The Human gC1qR/p32 Gene, C1qBP (2001)

gC1qR is an ubiquitously expressed cell protein that interacts with the globular heads of C1q (gC1q) and many other ligands. In this study, the 7.8-kilobase pair (kb) human gC1qR/p32 (C1qBP) gene was cloned and found to consist of 6 exons and 5 introns. Analysis of a 1.3-kb DNA fragment at the 5′-flanking region of this gene revealed the presence of multiple TATA, CCAAT, and Sp1 binding sites. Luciferase reporter assays performed in different human cell lines demonstrated that the reporter gene was ubiquitously driven by this 1.3-kb fragment. Subsequent 5′ and 3′ deletion of this fragment confined promoter elements to within 400 base pairs (bp) upstream of the translational start site. Because the removal of the 8-bp consensus TATATATA at −399 to −406 and CCAAT at −410 to −414 did not significantly affect the transcription efficiency of the promoter, GC-rich sequences between this TATA box and the translation start site may be very important for the promoter activity of the C1qBP gene. One of seven GC-rich sequences in this region binds specifically to PANC-1 nuclear extracts, and the transcription factor Sp1 was shown to bind to this GC-rich sequence by the supershift assay. Primer extension analysis mapped three major transcription start regions. The farthest transcription start site is 49 bp upstream of the ATG translation initiation codon and is in close proximity of the specific SP1 binding site. gC1qR is an ubiquitously expressed cell protein that interacts with the globular heads of C1q (gC1q) and many other ligands. In this study, the 7.8-kilobase pair (kb) human gC1qR/p32 (C1qBP) gene was cloned and found to consist of 6 exons and 5 introns. Analysis of a 1.3-kb DNA fragment at the 5′-flanking region of this gene revealed the presence of multiple TATA, CCAAT, and Sp1 binding sites. Luciferase reporter assays performed in different human cell lines demonstrated that the reporter gene was ubiquitously driven by this 1.3-kb fragment. Subsequent 5′ and 3′ deletion of this fragment confined promoter elements to within 400 base pairs (bp) upstream of the translational start site. Because the removal of the 8-bp consensus TATATATA at −399 to −406 and CCAAT at −410 to −414 did not significantly affect the transcription efficiency of the promoter, GC-rich sequences between this TATA box and the translation start site may be very important for the promoter activity of the C1qBP gene. One of seven GC-rich sequences in this region binds specifically to PANC-1 nuclear extracts, and the transcription factor Sp1 was shown to bind to this GC-rich sequence by the supershift assay. Primer extension analysis mapped three major transcription start regions. The farthest transcription start site is 49 bp upstream of the ATG translation initiation codon and is in close proximity of the specific SP1 binding site. kilobase pair(s) transcription factor activator protein base pair(s) gC1qR is a biologically important, widely distributed, multiligand-binding and multifunctional protein (1Ghebrehiwet B. Peerschke E.I. Immunobiology. 1998; 199: 225-238Crossref PubMed Scopus (75) Google Scholar). Numerous reports have claimed that gC1qR and its homologue could be isolated or identified in various cellular compartments, including plasma membrane, cytoplasm, mitochondria and nucleus. gC1qR isolated from the plasma membrane was originally characterized as a high affinity C1q-binding protein (2Ghebrehiwet B. Lim B.L. Peerschke E.I. Willis A.C. Reid K.B. J. Exp. Med. 1994; 179: 1809-1821Crossref PubMed Scopus (317) Google Scholar), and later many reports showed that gC1qR could interact with several proteins of the intrinsic coagulation/bradykinin-forming cascade, including high molecular weight kininogen (3Joseph K. Ghebrehiwet B. Kaplan A.P. Clin. Immunol. 