compounds bind to the TRAF-3 isoforms. These compounds are lead compounds and are then tested for their ability to inhibit cellular activation.
As used herein “small molecule inhibitors” is encompassed by the following characteristics: capable of being administered to a subject with bioavailability so that it is effective at the intracellular level. For example, a nucleic acid molecule that is capapble of being transfected into cells and ultimately inhibiting CD40 mediated cell activation is a small molecule inhibitor. Another example, is a small peptide, a peptidemimetic; a fragment of an antibody, or a small organic molecule which mimics a region of a TRAF-3 deletion isoform. A small molecule inhibitor also includes a chiral form of a small molecule when the molecule is found to be a racemic mixture.
The present invention provides for an assay for identifying an agent which inhibits CD40 mediated cell activation which comprises contacting a cell capable of undergoing CD40 mediated cell activation with an agent; determining whether the cell is activated in the presence of the agent under conditions which normally produce cell activation; identifying the agent as either capable of inhibiting cell activation or not so capable.
The present invention provides for an agent which inhibits the CD40 cell activation pathway. In one embodiment of the invention, the agent is be a small molecule. In another embodiment, the agent is a mimic of a region of TRAF-3 Δ130 protein or TRAF-3 Δ221 protein sequence as shown in FIGS. 16 and 18. In one embodiment, these isoforms are used in an assay to identify agents which modulate the immune response in a subject. In one embodiment of the present invention, the agent is administered to a subject in a therapeutically effective amount to treat an autoimmune disease or to treat transplant rejection.
This invention provides a method of inhibiting activation by CD40 ligand of cells bearing CD40 on the cell surface, comprising providing the cells with a TRAF-3 deletion isoform protein, the protein being present in an amount effective to inhibit activation of the cells, wherein the TRAF-3 deletion isoform protein is either Δ130 or Δ221 as shown in FIGS. 16 or 18 respectfully.
The present invention further provides a method of inhibiting activation by CD40 ligand of cells expressing CD40 on the cell surface, comprising providing the cells with the peptide of the invention, the peptide being present in an amount effective to inhibit activation of the cells.
In one embodiment, the cells are provided with the peptide by introducing into the cells a nucleic acid sequence encoding the peptide under conditions such that the cells express an amount of the peptide effective to inhibit activation of the cells.
In another embodiment, the nucleic acid sequence is operably linked to a transcriptional control sequence recognized by the cell. The nucleic acid sequence may be a plasmid. The CD40-bearing cells may be selected from the group consisting of B cells, fibroblasts, endothelial cells, epithelial cells, T cells, basophils, macrophages, Reed-Steinberg cells, dendritic cells, renal cells, and smooth muscle cells. The B cells may be resting B cells, primed B cells, myeloma cells, lymphocytic leukemia B cells, or B lymphoma cells. The epithelial cells may be keratinocytes. The fibroblasts may be synovial membrane fibroblasts, dermal fibroblasts, pulmonary fibroblasts, or liver fibroblasts.
The renal cells may be selected from the group consisting of glomerular endothelial cells, mesangial cells, distal tubule cells, proximal tubule cells, parietal epithelial cells, visceral epithelial cells, cells of a Henle limb, and interstitial inflammatory cells.
The parietal epithelial cells may be crescent parietal epithelial cells.
The smooth muscle cells may be smooth muscle cells of the bladder, vascular smooth muscle cells, aortic smooth muscle cells, coronary smooth muscle cells, pulmonary smooth muscle cells, or gastrointestinal smooth muscle cells.
The gastrointestinal smooth muscle cells may be esophageal smooth muscle cells, stomachic smooth muscle cells, smooth muscle cells of the small intestine, or smooth muscle cells of the large intestine.
The invention provides a method of providing a subject with an amount of a TRAF-3 deletion isoform peptide effective to inhibit activation by CD40 ligand of cells bearing CD40 on the cell surface in the subject, comprising: introducing into CD40-bearing cells of the subject, a nucleic acid sequence encoding the TRAF-3 deletion isoform peptide, under conditions such that the cells express in the subject an activation inhibiting effective amount of the peptide.
The introducing of the nucleic acid into cells of the subject may comprise a) treating cells of the subject ex vivo to insert the nucleic acid sequence into the cells; and b) introducing the cells from step a) into the subject.
The subject may be a mammal. The mammalian subject may be a human.
An embodiment of the present invention is a method of treating a condition characterized by an aberrant or unwanted level of CD40-mediated intracellular signaling, in a subject, comprising providing the subject with a therapeutically effective amount of a TRAF-3 deletion isoform peptide capable of inhibiting CD40-mediated intracellular signaling in cells bearing CD40 on the cell surface.
The peptide may be provided by introducing into CD40-bearing cells of the subject, a nucleic acid sequence encoding the peptide under conditions such that the cells express the peptide according to this method.
The condition may be organ rejection in a subject receiving transplant organs, or an immune response in a subject receiving gene therapy. The transplant organ may be a kidney, heart or liver. The condition may be a CD40-dependent immune response. The CD40-dependent immune response may be an autoimmune response in a subject suffering from an autoimmune disease.
The autoimmune disease may comprise rheumatoid arthritis, Myasthenia gravis, systemic lupus erythematosus, Graves' disease, idiopathic thrombocytopenia purpura, hemolytic anemia, diabetes mellitus, a drug-induced autoimmune disease, psoriasis, or hyper IgE syndrome.
The drug-induced autoimmune disease may be drug-induced lupus. The immune response may comprise induction of CD23, CD80 upregulation, rescue from CD95-mediated apoptosis, rescue from apoptosis in a subject undergoing chemotherapy against a tumor, or autoimmune manifestations of an infectious disease.
The autoimmune manifestations may be derived from Reiter's syndrome, spondyloarthritis, Lyme disease, HIV infections, syphilis or tuberculosis. The condition may be an allergic response. The allergic response may be hay fever or a penicillin allergy. The condition may be dependent on CD40 ligand-induced activation of fibroblast cells. The condition may be selected from the group consisting of arthritis, scleroderma, and fibrosis. The arthritis may be rheumatoid arthritis, non-rheumatoid inflammatory arthritis, arthritis associated with Lyme disease, or osteoarthritis. The fibrosis may be pulmonary fibrosis, hypersensitivity pulmonary fibrosis, or a pneumoconiosis. The pulmonary fibrosis may be pulmonary fibrosis secondary to adult respiratory distress syndrome, drug-induced pulmonary fibrosis, idiopathic pulmonary fibrosis, or hypersensitivity pneumonitis. The pneumoconiosis may be asbestosis, siliconosis, or Farmer's lung. The fibrosis may be a fibrotic disease of the liver or lung. The fibrotic disease of the lung may be caused by rheumatoid arthritis or scleroderma. The fibrotic disease of the liver may be selected from the group consisting of: Hepatitis-C; Hepatitis-B; cirrhosis; cirrhosis of the liver secondary to a toxic insult; cirrhosis of the liver secondary to drugs; cirrhosis of the liver secondary to a viral infection; and cirrhosis of the liver secondary to an autoimmune disease. The toxic insult may be alcohol consumption. The viral infection may be Hepatitis B, Hepatitis C, or hepatitis non-B non-C. The autoimmune disease may be primary biliary cirrhosis, or Lupoid hepatitis. The condition may he dependent on CD40 ligand-induced activation of endothelial cells. The condition may be selected from the group consisting of atherosclerosis, reperfusion injury, allograft rejection, organ rejection, and chronic inflammatory autoimmune diseases.
The atherosclerosis may be accelerated atherosclerosis associated with organ transplantation.
The chronic inflammatory autoimmune disease may be vasculitis, rheumatoid arthritis, scleroderma, or multiple sclerosis.
The condition may be dependent on CD40 ligand-induced activation of epithelial cells.
In one embodiment of the invention, the epithelial cells are keratinocytes, and the condition is psoriasis.
The condition may be an inflammatory kidney disease. The inflammatory kidney disease may not be initiated by autoantibody deposition in kidney. The kidney disease may be selected from the group consisting of: membranous glomerulonephritis; minimal change disease/acute tubular necrosis; pauci-immune glomerulonephritis; focal segmental glomerulosclerosis; interstitial nephritis; antitissue antibody-induced glomerular injury; circulating immune-complex disease; a glomerulopathy associated with a multisystem disease; and drug-induced glomerular disease.
The antitissue antibody-induced glomerular injury may be anti-basement membrane antibody disease.
The present invention provides for an isolated nucleic acid molecule encoding a TRAF-3 deletion isoform peptide. The nucleic acid molecule may be DNA, RNA, cDNA, recombinant DNA, or mRNA.
One embodiment of the invention is a vector comprising the TRAF-3 deletion isoform nucleic acid molecule or variants or isoforms thereof operably linked to a transcriptional control sequence recognized by a host cell transformed with the vector. The vector may be a plasmid.
The present invention provides for a method of providing a subject with an immunosuppressant effective amount of an abnormal TRAP-3, comprising:introducing into CD40-bearing cells of the subject, a nucleic acid sequence encoding an abnormal TRAF-3 polypeptide, under conditions such that the cells express in the subject an immunosuppressant effective amount of the abnormal TRAF-3.
In embodiments of the methods described herein, the CD40-bearing cells are selected from the group consisting of B cells, fibroblasts, endothelial cells, epithelial cells, T cells, basophils, macrophages, Reed-Steinberg cells, dendritic cells, myeloma cells, renal cells, and smooth muscle cells.
In a more specific embodiment the B cells are resting B cells, primed B cells, myeloma cells, lymphocytic leukemia B cells, or B lymphoma cells. In another specific embodiment the epithelial cells are keratinocytes. In another embodiment the fibroblasts are synovial membrane fibroblasts, dermal fibroblasts, pulmonary fibroblasts, or liver fibroblasts. In another specific embodiment the renal cells are selected from the group consisting of glomerular endothelial cells, mesangial cells, distal tubule cells, proximal tubule cells, parietal epithelial cells (e.g., crescent parietal epithelial cells), visceral epithelial cells, cells of a Henle limb, and interstitial inflammatory cells. In another embodiment the smooth muscle cells are smooth muscle cells of the bladder, vascular smooth muscle cells, aortic smooth muscle cells, coronary smooth muscle cells, pulmonary smooth muscle cells, or gastrointestinal smooth muscle cells. In a more specific embodiment the gastrointestinal smooth muscle cells are esophageal smooth muscle cells, stomachic smooth muscle cells, smooth muscle cells of the small intestine, or smooth muscle cells of the large intestine.
In an embodiment of this invention the introducing of the nucleic acid into cells of the subject comprises: a) treating cells of the subject ex vivo to insert the nucleic acid sequence into the cells; and b) introducing the cells from step a) into the subject.
The subject which can be treated by the methods described herein is an animal. Preferably the animal is a mammal. Subjects specifically intended for treatment with the method of the invention include humans, as well as nonhuman primates, sheep, horses, cattle, goats, pigs, dogs, cats, rabbits, guinea pigs, hamsters, gerbils, rats and mice, as well as the organs, tumors, and cells derived or originating from these hosts.
In an embodiment the condition is organ rejection in a subject receiving transplant organs. Examples of suitable transplant organs include a kidney, heart or liver, as well as others known to those of skill in the art. In another embodiment the condition is an immune response in a subject receiving gene therapy. One difficulty encountered in gene therapy is an immune response by the patient to the gene therapy vector and the proteins it expresses (Yang, Y. et al. (1996) J. Virol. 70:6370). Because the protein of this invention inhibits the immune response, gene therapy with the protein of this invention does not trigger an immune response. Its immunosuppressant effect also makes it useful as an adjunct to other forms of gene therapy. For example, at the same time that a vector being administered to provide a gene therapy patient with a desired gene product, the patient is also administered a vector which provides the protein of this invention.
In an embodiment of this invention the immune response is autoimmune manifestations of an infectious disease. In more specific embodiments the autoimmune manifestations are derived from Reiter's syndrome, spondyloarthritis, Lyme disease, HIV infections, syphilis or tuberculosis.
This invention provides a nucleic acid molecule encoding the protein of this invention. The nucleic acid may be DNA (including cDNA) or RNA. It may be single or double stranded, linear or circular. It may be in the form of a vector, such as a plasmid or viral vector, which comprises the nucleic acid molecule operably linked to a transcriptional control sequence recognized by a host cell transformed with the vector. In one embodiment the DNA molecule comprises the coding strand of the TRAF-3 deletion isoform Δ130 or Δ221 clone (nucleotide sequence shown in FIGS. 15 and 17, respectfully). In another embodiment the DNA molecule is complementary to the coding strand of the TRAF-3 clone of FIG. 15 or 17.
This invention also provides a method of providing a subject with an immunosuppressant effective amount of an abnormal TRAF-3, comprising: introducing into CD40-bearing cells of the subject, a nucleic acid sequence encoding an abnormal TRAF-3 deletion isoform polypeptide, under conditions such that the cells express in the subject an immunosuppressant effective amount of the abnormal TRAF-3.
Gene therapy for providing a subject with a protein encoded by a gene are described in U.S. Pat. No. 5,399,346, issued Mar. 21, 1995 (Anderson, et al.). A nucleic acid sequence encoding the protein of interest can be inserted into cells of the subject in vivo. Alternatively the nucleic acid can be inserted into cells ex vivo and the transfected cells can then be introduced into the subject. Accordingly, in an embodiment the introducing of the nucleic acid into cells of the subject comprises: a) treating cells of the subject ex vivo to insert the nucleic acid sequence into the cells; and b) introducing the cells from step a) into the subject.
This invention will be better understood from the Experimental Details which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter.
EXPERIMENTAL DETAILS
Example 1
TRAF-3 mRNA Splice-Deletion Variants Encode Isoforms that Induce NF-κB Activation
[Abbreviations used: TRAF, TNF receptor-associated factor; TNF-R,Tumor Necrosis Factor Receptor; Zn finger, zinc finger; ORF, open reading frame; aa, amino acid; Iscove's Modified Dulbecco Medium, IMDM.]
Although TRAF-3 gene products are required for signaling in T-B cell collaboration, full-length TRAF-3 appears to lack signaling function in transient transfection assays that measure NF-κB activation. However, the TRAF-3 gene also encodes at least 3 mRNA splice-deletion variants that predict protein isoforms (Δ25aa, Δ52aa and Δ56aa) with altered zinc (Zn) finger domains and unknown functional capacities. To determine whether TRAF-3 splice-deletion variants may transmit activating receptor signals to the nucleus, cDNAs for 5 additional splice-variant isoforms (Δ27aa, Δ83aa, Δ103aa, Δ130aa and Δ221aa) were cloned from a TRAF-3+ lymphoma and the expression and function of each of the 8 TRAF-3 splice-deletion variants was analyzed. Among the splice-deletion variants, TRAF-3 Δ130 mRNA is expressed by tonsilar B cells and by each of a panel of B and T cell lines. TRAF-3 Δ221 protein is expressed by tonsilar B cells and by each of the lymphocytic lines. The functional effect of over-expressing each TRAF-3 splice-deletion variant on NF-κB activation was studied in 293T cells. Seven of the TRAF-3 splice-deletion variants, such as TRAF-3 Δ130, induce substantial NF-κB-driven luciferase activity (80-500 fold). In contrast, TRAF-3 Δ221 (in which the complete Zn finger domain is absent) fails to induce NF-κB activation. Although full-length TRAF-3 alone is inactive, it augments the functional effects of the 7 activating TRAF-3 splice-deletion variants (1.4-5 fold). These data indicate that alterations of the Zn finger domains render the TRAF-3 splice-deletion variants capable of inducing NF-κB activation and that full-length TRAF-3 augments their signaling.
1. Introduction
TRAF-3 gene products are signaling molecules that interact with the cytoplasmic tails of CD40 (Cheng et al., 1995; Hu et al., 1994; Sato et al., 1995), other Tumor Necrosis Factor-Receptor (TNF-R) family members (e.g. LTβ-R, CD30, CD27, Ox40) (Mosialos et al., 1995; Gedrich et al., 1996; Boucher et al., 1997; Yamamoto et al., 1998; Vanarsdale et al., 1997; Arch and Thompson, 1998; Kawamata et al., 1998) and the Epstein-Barr virus latent membrane protein, LMP1 (Mosialos et al., 1995). The finding that TRAF-3(−/−)/ lymphocytes are specifically defective in T-B lymphocyte collaboration, indiates that TRAP-3 gene products are required for signaling events that underlie this function (Xu et al., 1996). Full-length TRAF-3 alone is unlikely to account for such signaling, since over-expression of full-length TRAF-3 fails to induce NF-κB activation (Rothe et al., 1995; Takeuchi et al., 1996; Dadgostar and Cheng, 1998). However, alternative splicing of TRAF-3 transcripts generates mRNA species that encode at least 3 putative isoforms with altered Zn finger domains that may participate in transmitting receptor signals to the nucleus (Sato et al., 1995; Krajewski et al., 1997; van Eyndhoven et al., 1998). Therefore, the present study addressed whether TRAF-3 mRNA splice-deletion variants encode TRAF-3 protein isoforms that are able to induce NF-κB activation.
TRAF-3 is a member of the TRAP (TNF Receptor-associated factor) family of proteins, of which six have been identified (TRAF-1 through 6) (Rothe et al., 1994; Regnier et al., 1995; Ishida et al., 1996; Ishida et al., 1996; Kashiwada et al., 1998; Cheng et al., 1995; Hu et al., 1994; Sato et al., 1995; Mosialos et al., 1995). TRAF-3, like other TRAFs, appears to lack intrinsic catalytic activity, which suggests that TRAF-3 functions as a docking or adaptor molecule for other proteins that mediate signaling events. TRAF family members are related by significant homology in their carboxy-terminal TRAF-C domains (Rothe et al., 1994; Cheng et al., 1995). The TRAF-3 TRAF-C domain is known to be important for the interaction of TRAF-3 with the cytoplasmic tails of TNF-R family receptors (Cheng et al., 1995; Force et al., 1997; Vanarsdale et al., 1997), homo-oligomerization (Cheng et al., 1995; Force et al., 1997; Sato et al., 1995; Pullen et al., 1998) and binding to cytoplasmic proteins such as I-TRAF/TANK (Rothe et al., 1996; Cheng and Baltimore, 1996) and NIK (Song et al., 1997; Malinin et al., 1997). In addition to the TRAF-C domain, TRAF-3 contains an amino-terminal RING finger domain, five atypical Zn finger motifs, an iso-leucine zipper domain and a TRAF-N domain (Cheng et al., 1995; Hu et al., 1994; Sato et al., 1995; Mosialos et al., 1995). In TRAF-2 and TRAF-5, the RING finger and Zn finger domains have been shown to play important roles in mediating NF-κB activation (Takeuchi et al., 1996; Dadgostar and Cheng, 1998). The functional potentials of the TRAF-3 RING finger and Zn finger domains remain enigmatic, since TRAF-3 itself fails to induce NF-κB activation (Rothe et al., 1995; Takeuchi et al., 1996; Dadgostar and Cheng, 1998). However, the TRAF-3 RING finger domain is capable of supporting NF-κB activation in chimeric TRAF-3/5 molecules (Dadgostar and Cheng, 1998).
Alteration of the TRAF-3 Zn finger domain by alternative mRNA splicing was suggested by analysis of the sequences of the initial TRAF-3 cDNA clones isolated. One TRAF-3 cDNA clone (CAP1) contains a 75 bp deletion, relative to 3 other TRAF-3 cDNA clones (termed CRAF1, CD40 bp and LAP1) (Cheng et al., 1995; Hu et al., 1994; Sato et al., 1995; Mosialos et al., 1995). In addition, 2 other TRAF-3 mRNA variants containing 156 bp and 168 bp deletions were recently identified (van Eyndhoven et al., 1998). Each of the 3 mRNA species with deleted elements, encodes a putative TRAF-3 protein isoform with an altered number and composition of the Zn fingers.
These putative isoforms have been termed, TRAF-3b (Δ25aa), TRAF-3c (Δ52aa) and TRAF-3d (Δ56aa) (Krajewski et al., 1997; van Eyndhoven et al., 1998) Characterization of the human TRAF-3 genomic structure indicated that these 3 TRAF-3 mRNA species result from alternative mRNA splicing (van Eyndhoven et al., 1998). Further analysis of the TRAF-3 gene suggested that a large number of additional splice variants may be generated, since each of the splice junctions of exons 4 through 10 are class “0”. Therefore, splice-deletion variants involving any or all of exons 5-10 would maintain open reading frames (ORFs) (van Eyndhoven et al., 1998). However, additional splice-deletion variants have not been characterized and the functions of the identified or predicted splice-deletion variants have not been studied.
