Patent Application: US-95286910-A

Abstract:
an isolated polypeptide comprises an amino acid sequence of seq id no . 1 or 2 or a variant or fragment thereof . the variant may comprise an amino acid sequence that is at least 70 % or 95 % identical to the amino acid sequence of seq id no . 1 or 2 . a fragment thereof may be a peptide comprising at least 12 contiguous amino acids of seq id no . 1 or 2 . the polypeptide exhibits toll - like receptor activity . the tlr has been named tlr1 4 . tlr receptors recognise a range of ligands and activate a series of signalling pathways that lead to the induction of immune and inflammatory genes .

Description:
we have identified a novel gene that shows remarkable homology with members of the toll - like receptor / interleukin - 1 receptor ( tlr ) family . in cell - based assays , this novel receptor activates the transcription factors nf - κb and irf3 and drives the production of the anti - viral cytokine , rantes . the protein interacts with the tlr2 , tlr4 and the universal tlr adapter , myd88 . we have named the receptor tlr14 . expression of this putative receptor is enhanced by microbial products , for example lps , suggesting that it may function as an immuno - modulator . in support of this , the transcription factors nf - κb and irf3 were activated when cells were transfected with a vector expressing tlr14 . as both nf - κb and irf3 are central in the elimination of bacterial and viral pathogens , inhibiting or activating tlr14 is a promising new approach for the treatment of inflammatory diseases . in addition , we have found high levels of tlr14 in serum . a soluble form of tlr2 comprising mainly of the ectodomain of this receptor is also found at high levels in serum and in breast milk . this form of tlr2 is protective in that it dampens over active immune responses to tlr2 ligands . the full length tlr14 polypeptide or the ectodomain itself may have similar biological properties and could therefore be considered a potential biotherapeutic . a microarray approach was used to identify genes that are regulated by lps and components of the tlr4 signalling pathway . as mentioned above , the adapter molecule mal is required to transmit signals from tlr2 and tlr4 following receptor stimulation . we used a gene - targeting vector to disrupt the gene encoding mal in embryonic stem cells . these cells were then treated with lps and differences in gene expression between knockout and wild - type cells were measured . in this way the gene that shows remarkable homology with members of the toll - like receptor / interleukin - 1 receptor ( tlr ) family was identified . the examples presented are illustrative only and various changes and modifications within the scope of the present invention will be apparent to those skilled in the art . hek 293 and u373 cells were cultured in dulbecco &# 39 ; s modified eagles medium ( dmem ) with 10 % fetal bovine serum ( fbs ), supplemented with 100 units / ml penicillin , 100 mg / ml streptomycin , and 2 mm l - glutamine . the chimeric tlr receptor cd4 - tlr4 , was a gift from r . medzhitov ( yale university , new haven , conn .). the vector containing the tlr14 cdna ( kiaa0644 ) was supplied by the kazusa dna research institute and used as target for subsequent pcr cloning . the primers used included restriction sites for hindiii and ecorv and were as follows : 5 ′- gcaagcttatggaggctgcccgcgccttg ( sense ) ( seq id no . 5 ); and 5 ′- gcgatatcggcctaagcgtagtctgggacgtcgtatgggtagtcggcaaatc gc ( antisense ) ( seq id no . 6 );. the antisense primer includes a sequence encoding a 9 amino acid hemagglutinin epitope tag in order to detect expression of the translated protein product in transfected cells . the resulting ecor1 - hindiii fragment was ligated into the multiple cloning site of the mammalian expression vector pcdna 3 . 1 ( invitrogen ). embryonic stem cells lacking the gene encoding mal were generated by homologous recombination . briefly , murine embryonic stem cells were electroporated with a targeting vector , in which a 700 by exon encoding most of the coding sequence of the mal gene was replaced with a neomycin resistance cassette . targeted cells were identified by southern blotting before being subjected to a second round of targeting in order to generate clones homozygous for the mal deletion . mutant and wild - type cells were stimulated with lps for various times and rna was extracted for microarray analysis . the complete nucleotide sequence of the human riken clone kiaa0644 and flanking regions was obtained from the national center for biotechnology information ( ncbi ) website at www . ncbi . nlm . nih . gov . identification of transcribed nucleotide sequences and repeat sequences in the genomic sequence was performed using the nix application ( http :// menu . hgmp . mrc . ac . uk ) and the program repeat - masker ( http :// searchlauncher . bcm . tmc . edu ) ( 20 ). transcription factor binding site predictions were performed using matinspector release professional ( www . genomatix . de / cgi - bin / matinspector / matinspector . pi ) ( 21 ). mrna was extracted from cells following treatment for various times with lps ( 1 μg / ml ). briefly , treated cells were pelleted and lysed in l ml of tri reagent ( sigma ). chloroform ( 0 . 