1999; 92: 246-255Crossref PubMed Scopus (72) Google Scholar), Factor XII (4Joseph K. Ghebrehiwet B. Peerschke E.I. Reid K.B. Kaplan A.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8552-8557Crossref PubMed Scopus (215) Google Scholar), fibrinogen (5Lu P.D. Galanakis D.K. Ghebrehiwet B. Peerschke E.I. Clin. Immunol. 1999; 90: 360-367Crossref PubMed Scopus (26) Google Scholar), and multimeric vitronectin (6Lim B.L. Reid K.B. Ghebrehiwet B. Peerschke E.I. Leigh L.A. Preissner K.T. J. Biol. Chem. 1996; 271: 26739-26744Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Conversely, intracellular gC1qR was shown to interact and subsequently down-regulate the surface expression of the α1b-adrenergic receptor (7Xu Z. Hirasawa A. Shinoura H. Tsujimoto G. J. Biol. Chem. 1999; 274: 21149-21154Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) as well as bind to the kinase domain of protein kinase Cμ and thus prevent its substrate phosphorylation activity (8Storz P. Hausser A. Link G. Dedio J. Ghebrehiwet B. Pfizenmaier K. Johannes F. J. Biol. Chem. 2000; 275: 24601-24607Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). In addition, gC1qR was also reported to bind to a nuclear splicing factor, SF2, and to many viral proteins, including HIV-1 Tat (9Yu L. Loewenstein P.M. Zhang Z. Green M. J. Virol. 1995; 69: 3017-3023Crossref PubMed Google Scholar) and Rev (10Luo Y., Yu, H Peterlin B.M. J. Virol. 1994; 68: 3850-3856Crossref PubMed Google Scholar), core protein V of adenovirus (11Mathews D.A. Russell W.C. J. Gen. Virol. 1998; 79: 1677-1685Crossref PubMed Scopus (161) Google Scholar), EBNA-1 of the Epstein-Barr virus (12Wang Y. Finnan J.E. Middeldorp J.M. Hayward S.D. Virology. 1997; 236: 18-29Crossref PubMed Scopus (137) Google Scholar), and open reading frame P of the herpes simplex virus (13Bruni R. Roizman B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10423-10427Crossref PubMed Scopus (71) Google Scholar), implying that gC1qR may play a role in virus-host interaction. The full-length cDNA of gC1qR encodes a pre-pro-protein of 282 residues from which a 73-residue-long N-terminal segment is removed by site-specific cleavage to generate the mature gC1qR (2Ghebrehiwet B. Lim B.L. Peerschke E.I. Willis A.C. Reid K.B. J. Exp. Med. 1994; 179: 1809-1821Crossref PubMed Scopus (317) Google Scholar). It was shown that the fusion of the residues 1–81 or 1–33 of the pre-pro-protein to the N terminus of the green fluorescent protein directed the fusion protein to mitochondria (14Dedio J. Jahnen-Dechent W. Bachmann M. Muller-Esterl W. J. Immunol. 1998; 160: 3534-3542PubMed Google Scholar). However, the findings of Dedioet al. (14Dedio J. Jahnen-Dechent W. Bachmann M. Muller-Esterl W. J. Immunol. 1998; 160: 3534-3542PubMed Google Scholar) do not exclude the possibility that gC1qR, like many other proteins, could be exported from the mitochrondria by an unknown mechanism (15Soltys B.J. Gupta R.S. Trends Biochem. Sci. 1999; 24: 174-177Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). This possibility is supported by a recent report showing that anti-gC1qR monoclonal antibody can reverse the anti-proliferation effects of hepatitis virus C core antigen on activated T cells (16Kittlesen D.J. Chianese-Bullock K.A. Yao Z.Q. Braciale T.J. Hahn Y.S. J. Clin. Invest. 2000; 106: 1239-1249Crossref PubMed Scopus (295) Google Scholar). The human C1qBP gene was assigned to human chromosome 17q13.3 (17Guo N. Weremowicz S. Lynch N. Lim B.L. Schwaeble W. Peerschke E.I. Morton C.C. Reid K.B. Ghebrehiwet B. Sastry K.N. Cytogenet. Cell Genet. 