Furthermore, 5 additional cDNA clones were identified that predict novel TRAF-3 protein isoforms with alterations in the Zn finger domains (Δ27 aa, Δ83 aa, Δ103 aa, Δ130 aa and Δ221 aa). To determine whether the splice-deletion variants are expressed at either the mRNA or protein level, RT-PCR and Western Blotting analysis of a panel of B and T cell lines and tonsilar B cells was performed. TRAF-3 Δ130 mRNA is expressed by tonsilar B cells and by each of the lymphocytic cell lines and TRAF-3 Δ130 protein is expressed by two lymphocytic cell lines, D1.1 and BA. TRAF-3 Δ221 protein is expressed by tonsilar B cells and by each of the lymphocytic lines. To study the effects of the TRAF-3 protein splice-deletion variants on the activation of NF-κB, each of the cDNAs encoding the 8 TRAF-3 splice-deletion variants was over-expressed in 293T cells and their effects on an NF-κB driven Luciferase reporter assay were studied. Over-expression of TRAF-3 Δ221 (in which the complete Zn finger domain is absent) fails to activate NF-κB, similar to full-length TRAF-3. However, over-expression of each of the 7 other TRAF-3 splice-deletion isoforms strongly activates NF-κB. In addition, co-expression of full-length TRAF-3 augments the activity of the NF-κB-inducing TRAF-3 splice-deletion variants 1.4 to 5 fold. Taken together, these data indicate that several TRAF-3 splice-deletion variants induce NF-κB activation, and that full-length TRAF-3 can augment their signaling.
2. Materials and Methods
2.1. Isolation and Characterization of TRAP-3 cDNA Clones
Poly (A)+ RNA was isolated from the D1.1 Jurkat T cell line using the Fast Track System (Invitrogen®, San Diego, Calif.). The Reverse Transcription (RT) reaction was performed with an oligo(dT) primer (5′-GGCCACGCGTCGACTAGTAC(T)17-3′) (SEQ ID NO.: 7) on 0.5 μg mRNA using SuperScript II Reverse transcriptase (Life Technologies, Gaithersburg, Md.) at 50° C., followed by treatment with 1 U of RNase H (Boehringer Mannheirn®, Indianapolis, Ind.) for 30 min at 37° C. and subsequently purified with the GLASSMAX® DNA Isolation Spin Cartridge System (Life Technologies). The Polymerase Chain Reaction CPCR) was performed with forward primer ZnRING. for (5′-GCGGAATTCGGTACCACCGTGGAGGACAAGTACAAGTG-3′) (SEQ ID NO.: 8) and reverse primer Coiled.rev (5′-CGCGGATCCAAGCTTCTAGTTCTGCCGGAAGGGCCGGATC-3′) (SEQ ID NO.: 9) using the Expand High Fidelity PCR System (Boehringer Mannheim). PCR conditions were 3 min at 95° C. (1 cycle); 30 sec at 95° C., 30 sec at 59° C., 1 min at 72° C. (35 cycles); 7 min at 72° C. (1 cycle). PCR products were separated on 0.6% agarose gels, followed by gel-isolation using the QIAQUICK® Gel Extraction System (Qiagen, Valencia Calif.) and cloning into the pCR2.1 vector (TA CLONING SYSTEM®, Invitrogen). Individual cDNA clones were sequenced on an automated ABI 373 DNA sequencer available through the DNA sequencing core facility at Columbia University. These cDNA clones were digested with appropriate restriction enzymes and ligated into the TRAF-3 (CRAF-1) cDNA clone IIIb/pCEP4, the sequence of which is deposited in GenThank under accession number: U21092 (Cheng et al., 1995).
2.2. Isolation of Tonsilar B Cells
Freshly isolated tonsil specimens were cut into 3-10 mm fragments and mashed through a metal sieve in a culture dish containing approximately 10 ml of Iscove's Modified Dulbecco Medium (IMDM) containing 10% fetal calf serum (ECS). The cell mixture was centrifuged at 2000×g for 15 min at room temperature on ficoll HISTOPAQUE-1077®, Sigma Diagnostics, St. Louis, Mo.) after which the buffy coat was isolated and washed 4 times with Minimal Essential Media (MEM) containing 5% FCS. The B cell population was enriched by depletion of T cells by rosetting with sheep red blood cells SRBCs) (Colarado Serum Company, Denver, Colo.). SRBCs were washed 3 times with PBS and allowed to interact with 2×107 tonsilar cells for 10 min at 37° C. before the mixture was centrifuged at 150×g for 5 mm at room temperature. After rosetting for approximately 15 hrs at 4° C., the cells were resuspended and centrifuged on ficoll (HISTOPAQUE-1077®, Sigma), followed by isolation of the non-rosetting population. The tonsilar B cells were analyzed by FACS and subsequently prepared for poly (A)+ or protein isolation as described below.
2.3. Expression of Alternatively Spliced TRAF-3 mRNA Species
Poly (A)+ RNA was isolated from tonsilar B cells and several B-cell lines (Ramos 2G6, Daudi, Raji, BA) and T-cell lines (D1.1, B2.7, H9, CEM) using the FAST TRACK® System (Invitrogen). RT was performed with the TRAF-3 specific primer 3Bend-2(749). R (5′-TTGAAACAAAATTGCACTCTTGAA-3′) (SEQ ID NO.: 10). RT and PCR were performed as described above. PCR reactions were separated on 1.5% agarose gels, blotted onto HYBOND-N® membranes (Amersham Life Science, Cleveland, Ohio), UV-crosslinked using a STRATALINKER® (Stratagene, La Jolla, Calif.) and hybridized to [γ-32P]ATP end-labeled oligonucleotide FF7GEN (5′-AAATGTACAGCGTGTCAAGAGAGCATCG-3′) (SEQ ID NO.: 11). Blots were incubated for 1.5 h at 42° C. in pre-hybridization solution (50% formamide, 5×SSCPE, 5× Denhart's solution, 1 mg/ml salmon sperm DNA and 0.1% SDS), followed by incubation for 15 h at 42° C. in hybridization solution (50% formamide, 5×SSCPE, 5× Denhart's solution, 100 μg/ml salmon sperm DNA and 10% Dextran Sulfate) containing the radiolabeled probe. The blots were washed twice for 15-20 min at 68° C. in 2×SSC, 1% SDS and the hybridization patterns were visualized by autoradiography BIOMAX MS® film, Eastman Kodak, Rochester, N.Y.)
2.4. Expression Constructs
In addition to the TRAF-3 cDNA isoforms, full-length human TRAF-2 cDNA was cloned by RT-PCR from Jurkat D1.1 mRNA. A plasmid containing human CD40 cDNA was used. Full-length TRAF-3, all TRAF-3 cDNA splice-deletion variants, TRAF-2 and CD40 were cloned into the expression vector pCEP4 (Invitrogen). The pRDII×4LUC Firefly Luciferase reporter construct was generated by digestion of pRDII×4CAT (Tran et al., 1997) after which the fragment representing the minimal Interferon-β promoter containing 4 upstream NF-κB sites was cloned 5′ to the Luciferase gene in the pGL3-ENHANCER® Luciferase reporter vector (Promega, Madison, Wis.). The pRL-TK Renilla Luciferase control reporter vector was obtained from Promega.
2.5. Luciferase Assay
Using the Mammalian Cell Transfection System (Specialty Media, Lavallette, N.J.), which allows transfection of mammalian cells by standard calcium phosphate precipitation, 0.5×106 293T cells/60 mm plate were transfected with 0.3 μg of pRDII×4LUC and 0.3 μg pRL-TK reporter constructs and either 3 μg pCEP4/TRAF-3 (full-length or isoforms), 3 μg pCEP4/TRAF-2 or 0.5 μg pCEP4/CD40 expression constructs. In all experiments the amount of DNA transfected was equalized by addition of empty pCEP4 expression vector. After transfection, the cells were cultured for approximately 40 h before harvesting. The NF-κB dependent Luciferase reporter assay was performed utilizing the Dual-Luciferase Reporter Assay System (Promega). Luciferase activity was measured using a Monolight 2010 luminometer (Analytical Luminescence Laboratory, Sparks, Md.). The amount of Renilla Luciferase served as an internal control for normalization of the experimental Firefly Luciferase activities. The results shown are representative of three independent experiments.
2.6. Western Blot Analysis
For Western blot analysis, 1.5×106 293T cells were transfected with 3 μg of TRAF-3 (full-length and/or isoforms) as described above. After 40 h cells were washed in ice-cold PBS and then lysed for 30 min at 4° C. in 250 μl of RIPA lysis buffer (containing 50 mM Tris, pH 8.0, 150 mM Sodium Chloride, 0.1 mM Sodium Orthovanadate, 50 mM Sodium Fluoride, 1% Nonidet P-40, 0.5% Deoxycholate, 0.1% SDS, 1 mM PMSF, 1 μg/ml Aprotinin, 1 μg/ml Leupeptin, 1 μg/ml Pepstatin). Subsequently, the samples were centrifuged at 14,000×g for 15 min at 4° C. to remove cellular debris. Protein concentration was determined using the Bio-Rad Detergent Compatible Protein Assay (Bio-Rad Laboratories, Hercules, Calif.). For Western blot analysis of endogenous TRAF-3 (full-length or isoforms) in tonsilar B cells or in the B-cell and T-cell lines, approximately 20×106 cells were washed in ice-cold PBS and then lysed for 30 mm at 4° C. in 500 μl of RIPA lysis buffer. Protein samples were boiled in reducing sample buffer for 5 min separated by SDS-PAGE (10% gel), transferred onto IMMOBILON-P® membranes (Millipore, Bedford, Mass.) using the Bio-Rad TRANSBLOT® System (Bio-Rad) and detected with rabbit polyclonal anti-TRAF-3 H-20 antiserum (Santa Cruz Biotechnology, Santa Cruz, Calif.) as the primary antibody (directed at amino acids 8-27 at the amino-terminus of TRAF-3) and anti-rabbit IgG conjugated to horse-radish peroxidase (Boehringer Mannheim, Indianapolis, Ind.) as the secondary antibody. Proteins were visualized using BM Chemiluminescence Blotting Substrate (Boehringer) and Fuji Medical X-ray film (Fuji Medical Systems U.S.A., Stamford, Conn.).
3. Results
3.1. Alternative Splicing Results in Distinct TRAF-3 mRNA Species that Predict TRAF-3 Protein Isoforms
To identify TRAF-3 mRNA splice variants, RT-PCR was performed using TRAF-3 specific primers (derived from nucleotide sequences in exons 3 and 11) on mRNA from the TRAF-3+ Jurkat D1.1 T cell line (van Eyndhoven et al., 1998). Several amplified bands were found to be TRAF-3 specific by Southern blotting using a TRAF-3 specific oligonucleotide probe. The PCR products were cloned and sequenced and found to represent 8 distinct TRAF-3 mRNA species containing deletions that correspond to precise excisions of exon-encoded segments by alternative splicing: Δ75 bp (Δexon 8; TRAF-3b), Δ81 bp (Δexon 7), Δ156 bp (Δexon 7+8; TRAF-3c), Δ168 bp (Δexon 8+9; TRAF-3d), Δ249 bp (Δexon 7−9), Δ309 bp (Δexon 8−10), Δ390 bp (Δexon 7−10) and Δ663 bp (Δexon 5−10) (FIG. 1). In all cDNA clones isolated, ORFs are maintained and therefore predict TRAF-3 protein isoforms with amino acid deletions; Δ25aa, Δ27aa, Δ52aa, Δ56aa, Δ83aa, Δ103aa, Δ130aa and Δ221aa, respectively (FIG. 1). The Zn finger domain is altered in all of the splice-variant isoforms, except TRAP-3 Δ221 in which it is completely absent (FIG. 1). An N-terminal portion of the predicted iso-leucine Zipper domain is deleted in TRAF-3 Δ103, Δ130 and Δ221, and in addition, a C-terminal portion ( ̃8aa) of the RING finger domain is deleted in TRAF-3 Δ221 (FIG. 1). These data suggest that at least 8 TRAF-3 mRNA splice-deletion variants are expressed by the D1.1 cell line that encode putative TRAF-3 protein isoforms, containing distinct alterations in the Zn finger domain.
3.2. Expression of Alternatively Spliced TRAF-3 mRNA Species in Lymphoid Cells
To provide some information about the relative levels of full-length TRAF-3 and the 8 TRAF-3 mRNA splice-deletion species cloned from Jurkat D1.1 mRNA, RT-PCR was performed on mRNA from Jurkat D1.1, using the same PCR primer pair (described above) and analyzed by Southern blotting using the same TRAF-3 specific probe (described above). The following statements are based on PCR based analysis. Example 4 herein below shows an improvement of this analysis by using RNase protection assays. As controls, cloned cDNAs representing the 9 TRAF-3 mRNA species were used as templates in parallel PCR reactions. Jurkat D1.1 cells express high levels of full-length TRAF-3 and Δ130 mRNAs and moderate levels of TRAF-3 Δ83 and Δ221 mRNAs (FIG. 2). This analysis did not discriminate between the PCR products from TRAF-3 Δ25 and Δ27 or between TRAF-3 Δ52 and Δ56 and therefore these amplification products are referred to as “Δ25/Δ27” and “Δ52/Δ56”, respectively. Jurkat D1.1 express moderate levels of both the Δ25/Δ27 and Δ52/Δ56 mRNA species (FIG. 2). Surprisingly, Jurkat D1.1 does not express detectable TRAF-3 Δ103 mRNA (FIG. 2), despite the fact that a cDNA encoding TRAF-3 Δ103 was cloned from D1.1 mRNA. Therefore, these data are consistent with the interpretation that the 9 cloned TRAF-3 cDNAs (8 splice-deletion variants plus full-length) are representative of the major TRAF-3 mRNA species in D1.1 except for TRAF-3 Δ103 which by PCR analysis appeared to be a relatively rare transcript in D1.1.
The analysis of the relative expression of TRAF-3 mRNA species was extended to examine mRNA from a panel of human B cell (Ramos 2G6, Daudi, Raji and BA) and T cell (B2.7, H9 and CEM) tumor lines. Jurkat B2.7 cells are similar to D1.1 in their pattern of expressing TRAF-3 mRNA species, except that B2.7 cells have a lower level of TRAF-3 Δ130 (FIG. 2). In each of the other lymphocytic cell lines studied, TRAF-3 Δ130 mRNA was the predominant product and serves as a reference for the relative expression of the other TRAF-3 mRNA species (FIG. 2). Relative to their expression of TRAF-3 Δ130 mRNA, substantially less of the longer TRAF-3 mRNA species are expressed (FIG. 2). The T cell lines, H9 and CEM, express full-length TRAF-3, which is barely detectable in the other cell lines (except Jurkat D1.1 and B2.7) (FIG. 2). TRAF-3 Δ221 is expressed by each of the cell lines, except the EBV transformed B cell BA (FIG. 2). The Δ25/Δ27 band is amplified in the B cell lines Ramos and Daudi and the T cell line CEM (FIG. 2). Together, these data indicate that TRAF-3 splice-deletion mRNAs are expressed in a variety of B and T cell tumor lines.
The analysis of the relative expression of TRAF-3 mRNA species was also extended to examine mRNA from human tonsilar B cells. Similar to the tumor cell lines, torsilar B cells express TRAF-3 Δ130 mRNA (FIG. 2). Tonsilar B cells express barely detectable full-length TRAF-3 mRNA (only evident at longer exposures). Tonsilar B cells do not express detectable RNA for TRAF-3 Δ221, which is similar to the EBV transformed B cell line BA, but different from the other tumor cell lines in which TRAF-3 Δ221 mRNA is relatively prominent (FIG. 2). Together, these data show that tonsilar B cells express the TRAF-3 splice-deletion mRNA species TRAF-3 Δ130, similar to several B and T cell tumor lines.
3.3. Expression of TRAF-3 Protein Isoforms in Lymphoid Cells
To determine the expression of TRAF-3 protein isoforms in tonsilar B cells and the panel of B cell and T cell tumor lines, cell lysates were examined by Western blotting using a polyclonal anti-TRAF-3 N-terminus antiserum (H-20) (FIG. 3). As controls, cDNAs encoding full-length TRAF-3 and the 8 splice-deletion variants were over-expressed in 293T cells and cell lysates from these transfected cells were examined in parallel. TRAF-3 protein that migrates as either full-length TRAF-3 or TRAF-3 Δ25/Δ27 is prominently detected as a specific band in all cells, except Ramos (FIG. 3). The absence of full-length TRAF-3 protein in Ramos cells has been previously reported (Krajewski et al., 1997) and is consistent with the undetectable full-length TRAF-3 mRNA in Ramos (FIG. 2). The expression of either full-length TRAF-3 or TRAF-3 Δ25/Δ27 in all of the other cells studied, is surprising given the relatively low expression of TRAF-3 mRNA species that encode either of these isoforms in the majority of these cells (FIG. 2). TRAF-3 protein that migrates as TRAF-3 Δ221 is expressed in all cells, and at low levels in tonsilar B cells (FIG. 3). The expression of protein that migrates as TRAF-3 Δ221 is generally consistent with the pattern of Δ221 TRAF-3 mRNA expression, except for BA cells that express TRAF-3 Δ221 protein, but lack detectable mRNA encoding TRAF-3 Δ221. Jurkat D1.1 cells, and to a lesser extent BA cells, express TRAF-3 protein that migrates as TRAF-3 Δ130 (FIG. 3). The lack of detectable TRAF-Δ130 protein in most cells examined is surprising, given the relatively prominent band for TRAF-3 Δ130 mRNA in PCR all of the cells and cell lines examined. However, the expression of TRAF-3 Δ130 protein by D1.1 and not B2.7 Jurkat cells is consistent with the relatively higher TRAF-3 Δ130 mRNA expression by D1.1 (FIG. 2). Together, these data show that the peptides that migrate as TRAF-3 Δ221 is expressed in a variety of cell lines and that peptides that migrate as TRAF-3 Δ130 is expressed in certain tumors.
3.4. Effect of Over-Expression of TRAF-3 Splice-Deletion Variants on NF-κB Activation in 293T Cells
To determine whether any of the 8 TRAF-3 splice-deletion variants are able to induce NF-κB activation, their functional effects were studied on the transcription of an NF-κB driven Luciferase reporter construct in transient assays in 293T cells. As positive controls for NF-κB activation, cDNAs encoding CD40 and TRAF-2 were over-expressed and substantial NF-κB activation was observed, as expected (Rothe et al., 1995) (FIG. 4). Over-expression of 7 of the TRAF-3 splice-deletion variants (Δ25 aa, Δ27 aa, Δ52 aa, Δ56 aa, Δ83 aa, Δ103 aa and Δ130 aa) potently activates NF-κB (between 80 to 500 fold). Among the NF-κB-inducing TRAF-3 splice-deletion variants, TRAF-3 Δ52 is relatively more potent (approximately 500 fold activation) and TRAF-3 Δ56 is relatively less potent (approximately 80 fold activation). In contrast, over-expression of TRAF-3 Δ221 fails to activate NF-κB (FIG. 4), which indicates that the NF-κB activating effect of the other cDNAs encoding TRAF-3 splice-deletion variants is specific. These data show that over-expression of certain TRAF-3 splice-deletion variants induces NF-κB activation, but full-length TRAF-3 and TRAF-3 Δ221 lack this effect.
3.5. Effect of Full-Length TRAF-3 on NF-κB Activation Induced by TRAF-3 Splice-Deletion Variants
Since full-length TRAF-3 has been reported to inhibit NF-κB activation induced by over-expression of CD40 (Rothe et al., 1995), the next series of experiments analyzed the effects of over-expressing full-length TRAF-3 on the NF-κB activation induced by over-expression of TRAF-3 splice-deletion variants. Consistent with previous reports (Rothe et al., 1995), over-expression of full-length TRAF-3 inhibits the NF-κB activation induced by CD40 over-expression (FIG. 5). The TRAP-3 mediated inhibition of CD40-induced NF-κB activation is specific, since over-expression of full-length TRAF-3 has minimal or no effect on NF-κB activation induced by TRAF-2 over-expression in 293T cells (FIG. 4). Co-expression of full-length TRAF-3 with each of the 7 TRAF-3 splice-deletion isoforms that activates NF-κB alone, results in augmentation of their NF-κB activation by 1.4 to 5 fold (FIG. 4). For example, in the case of TRAF-3 Δ130, the augmentation by full-length TRAF-3 is greater than 2 fold (FIG. 4). In contrast, co-expression of full-length TRAF-3 with TRAF-3 Δ221 (which does not activate NF-κB alone) does not confer TRAF-3 Δ221 with an ability to induce NF-κB activation (FIG. 4). Therefore, these data indicate that full-length TRAF-3, which lacks intrinsic signaling ability, augments signaling by the NF-κB-inducing TRAF-3 splice-deletion variants.
4. Discussion
In the present study, full-length TRAF-3 and 8 TRAF-3 splice-deletion mRNA species (Δ25aa, Δ27aa, Δ52aa, Δ56aa, Δ83aa, Δ103aa, Δ130aa, and Δ221aa) were analyzed for their expression in tonsilar B cells and in a panel of B and T cell tumor lines and for their potential to induce NF-κB activation. Consistent with previous studies, full-length TRAF-3 protein is expressed by tonsillar B cells and by each of the B and T cell tumors studied (except Ramos), but lacks the ability to activate NF-κB when over-expressed. Of the splice-deletion species, TRAF-3 Δ130 mRNA is expressed by tonsillar B cells and by each of the B and T cell tumors and was found to have the ability to activate NF-κB when over-expressed in 293T cells. TRAF-3 Δ221 protein is expressed at low levels by tonsilar B cells and at higher levels by each of the B and T cell tumors, but lacks the ability to induce NF-κB activation. Full-length TRAF-3 augments the NF-κB activation induced by over-expression of TRAF-3 Δ130 or the six other activating splice-deletion variants, but does not confer activity to TRAF-3 Δ221. Together, these data indicate that alternative splicing of TRAF-3 results in splice-deletion variants that induce NF-κB activation, and that full-length TRAF-3 has the potential to augment their function.
Alternative splicing of the TRAP-3 mRNA elements that encode the Zn finger domain was previously described by the identification of cDNAs encoding TRAF-3 Δ25aa, Δ52aa and Δ56aa and by the characterization of the genomic structure of TRAF-3 which showed that these mRNA species correspond to specific deletions of exon-encoded segments (Sato et al., 1995; Krajewski et al., 1997; van Eyndhoven et al., 1998). The genomic structure also revealed that exon/intron junctions of exons 4−10 all have type “0” intron junctions, which would allow for the variable presence or absence of mRNA segments encoded by exons 5−10 in mRNA species that maintain ORFs (van Eyndhoven et al., 1998). This finding indicates that it is possible that 26 (or 64) distinct mRNA species that maintain ORFs could be generated by alternative mRNA splicing of exons 5—10. Therefore, the approach taken to studying potential isoforms was to isolate cDNAs from the TRAF-3+ cell line, Jurkat D1.1 and then to determine whether these mRNA species are expressed by other tumor cell lines or by tonsilar B cells. This approach yielded cDNAs encoding five novel TRAF-3 splice-deletion variants (Δ27aa, Δ83aa, Δ103aa, Δ130aa and Δ221) in addition to the three previously described. All of the 8 splice-deletion variants encode putative TRAF-3 isoforms that maintain ORFs with altered Zn finger domains. It should be noted that not all TRAP-3 isoforms were analyzed in these experiments. For example, splice-deletion variants of other exon-encoded elements may exist and one example of such a variant was reported that predicted translation initiation at Methionine 350 (encoded by exon 11), which predicts a peptide containing only the TRAF-N and TRAF-C domains (Mosialos et al., 1995). All of the TRAF-3 splice-deletion variants identified, contain the TRAF-C domain, which is known to be critical for binding to the cytoplasmic tails of CD40 and other TNF-R family receptors (Cheng et al., 1995; Force et al., 1997; Vanarsdale et al., 1997), homo-oligomerization (Cheng et al., 1995; Force et al., 1997; Sato et al., 1995; Pullen et al., 1998) and binding to cytoplasmic proteins such as I-TRAF/TANK (Rothe et al., 1996; Cheng and Baltimore, 1996) and NIK (Song et al., 1997; Malinin et al., 1997).