2 ml ) was added to the sample and the mixture was centrifuged at 12 , 000 g for 15 minutes . the rna containing aqueous phase was removed and the total rna was precipitated from the mixture with the addition of an equal volume of isopropanol . following centrifugation at 12 , 000 g for 10 minutes , the rna containing pellet was washed with 500 μl of 75 % ethanol . any traces of ethanol were then removed and the pellet was left to dry at room temperature for 10 minutes . the pellet was resuspended in 30 μl of rnase free water and stored at − 80 ° c . rt - pcr was carried out using the promega impromptii rt - pcr kit . the reverse transcription reaction was carried out in two steps , a pcr reaction was then carried out on the synthesised cdna . step 1 : 1 μl of random primers were added to 4 μl of rna in a thin walled 500 μl pcr micro centrifuge tube . the tube was placed in a thermal cycler at 70 ° c . for 5min and 4 ° c . for 5 min . step 2 : a second set of components were added ; 1 μl deoxynucleotide mix ( dntps mix ) ( 500 μm each dntp ), 5 . 5 μl of pcr reagent water , 4 . 0 μl of 10 × buffer , 3 . 0 μl of magnesium chloride , 1 μl rnase inhibitor ( 1 units / μl ), 1 μl of rt ( 1 units / μl ). this brought the total volume of the pcr tube to 20 μl . the tube was placed in a thermal cycler for the following times and temperatures , 25 ° c . for 5 min , 42 ° c . for 60 min , 70 ° c . for 15 min . the following was added to a thin walled 500 μl pcr microcentrifuge tube on ice : 5 μl of 10 × buffer , 1 μl dntps ( 200 μm each dntp ), 1 μl pcr primers ( 0 . 4 μof each ), 2 - 5 μl template dna ( cdna ), 1 μl taq dna polymerase mix ( 0 . 05 units / μl ) and a sufficient volume of pcr reagent water to make a total volume in the pcr tube of 50 μl . the amplification temperatures were as follows , denaturation / rt inactivation ( step 1 ) 94 ° c . for 2 min , denaturation ( step 2 ) 94 ° c . for 15 sec , annealing ( step 3 ) 55 ° c . for 30 sec , extension 68 ° c . for 1 min ( step 2 , 3 and 4 were repeated 35 times ), final extension ( step 5 ) 68 ° c . for 5 min . the pcr products were then electrophoresed on a 1 % agarose gel and visualised on a uv transluminator . a peptide antibody directed at the c - terminus of the putative protein was synthesised by eurogentec , liege science park , belgium . the peptide used for immunization is composed of the following amino acids — cgslrredrllqrfad ( seq id no . 7 );. cell lines were treated for various times with tlr ligands as indicated . stimulations were stopped with the addition of cold pbs and cells were lysed in sds - page sample buffer . for western blotting , the tlr14 antibody was diluted 1 : 1000 in tris buffered saline containing 0 . 5 % tween 20 . hek 293 cells or u373 cells were seeded into 96 - well plates ( 2 × 10 4 cells per well ) and transfected the next day with expression vector and reporter plasmids . genejuice ™ ( novagen ) was used for transient transfections , according to the manufacturer &# 39 ; s instructions . for experiments involving nf - κb or irf3 , 80 ng of the nf - κb - or isre - luciferase reporter gene ( stratagene ) were transfected into cells along with 40 ng of the renilla luciferase internal control plasmid ( promega ). after 24 h cells were harvested in passive lysis buffer ( promega ) and reporter gene activity was measured in a luminometer . in cases where cells were stimulated , lps ( sigma ) was added to the cells at a final concentration of 1 μg / ml 6 h prior to harvesting . data are expressed as mean fold induction ± s . d . relative to control levels , for a representative experiment from a minimum of three separate experiments , each performed in triplicate . u373 cells were transfected with increasing doses ( 1 , 10 and 100 ng ) of the tlr14 expression plasmid . the cells were incubated at 37 ° c . for 24 h . a 96 well microtitre plate was coated with the capture antibody ( mouse anti - human rantes ) at a final concentration of 40 ng / ml . after 24 hours the plates were washed with pbs containing 0 . 05 % tween 20 . the plates were then blocked for 1 h at room temperature in pbs containing 1 % bsa and 5 % sucrose . cell supernantant ( 100 μl ) was added to each well and the plates were incubated for 2 h at room temperature . detection antibody ( biotinylated goat anti - human rantes ) was then added to the wells at a final concentration of 10 ng / ml . the plates were again incubated for 2 h at room temperature . after washing , 100 μl of streptavidin - hrp was added to each well , the plates were covered and incubated for 20 minutes at room temperature . finally , 100 μl of substrate solution ( r & amp ; d systems , catalog # dy999 ) was added to the wells followed by 50 μl of stop solution ( 2n h 2 so 4 ). the optical density of each well was measured in a microplate reader set to 450 nm . hek293 cells were seeded on 10 cm plates at 1 × 10 5 cells / ml . the following day , cells were transfected with 3 μg of flag - tagged tlr2 , tlr4 or myc - tagged myd88 . after 24 hrs the cells were lysed in hepes buffer containing 1 % np40 . the cell lysates were then incubated with m2 anti - flag agarose beads ( sigma ). after three hours the beads were washed x3 with hepes buffer and resuspended with 20 μl of sds - page sample buffer . the protein samples were run on 10 % sds - page gels and transferred to nitrocellulose for western blotting . the resulting blots were probed with anti - tlr14 and anti - flag antibodies . cells were seeded in 10 cm dishes at 1 × 105 cells / ml 24 hours prior to stimulation with lps . membrane and cytosolic fractions were prepared by ultracentifugation and subjected to sds - page and western blotting in order to determine the localisation of tlr14 . medium ( dmem ) containing 10 % fcs was blotted for the presence of tlr14 following sds - page . preliminary microarray analysis identified six genes that exhibit lower expression levels in mal knockout cells . five of the genes identified have been characterised to some extent while the remaining gene is novel and characterised herein . the sequence of this gene is available on the huge ( human unidentified gene - encoded large proteins ) protein database as part of the human cdna project at the kazusa dna research institute ( www . kazusa . or . jp ). we have named this novel gene tlr14 for reasons outlined below . we have mapped the gene to human chromosome 7 using the map viewer tool available from ncbi ( fig1 a ). the gene is 4 . 