1997; 77: 283-284Crossref PubMed Google Scholar). A high degree of amino acid identity exists between the human, rat, and mouse gC1qR cDNA sequences (18Lynch N.J. Reid K.B. van-den-Berg R.H. Daha M.R. Leigh L.A. Ghebrehiwet B. Lim W.B. Schwaeble W.J. FEBS Lett. 1997; 418: 111-114Crossref PubMed Scopus (26) Google Scholar). In this study, the full-length gene of human gC1qR was cloned, and its exon-intron boundaries were revealed. Furthermore, the transcription start site and its promoter elements were also mapped and characterized. A human genomic library in bacteriophage EMBL3 was purchased fromCLONTECH (cat. no. HL1067J). The human cDNA of the C1qBP gene was used as the probe for library screening (2Ghebrehiwet B. Lim B.L. Peerschke E.I. Willis A.C. Reid K.B. J. Exp. Med. 1994; 179: 1809-1821Crossref PubMed Scopus (317) Google Scholar). The cDNA insert was released from its vector and gel-purified before radiolabeling with [32P]dATP by the random priming method. Positive plaques were picked, replated, and rescreened until single positive plaques were picked. The insert in a positive plaque was digested with various restriction enzymes, and the positive fragments were mapped by Southern blot analysis. Six overlapping subclones (Fig. 1) were obtained by inserting the positive fragments into the plasmid pBluescript. Each subclone was sequenced by the primer walking method using the ABI Prism 310 Genetic Analyzer (Applied Biosystems, Hong Kong Limited, Hong Kong). Sequence data were assembled and analyzed by DNA processing software including MAC DNASIS (Hitachi, Japan) and DNA Strider (Christian Marck, Service de Biochimie, Department de Biologie, Institut de Recherche Fondamentale, CEA, Paris, France). Promoter analysis was performed using MatInspector software (version 2.2) (19Quandt K. Frech K. Karas H. Wingender E. Werner T. Nucleic Acids Res. 1995; 23: 4878-4884Crossref PubMed Scopus (2427) Google Scholar) obtained from the German Research Center for Biotechnology, Braunschweig, D-38124, Germany. A transcription factor data base (TRANSFAC, version 4.0) was employed for the search of promoter elements (20Heinemeyer T. Wingender E. Reuter I. Hermjakob H. Kel A.E. Kel O.V. Ignatieva E.V. Ananko E.A. Podkolodnaya O.A. Kolpakov F.A. Podkolodny N.L. Kolchanov N.A. Nucleic Acids Res. 1998; 26: 364-370Crossref Scopus (1325) Google Scholar). Various DNA fragments of the 5′-upstream region of the human gC1qR gene (C1qBP) were obtained by polymerase chain reaction using subclone gI as the template. Restriction sitesBglII or SacI were added to the 5′-end of the primers (Table I) so that the polymerase chain reaction products could easily be subcloned into the corresponding restriction sites in the dual-luciferase reporter vector, pGL3-Basic (Promega, Hong Kong). A nested family of 5′ and 3′ deletion clones (Table II) was generated in this manner (Fig. 4).Table IPrimers designed for construction of recombinant plasmids and gel shift assaysPrimersPrimer sequencesPosition in 5′-flanking regionFor recombinant plasmids1-aFour bases and a restriction site were added at the 5′ end.Pro0Sac5′ attaGAGCTC CTGCCCTTGAGGATG 3′−1314 to −1300Pro200Sac5′ attaGAGCTC AACAATCAACACAGAAGACTT 3′−1121 to −1101Pro400Sac5′ attaGAGCTC CGACCAACCCGCTTCA 3′−916 to −901Pro600Sac5′ attaGAGCTC CAGGAACCTCTACG 3′−713 to −702Pro800Sac5′ attaGAGCTC GTTGTTTCCTGAGGCCT 3′−506 to −489Pro954Sac5′ attaGAGCTC GTGGAAGGGTCTGGT 3′−364 to −350G1A2Bgl5′ attaGATCTC GCGGAAACGACTGCGAA 3′−18 to −1 (inverted)Pro0Bgl5′ attaGATCTC CTGCCCTTGAGGATG 3′−1314 to −1300G1A2Sac5′ attaGAGCTC GCGGAAACGACTGCGAA 3′−18 to −1 (inverted)For gel shift assays 1-bAll primers were designed from their GC with respect to their location in the 5′ flanking region of the human gClqBP gene. “S” indicates sense primers, and “A” is the antisense version of its sense primer. L8S