Analysis of tonsillar B cells and a panel of B and T cell tumor cell lines for mRNA and protein corresponding to the TRAF-3 splice-deletion variants indicates that, in addition to full-length TRAF-3, certain splice-deletion variants are expressed (e.g., TRAF-3 Δ130 and Δ221). However, discrepancies between the amounts of mRNA and corresponding proteins detected, suggest that mRNA and/or protein stability varies between the isoforms. For example, full-length TRAF-3 protein was expressed by all cells (except Ramos), but full-length TRAF-3 mRNA was expressed at low or undetectable levels in the majority of cells studied. These data are consistent with the possibility that full-length TRAF-3 protein is stable and/or that full-length TRAP-3 mRNA is short-lived. The protein and mRNA corresponding to TRAF-3 Δ221 was found to correlate, except for the case of the EBV transformed B cell BA, in which high level protein was detected in the setting of undetectable mRNA. These data are consistent with the possibility that TRAF-3 Δ221 protein is stable and that the corresponding mRNA may be short lived, at least in BA cells. The PCR product for Δ130 TRAF-3 mRNA was relatively abundant in all cells examined, but the corresponding protein was detected only in Jurkat D1.1 and BA. These data are consistent with the possibility that TRAF-3 Δ130 protein is short lived and/or the corresponding mRNA is stable. Another possibility is that the translation of TRAF-3 Δ130 is regulated. Future studies will address whether the stability of mRNA and protein affect the expression of TRAF-3 splice variants.
The finding that certain TRAF-3 splice-deletion variants have the capacity to induce NF-κB activation suggests a structural basis for this effect. First, comparison of splice-deletion variants that induce NF-κB activation with full-length TRAF-3 that lacks activating capacity, shows that alterations of the Zn finger domain (relative to full-length TRAF-3) are required for inducing NF-κB activation. Second, comparison of splice-deletion variants that activate NF-κB (TRAF-3 Δ25aa, Δ27aa, Δ52aa, Δ56aa, Δ83aa, Δ103aa, and Δ130aa) with the variant that lacks activating capacity (TRAF-3 Δ221) shows the importance of the Zn finger domain in inducing NF-κB activation, because TRAF-3 Δ221 lacks the entire Zn finger domain. However, TRAF-3 Δ221 also lacks approximately 8 aa from the C-terminal portion of the RING finger domain, which may contribute to its inactivity, since portions of the TRAF-3 RING finger domain support NF-κB activation in chimeric TRAF-3/5 molecules (Dadgostar and Cheng, 1998). These data suggest that the TRAF-3 Zn finger domain and perhaps the C-terminal portion of the RING domain are required for inducing NF-κB activation, but indicate that the Zn finger domain must be of a form encoded by certain of the splice-deletion variants. In this regard, the fact that exons 7, 8 and 9 encode portions of distinct fingers, indicates that the fingers encoded by the splice-deletion variants have altered compositions, relative to full-length TRAF-3, which may confer distinct binding specificites to these motifs.
Another TRAF-family member, TRAF-2, has been shown to utilize alternative mRNA splicing to generate a protein isoform with altered function (Brink and Lodish, 1998). In this case, the isoform termed “TRAF-2” that induces NF-κB activation, is a splice-deletion- variant of a full-length TRAF-2 (termed “TRAF-2a”) that lacks NF-κB activating capacity (Brink and Lodish, 1998). The activating splice-deletion variant (TRAF-2) lacks 21 bp corresponding to a deletion of 7 aa in the C-terminal portion of the RING finger domain (Brink and Lodish, 1998). Therefore, alternative splicing appears to be a mechanism used by both TRAF-2 and TRAF-3 genes to regulate signaling events, since in both cases splice-deletion variants induce NF-κB activation, but full-length forms do not. Future studies will address whether TRAF-3 mRNA splicing is controlled by activation and differentiation.
Changes in mRNA splicing in response to cellular activation and differentiation have been observed in many systems, such as the change from surface to secreted Ig molecules (the IgH gene locus (Alt et al., 1980)). In addition, a TNF-R gene family member, LARD utilizes alternative mRNA splicing to generate at least 11 isoforms. (Screaton et al., 1997). Naive B and T cells predominantly express LARD isoforms with internal deletions, but upon activation by PHA, a shift in the splicing pattern results in a predominant expression of full-length LARD-1 (Yang et al., 1998). It is also of interest that TNF-α mRNA changes its pattern of mRNA splicing after lymphocyte activation (Yang et al., 1998), which suggests that “ligands”, “receptors” and signaling molecules, in the TNF-α family, TNF-R family and TRAF-family, respectively, may utilize alternative mRNA splicing to regulate signaling outcomes.
Although over-expression of full-length TRAF-3 alone does not activate NF-κB, co-transfection of full-length TRAF-3 augments the NF-κB activation induced by splice-deletion variants. It is unclear how full-length TRAF-3 mediates this effect, but it interesting to consider the possibility that full-length TRAF-3 may form complexes with splice-deletion isoforms. In this regard, all of the TRAF-3 isoforms contain TRAF-C domains (and at least, the carboxy-terminal portion of the isoleucine zipper). Therefore, based on the structures encoded by the activating splice-deletion variants, they are expected to form complexes with full-length TRAF-3 (Cheng et al., 1995; Force et al., 1997; Pullen et al., 1998). It will be of interest to determine whether such complexes are formed and whether full-length TRAF-3 stabilizes the activating splice-deletion variants, such as TRAF-3 Δ130, for which protein is undetectable in tonsilar B cells, despite relatively abundant mRNA. In the case of TRAF-2, co-transfection of the splice-deletion variant (TRAF-2) increases the stability of the full-length form (TRAP-2a) (Brink and Lodish, 1998). Future studies will address whether full-length TRAF-3 modulates the expression or stability of activating splice-deletion variants.
The finding that certain TRAF-3 splice-variants are expressed by tonsilar B cells and have the capacity to induce NF-κB activation may reconcile previous views of the role of TRAF-3 gene products in T-B cell interactions that depend on CD40 signaling. Two lines of evidence strongly suggest that TRAF-3 gene products play important roles in CD40 signaling. First, over-expression of a truncated form of TRAF-3 (termed C26), which contains only the TRAF-N and TRAF-C domains, inhibits CD40-mediated CD23 upregulation (Cheng et al., 1995). The C26 TRAF-3 fragment binds the CD40 cytoplasmic tail, but C26 lacks the Zn RING and Zn finger domains that are required by other TRAFs for downstream functions, such as activating NF-κB (Cheng et al., 1995; Takeuchi et al., 1996; Dadgostar and Cheng, 1998). Second, reconstitution of lethally irradiated mice with TRAF-3−/− fetal liver cells resulted in mice with a profound, selective defect in T-dependent antibody responses (Xu et al., 1996). Since this phenotype is similar to that observed in CD154 (CD40-L)−/− (Xu et al., 1994; Renshaw et al., 1994) or CD40−/− mice (Kawabe et al., 1994; Castigli et al., 1994), these data suggest that TRAF-3 gene products play important positive roles in CD40 mediated signaling.
However, the inability of full-length TRAF-3 over-expression to activate NF-κB raised questions about whether TRAF-3 participated in transmitting CD40 signals to the nucleus (Rothe et al., 1995). In light of the new finding that TRAF-3 splice-deletion variants are capable of inducing NF-κB activation, it may be appropriate to reconsider the inactivity of full-length TRAF-3 alone in the context that other TRAF-3 gene products may play dominant roles in transmitting activating CD40 signals to the nucleus. First, the C26 fragment is expected to compete with all of the activating TRAF-3 isoforms for CD40 cytoplasmic tail binding and may form dysfunctional oligomers with these isoforms, which would be consistent with its inhibitory effects (Cheng et al., 1995). Second, the targeted disruption of TRAF-3 prevents the expression of any of the NF-κB-inducing TRAF-3 isoforms, which is consistent with the specific defect of TRAF-3−/− lymphocytes in generating T-dependent antibody responses (Xu et al., 1996). Third, full-length TRAF-3 augments the activating potential of TRAF-3 splice-deletion variants, which suggests that over-expression of full-length TRAF-3 alone is not sufficient to reveal its functional roles. Taken together, these considerations strongly suggest that TRAF-3 splice-deletion variants, which are capable of inducing NF-κB activation, may contribute to CD40 signaling in T-B cell interactions. An important goal of future studies will be to determine whether certain TRAF-3 isoforms, such as TRAF-3 Δ130, have special roles in CD40 signaling.
Example 2
A Single Gene for Human TRAF-3 at Chromosome 14q32.3 Encodes a Variety of mRNA Species by Alternative Polyadenylation, mRNA Splicing and Transcription Initiation
[Abbreviations used: TRAF, TNF receptor-associated factor; TNF-R, Tumor Necrosis Factor Receptor; NIK, NF-κB-inducing kinase; Zn finger, zinc finger; IGHC, Immunoglobulin heavy chain constant region gene complex, ORF, open reading frame; UTR, untranslated region; RACE, rapid amplification of cDNA ends; RT, reverse transcriptase; FISH, fluorescence in situ hybridization; LOH, loss of heterozygosity; aa, amino acid; His-tagged, poly-histidine fusion construct; EST, Expressed Sequence Tag; cR, centiRays; Mb, megabase; kb, kilobase; HIGM, hyper-IgM syndrome.]
Human TRAF-3 is a signaling molecule that interacts with the cytoplasmic tails of CD40 and other TNF-receptor family members. TRAF-3 mRNA is expressed as two major classes of approximately 2 and 8 kb and a number of TRAF-3 encoding cDNA clones differ in discrete gene segments. Because this variety of mRNA species could result from mRNA processing events and/or multiple genes, the structure and localization of TRAP-3 encoding gene elements were determined. FISH and radiation hybrid mapping demonstrated that TRAF-3 is located at chromosome 14q32.3, approximately 1 Mb centromeric to the Ig heavy chain gene complex. Physical mapping of 4 overlapping genomic PAC clones established that TRAF-3 transcripts are encoded by a single gene, comprised of 13 exons spanning 130 kb. Alternative polyadenylation in the mRNA segment encoded by exon 12 accounts for the difference between the 2 kb and the 8 kb classes of transcripts. Alternative mRNA splicing in the coding region (encoded by exons 3−12) generates transcripts which delete exons 8 (75 nt), 7+8 (156 nt) or 8+9 (168 nt) and that encode distinct protein isoforms (Δ25, Δ52 and Δ56 aa, respectively). Alternative splicing of exon 2 (139 nt) and alternative transcriptional initiation result in mRNA species with distinct 5′UTRs. Together, these data indicate that a single TRAF-3 gene encodes a variety of mRNA species by a combination of alternative polyadenylation, alternative mRNA splicing and/or alternative initiation.
1. Introduction
Human TRAF-3 is a 568 amino acid (aa) cytoplasmic signaling molecule that interacts with the cytoplasmic tails of CD40 (Hu et al. 1994; Cheng et al. 1995; Mosialos et al. 1995; Sato et al. 1995) and other Tumor Necrosis Factor-Receptor (TNF-R) family members (e.g., LTβ-R, CD30 and Ox40) (Mosialos et al. 1995; Vanarsdale et al. 1997; Gedrich et al. 1996; Ansieau et al. 1996; Boucher et al. 1997; Arch and Thompson, 1998). Although the understanding of the role of TRAF-3 in signaling is still evolving, TRAF-3 is known to participate in CD40-mediated signaling (Cheng et al. 1995) and to play an important role in T helper function for B cells (Xu et al. 1996). Dissecting the role of TRAF-3 in signaling has been complicated, in part, due to the expression of mRNA transcripts encoding more than one TRAF-3 isoform (Mosialos et al. 1995; Sato et al. 1995; Krajewski et al. 1997). Therefore, as part of a larger effort to analyze the function of TRAF-3 gene products in signaling, the present study characterized the structure of TRAF-3 mRNA transcripts and the organization of the genetic elements that encode them.
TRAF-3 is a member of the TNF receptor-associated factor (TRAF) family of signal transducing molecules (Rothe et al. 1994), of which six members currently have been identified (TRAF-1 through 6) (Rothe et al. 1994; Regnier et al. 1995; Ishida et al. 1996; Nakano et al. 1996; Ishida et al. 1996; Kashiwada et al. 1998). The characteristic feature of TRAF-family members is the TRAF-C domain (Rothe et al. 1994; Cheng et al. 1995) and mutational analyses of the TRAF-3 TRAF-C domain show that it is critical for interactions with the cytoplasmic tails of the TNF-R family members (Cheng et al. 1995; Force et al. 1997) and with cytoplasmic proteins including, other TRAF-3 molecules (Cheng et al. 1995; Force et al. 1997), I-TRAF/TANK (Cheng and Baltimore, 1996; Rothe et al. 1996) and NF-κB-inducing kinase (NIK) (Regnier et al. 1997; Song et al. 1997; Malinin et al. 1997). Among the TRAF family members, TRAF-5 shares the closest homology with TRAF-3 and both interact with the same set of TNF-R family members' cytoplasmic tails (Ishida et al. 1996; Nakano et al. 1996; Aizawa et al. 1997; Kawamata et al. 1998). TRAF-2 and TRAF-1 share less homology with TRAF-3, but TRAF-2 binds several of the same TNF-R family members' cytoplasmic tails (Rothe et al. 1994; Rothe et al. 1995; Gedrich et al. 1996; Ansieau et al. 1996; Boucher et al. 1997; Arch and Thompson, 1998; Lee et al. 1996). Finally, TRAF-6 and TRAF-4 are most distantly related to TRAF-3 (Ishida et al. 1996; Kashiwada et al. 1998; Regnier et al. 1995). Although TRAF-6 interacts with the cytoplasmic tail of CD40, its binding site on the CD40 tail is distinct from that of TRAF-2, 3 or 5 (Ishida et al. 1996; Kashiwada et al. 1998). The genes for TRAP-1, 4 and 5 have been localized and are dispersed in the genome on different chromosomes (Regnier et al. 1995; Siemienski et al. 1997; Nakano et al. 1997). Although the genomic localization of human TRAF-3 is unknown, the murine TRAF-3 genomic structure has been partially characterized and localized to the distal region of chromosome 12, which has synteny with human 14q32 (Wang et al. 1996).
The functional roles of TRAF-3 in receptor-mediated signaling have been studied in cell lines by over-expression of full-length or truncated TRAF-3 peptides and in vivo by targeted disruption of the TRAF-3 gene. In this regard, over-expression of particular truncated forms of TRAF-3, that include the TRAF-C domain, inhibit signaling by CD40 or LTβ-R in a dominant-negative fashion (Cheng et al. 1995; Force et al. 1997; Eliopoulos et al. 1996). Further evidence for signaling by TRAF-3 gene products was derived from targeted disruption of the TRAF-3 gene in mice (Xu et al. 1996). Fetal liver cells from TRAF-3 deficient (−/−) embryos are capable of reconstituting all hematopoietic lineages after transfer to lethally irradiated mice (Xu et al. 1996). In these mice, the deficiency of TRAF-3 in lymphoid cells results in a severe and selective defect in the generation of antibody responses to T cell dependent antigens (Xu et al. 1996). This phenotype is similar to that of CD154 (CD40-L) deficient (Xu et al. 1994; Renshaw et al. 1994) and CD40 deficient (Kawabe et al. 1994; Castigli et al. 1994) lymphocytes in vivo, which suggests that TRAF-3 plays a role in CD40-mediated aspects of T cell-B cell collaboration. However, some CD40-mediated effects are preserved in TRAF-3 (−/−) lymphocytes, such as CD23 upregulation (Xu et al. 1996). A functional redundancy of TRAF-5 for TRAF-3 may account for this preserved aspect of CD40-signaling in TRAF-3 (−/−) lymphocytes, since over-expression of truncated TRAF-5 constructs inhibits CD40-mediated CD23 upregulation (Ishida et al. 1996). Nevertheless, the fact that TRAF-3 (−/−) lymphocytes are deficient in T cell-B cell collaboration in vivo is notable because a variety of other molecules are implicated in CD40-mediated signaling, including TRAF-2, 5 and 6 (Rothe et al. 1995; Ishida et al. 1996; Kashiwada et al. 1998), as well as other cytoplasmic factors (Morio et al. 1995; Hanissian and Geha, 1997).
Transient over-expression of full length TRAF-3 cDNA clones in cell lines inhibits NF-κB activation mediated by over-expression of CD40, TRAF-2 or TRAF-5 (Duckett et al. 1997; Rothe et al. 1995; Ishida et al. 1996; Devergne et al. 1996; Kawamata et al. 1998). The RING finger domain of TRAF-2, but not that of TRAF-3, confers NF-κB activating capacity to chimeric molecules (Takeuchi et al. 1996). However, the conclusion that TRAF-2 is an activator and TRAP-3 is an inhibitor of NF-κB activation may be premature, since an isoform of TRAF-2 (TRAF-2A) that contains an additional 7 aa in the RING finger domain has recently been isolated and found to be an inhibitor of TRAF-2 induced NF-κB activation (Brink and Lodish, 1998). The fact that “TRAF-2” is apparently a splice deletion variant of the “full length” TRAF-2A suggests that alternative mRNA splicing of TRAF-2 gene products regulates NF-κB activating capacity. These considerations suggest that regulation of signaling function by alternative splicing may be a feature of other members of the TRAF-family, such as TRAP-3. It is unknown if TRAF-3 isoforms exist that activate NF-κB, but this possibility is suggested by the observation that TRAF-3 binds NIK similarly to TRAF-1, 2, 5 and 6 (Regnier et al. 1997; Song et al. 1997; Malinin et al. 1997). Together, these findings indicate that the roles of TRAF-3 may be elucidated by understanding the precise structure of TRAF-3 isoforms.
Evidence for diverse TRAF-3 mRNA transcripts was first obtained from Northern blot analysis (Cheng et al. 1995; Mosialos et al. 1995; Wang et al. 1996) and comparison of the sequences of various TRAF-3 cDNA clones (Hu et al. 1994; Cheng et al. 1995; Sato et al. 1995; Mosialos et al. 1995). Both human and mouse TRAF-3 mRNA are expressed as two major classes of 2 and 8 kb (Cheng et al. 1995; Mosialos et al. 1995; Wang et al. 1996). Isolation of a 7.3 kb murine TRAF-3 cDNA clone (TRAFamn) and partial characterization of a murine TRAF-3 gene suggested that alternative polyadenylation controls the addition of 5 kb of 3′UTR to TRAF-3 transcripts (Wang et al. 1996). In addition, comparison of the reported sequences of the human TRAF-3 cDNA clones suggests that alternative splicing occurs in at least two regions. For example, comparison of the reported sequences of TRAF-3 (CD40 bp) (Hu et al. 1994) and TRAF-3 (CRAF1) (Cheng et al. 1995) reveals that a region of 139 bp in the 5′UTR is not included in a subset of TRAF-3 cDNA clones. In addition, comparison of TRAF-3 (CAP-1) (Sato et al. 1995; Krajewski et al. 1997) with other TRAF-3 cDNA clones reveals that a 75 bp region is deleted in the coding region. Because TRAF-3 (CAP-1) encodes a protein with a 25 aa deletion in the Zn finger complex, it has been termed TRAF-3b (Krajewski et al. 1997). These studies demonstrate that a variety of TRAF-3 mRNA transcripts are expressed in cells. However, the mechanisms that generate these various TRAF-3 transcripts have not been studied systematically. Moreover, the structure and organization of the genetic elements encoding TRAF-3 have not been characterized and some of the variety of TRAF-3 transcripts could result from the existence of more than one TRAF-3 gene.
The present work characterized a number of TRAF-3 mRNA transcripts and the molecular structure and organization of the genetic elements that encode them. The structure and localization of the human TRAF-3 gene was determined by fluorescence in situ hybridization (FISH) analysis, radiation hybrid mapping and characterization of 4 overlapping genomic PAC clones. A single TRAF-3 gene was identified that contains 13 exons and spans approximately 130 kb at chromosome 14q32.3. Alternative polyadenylation in the mRNA encoded by exon 12 appends either 0.5 or 5.8 kb of 3′-untranslated region (UTR) and accounts for the difference between the 2 and 8 kb transcripts. Alternative mRNA splicing accounts for the TRAF-3b transcript (Sato et al. 1995; Krajewski et al. 1997) as well as for two other TRAF-3 transcripts that encode isoforms with 52 and 56aa deletions in the Zn finger region, which alter the number and composition of the Zn fingers. Alternative splicing and alternative transcription initiation result in TRAF-3 transcripts with distinct 5′UTR sequences. Together, these studies demonstrate that a single human TRAF-3 gene accounts for the diverse TRAF-3 mRNA species, which are generated by alternative polyadenylation, alternative splicing and/or alternative transcriptional initiation.
2. Materials & Methods
2.1. Isolation and Characterization of TRAF-3 cDNA Clones
Hybridization screening of a Raji B cell λ-gt11 cDNA library (Clontech Laboratories, Palo Alto, Calif.) yielded several TRAF-3 cDNA clones, including IIIb, Ib and 2-1. The cDNA inserts were cloned into the pBluescript SK(+) cloning vector (Stratagene, La Jolla, Calif.). In addition, 5′-rapid amplification of cDNA ends (RACE) was used to identify 5′-sequences of TRAF-3 with mRNA from D1.1, Raji or EBV-transformed B cells as templates (Boehringer Mannheim, Indianapolis, Ind.; Life Technologies, Gaithersburg, Md.; Clontech Laboratories). Although a number of TRAF-3 clones were identified and cloned into the pCR2.0 or pCR2.1 TA cloning vectors (Invitrogen, San Diego, Calif.) or the pBluescript SK(+) cloning vector (Stratagene), sequences further upstream of the 5′-termini of the λ-gt11 clones were not present in any of these 5′-RACE products. The sequence of the previously reported TRAF-3 (CRAF1) cDNA clone IIIb is deposited in GenBank under accession number: U21092 (Cheng et al. 1995). The nucleotide positions (nt) of the IIIb clone are used in this study.
The extended 5.8 kb TRAF-3 3′UTR was cloned as two overlapping cDNA fragments of approximately 4.7 kb (upstream) and 1.8 kb (downstream). The 4.7 kb cDNA clone was generated by reverse transcriptase (RT)-PCR, in which the RT reaction was primed with oligo(dT) and PCR amplification utilized the gene specific oligonucleotide primers, 3B1850F (5′ GTCTTTGTGGCCCAAACTGTTCTAGAAAATGG 3′) (SEQ ID NO.:12) and EST/Mu-CRAF1.R (5′ GATTTGGGTGACAGACCCTCATT 3′) (SEQ ID NO.:13). The 1.8 kb cDNA clone was generated by 3′-RACE (Life Technologies) utlilizing an oligo(dT) primer (5′ GGCCACGCGTCGACTAGTAC(T)17 3′) (SEQ ID NO.: 7) and a gene specific forward oligonucleotide primer 5′CRAF-EST2(186).F (5′ ATGCAGTTCTAGGCACAGCC 3′) (SEQ ID NO.: 14). The PCR products (4,691 and 1,845 bp that overlap in a 672 bp segment) were cloned into the pCR2.0 or pCR2.1 TA cloning vectors (Invitrogen, San Diego, Calif.) or the pBluescript SK(+) cloning vector (Stratagene). The 5864 bp (4.7 and 1.8 kb) cDNA clones together are deposited in GenBank under accession number AF110908. (Van E. 1998, pg. 1192, first sentence).
The cDNA clones containing deletions in the region coding for the Zn fingers were isolated by RT-PCR, in which the RT reaction was primed by oligo(dT) and PCR amplification utilized gene specific oligonucleotide primers. The PCR products were cloned into the pCR2.0 or pCR2.1 TA cloning vectors (Invitrogen).
2.2. Isolation and Characterization of Human Genomic λ-Phage Clones
Three clones from a λ-FIX II genomic library (Stratagene) were identified by hybridization screening using the full length IIIb cDNA clone as a probe. All three phage clones were analyzed by restriction digestion, partial sequencing and PCR using primers derived from the IIIb cDNA sequence. Two of these clones (#4 and #9) overlap and represent the 3′-coding region and 3′UTR (IIIb nt 975-2433), while the third clone (#6) contains genomic sequence representing the 5′-coding region of TRAF-3 (IIIb nt 200-787).
2.3. Isolation of TRAF-3′ Human Chromosome 14q22.3 DNA Clones
PAC clones, representing the genomic region of TRAF-3, were identified by hybridization screening of segment RPCI1 of an arrayed human genomic PAC library). This library is described at the BacPac Resource Center at the Department of Human Genetics at Roswell Park Cancer Institute (http://bacpac.med.buffalo.edu/libchar.htm). Two PAC clones (PAC 167m5 and PAC 20605) were identified by hybridization to 32P labeled TRAF-3 genomic PCR products (generated from the λ-FIX II clones). Two additional PAC clones (PAC 342f5 and PAC 373n17) were isolated by genomic walking using a probe representing the 5′-end of the PAC 167m5 insert.
2.4. DNA Preparation
The PAC clones were grown in LB media containing 25 μg ml−1 Kanamycin (Sigma Chemical, St. Louis, Mo.). Individual colonies were grown for 15 h at 37° C. to saturation in a volume of 2 ml, followed by transfer to 30 ml of fresh media and grown for 4 h. Subsequently, this 30 ml culture was transferred to 1 liter of fresh media, grown for 1.5 h after which it was supplemented with 0.5 mM IPTG (Denville Scientific, Metuchen, N.J.) to increase the copy number. After an additional 5 h the bacteria were harvested and plasmid DNA was isolated using standard alkaline lysis and CsCl (Sigma) gradient ultra-centrifugation (Maniatis et al. 1982).
Human genomic DNA was extracted from the Ramos 2G6 B cell line (5×107 B cells), grown in Iscove's Modified Dulbecco's Medium (Life Technologies), supplemented with 10% fetal calf serum (Summit Biotechnology, Ft. Collins, Colo.) and 1% Penicillin/Streptomycin (Sigma). Cells were pelletted at 500×G for 5 min and washed in PBS twice, resuspended in 0.5 ml digestion buffer (100 mM NaCl, 10 mM TrisCl pH 8.0, 25 mM EDTA pH 8.0, 0.5% SDS and 0.1 mg/ml proteinase K), incubated with gentle shaking at 50° C. for 18 h, extracted three times with phenol-chloroform and dialyzed against TE for 24 h (Maniatis et al. 1982).
2.5. Genomic DNA Isolation and Subcloning
Mapping and analysis of genomic DNA was performed by PER and restriction enzyme digestion. Genomic PCR was carried out using the Expand Long Template PCR System (Boehringer Mannheim) in a DNA Thermal Cycler 480 (Perkin Elmer/Cetus, Branchburg, N.J.). Typical PCR amplification reaction conditions were: initial denaturation for 4 min at 94° C. (1 cycle); denaturation for 30 sec at 94° C., annealing for 30 sec at 62° C., extension for 15 min at 68° C. (10 cycles); denaturation for 30 sec at 94° C., annealing for 30 sec at 62° C., extension for 15 min at 68° C. with an additional 20 sec per cycle (30 cycles) and final extension for 7 min at 68° C. Typically, PCR products were separated on a 1% agarose gel and visualized by ethidium bromide staining, followed by gel-isolation using the QIAquick® Gel Extraction System (Qiagen, Valencia, Calif.) and cloned into the pCR2.0 or pCR2.1 cloning vector (TA Cloning System, Invitrogen). Restriction enzyme digestion of PAC clones was performed using NotI and SalI (New England Biolabs, Beverly, Mass.) alone or in combination. DNA fragments were separated by Field Inversion Gel Electrophoresis (FIGE mapper system, Bio-Rad Laboratories, Hercules, Calif.) on a 1% agarose gel (SeaKem® gold, FMC BioProducts, Rockland, Me.) and blotted onto Hybond-N membranes (Amersham Life Science, Cleveland, Ohio) for Southern blot analysis (see below).
The 5′UTR exons and upstream genomic sequences were subcloned by EcoRI (New England Biolabs) digestion of PAC 206o5 DNA and ligation of two genomic DNA fragments, 7 kb and 3 kb in length, that contain exons 1a and 1b, and exon 2, respectively, into pBluescript SK(+) cloning vector (Stratagene) using T4 DNA ligase (New England Biolabs) 15 h at 16° C. The cloned 7 kb genomic EcoR1 fragment, containing exons 1a and 1b, is deposited in GenBank under accession number AF110907 (Van E. 1998, pg. 1192).
2.6. mRNA Isolation and Characterization
Poly (A)+ RNA was isolated from B cell lines Daudi, Ramos 2G6 (Siegel and Mostowski, 1990; Lederman et al. 1992), Raji and BA, and from T cell lines B2.7, D1.1 (Yellin et al. 1991), CEM and H9, using the Fast Track® System (Invitrogen). Poly (A)+ RNA (4 μg) was run on a 0.8% agarose/formaldehyde gel for 5.5 h (4 Volts/cm) at 15° C. and blotted onto Hybond-N membranes (Amersham Life Science) for Northern blot analysis. RNA blots containing 2 μg poly (A)+ RNA per lane from fifteen tissues were obtained from Clontech Laboratories for Northern analysis (see below).
2.7. Southern/Northern Blot Analysis
DNA or RNA on membranes was UV-crosslinked using a Stratalinker (Stratagene) and hybridized to [α-32P] dCTP random labeled dsDNA probes (NEBlot® System, New England Biolabs) or to [γ-32P]ATP end-labeled oligonucleotide probes (polynucleotide kinase, Boehringer Mannheim). Unincorporated nucleotides were removed from the labeled probes using G-50 Sephadex® Quick Spin Columns (Boehringer Mannheim). Subsequently, the random labeled probes were denatured by boiling (5 min). Blots were incubated for 1.5 h at 42° C. in prehybridization solution (50% formamide, 5×SSCPE, 5×Denhart's solution, 1 mg ml−1 salmon sperm DNA and 0.1% SDS), followed by incubation 15 h at 42° C. in hybridization solution (50% formamide, 5×SSCPE, 5× Denhart's, 100 μg ml−1 salmon sperm DNA and 10% Dextran Sulfate) containing the radiolabeled probe. The blots were washed twice at 68° C. for 15-20 min in 2×SSC, 1% SDS and the hybridization patterns were visualized by autoradiography (BioMax MS® film, Eastman Kodak, Rochester, N.Y.).
2.8. DNA Sequencing and Sequence Analysis
Isolated PAC clones were sequenced manually by cycle sequencing (dsDNA Cycle Sequencing System®, Life Technologies). Sequence reactions were analyzed by denaturing PAGE (6% polyacrylamide/7 M Urea) and autoradiography (BioMax MS® film, Eastman Kodak). DNA fragments, cloned into the pCR2.0 or pCR2.1 TA cloning vector (Invitrogen) or the pBluescript SK(+) cloning vector (Stratagene), were sequenced on a automated ABI 373 DNA sequencer, by the DNA sequencing core facility at Columbia University or at the Molecular Resource Facility, New Jersey Medical School, UMDNJ. All sequence information was submitted to the BLAST server available through the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.govlBLAST) or the DNA analysis programs of the Genetics Computer Group (GCG, Madison, Wis.) for alignment with additional sequences, identification of repetitive elements or any other homology. Unique sequences were selected and submitted to the Primer3 server available through the Whitehead Institute for Biomedical Research/M.I.T. Center for Genome Research (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3.cgi) or Amplify 1.2 (University of Wisconsin, Madison, Wis.) to design oligonucleotides (obtained from Life Technologies) used for PCR or sequencing.
2.9. FISH Analysis
Metaphase spreads of normal human PBL or the B cell line Ramos 2G6 were probed with genomic TRAF-3 λ-FIX II clone #6 as well as a digoxigenin labeled 14q32.3 telomeric probe (Oncor, Gaithersburg, Md.). Chromosome spreads were counterstained using DAPI. The genomic TRAF-3 probe was labeled with biotin by nick translation and detected with either streptavidin-FITC or streptavidin-rhodamine as previously described (Yu et al. 1996). Images were captured using the Applied Imaging Probevision system (Applied Imaging, Pittsburgh Pa.) and chromosomes were identified using an enhanced DAPI image.
2.10. Radiation Hybrid Screening
Radiation Hybrid screening was performed by Research Genetics (Huntsville, Ala.) on the Genebridge 4 panel using primers: Flgen (5′ GAGAGAAATTCTGGCTCTTCAGATCTATTGTCGG 3′) (SEQ ID NO:5), located in exon 3, and FR8gen (5′ TGCAGTCAGGACGCACACATGGAAG 3′) (SEQ ID NO.: 6), located in exon 4. Vector data were submitted to the Whitehead Institute for Biomedical Research server for placement on the Whitehead framework map (http://www.genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl).
3. Results
3.1. Characterization of TRAF-3 Encoding mRNA Species
TRAF-3 mRNA species were analyzed by hybridizing RNA blots from different human tissues (FIG. 7) with a probe (“P55”, FIG. 2c) that represents the complete TRAF-3 open reading frame (ORF). Similar to previous reports (Cheng et al. 1995; Mosialos et al. 1995; Wang et al. 1996), TRAF-3 hybridizing mRNA species are expressed predominantly in lymph node and PBL and migrate as two major classes of transcripts, a 2 kb class (migrating as two bands of 2.2 and 2.6 kb) and an 8 kb class (migrating at 8.5 kb) (FIG. 7). The expression of TRAF-3+ transcripts in spleen and thymus is notably low, consistent with recent immunohistochemical analysis showing low levels of TRAF-3 protein in these tissues (Krajewski et al. 1997). The “P55” TRAF-3 probe also hybridized with the 2 kb class and the 8 kb class (migrating as two bands of 8.3 and 8.7 kb) of TRAF-3 mRNA transcripts from certain T and B cell tumor lines (FIG. 2a). The observed TRAF-3 specific hybridization pattern of mRNA from these cell lines is consistent with results from previous studies. For example, similar to the results in FIG. 2a, Ramos cells are known to have barely detectable TRAF-3 mRNA (Cheng et al. 1995) and protein (Krajewski et al. 1997). In addition, EBV transformed B cells, like BA (FIG. 8A), have high levels of TRAF-3 mRNA (Mosialos et al. 1995). Since the Raji B cell line, Jurkat D1.1 T cell line and EBV transformed B cells express both the 2 kb and 8 kb classes of TRAF-3 transcripts, further studies of TRAF-3 mRNA (below) were based on mRNA from Raji, Jurkat D1.1 or EBV transformed B cells.
3.2. Characterization of an Extended 3′UTR in 8 kb TRAF-3 cDNA
All previously reported human and murine TRAF-3 cDNA clones are approximately 2.4 kb in length and terminate in a similar region of the 3′UTR, except for the murine TRAF-3 cDNA clone (TRAFamn) reported by Lacy and coworkers (Wang et al. 1996), which is 7.3 kb in length and contains a 5 kb 3′UTR. During the course of the human genomic analysis (described below), a genomic sequence located 3′ to the region encoding human TRAF-3 was noted to be homologous to a segment of the 3′UTR of the murine TRAF-3 (TRAFamn) cDNA clone. In addition, the human genomic sequence was identical to sequence elements of three deposited human Expressed Sequence Tags (ESTs) (GenBank accession numbers: AA035757, AA159960 and AA375889). Primers, derived from these sequences, were used in RT-PCR and 3′-RACE experiments to clone two overlapping cDNA clones (4.7 and 1.8 kb) that together represent a 5.8 kb cDNA segment, containing IIIb nt 1823 to 2433 followed by 5.3 kb of novel sequence that terminates in a poly-A+ tail. Sequence analysis revealed that the 8 kb class transcripts utilize a canonical polyadenylation signal, AAUAAA (located at nt 7617), 24 bp upstream of the start of the poly-A+ tail. Together, these data suggest that these cDNA clones represent an extended human TRAF-3 3′UTR. To confirm that the 8 kb class TRAF-3+ transcripts, as observed in FIGS. 7 and 8a, contain the novel 5.3 kb 3′UTR, RNA blots derived from the T and B cell lines were probed with a DNA segment from the 3′-end of the extended 3′UTR. The 8 kb class transcripts, but not the 2 kb transcripts, hybridize with the “3′UTR” probe (FIG. 8b). Taken together, these data show that the 8 kb class of TRAF-3 mRNA transcripts are distinguished by the presence of a 5.3 kb extended 3′UTR.
The sequences of the human TRAF-3 3′UTRs revealed segments of homology with murine TRAF-3 and certain regulatory elements. Comparison of the 3′UTR sequences of both the human and murine cDNA clones revealed two segments in the 3′UTR of both species that share 75% or higher identity. The first segment of high homology (>75%) is 2.0 kb and is located immediately after the stop codon, whereas the second is 0.5 kb and is located near the 3′-end of the extended 3′UTR. Interestingly, these two segments of relatively high homology are interrupted with a region of 3.3 kb with less than 50% homology with murine TRAF-3 (FIG. 8C). In addition, the human TRAP-3 3′UTR contains 6 AUUUA elements (IIIb nt 1944, 2133, 2335, 2529, 2774 and 7502), three of which are present exclusively in the extended 3′UTR of the 8 kb transcripts (2529, 2774 and 7502). Such elements have been implicated in transcript instability and/or suppression of mRNA translation of proto-oncogenes, transcription factors and cytokines (Chen and Shyu, 1995; Han et al. 1990; Kruys et al. 1987; Wilson and Treisman, 1988; Shaw and Kamen, 1986). The utility of sequence analysis to predict functional AUUUA elements is strengthened when these elements are flanked by U nucleotides (Chen and Shyu, 1995; Han et al. 1990; Kruys et al. 1987; Wilson and Treisman, 1988; Shaw and Kamen, 1986), which is the case for the AUUUA elements located at nt 2133 and 7502. In addition, both of these elements, as well as the AUUUA element at nt 2529, are conserved between human and mouse (Wang et al. 1996). Together, these data suggest that the 3′UTRs of both the 2 and 8 kb classes of TRAF-3 mRNA transcripts contain sequence elements that may regulate mRNA expression.
3.3. Diversity in the Region of cDNA Clones Encoding the Zn Finger Domain
In the coding region of the TRAF-3 cDNA, evidence for alternative splicing was first observed by the comparison of the sequences of TRAF-3b (CAP-1) cDNA (Sato et al. 1995) with the other reported TRAF-3 cDNA clones (Hu et al. 1994; Cheng et al. 1995; Mosialos et al. 1995). This analysis revealed that TRAF-3b (CAP-1) is identical to TRAF-3 except for a 75 nucleotide deletion (resulting in Δ25 aa (Δaa 218-242)) in a region of TRAF-3 predicted to encode five atypical Zn fingers (FIG. 9). To examine the extent to which TRAF-3 mRNA species vary in this region, RT-PCR was performed using gene specific primers that amplify this region. These experiments revealed three distinct TRAF-3 PCR products that were cloned and characterized. Sequence analysis of these variant amplification products revealed deletions of 75 bp (IIIb nt 869-943), 156 bp (IIIb nt 788-943) and 168 bp (IIIb nt 869-1036) in the Zn finger-like region. In all three cases, the mRNA species maintain the ORF and therefore predict TRAF-3 isoforms (FIG. 9). The TRAF-3 cDNA clone containing the 75 bp (IIIb nt 869-943) deletion is identical to the previously reported TRAF-3b (CAP-1) (Sato et al. 1995). However, the two novel cDNA clones predict TRAF-3 isoforms which contain deletions of 52 aa (Δ_aa 191-242) and 56 aa (Δ_aa 218-273), respectively, in the Zn finger-like region. All three isoforms can be distinguished by the number of Zn fingers and the aa sequence of the most C-terminal finger. The Δ25 aa (TRAF-3b) isoform contains four Zn fingers with a C-terminal finger formed by the fusion of the N-terminal half of the 4th and the C-terminal half of the 5th finger (the numbering of segments in hybrid fingers refers to the location of these aa sequences in the 5 Zn fingers of full length TRAF-3) (FIG. 9). The Δ52 aa isoform contains three Zn fingers with a C-terminal finger formed by the fusion of the N-terminal half of the 3rd and the C-terminal half of the 5th finger (FIG. 9). The Δ56 aa isoform contains three complete Zn fingers and the N-terminal portion of the 4th finger (FIG. 9). Following the nomenclature of Krajewski et al. (Krajewski et al. 1997), the two novel_putative proteins are designated TRAF-3c (Δ52 aa) and TRAF-3d (Δ56 aa).
3.4. Characterization of TRAF-3 cDNA Clones with Distinct 5′UTR Elements
Comparison of TRAF-3 cDNA clones, isolated from a Raji B cell cDNA library or from 5′-RACE experiments using Raji or D1.1 mRNA, with each other and with TRAF-3 sequences previously reported (Hu et al. 1994; Cheng et al. 1995; Mosialos et al. 1995; Sato et al. 1995), revealed that distinct cDNA sequence elements were variably included in the 5′UTR of different clones (FIG. 10). For example, the IIIb and Ib cDNA clones differ by the presence, in the latter type, of a discrete 139 bp DNA segment, positioned 18 nt upstream of the translational start site (FIG. 10). Semi-quantitative analysis of 62 isolated cDNA clones, revealed that 85% contain the 139 bp segment while 15% lack this segment. Together, these data suggest that the 139 bp element is encoded by an exon that is variably included, resulting in mRNA species with distinct 5′UTRs.
Additional diversity in the 5′UTR of TRAF-3 was manifested by the characterization of the 2-1 and Ib cDNA clones that contain distinct sequences upstream of the 139 bp segment (FIG. 10). In the 2-1 cDNA clone, the 139 bp segment is preceded by all or part of a 141 bp element that contains a high G-C content (76%), while in the Ib cDNA clone, the 139 bp segment is preceded by all or part of a 200 bp element that is also G-C rich (82%) (FIG. 10). In both cases (the 141 bp element and the 200 bp element), 5′-RACE experiments did not yield additional upstream sequences, perhaps due to the G-C rich nature of these 5′UTR elements. The fact that the two distinct G-C rich elements were not observed in the same cDNA clones suggests that TRAF-3 transcripts initiate from two distinct start sites. Together, these data indicate that in addition to alternative mRNA splicing, alternative transcription initiation contributes to the diversity in the 5′UTR of TRAF-3 transcripts. To clarify the mechanism(s) by which these distinct TRAF-3 transcripts are generated, the next series of experiments characterized the genomic structure and localization of the genetic elements that encode them.
3.5. Genomic Localization of TRAF-3 by FISH and Radiation Hybrid Analysis
To identify the chromosomal localization of the TRAF-3 encoding gene(s), FISH analysis was performed by hybridization of normal human PBL metaphase spreads with a genomic TRAF-3 λ-FIXII clone #6 (containing IIIb nt 200-787). This probe bound specifically to the most telomeric region of the long arm of human chromosome 14, in band 14q32.3 (FIG. 11A), consistent with a single gene or gene cluster near the telomere of chromosome 14q. Since this region is known to contain the well-characterized breakpoint in Burkitt's lymphoma t(8;14) (q24.1; q32.3), TRAF-3 was localized with respect to this chromosomal breakpoint in the Ramos cell line. Two-color FISH analysis (Yu et al. 1996) was performed on metaphase spreads from Ramos 2G6 using a TRAF-3+ λ-phage probe and a chromosome 14 telomere probe (FIG. 11B). The two probes bound in very close proximity on the normal chromosome 14. However, the TRAF-3 probe remained on the derivative chromosome 14, proximal to the breakpoint, whereas the chromosome 14 telomere probe was detected on the derivative chromosome 8, consistent with the translocation of this telomere to chromosome 8 in Ramos (FIG. 11B). Taken together, these data indicate that the genetic elements encoding TRAF-3 are located centromeric to the Ramos breakpoint on 14q32.3, which has been mapped to a precise location in the human Ig heavy chain gene complex (the Switch-μ region) (Wiman et al. 1984).
Radiation Hybrid mapping confirmed and extended this analysis. Using PCR primers derived from the region encoding the RING finger domain that generate a 740 bp fragment (containing an intron) from genomic DNA, the position of a gene for TRAF-3 was determined more precisely on chromosome 14q32.3. The TRAF-3 gene is located 3.46 centiRays (cR) from framework marker FB19F11, which is 354.28 cR from the top of chromosome 14 on the Whitehead radiation hybrid map (FIG. 12). Together with the FISH analysis, these data demonstrate that TRAF-3 is encoded by a single gene or gene cluster located at 14q32.3.
3.6. Analysis of Intron-Exon Structure of the TRAF-3 Gene.