and L8A, which do not contain GC-rich sequences, were designed as negative controls.L1S5′ AGAGCTGGCG GGAGACAA5′ −348 to −331L1A3′ TTGTCTCCCG CCAGCTCT3′ −331 to −348L2S5′ AGGCTTCCCG GGCTGCAC5′ −188 to −171L2A3′ GTGCAGCCCG GGAAGCCT3′ −171 to −188L3S5′ AGCAGAGGGC GCGATTCCG5′ −160 to −142L3A3′ CGGAATCGCG CCCTCTGCT3′ −142 to −160L4S5′ GCGATTCCGC CCGCTGGC5′ −150 to −133L4A3′ GCCAGCGGGC GGAATCGC3′ −133 to −150L5S5′ GAGCGCCGGC GCGGGAAGC5′ −120 to −102L5A3′ GCTTCCCGCG CCGGCGCTC3′ −102 to −120L6S5′ CGGGCAGGGG CGGGGCTTCC G5′ −96 to −76L6A3′ CGGAAGCCCC GCCCCTGCCC C3′ −76 to −96L7S5′ TCAGGTCGCG GGGCGCCTAG5′ −67 to −48L7A3′ CTAGGCGCCC CGCGACCTGA3′ −48 to −67L8S5′ GGCCACTAGA GGGAATTAAA5′ −264 to −245L8A3′ TTTAATTCCC TCTAGTGGCC3′ −245 to −2641-a Four bases and a restriction site were added at the 5′ end.1-b All primers were designed from their GC with respect to their location in the 5′ flanking region of the human gClqBP gene. “S” indicates sense primers, and “A” is the antisense version of its sense primer. L8S and L8A, which do not contain GC-rich sequences, were designed as negative controls. Open table in a new tab Table IIExpression plasmids for luciferase promoter assayConstructsPrimers employedRestriction sites at 5′-endRestriction sites at 3′-endFragment sizeDeletion fromkbERES 1Pro0Bgl + G1A2SacBglIISacI1.3Nil (reverse)2-aNil, no deletion.ERES 2Pro0Sac + G1A2BglSacIBglII1.3NilERES 3Pro200Sac + G1A2BglSacIBglII1.15′ERES 4Pro400Sac + G1A2BglSacIBglII0.95′ERES 5Pro600Sac + G1A2BglSacIBglII0.75′ERES 6Pro800Sac + G1A2BglSacIBglII0.55′ERES 7Pro954Sac + G1A2BglSacIBglII0.45′ERES 2HPro0Sac + G1A2BglSacIHindIII0.673′ERES 2PPro0Sac + G1A2BglSacIPsPAI1.13′2-a Nil, no deletion. Open table in a new tab The human cell lines PANC-1 (ATCC no. CRL-1469), MDA-MB-231 (ATCC no. HTB-26), SVG P12 (ATCC no. CRL-8621), HuTu80 (ATCC no. HTB-40), and 293-EBNA (Invitrogen, no. R620-07) were selected for transient transfection studies. SVG P12 and HuTu80 cells were cultured in minimum essential medium (Life Technologies, Inc.); PANC-1, MDA-MB-231, and 293-EBNA cells were cultured in high glucose Dulbecco's modified Eagle's medium (Life Technologies, Inc.). All culture media were supplemented with 10%(v/v) fetal bovine serum (50 ml in 500 ml of medium) and 1× antibiotic-antimycotic mixture (Life Technologies, Inc.) at 37 with DNA used for transfection was using the Hong and with before transient cells were Inc.) at a of was performed later using the (Life Technologies, Inc.) with performed to the The of the recombinant plasmid to be and the plasmid (Promega, Hong was at in the cells were in the medium serum and at 37 with for before to The cells were at by the cells with 1× by the of reporter (Promega, Hong Kong). The dual-luciferase reporter was performed to the reporter Hong using a as the cellular was isolated from human cell MDA-MB-231 by (Life Technologies, Inc.). was from of using the of the primer and primer extension reaction was performed using of with of the antisense primer the for the Primer of the primer extension reaction was analyzed on a The of the primer extension was by with a DNA sequence generated with the primer using plasmid as a template. The human cell PANC-1 was selected for gel shift were cultured to as and of (Table I) was using the kinase and and gel shift assays were performed as A. Green M.R. PubMed Scopus Google R. K. in Scholar). is a of the genomic of the including its and the codon of the initiation to the codon of the the gene bp (Fig. of the cDNA and the genomic sequences, boundaries were 6 exons and 5 in the C1qBP gene. The of the exons from to bp and that of the from to bp acid by and at the of their (Table A is bp from the The genomic sequence in the no. boundaries of the human C1qBP on 5′ 