To further characterize the genetic elements encoding TRAF-3, hybridization screening of a λ-FIX II and a PAC library yielded 3 genomic phage clones and 4 overlapping genomic PAC clones which were analyzed by PCR, Southern blotting and partial sequencing. These studies established that the phage and PAC clones contain all of the TRAF-3 encoding genetic elements represented in the TRAF-3 cDNA clones isolated. Furthermore, comparison of the genomic cDNA sequences allowed the establishment of the intron-exon junctions of exons 2 through 12 as well as the 3′ boundaries of exons 1a and 1b (FIG. 14). All intron-exon junctions conform to the GT/AG rule (Breathnach and Chambon, 1981). The establishment of the intron-exon structure also allowed the superimposition of the five predicted protein domains of TRAF-3 onto the exon structure (FIG. 9). The RING finger (IIIb nt 353-523) is encoded by exons 3 through 5; the Zn finger domain (nt 545-1009) by exons 5−9; the isoleucine zipper domain (nt 1076-1243) by exons 10 and 11; The TRAF-N domain (nt 1283-1462) by exons 11 and 12; and the TRAF-C domain (nt 1463-stop solely by exon 12 (FIG. 9). In addition, sequence analysis of the extended 5.8 kb 3′UTR (described above) and of the genomic region that encodes the extended 3′ UTR, revealed that the cDNA is co-linear with the genomic sequence until the 3′-polyadenylation site (nt 7640) in exon 12, after which the sequences diverge (FIG. 9). The 2 kb class of transcripts appear to utilize either of two signals; a non-canonical polyadenylation signal, AGUAAA (nt 2323), or a canonical signal, AAUACA (nt 2401) (Hu et al. 1994; Cheng et al. 1995; Sato et al. 1995; Mosialos et al. 1995; Parthasarathy et al. 1997). Together, these data indicate that alternative polyadenylation is the mechanism that generates the 2 kb or 8 kb classes of TRAF-3 transcripts.
The identification and analysis of the intron-exon borders revealed that alternative mRNA splicing accounts for several of the mRNA species observed (described above). In this regard, the mRNA encoded by the 139 bp exon 2 is variably included in certain TRAF-3 cDNA clones, such as Ib and 2-1 (FIG. 10). In addition, the TRAF-3b encoding transcript (CAP-1) lacks exon 8 and the transcripts encoding the putative TRAF-3c and TRAF-3d protein isoforms result from deletion of exon 7 and 8 (Δ52 aa) or deletion of exon 8 and 9 (Δ56 aa), respectively (FIG. 9). Alternative splicing of these exons does not disrupt the ORF, since the splice junctions between exons 7 and 9 (TRAF-3b), exons 6 and 9 (TRAF-3c) and exons 7 and 10 (TRAF-3d) are of the same class (class 0) as each of the other splice junctions between exons 5 through 10 (FIG. 14).
3.7. Physical Map of the TRAF-3 Gene Locus
The analysis of the 4 genomic PAC clones provides a physical map of the genomic locus of the TRAF-3 gene (FIG. 13). The gene for TRAF-3 consists of 13 exons that span a genomic region of 130 kb. Exons 1a and 1b are closely spaced ( ̃800 bp) in a region with a high G-C content ( ̃80%) and characteristics of a CpG island, which are found at the 5′-end of most housekeeping genes and of many other genes that are expressed in a tissue specific pattern (Wenger et al. 1998; Cross and Bird, 1995; Gardiner-Garden and Frommer, 1987). In contrast, exons 1a and 1b are separated from exon 2 by a 54 kb intron and exon 2 is separated from exon 3 by a 32 kb intron. The coding exons (exons 3-12) are relatively compactly situated in a genomic region of 41 kb, with the nine introns between exons 3 through 12 ranging in size from approximately 0.6 to 9.5 kb.
The next series of experiments sought to physically link the TRAF-3 gene with other genomic markers. Two other genomic markers, SGC30775 and D14S272, are mapped at a similar distance as TRAF-3 from framework marker FB19F11 on the Whitehead radiation hybrid map. Therefore, to determine whether these two genomic markers are represented on the PAC clones, PCR was performed with primers derived from the sequences of these two markers using the PAC clones as templates. These studies revealed that SGC30775 is located on PAC 167m5, whereas D14S272 was not be detected on any of the PAC clones. The presence of SGC30775 on PAC 167m5 was confirmed by directly sequencing PAC 167m5 in this region. In addition, Southern blot analysis positioned SGC30775 approximately 100 kb downstream of TRAF-3 exon 12.
Since D14S272 was not detected on any of the PAC clones, the PAC clones were also analyzed for the presence of other genomic markers that are closely linked to D14S272 on the Human Transcript Map (available through the NCBI server at http://www.ncbi.nlm.nih.gov/cgi-bin/SCIENCE96). Primers derived from the 3′-end sequence of one of these genomic markers, a 5241 nt cDNA clone KIAA0071 (GenBank accession number: D31888), specifically amplify the predicted product from PAC 20605. The presence of KIAA0071 on PAC 20605 was confirmed by-directly sequencing PAC 20605 in this region. Subsequently, PCR and Southern blot analysis revealed that the 3′-end of KIAA0071 is located approximately 40 kb upstream of TRAF-3 exon 1a and that this gene is situated in the same transcriptional orientation as TRAF-3 (FIG. 13). The unambiguous radiation hybrid mapping of the 740 bp TRAF-3 exon 3-exon 4 PCR product to a single region of 14q32.3 and the physical mapping of the 4 PAC clones strongly suggest that TRAF-3 is a single gene. Together these data establish the physical relationship of TRAF-3 with two genomic markers at 14q32 and indicate that TRAF-3 is encoded by a single gene located in the subtelomeric region.
As stated above, radiation hybrid mapping showed that the TRAF-3 gene is located 3.46 centiRays (cR) from framework marker FB19F11, which is 354.28 cR from the top of chromosome 14 on the Whitehead radiation hybrid map (FIG. 12). From this analysis, TRAF-3 could be located at 357.74 cR or at 350.82 cR. The physical linkage of TRAF-3 to SGC30775, which is at 357.63 cR, and located approximately 100 kb downstream of TRAF-3, establish the location of TRAF-3 at 357.74 cR from the top of chromosome 14 and approximately 13.5 cR from the telomere (FIG. 12). In addition, the radiation hybrid and physical mapping indicate that the TRAF-3 gene is situated at a distance of 6.29 cR from genomic marker SGC33974 (364.03 cR), the sequence of which is identical to a portion of the human IgD segment locus (GenBank accession number: X97051). Together with the fact that the IgD segment locus is approximately 400 kb telomeric to the 3′-end of the Ig heavy chain constant region complex (IGHC) (Hofker et al. 1989), these data suggest that the TRAF-3 gene is located at a distance of approximately 1 Mb from the 3′-end of the IGHC.
4. Discussion
The genomic structure of TRAF-3 was characterized by FISH analysis, radiation hybrid mapping and characterization of four overlapping PAC clones which demonstrate that a single TRAF-3 gene is located on chromosome 14q32.2, contains 13 exons, and spans 130 kb within 13.5 cR of the telomere. To generate a variety of mRNA transcripts, the TRAF-3 gene utilizes two alternative starting exons (1a and 1b, respectively), alternative splicing of a non-coding exon 2, alternative splicing of certain of the 10 coding exons (exons 7−9) and alternative processing of a 3′UTR of approximately 5.8 kb (accounting for the 2 and 8 kb classes of mRNA transcripts). Together, these data indicate that a single TRAF-3 gene codes for diverse transcripts by a combination of alternative initiation, alternative splicing and alternative polyadenylation.
A number of TRAF-3 mRNA species are generated by alternative splicing of coding exons (7, 8, and 9) which lead to the generation of mRNA species that predict TRAF-3 peptides which have distinct aa sequences in the region encoding Zn fingers. Evidence for this type of posttranslational modification was first observed by the isolation of a cDNA encoding TRAF-3b (Δ25) by Sato et al. (Sato et al. 1995). The data presented in this paper suggest the existence of additional isoforms; Δ52 and Δ56. Following the nomenclature of Reed (Krajewski et al. 1997), the new putative TRAF-3 isoforms have been termed TRAF-3c and TRAF-3d, respectively. Like TRAF-3b, both TRAF-3c and TRAF-3d predict proteins with a reduced number of Zn fingers and a distinct composition of the most C-terminal Zn finger domains as compared to TRAF-3. The fact that removal or insertion of one or a combination of certain exons (7−9) encoding the Zn fingers does not alter the ORF is due to the common class of splice junctions (class 0, the junction of two exons falls between two codons). It is of potential interest that the splice junctions between exons 4 through 11 are all of class 0. The common splice junctions of these exons suggests that additional splice variants involving exons 5, 6 and/or 10 may yet be identified, perhaps in combinations with deletions of exons 7-9, that would encode novel TRAF-3 proteins. In this regard, deletions of exon 5 would alter the RING finger domain and may affect signaling function.
It is unknown whether these or other TRAF-3 isoforms are expressed as proteins and if so, whether they have functional significance. However, the possible functional roles of such splice deletion variants are intriguing, because RING finger and Zn finger domains are known to mediate either protein-protein and protein-DNA interactions. In this regard, it is of interest that alternative mRNA splicing results in a TRAF-2 isoform (TRAF-2A) that contains an insertion of 7 aa in the RING finger domain, and inhibits NF-κB activation by TNF-R stimulation (Brink and Lodish, 1998). These data indicate that the inhibitory isoform, TRAF-2A, is “full length” and that the NF-κB activating isoform, “TRAF-2”, is a splice deletion variant (Brink and Lodish, 1998). Therefore, the fact that full length TRAF-3 inhibits NF-κB activation must be considered in the context that cDNA clones encoding smaller TRAF-3 isoforms have been identified, but not yet studied for their functional capabilities in receptor-mediated signaling. Therefore, future studies will address whether such isoforms are expressed and if so, whether such isoforms regulate TRAF-3 functions by regulating its interaction with other constituents of the cytoplasmic signaling apparatus.
In addition to the splice variants identified in this paper, Kieff and coworkers (Mosialos et al. 1995) reported the characterization of a TRAF-3 cDNA that predicts a peptide which initiates translation at methionine 350 (aa350-568). The peptide predicted from such a transcript would be 26 aa longer than the His-tagged, truncated form, C26 (containing murine aa324-567)) that we previously showed to inhibit CD40-mediated upregulation of CD23 (Cheng et al. 1995) and 29 aa longer than a related truncated form (containing murine aa 327-567) that inhibits CD40-mediated growth inhibition of epithelial cells (Eliopoulos et al. 1996). If a TRAF-3 isoform is expressed by the mRNA splice variant initiating translation at methionine 350 (aa350-568), such an isoform would contain the TRAF-C domain, but would lack both the RING and the Zn finger domains.
In the 5′UTR, further variety in TRAF-3 transcripts was found to be mediated by alternative splicing of exon 2 in a subset of transcripts and by utilization of two alternative 5′UTR encoding exons, 1a and 1b. Because the sequence elements encoded by exons 1a and 1b were not observed in the same cDNA clone, these two exons appear to represent alternative initiating exons. However, it should be noted that the precise transcriptional start sites were not identified, perhaps due to the G-C rich nature of exons 1a and 1b (76% and 82%, respectively). The function of the diversity generated by alternative splicing and alternative transcriptional initiation in the 5′UTR may regulate the level of TRAF-3 transcripts or protein expression. For instance, the high G-C content in the two initiating exons may regulate TRAF-3 expression by maintaining a “closed” conformation that does not allow the translation machinery to proceed through this area in the transcript (Kozak, 1989a; Kozak, 1989b; Kim et al. 1992; Romeo et al. 1993). In this light, it should be noted that upstream of the TRAF-3 initiating exons no conventional promoter with a “TATA-box” and/or “CAAT-box” was identified. This sequence analysis suggests that the gene for TRAF-3 contains “TATA-less” promoters, upstream of exon 1a and 1b, which is consistent with the overall high G-C content of the genomic region in which exon 1a and 1b are located. Moreover, this region contains typical characteristics of a CpG island (Gardiner-Garden and Frommer, 1987), which are found in the 5′-ends and promoters of most housekeeping genes and in many genes with tissue-specific expression (Wenger et al. 1998; Cross and Bird, 1995; Gardiner-Garden and Frommer, 1987). Therefore, it is possible that the differences in the 5′UTR lead to regulation of the level of TRAF-3 expression in a lineage- and/or differentiation-specific fashion (Krajewski et al. 1997).
Results of chromosomal FISH analysis, radiation hybrid mapping and physical characterization of the 4 PAC clones in this study indicate that TRAF-3 is a single copy gene located on chromosome 14q32.3. In this regard, the genes for human TRAF-1, 4 and 5 have been mapped and are dispersed in the genome at chromosomes 9q33-34, 17q11-12 and 1q32, respectively (Nakano et al. 1997; Regnier et al. 1995; Siemienski et al. 1997). In addition, the genomic organizations of TRAF-1 and 4 have been determined and comparison between TRAF-3 and TRAF-3 and 4 reveal both similarities and differences. First, the TRAF-3 gene spans approximately 130 kb and is substantially larger than TRAF-1 (12 kb) or TRAF-4 (5.5 kb) genes. Second, the TRAF-C domains of TRAF-3 and TRAP-4 are contained on single exons, whereas the TRAF-C domain of TRAF-1 is divided into 4 exons (Nakano et al. 1997; Regnier et al. 1995; Siemienski et al. 1997). In this regard, the differences in genomic organization between TRAP-3 and TRAF-1 could reflect the fact that, based on protein and DNA homology, TRAF-3 is relatively distantly related to TRAF-1. It will be of interest to determine the genomic organizations of TRAF-2, 5 and 6 to understand the phylogenetic relationships of these genes in more detail.
The localization of human TRAF-3 to 14q32.3 is of interest, because murine TRAF-3 is located on the distal region of murine chromosome 12, which has synteny with human 14q32 (Wang et al. 1996). In both species, the proximity of TRAF-3 to the IGHC is maintained. The region of human chromosome 14q32.3, that contains the TRAF-3 gene, is known to have a relatively high rate of recombination (Hofker et al. 1990). In addition, 14q32.3 is the site of many translocation breakpoints. In this regard, several genes that contribute to the transformed phenotype of lymphoid malignancies have been identified that translocate into the IGHC including; myc (Taub et al. 1982), CCND1 (BCL-1) (Motokura et al. 1991), BCL-2 (Tsujimoto et al. 1984), BCL-3 (Ohno et al. 1990), BCL-6 (Ye et al. 1993) and lyt-10 (Neri et al. 1991). However, TRAF-3 is approximately 1 Mb centromeric to the IGHC and is not involved-in translocations associated with these lymphoid malignancies, since these translocation junctions have been well characterized. However, in a type of childhood acute leukemia in which blasts have both lymphoid and myeloid (“mixed lineage”) features (Hayashi et al. 1990), the translocation junction in 14q32.3 occurs centromeric to IgH locus and the involvement of TRAF-3 has not been excluded. In addition, 14q telomeric deletions, involving 14q32.3, have been observed as loss of heterozygosity (LOH) in renal oncocytomas (Schwerdtle et al. 1997), non-papillary renal cell carcinomas (Schullerus et al. 1997) and malignant meningiomas (Simon et al. 1995; Tse et al. 1997; Menon et al. 1997). Future studies will address whether such translocations or LOH involves TRAF-3, and if so, whether alterations in the level of TRAF-3 expression contributes to a transformed phenotype, as would be the case for a proto-oncogene or a tumor suppressor gene.
The characterization of the PAC clones presented in this study is also of interest because this region has not yet been mapped by yeast artificial chromosomes (YACs) (Hofker et al. 1990; Chen et al. 1995). It is possible that the high rate of recombination in this region affects the stability of such large clones (Wintle et al. 1997). In this regard, the PAC clones described in this study may contain segments of DNA that were considered “unclonable” (Kang and Cox, 1996). Therefore, the PAC clones and the physical map of TRAF-3 described in this work may be useful, not only in the establishment of the TRAF-3 transcriptional orientation and the physical linkage of TRAF-3 with respect to the IGHC, but also in the further characterization of 14q32.3 (Cox et al. 1995).
Furthermore, the chromosomal localization of TRAF-3 and the structural characterization of the TRAF-3 gene may be useful in studying genetic immunodeficiency syndromes. For example, a subset of individuals with hyper-IgM syndrome (HIGM) have normal CD154 (Callard et al. 1994; Conley et al. 1994) and the role of TRAF-3 in CD40 signaling suggests that TRAF-3 defects might result in similar phenotypes. In this regard, TRAF-3 deficient lymphocytes share features with CD154 deficient (Xu et al. 1994) or CD40 deficient (Kawabe et al. 1994; Castigli et al. 1994) lymphocytes in mice in vivo (Xu et al. 1996). In addition, the fact that truncated forms of TRAF-3 inhibit CD40 signaling suggests that mutations affecting TRAF-3 splicing or that translocation events involving TRAF-3 may result in abnormal CD40 signaling that might manifest as HIGM. Therefore, the chromosomal localization of TRAF-3, and the physical mapping reported in this paper, will be useful in characterizing the structure of TRAF-3 genes in such individuals.
Example 3
Gene Therapy
The invention features expression vectors for in vivo transfection and expression in particular cell types of TRAF-3 deletion isoform mutants so as to antagonize the function of wild type TRAF-3 in an environment in which the wild-type protein is expressed (i.e., introduce abnormal TRAF-3 deletion isoform that acts as a dominant negative protein to inhibit CD40 signaling).
Expression constructs of TRAF-3 deletion isoform polypeptides may be administered in any biologically effective carrier that is capable of effectively delivering a polynucleotide sequence encoding the TRAF-3 isoform to cells in vivo. Approaches include insertion of the subject gene in viral vectors including recombinant retroviruses, baculovirus, adenovirus, adeno-associated virus and herpes simplex virus-1, or recombinant bacterial or eukaryotic plasmids. Viral vectors transfect cells directly, plasmid DNA can be delivered with the help of, for , example, cationic liposomes or derivatized (e.g., antibody conjugated) polylysine conjugates, gramacidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaPO4 precipitation carried out in vivo.
Any of the methods known in the art for the insertion of polynucleotide sequences into a vector may be used. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989) and Ausubel et al., Current Protocols in Molecular Biology, J. Wiley & Sons, N.Y. (1992), both of which are incorporated herein by reference. Conventional vectors consist of appropriate transcriptional/translational control signals operatively linked to the polynucleotide sequence for a particular anti-fibrotic polynucleotide sequence Promoters/enhancers may also be used to control expression of anti-fibrotic polypeptide. Promoter activation may be tissue specific or inducible by a metabolic product or administered substance. Such promoters/enhancers include, but are not limited to, the native E2F promoter, the cytomegalovirus immediate-early promoter/enhancer (Karasuyama et al., J. Exp. Med., 169: 13 (1989)); the human beta-actin promoter (Gunning et al., Proc. Natl. Acad. Sci. USA, 84: 4831 (1987); the glucocorticoid-inducible promoter present in the mouse mammary tumor virus long terminal repeat (MMTV LTR) (Klessig et al., Mol. Cell. Biol., 4: 1354 (1984)); the long terminal repeat sequences of Moloney murine leukemia virus (MuLV LTR) (Weiss et al., RNA Tumor Viruses, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1985)); the SV40 early region promoter (Bernoist and Chambon, Nature, 290:304 (1981)); the promoter of the Rous sarcoma virus (RSV) (Yamamoto et al., Cell, 22:787 (1980)); the herpes simplex virus (HSV) thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA, 78: 1441 (1981)); the adenovirus promoter (Yamada et al., Proc. Natl. Acad. Sci. USA, 82: 3567 (1985).
Expression vectors compatible with mammalian host cells for use in gene therapy of tumor cells include, for example, plasmids; avian, murine and human retroviral vectors; adenovirus vectors; herpes viral vectors; and non-replicative pox viruses. In particular, replication-defective recombinant viruses can be generated in packaging cell lines that produce only replication-defective viruses. See Current Protocols in Molecular Biology: Sections 9.10-9.14 (Ausubel et al., eds.), Greene Publishing Associcates, 1989.
Specific viral vectors for use in gene transfer systems are now well established. See for example: Madzak et al., J. Gen. Virol., 73: 1533-36 (1992: papovavirus SV40); Berkner et al., Curr. Top. Microbiol. Immunol., 158: 39-61 (1992: adenovirus); Moss et al., Curr. Top. Microbiol. Immunol., 158: 25-38 (1992: vaccinia virus); Muzyczka, Curr. Top. Microbiol. Immunol 158: 97-123 (1992: adeno-associated virus); Margulskee, Curr. Top. Microbiol. Immunol., 158: 67-93 (1992: herpes simplex virus (HSV) and Epstein-Barr virus (HBV)); Miller, Curr. Top. Microbiol. Immunol., 158: 1-24 (1992: retrovirus); Brandyopadhyay et al., Mol. Cell. Biol., 4: 749-754 (1984: retrovirus); Miller et al., Nature, 357: 455-450 (1992: retrovirus); Anderson, Science, 256: 808-813 (1992: retrovirus), all of which are incorporated herein by reference.
Several methods of transferring potentially therapeutic genes to defined cell populations are known. See, e.g., Mulligan, “The Basic Science of Gene Therapy”, Science, 260, pp. 920-31 (1993). These methods include:

    1) Direct gene transfer. See, e.g., Wolff et al., “Direct Gene transfer Into Mouse Muscle In Vivo”, Science, 247, pp. 1465-68 (1990);
    2) Liposome-mediated DNA transfer. See, e.g., Caplen et al., “Liposome-mediated CFTR Gene Transfer To The Nasal Epithelium of Patients With Cystic Fibrosis”, Nature Med., 3, pp. 39-46 (1995); Crystal, “The Gene As A Drug”, Nature Med., 1, pp. 16-17 (1995); Gao and Huang, “A Novel Cationic Lipoma Reagent For Efficient Transfection Of Mammalian Cells”, Biochem. Biophys. Res. Comm., 179, pp. 280-85 (1991);
    3) Retrovirus-mediated DNA transfer. See, e.g., Kav et al., “In Vivo Gene Therapy Of Hemophilia B: Sustained Partial Correction In Factor IX-Deficient Dogs”, Science, 262, pp. 117-19 (1993); Anderson, “Human Gene Therapy”, Science, 256, pp. 808-13 (1992);
    4) DNA Virus-mediated DNA transfer. Such DNA viruses include adenoviruses (preferably Ad-2 or Ad-0 based vectors), herpes viruses (preferably herpes simplex virus based vectors), baculoviruses, and parvoviruses (preferably “defective” or non-autonomous parvovirus based vectors, more preferably adeno-associated virus based vectors, most preferably AAV-2 based vectors). See, e.g. Ali, et al., “The Use Of DNA Viruses As Vectors For Gene Therapy”, Gene Therapy,1, pp. 367-84 (1994); U.S. Pat. No. 4,797,368, incorporated herein by reference, and U.S. Pat. No. 5,139,941, incorporated herein by reference.