3′ 5′ 3′ acid exon-intron boundaries the by the Open table in a new tab The exon-intron boundaries the by the A 1.3-kb sequence upstream of the ATG initiation codon of the gene was analyzed using the transcription factor data promoter and elements including TATA CCAAT Sp1 binding sequences, and elements were identified by the software TATA to to to and −399 to and three CCAAT to to and −410 to The 8-bp consensus TATA box with the sequence TATATATA at −399 to −406 is the TATA found in the and is in close proximity to a CCAAT box at −410 to Sp1 binding GC-rich also found the 5′-flanking region of the gene at to to to to to to to to to to and to A of consensus elements for transcription also including an site for the domain factor to and several sites for the transcription factor which is expressed in high in the and cells A. G. Y. F. P. 1997; PubMed Scopus Google Scholar). sites for elements and Cell Biochem. 2000; PubMed Google Scholar), factor of activated T J. T. H. J. Exp. Med. 2000; PubMed Scopus Google Scholar), transcription factor B. 2000; PubMed Scopus Google Scholar), and transcription factor R. 1998; PubMed Scopus Google Scholar) also identified in the 5′-flanking A primer extension analysis was performed to the transcription start site of the gene. shown in three with and region was found with a of start sites. the major transcription start sites. The farthest extension was at 49 bp upstream of the ATG translation initiation codon and is an This is to an SP1 binding site found in the promoter All of within bp upstream to the translation initiation the essential promoter region for human gC1qR gene several 5′ and 3′ deletion clones were by various restriction fragments by polymerase chain reaction into a reporter vector, Because fragments were cloned upstream to a luciferase reporter of the could be by various clones to the human cell PANC-1 It was found that the essential region for transcription is very close to the start site of luciferase between to and that of with and the essential promoter region could be mapped to a region the translation start site to This is supported by the that no in the luciferase was deletion of the 5′ sequence from to However, in the in which the 3′ region was the luciferase were Because no 5′ deletion were and the essential promoter elements in the −1 to region upstream of the translation start site. data also showed that the 1.3-kb region the transcription and were to the other human cell lines to the cell of the promoter as well as to the All of the cell MDA-MB-231, SVG and showed of luciferase for and from to on the cell lines (Fig. However, to the obtained in PANC-1, deletion of the 3′ region of the promoter the luciferase in of cell It was that the removal of the TATA box at −399 to −406 and the CCAAT box at −410 to −414 in did not significantly affect the transcription efficiency of the This that promoter elements in the −1 to region of the 5′-flanking In the −1 to seven GC-rich sequences with high to the consensus SP1 binding site were identified (Fig. is nuclear factor with GC-rich sequences, gel shift assays were performed with nuclear of PANC-1 pairs of in sense and antisense were designed on GC-rich the pairs a negative that is not the probe with the nuclear In the a of the was (Fig. the of the probe were that of the an DNA probe was used for the 1) of the also showed a that is a the identity of the monoclonal Inc.) and bovine serum were with the in a supershift assay. It was that the was by not by bovine serum that is a between and Sp1 (Fig. was performed using the as a of were added to the the of from its shown no antibody was the other three were be a of and the transcription factor serum was used as the negative in this which was to the mouse the human gene exons and and the of the exons do not for 6 B.L. Schwaeble W. Lynch N.J. Peerschke E.I. Reid K.B. Ghebrehiwet B. 1998; PubMed Scopus Google Scholar). 