The choice of a particular vector system for transferring the gene of interest will depend on a variety of factors. One important factor is the nature of the target cell population. Although retroviral vectors have been extensively studied and used in a number of gene therapy applications, these vectors are generally unsuited for infecting non-dividing cells. In addition, retroviruses have the potential for oncogenicity.
Example #4
Expression and Function of TRAF-3 Splice-Variant Isoforms in Human Lymphoma Cell Lines
[Abbreviations used)]: TRAF, TNF receptor-associated factor; NF-κB, Nuclear factor-κB; TNF-R, Tumor Necrosis Factor Receptor; IVT, in vitro transcription; NIK, NF-κB-inducing kinase; ASK1, Apoptosis Signaling-regulated Kinase 1; IκE, Inhibitor of NF-KκB; IKK, IκB Kinase; N-terminal, amino-terminal; C-terminal, carboxy-terminal; ORF, open reading frame; RT, reverse transcriptase; β-gal, β-galactosidase; His, histidine; aa, amino acid; HA-tagged, hemagglutinin epitope tagged fusion construct; His-tagged, poly-histidine fusion construct; TBST, Tris buffered saline with 0.1% Tween-20; IMDM, Iscove's Modified Dulbecco's Media.
Abstract
TRAF-3 gene products are signaling adaptor molecules required for lymphocytes to mediate T-dependent antibody responses in vivo. Previous work identified 8 splice-variant TRAF-3 mRNA species by RT-PCR that have the potential to encode novel isoforms, seven of which induce NF-κB activation when over-expressed in 293 cells. To characterize the expression of TRAF-3 splice-variant mRNAs in lymphoma cell lines, RNAse protection assays were performed using nine antisense probes that correspond to full length TRAF-3 and each of 8 splice-variants. The T cell line Jurkat D1.1 and the B cell lines BJAB, Daudi, and Raji each expressed mRNA encoding TRAF-3 splice-variants in approximately the same rank order (from highest to lowest); TRAF-3 Δ103aa, Δ83aa, full-length, Δ25aa, Δ52aa, Δ56aa, Δ27aa, and Δ221aa mRNA. The TRAP-3 Δ130aa mRNA was not detectable in any of the cell lines examined. The functional effect of over-expressing each TRAF-3 splice-variant on NF-κB activation was studied in the TRAF-5-responsive B cell line, BJAB. Of the seven TRAF-3 splice-variant isoforms that induce NF-κB activation in 293 cells, only TRAF-3 Δ27aa, Δ103aa, or Δ130aa induce NF-κB activation in BJAB cells. Together, these data indicate that a number of TRAF-3 splice-variant mRNAs are expressed and function in B and T lymphoma lines, which suggests that certain TRAF-3 splice-variant isoforms may participate in mediating the known functions of the TRAF-3 gene in lymphocytes.
1. Introduction
TRAF-3 gene products are signaling adaptor molecules that interact with the cytoplasmic tails of CD40 (Hu et al., 1994; Cheng et al., 1995; Sato et al., 1995; Mosialos et al., 1995) and other Tumor Necrosis Factor-Receptor (TNF-R) family members (e.g. LTβ-R, CD30, CD27, OX40) (Mosialos et al., 1995; Gedrich et al., 1996; Boucher et al., 1997; Vanarsdale et al., 1997; Arch and Thompson, 1998; Kawamata et al., 1998). Eight TRAF-3 mRNA splice-variants were identified by RT-PCR that have the potential to encode isoforms with altered Zn finger domains (Sato et al., 1995; Krajewski et al., 1997; van Eyndhoven et al., 1998; van Eyndhoven et al., 1999). In addition, over-expression of seven of the eight putative TRAF-3 splice-variant isoforms induces NF-κB activation in 293 cells (van Eyndhoven et al., 1999). However neither the expression nor function of mRNA for these splice-variants has been studied systematically in lymphoid cells. Therefore, the present study addressed whether TRAF-3 mRNA splice-variants are expressed or induce NF-κB activation in B or T lymphoma cells.
Understanding of the signaling function of TRAP proteins has been inferred largely from the effects of transient over-expression of TRAFs in 293 cells, an adenovirus transformed human kidney epithelial cell line (Rothe et al., 1995). By these criteria, TRAF-2 and TRAF-5 were thought to be NF-κB activating and TRAF-3 to be inhibitory (Rothe et al., 1995; Nakano et al., 1996; Ishida et al., 1996). However, subsequent work demonstrated that neither TRAF-3 nor TRAF-2 could be considered to be genes that encode simply inhibitory or stimulatory functions, since over-expression of seven TRAF-3 splice-variant isoforms, individually, induces NF-κB activation in 293 cells (van Eyndhoven et al., 1999) and a TRAF-2A splice-variant isoform (which might be considered a “full-length TRAF-2”) inhibits NF-κB activation (Brink and Lodish, 1998). These findings suggest that each TRAF-3 or TRAF-2 splice-variant isoform may have distinct functional potentials. In addition, it is unclear whether functional effects in 293 cells corresponds to functional potential in lymphocytes, where TRAF-3 is predominantly expressed (Krajewski et al., 1997; van Eyndhoven et al., 1998).
The present study analyzed the expression of full-length TRAF-3 and eight characterized mRNA splice-variants by RNAse protection assays in a panel of B and T lymphoma lines. Seven of 8 TRAF-3 mRNA splice-variants, as well as full-length TRAF-3, were expressed by the TRAF-3+ lymphoma cells and each variant contributed to the total TRAF-3 mRNA in a similar rank order in the different lymphomas. In addition, none of the cell lines expressed detectable levels of mRNA encoding TRAF-3 Δ130aa. The functional effect of over-expressing each TRAF-3 mRNA splice-variant isoform on NF-κB activation was studied in the B cell line, BJAB. Of the seven TRAF-3 splice-variant isoforms that induce NF-κB activation in 293 cells, only TRAF-3 Δ27aa, Δ103aa, or Δ130aa induced NF-κB activation in BJAB cells. Together, these data indicate that a number of TRAF-3 splice-variant mRNAs are expressed and function in B and T lymphoma lines.
2. Materials and Methods
2.1 Plasmid Constructs
DNA fragments encoding subdomains of full-length TRAF-3 (FL) and 8 TRAF-3 splice-variant isoforms (Δ25, Δ27, Δ52, Δ56, Δ83, Δ103, Δ130, Δ221) which had been generated by RT-PCR amplification (van Eyndhoven et al., 1999), were isolated by restriction digestion with EcoR I and BamH I (Roche Molecular Biochemicals, Indianapolis, Ind.) and ligated into pBluescript II SK+ (Stratagene, La Jolla, Calif.) for use in in vitro transcription (IVT) reactions. These constructs are termed; pIVT-FL, pIVT-Δ25, pIVT-Δ27, pIVT-Δ52, pIVT-Δ56, pIVT-Δ83, pIVT-Δ103, pIVT-Δ130, and pIVT-Δ221.
To generate an internal control, TRAF-3 sense RNA generating construct, PCR was used to amplify an exon 11 encoded fragment, TRAF-3 nt 1190-1246 (numbering from TRAF-3 CCRAF1) GenBank accession number U21092), using the primers Exon 11. for (5′-ATCGAATTCGGTACCAGCCAAGCAGACAAACTGAAG-3 ′) (SEQ ID NO:38) and Coiled. rev (5′-CGCGGATCCAAGCTTCTAGTTCCTCTAGTTCTGCCGGAAGGGCCGGATC-3′) (SEQ ID NO:39). This reaction utilized the Expand High Fidelity PCR System (Roche Molecular Biochemicals) and PCR conditions; 2 min. 94° C. (1 cycle); 15 sec. 94° C., 30 sec. 55° C., 45 sec. 72° C. (10 cycles); 15 sec. 94° C., 30 sec. 55° C., 1 min. 72° C. (10 cycles); 7 min. 72° C. (1 cycle). The PCR product was digested with EcoR I and BamH I and ligated into pBluescript II SK+ yielding pIVT-Exon-11/Sense.
Mammalian expression constructs in pCEP4 (Invitrogen, Carlsbad, Calif.) encoding TRAF-2, full-length TRAF-3, and TRAF-3 splice-variant isoforms, as well as the NF-κB dependent luciferase reporter PRDII×4 Luc, have been described previously (van Eyndhoven et al., 1999). The β-galactosidase (β-gal) open reading frame (ORF) was amplified using gene specific primers from pEBVHis LacZ (Invitrogen), digested with Xba I and BamH I (Roche Molecular Biochemicals) and ligated into the hemagglutinin (HA)-epitope tag expression vector pCGN (pCGN/LacZ). TRAF-5 was amplified by RT-PCR from Jurkat D1.1 mRNA with gene specific primers, ligated into pCR2.1 (Invitrogen), and sequenced. The TRAF-5 open reading frame was released from pCR2.1 by digestion with Sma I and BamH I (Roche Molecular Biochemicals) and ligated into corresponding sites in pCGN (pCGN/TRAF-5). Fragments encoding a portion of the CMV promoter and the HA tagged β-gal ORF or a portion of the CMV promoter and the HA tagged TRAF-5 ORF were released from pCGN by digestion with SnaB I and BamH I and ligated into the corresponding sites in pCEP4 (pCEP4/HA-LacZ and pCEP4/HA-TRAF-5). pTRI-GAPDH Human Antisense Control Template encoding nt 366-680 of the human GAPDH cDNA was obtained from Ambion (Austin, Tex.).
2.2 In Vitro Transcription
The pIVT-Δ221 construct was linearized with EcoR I. All other pIVT-TRAF-3 probe constructs were linearized with BstAP I (New England Biolabs, Beverly, Mass.), then blunted by addition of 3 units of T4 DNA Polymerase (New England Biolabs) in the presence of dNTPs (Roche Molecular Biochemicals) at a final concentration of 100 μM each, followed by incubation at 12° C. for 20 min. Samples were separated by agarose gel electrophoresis and gel purified using the Qiaquick Gel Extraction Kit (Qiagen, Valencia, Calif.) according to the manufacturer's instructions. Samples were ethanol precipitated and resuspended in distilled water at a concentration of approximately 0.5 μg/μl.
In vitro transcription (IVT) reactions were performed using the Riboprobe in vitro Transcription Systems kit (Promega, Madison, Wis.) according to the manufacturer's instructions using 50 μCi of α-32P CTP (NEN Life Science Products, Boston, Mass.) per reaction. Each of the pIVT TRAF-3 constructs were transcribed using approximately 1 μg of linearized DNA and T3 RNA polymerase to generate anti-sense radiolabeled probes. pTRI-GAPDH was transcribed using 500 ng of linearized DNA and T7 RNA polymerase. Probes were resolved by denaturing 6% PAGE, excised from the gel following autoradiography, and extracted at 37° C. for 2 h in buffer containing 2 M ammonium acetate (Sigma, St. Louis, Mo.), 1% SDS (Roche Molecular Biochemicals), and 25 μ/ml yeast tRNA (Sigma). Extracted probes were ethanol precipitated, washed with cold 70% ethanol, dried, and resuspended in 50 μl of DEPC (Sigma) treated distilled water. The radioactivity of a 1 μl aliquot of each probe was measured in a Bioscan QC 2000 (Bioscan, Washington, D.C.), and probes were diluted with DEPC treated distilled water to 25,000 cpm/μl.
A non-radioactive control sense RNA was synthesized from the pIVT-Exon-11/Sense construct using T7 RNA polymerase. Diluted aliquots of the reaction product were stored at −80° C. until use. Control hybridization reactions were performed on serial dilutions of the sense RNA in order to determine the amount to add to samples as an internal hybridization control during experimental hybridizations.
2.3 RNAse Protection Assays
Total RNA was isolated from the B cell lines Ramos CC, BJAB, Daudi, and Raji, and the T cell line Jurkat D1.1 using the RNEasy Mini Kit (Qiagen) according to the manufacturer's instructions. The concentration of RNA was measured by the 260 nm Absorbance in a Beckmann DU-65 Spectrophotometer (Beckmann Instruments, Palo Alto, Calif.) and stored in distilled water at −80° C. until use.
Three hundred μg aliquots of yeast tRNA or total RNA from the indicated cell lines were mixed with 2 μl of a 1:10000 dilution of the TRAF-3 transcript from pIVT-Exon 11/Sense, 0.15 volumes of 2M sodium acetate, and ethanol precipitated. Pellets were washed with cold 70% ethanol, then dried for 5 min at room temperature. Pellets were resuspended in 300 μl of Hybridization Buffer containing 80% Formamide, 40 mM Pipes, 0.4 M sodium chloride (all Fisher Scientific, Pittsburgh, Pa.), and 1 mM EDTA (Digene Diagnostics, Beltsville, Md.) and heated briefly at 80° C. to facilitate resuspension. Hybridization reactions containing 30 μg of resuspended RNA and 2 μl (50,000 cpm) of probe were heated 5 min at 80° C. then immediately transferred to 45° C. and hybridized overnight. Three hundred μl of RNAse digestion mixture containing 10 mM Tris pH 7.5, 300 mM sodium chloride, 5 mM EDTA, and 40 μg of RNAse A (Roche Molecular Biochemicals) and 1500 U RNAse T1 (Roche Molecular Biochemicals) per ml was added per reaction and samples were digested at 37° C. for 60 min. RNAses were inactivated by addition of 20 μl of 10% SDS (Roche Molecular Biochemicals), 160 μg of Proteinase K (Roche Molecular Biochemicals), and incubation at 37° C. for 30 min. Samples were extracted with phenol:chloroform. Supernatants were ethanol precipitated with 20 μg of yeast tRNA as a carrier. Pellets were rinsed with 70% ethanol, dried for 5 min, and resuspended in 5 μl of RNA loading buffer containing 80% formamide, 1 mM EDTA pH 8.0, 0.1% Bromphenol Blue (Malinckrodt, Phillipsburg, N.J.), and 0.1% Xylene Cyanol (International Biotechnologies, New Haven, Conn.). Samples were heated to 80° C. for 5 min and loaded on prewarmed 6% denaturing polyacrylamide sequencing gels. Gels were run at 60 W for 130 min. then dried and exposed to Biomax MS film (Kodak, Rochester, N.Y.) or PhosphorImager screens (Molecular Dynamics, Sunnyvale, Calif.). PhosphorImager screens were scanned on 445SI and Storm 820 scanners (Molecular Dynamics).
Image analysis was performed using ImageQuant software version 1.2 for the Macintosh (Molecular Dynamics). Ten pixel wide polylines were drawn over sample lanes. The Peak Finder function was used to identify primary fragment signal peaks and eliminate non-specific peaks present in the yeast tRNA control. Background signal intensities observed for Ramos CC samples were measured at the corresponding positions to peaks observed for Jurkat D1.1 samples on the same gel. Signal intensities for primary fragments protected by the TRAF-3 Δ130 probe (from pIVT-Δ130) were measured at a position corresponding to the expected size of protected fragment, which is not visible to the eye. Peak areas were corrected for fragment size and normalized to the intensity of the 72 nt peak corresponding to protection of probe by the artificial TRAF-3 Exon 11 sense transcript, from pIVT-exon-11/sense. Finally, signals for each cell line were normalized to the level of GAPDH expression measured in parallel reactions to allow comparison of signal intensities between different cell lines. The data in FIG. 20 are scaled to a metric in which the signal for full length TRAF-3 mRNA in Jurkat D1.1 has a value of 100. The data in Table C represent the percentage contributions of individual TRAF-3 splice-variant mRNAs to total TRAF-3 mRNA. These percentages were obtained by dividing the variant expression in each individual cell line (background subtracted) and dividing by the total TRAF-3 expression for all splice-variants (background subtracted) in that cell line.
2.4 Cell Culture
Ramos CC and Jurkat D1.1 cells were cultured in IMDM (Life Technologies) supplemented with 10% Fetal Bovine Serum (Summit Biotechnologies) and 50 U/ml Penicillin, 50 μg/ml Streptomycin (Sigma). BJAB cells were kindly provided by Dr. Ricardo Dalla Fevera, Columbia University. Daudi, and Raji cells were purchased from ATCC (Rockville, Md.). EJAB, Daudi, and Raji cells were cultured in RPMI 1640 (Cellgro) supplemented with 17.8 mM sodium bicarbonate (Fisher Scientific), 10 mM Hepes (Fisher Scientific), 1 mM sodium pyruvate (Sigma), 2 mM glutamate (Life Technologies), 25 mM glucose (Fisher Scientific), and 50 U/ml Penicillin, 50 μg/ml Streptomycin (Sigma). Cells were cultured at 37° C. and 5% CO2 in humidified incubators.
2.5 Transient Transfections and Luciferase Assays
BJAB cells were harvested and resuspended in serum free RPMI at 5 million per 300 μl. A 300 μl aliquot was mixed with 10 μg of the indicated expression vector, 1 μg of PRDII×4 Luc, and 250 ng of pRLtk. Cells were transferred to 0.4 cm Gene Pulser Cuvettes (BioRad, Hercules, Calif.) and electroporated at 270 V and 975 μF using a Gene Pulser II with a Pulse Controller Plus and Capacitance Extender Plus (BioRad). Cells were harvested following electroporation by adding 1 ml of normal culture medium to the cuvette, then washing with an additional 1.7 ml normal culture medium and pooling the sample in one well of a six well tissue culture dish. Cells were cultured for approximately 36 hours, then harvested, washed with 1×PBS, and pelleted for measurement of luciferase activity. Samples were lysed in 200 μl of 1×Passive Lysis Buffer and assayed using the Dual Luciferase Assay Kit (Promega, Madison, Wis.) according to the manufacturer's instructions. Firefly luciferase reporter levels were normalized for transfection efficiency with the internal Renilla luciferase control. Values were scaled to the level of Firefly luciferase activity observed in samples transfected with the LacZ control. Data shown are representative of 3 independent experiments and error bars represent the standard deviation of measurements from triplicate samples.
3. Results
3.1 RNAse Protection Analysis of TRAF-3 Splice-Variant Isoform Expression.
In addition to full-length TRAF-3, eight alternatively spliced TRAF-3 mRNA species were recently identified by RT-PCR (van Eyndhoven et al., 1999). In order to determine whether these TRAF-3 mRNA splice-variants are expressed in lymphoma cells, RNAse protection assays were performed using nine unique probes that were designed to protect distinct fragments when hybridized to the corresponding complementary splice-variant mRNA (Table B and FIG. 19A). As a positive control, a short, artificial sense TRAF-3 RNA (from exon 11) was added to each hybridization reaction to allow comparison of the signal intensities detected by the different probes. As negative controls, tRNA from yeast and RNA from the TRAF-3− lymphoma line Ramos CC were examined in parallel (Krajewski et al., 1997; van Eyndhoven et al., 1998). As expected, each probe protected the internal positive control (FIG. 19C and 19D) and neither yeast tRNA nor Ramos CC mRNA protected significant amounts of any probe (FIG. 19C), which showed that the RNAse protection assay was specific for TRAF-3 mRNA species.
In the RNAse protection assay, RNA from the T cell line Jurkat D1.1 and the B cell lines BJAB, Daudi, and Raji protected the predicted primary (or largest) fragments of each probe (listed in Table B) corresponding to TRAF-3 full-length, Δ25aa, Δ27aa, Δ52aa, Δ56aa, Δ83aa, Δ103aa, and Δ221aa mRNA splice-variants (indicated by * in FIGS. 19C and 19D). One unexpected finding was that none of the RNA samples protected the predicted fragment from the TRAF-3 Δ130aa probe (expected position indicated by * in FIGS. 19C and 19D).
This finding did not appear to result from degradation of the TRAF-3 Δ130aa probe, since this probe migrated appropriately and protected the internal control TRAF-3 RNA at a level similar to the other probes. Thus the inability of any RNA sample to protect the expected fragment for TRAF-3 Δ130aa indicates that the TRAF-3 Δ130aa splice-variant is expressed below the limits of sensitivity for the assay. Together, these data indicate that a number of TRAF-3 mRNA species, including full-length and seven of eight characterized splice-variants, are expressed in the lymphoma lines Jurkat D1.1, BJAB, Daudi, and Raji.
In addition to the primary protected fragment of each probe to its corresponding mRNA splice-variant, secondary bands were predicted to occur from hybridization of each probe to other splice-variant mRNA species (e.g., secondary bands predicted to arise from protection of the full-length TRAF-3 probe are listed in FIG. 19A). Most of these predicted secondary bands were observed in each TRAF-3 expressing cell line. As an example, a 304 nt band corresponding to protection of-mRNA encoded by exons 8 through 10 was detected by the probes representing TRAF-3 full length, Δ25, Δ27, and Δ52 but not by other probes (FIGS. 19C and 19D). Therefore, the protection of secondary bands confirmed and extended the analysis based on primary bands. However, an unexpected band of approximately 165 nt was detected by six probes (indicated by  in FIGS. 19C and 19D), which suggests that an additional TRAF-3 splice-variant mRNA exists that contains sequence from exons 10 and 11 (sequence common to all of the probes with a 165 nt protected fragment). Together, these data strongly suggest that, except for one additional band that seems to represent a single novel splice variant, the eight cloned cDNAs account for all of the alternatively spliced TRAF-3 mRNA species in these lymphoma lines that hybridize to these nine probes.
The intensities of the protected fragments in FIGS. 19C and 19D were measured using a PhosphorImager and normalized to GAPDH expression levels (FIG. 19B) to allow comparison of TRAF-3 splice-variant mRNA expression between cell lines (FIG. 20). The level of TRAF-3 mRNA varied approximately four-fold between the cell lines and was highest in Jurkat D1.1 and Raji (relative to GAPDH), and relatively lower in BJAB and Daudi (FIG. 20). The relative contribution of individual splice-variants to the total amount of TRAF-3 mRNA within each cell line was similar (Table C). The splice-variants contributing most to TRAF-3 mRNA expression are Δ103aa and Δ83aa, which together comprise more than 40% the TRAF-3 mRNA in any of the lines (Table C). Full-length TRAF-3 and Δ25aa are approximately twice as abundant as the Δ52aa and Δ56aa splice-variants. The splice-variants Δ27aa and Δ221aa each comprise less than 8% of the TRAF-3 mRNA in any cell line. TRAF-3Δ130aa was undetectable above background signals from yeast tRNA or Ramos CC, even by phosphorimaging analysis of the gel position estimated to coincide with the primary probe fragment that would be protected by any TRAF-3Δ130aa mRNA (FIG. 20 and Table C). Thus, these data indicate that relative contributions made by individual splice-variants to the total amount of TRAF-3 mRNA appear to be similar in different lymphoma cell lines.
3.2 Effects of Over-Expressing TRAF-3 Splice Deletion Variants on NF-κB Activation in BJAB.
The next experiments addressed whether the TRAF-3 splice deletion isoforms are capable of inducing NF-κB activation in the B cell line BJAB. BJAB cells were selected for functional studies due to their relatively efficient transfection by electroporation and their low background of NF-κB reporter gene expression. The effects of over-expressing TRAF-3 splice-variants or full-length TRAF-3 were compared with those of over-expressing TRAF-2 and TRAF-5, which are known to induce NF-κB activation in 293 cells (Rothe et al., 1995; Nakano et al., 1996; Ishida et al., 1996). In these experiments, the background NF-κB reporter gene activity was determined from parallel samples transiently transfected with β-galactosidase (indicated by LacZ in FIG. 21). As expected, over-expression of TRAF-5 induces NF-κB reporter gene expression, whereas full-length TRAF-3 or TRAF-3 Δ221aa are inactive (FIG. 21). In addition, over-expression of TRAF-3 Δ27aa, Δ103aa, or Δ130aa induces NF-κB activation in BJAB cells, 2-fold, 4-fold, and 11-fold, respectively (FIG. 21), similar to their ability to induce NF-κB activation in 293 cells (van Eyndhoven et al., 1999). Surprisingly, over-expression of TRAF-3 Δ25aa, Δ52aa, Δ56aa, Δ83aa, or TRAF-2 failed to induce NF-κB activation, despite the ability of these constructs to induce strong activation of NF-κB in 293 cells (van Eyndhoven et al., 1999). Together, these data suggest that only certain TRAF-3 splice-variant isoforms induce NF-κB activation in B lymphoma cells.
4. Discussion
Previous work identified eight TRAF-3 mRNA splice-deletion variants by RT-PCR (van Eyndhoven et al., 1999). In the present study, RNAse protection analysis established that, in addition to full-length TRAF-3, seven of these eight alternatively spliced TRAF-3 mRNA species are expressed in the lymphoma cell lines BJAB, Daudi, Raji, and Jurkat D1.1, and that each species contributed a similar proportion to the total TRAF-3 mRNA level in the cell lines. Although seven of the TRAF-3 splice-variants had been previously shown to induce NF-κB activation when over-expressed in 293 cells, only three TRAF-3 splice-variant isoforms (Δ27aa, Δ103aa, and Δ130aa) induced NF-κB activation in BJAB cells.
The present study confirmed by RNAse protection analysis that seven of eight mRNA splice-variant species previously cloned by RT-PCR are expressed in lymphoma lines, but also showed that TRAF-3 Δ221aa mRNA was expressed at very low levels and, even by phosphorimaging analysis, TRAF-3 Δ130aa did not appear to be expressed above background signals. The RNAse protection assay provides a more accurate measure of steady state mRNA levels than RT-PCR because RNAse protection does not rely on amplification. Therefore, the disproportionate intensity of RT-PCR bands for Δ221aa and Δ130aa may represent preferential amplification of these relatively small products during PCR (van Eyndhoven et al., 1999). In addition, the finding by RNAse protection analysis that Δ221aa is expressed at very low levels and Δ130aa mRNA is undetectable, suggests that peptide bands previously associated with these mRNA species may represent proteolytic TRAF-3 fragments or proteins encoded by additional, uncharacterized TRAF-3 splice-variant mRNAs (van Eyndhoven et al., 1999).
Although this study examined the expression and signaling properties of full-length TRAF-3 and eight splice-variant mRNAs, data exist in support of additional TRAF-3 splice-variants. For example, a cDNA clone has been described that is predicted to encode a TRAF-3 isoform lacking the N-terminal RING and Zn fingers due to translation initiation at an internal methionine within the coiled-coil domain (Mosialos et al., 1995). In addition, the RNAse protection analysis described in this work revealed a TRAF-3 specific band of approximately 165 nt that did not correspond to any of the previously cloned splice-variant mRNAs (indicated by  in FIGS. 19C and 19D). Since an apparently identical 165 nt band was protected by the probes representing full-length, Δ25aa, Δ27aa, Δ52aa, Δ56aa, and Δ83aa splice-variants, this band may represent a single, novel splice-variant mRNA that contains sequences from exon 10 and 11. Although the identity of the protected fragment is not certain, the data are consistent with an mRNA the results from a cryptic splice acceptor encoded in exon 10. It is interesting to note that splicing of exon 3 to this putative splice acceptor site could maintain an open reading frame and encode a polypeptide that lacks part of the RING finger, all 5 Zn fingers, and a large proportion of the coiled-coil domain of TRAF-3. A more complete understanding of TRAF-3 signaling will require further studies of TRAF-3 mRNA splicing and the resulting expression of TRAF-3 splice-variant isoforms.
The levels of TRAF-3 mRNA expression were found to vary approximately 4-fold between the four TRAF-3+ lymphoma lines and TRAF-3 mRNA was undetectable in Ramos CC. It is interesting to consider the possibility that TRAF-3 expression varies in these different lymphoma lines in a manner that may model its regulation in normal lymphocytes. In support of this idea, Jurkat D1.1, which has the phenotype of an activated T cell (Lederman et al., 1992), expresses relatively high levels of TRAF-3 mRNA, which may be analogous to the finding that CD3 cross-linking induces TRAF-3 expression in PBL (Krajewski et al., 1997).
Despite the variation in levels of TRAF-3 mRNA, the four TRAF-3+ cell lines were found to express TRAF-3 splice-variant mRNAs in a similar rank order. To the extent that lymphoma cell lines model features of different cell lineages or stages of differentiation, these data suggest that alternative splicing to each mRNA variant may occur in a fixed proportion in lymphoid cells and may not be regulated. However, in addition to lymphoid cells, TRAF-3 mRNA is expressed by a wide variety of other tissues and cell types (Cheng et al., 1995; Krajewski et al., 1997; van Eyndhoven et al., 1998). Therefore, it will be of interest to determine whether or not TRAF-3 expressing cells of different lineages or stages of differentiation contain the same relative proportions of TRAF-3 mRNA splice-variants observed in lymphoma lines. Since each TRAF-3 splice-variant isoform may possess differing signaling abilities, changes in the either the absolute amount or relative proportion of a particular splice-variant in a cell may affect the threshold of receptor stimulation required to initiate downstream signaling.
The finding that TRAF-3 Δ130aa isoform induced the highest NF-κB activation after over-expression in BJAB cells, is interesting in light of the fact that Δ130aa encoding mRNA was undetectable in any of the resting lymphoma lines. Since TRAF-3 Δ130aa encoding mRNA was cloned from Jurkat D1.1 (van Eyndhoven et al., 1999), these data suggest that Δ130aa is expressed under certain circumstances, and may be both potent and closely regulated. It remains unclear how TRAF-3 over-expression models receptor-induced signaling, but the possibility that receptor aggregation (by ligand) liberates TRAF-3 from receptor tails is suggested by consideration that high local concentrations of receptor tails and TRAF-3 homotrimers would favor TRAF-3 homotrimers either binding three or no receptor tails (Pullen et al., 1999). The downstream events in TRAF-3 signaling may be cell type restricted since four TRAF-3 splice-variants which induce NF-κB activation in 293 cells (van Eyndhoven et al., 1999) failed to induce NF-κB activation in BJAB cells. The basis of this cell-type restricted signaling are not yet understood, but may relate to differences in expression of TRAF binding kinases that stimulate the IKK complex, such as NIK, MEKK1, GCK, and GCKR (Malinin et al., 1997; Song et al., 1997; Baud et al., 1999; Yuasa et al., 1998; Shi and Kehrl, 1997; Chin et al., 1999). TRAF-2 signaling also appeared to be cell-type restricted since over-expression of TRAF-2 failed to induce NF-κB activation in BJAB whereas TRAF-5 was active.
The observation that certain TRAP-3 splice-variants and TRAF-5 share the ability to induce NF-κB activation in BJAB cells is interesting in light of recent reports that TRAF-3 and TRAP-5 may physically interact with each other and that such interactions may be required to recruit TRAF-5 to CD40 cytoplasmic tails (Pullen et al., 1998; Leo et al., 1999). An additional similarity between activating TRAF-3 splice-variants and TRAF-5 is that they are thought to functionally interact with full-length TRAF-3, since their ability to induce NF-κB activation is augmented by co-expression with full-length TRAF-3 (van Eyndhoven et al., 1999; Leo et al., 1999). These data suggest that TRAF-3 splice-variants or TRAF-5 might function together with full-length TRAF-3 in receptor-triggered signaling. Although the physiological roles for TRAP-3 splice-variants are not completely understood, redundant functional characteristics of TRAF-3 splice-variants and TRAF-5 may account for the preservation of CD40 triggered NF-κB activation in TRAF-5 deficient B cells (Nakano et al., 1999). In addition, if TRAF-5 participation in CD40 signaling depends on TRAF-3 mediated recruitment, the more severe phenotype of the TRAF-3 deficient mouse with respect to T-dependent antibody responses might arise from a functional disruption of both TRAF-3 and TRAF-5 dependent CD40 signaling (Xu et al., 1996; Nakano et al., 1999).
The finding that certain TRAF-3 mRNA splice-variants are expressed and are capable of inducing NF-κB activation in lymphoma lines, suggests that TRAF-3 splice-variant isoforms may functionally participate in transduction of positive signals from CD40 into the nucleus in lymphocytes. Thus, the biology of TRAF-3 appears to be intimately associated with alternative splicing and the function of splice products. Since abnormal mRNA splicing accounts for a substantial proportion of genetic diseases (Cooper and Mattox, 1997), it will be of interest to determine whether abnormalities in TRAF-3 splicing might underlie defects in CD40 signaling in certain individuals with Hyper-IgM syndrome and normal CD154 (Durandy et al., 1997).







TABLE B







Splice-Variant Specific Anti-sense


RNA Probes and Expected Sizes for Protected


Fragments from RNAse Protection Analysis










Splice
Exons Omitted
Probe
Protected


Isoform
by Splicing
(nt)
Fragment (nt)





FL
—
555
494


Δ25
8
480
419


Δ27
7
474
413


Δ52
7, 8
399
338


Δ56
8, 9
387
326


Δ83
7-9 
306
251


Δ103
8-10
246
185


Δ130
7-10
165
110


Δ221
5-10
304
232
















TABLE C







Contribution of Individual TRAF-3 Splice-Variant


mRNAs to Total TRAF-3 mRNA in Lymphoma Cell Lines (%)
















Cell











Line
FL
Δ25
Δ27
Δ52
Δ56
Δ83
Δ103
Δ130
Δ221



















D1.1
18.3
16.7
3.3
8.5
9.4
15.4
26.3
0.3
1.9


BJAB
11.2
2.3
2.5
6.7
6.3
27.5
43.5
N.D.
N.D.


Daudi
16.1
8.8
7.7
6.7
5.5
20.4
28.8
N.D.
6.0


Raji
11.7
10.6
1.7
7.3
10.5
21.0
34.2
0.3
2.7





N.D., not detectable above background observed for Ramos CC 






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## Claims

1. An isolated protein comprising a TRAF-3 deletion isoform which comprises the amino acid sequence shown in FIG. 16 (SEQ ID NO:2).

2. A method for identifying an agent that inhibits CD40-mediated cellular signaling in a cell which comprises:
(a) contacting the cell with an agent under conditions wherein CD40-mediated cell activation occurs; and 
(b) determining whether CD40-mediated signaling is inhibited in the cell in the presence of the agent so as to identify whether the agent inhibits CD40-mediated cellular signaling, 
wherein the cell in step (a) is transfected with a TRAF-3 deletion isoform 130 protein having the amino acid sequence shown in FIG. 16 (SEQ ID NO:2).

3. The method of claim 2, wherein the cell is a 293T cell.