6 in the gene is bp in the mouse gene of a region by the sites. the of the exons between the human and mouse the three of the of the human C1qBP gene have in with their mouse The of in the human and mouse is and which the human gene the mouse gene. The promoter elements of the human gene were identified by analysis and transfection studies. TATA and CCAAT were found on the 1.3-kb 5′-flanking The TATA box is a of the core promoter and is bp upstream of the transcription start the upstream promoter that a location with respect to the transcription start site. The CCAAT close to the TATA can at that from the transcription start site in have that the CCAAT box a role in the efficiency of the promoter, and its promoter The transcription start sites in close proximity to the TATA elements in the of However, the transcription start sites to of the gene bp from the TATA found in the 5′-flanking region of the gene. The 5′-flanking region of the human gene an 8-bp consensus TATA box with the sequence TATATATA at −399 and a CCAAT box at −410 in the mouse the TATA box to the ATG start codon is at J. Zhang Y. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). However, in the promoter the which did not TATA or CCAAT transcription that of the TATA or was essential for the transcription of luciferase in the cell lines deletion in cell lines showed of the activity the bp to the initiation was This promoter to the of GC-rich sequence in the mouse from −1 to + C B.L. Schwaeble W. Lynch N.J. Peerschke E.I. Reid K.B. Ghebrehiwet B. 1998; PubMed Scopus Google Scholar), seven GC-rich Sp1 which to the of a TATA no TATA was found to be essential for the activity of the promoter, Sp1 sites in the upstream region close to the transcription start site to be very important for the promoter activity of the gene. In of seven GC-rich Sp1 sites to was found to bind specifically to PANC-1 nuclear proteins in gel shift and of nuclear was to be Sp1 binding factor in supershift assays that binding of Sp1 to the SP1 binding site at bp upstream to the translation initiation codon may play an important role in transcription in human to the of various transcription elements in the 5′-flanking may be that different transcription elements essential for the gene expression in different human The of gC1qR was revealed by The mature protein N-terminal by seven and J. Zhang Y. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). a with an in the boundaries and the is encodes the amino acid residues the sequence (14Dedio J. Jahnen-Dechent W. Bachmann M. Muller-Esterl W. J. Immunol. 1998; 160: 3534-3542PubMed Google encodes the N-terminal and the encodes encodes and 5 encodes 6 and and the encodes for the gC1qR was shown to be a multiligand-binding be to the binding sites on gC1qR to various ligands. gC1qR was found to as a receptor for the protein of L. Ghebrehiwet B. P. J. 2000; PubMed Scopus Google Scholar) and to bind to protein A T. Ghebrehiwet B. Peerschke 2000; Scopus Google Scholar). report showed that the hepatitis virus C core antigen can interact with gC1qR, and the anti-proliferation effects of hepatitis virus C core antigen on activated T cells can be by anti-gC1qR monoclonal (16Kittlesen D.J. Chianese-Bullock K.A. Yao Z.Q. Braciale T.J. Hahn Y.S. J. Clin. Invest. 2000; 106: 1239-1249Crossref PubMed Scopus (295) Google Scholar). All of reports that gC1qR is a biologically important, widely distributed, multiligand-binding and multifunctional protein (1Ghebrehiwet B. Peerschke E.I. Immunobiology. 1998; 199: 225-238Crossref PubMed Scopus (75) Google Scholar).