7 kb in length and is flanked by creb5 and cpvl carboxypeptidase . the nucleotide sequences for human and murine tlr14 are shown in fig1 b and 1c , respectively . the predicted protein is 811 amino acids in length ( fig1 d ) and contains an n - terminal signal sequence , a feature common to all membrane localised proteins . the n - terminus of the putative protein also contains at least 6 leucine rich repeats and is highly homologous to the extracellular region of several tlrs ( tlr4 is given as an example in fig1 e ). expression profiles reveal a high abundance of the gene product in brain , kidney and ovary as shown in fig2 a ( information obtained from kazusa dna research institute ). we have generated a polyclonal antibody to the c - terminus of tlr14 . the peptide used for immunization comprises the amino acids cgslrreddrllqrfad ( seq id no . 7 );. the antibody detected a protein at approximately 81 kda in human brain and lung tissue ( fig2 b ). as described above , members of the tlr family all contain a cytosolic tir domain . this domain spans about 200 amino acids , with varying degrees of sequence similarity among family members . three particular boxes can be identified which are highly conserved among family members . box1 is considered the signature sequence of the family whereas boxes 2 and 3 contain amino acids critical for signalling . the crystal structure of the tir domains of tlr1 and tlr2 has revealed a core structural element centered around box 2 ( 22 ). this region , termed the bb loop , forms an exposed surface patch and contains a critical proline or arginine residue . these amino acids are located at the tip of the loop and are thought to form a point of contact with downstream signalling components . close inspection of tlr14 reveals that it also contains a highly conserved box 2 and an identifiable box 1 and 3 ( fig3 ) suggesting that this novel protein belongs to the tlr superfamily . expression of tlr14 is induced following treatment of cells with tlr2 and tlr4 ligands . as described above , tlr14 expression was abolished in cells lacking mal following exposure to lps . this indicates that the gene in question is regulated by lps and possibly other tlr ligands . in order to address this issue further , we identified the promoter region of tlr14 and possible transcription factor binding sites using the nix application ( http :// menu . hgmp . mrc . ac . uk ) and matinspector release professional ( www . genomatix . de / cgi - bin / matinspector / matinspector . pi ). it is likely that the functional tlr14 promoter is contained within the 4 kb region proximal to exon 1 . further analysis of this region revealed putative binding sites for several transcription factors , such as nf - κb , irf7 and ets - 1 ( fig4 ). the induction of tlr14 mrna expression was analysed by rt - pcr following treatment of cells with inflammatory stimuli . as shown in fig5 a , tlr14 mrna expression is induced in brain astrocytoma cells ( u373s ) and primary murine embryonic fibroblasts ( mefs ) with time following exposure to lps . a striking increase was also detected in the levels of tlr14 mrna prepared from the brains of mice treated with lps ( fig5 b ). induction of expression was also detected at the protein level in the humal glioma cell line , a172 , following treatment with the tlr2 ligand pam 3 cys , as shown in fig6 a . a similar effect was seen in hek - 293 cells stably transfected with tlr4 following treatment with lps ( fig6 b ). in addition , an increase in tlr14 protein expression was seen in the brains of mice injected with lps as shown in fig6 c . as described above , nf - κb is activated by most members of the tlr superfamily while irf3 activation is restricted to tlr3 and tlr4 . in order to address whether tlr14 can also activate these factors and therefore modulate immune responses , we cloned the cdna encoding the protein into the mammalian expression vector pcdna 3 . 1 and performed functional assays using luciferase reporter constructs containing elements of dna to which nf - κb and irf3 bind . the protein contains a tag encoding hemaglutinin ( ha ) and expression was detected in various cell lines using an anti - ha antibody ( data not shown ). when the tlr14 expression plasmid was transfected into cells along with the κb and isre reporter constructs , luciferase activity was enhanced ( fig7 ) suggesting that tlr14 , like tlr4 , activates both nf - κb and irf3 . preliminary elisas have also shown an increase in rantes production ( an irf3 inducible cytokine ) in cells transfected with tlr14 ( fig8 ). a common feature of tir domain containing proteins is their ability to homo - or heterodimerize with other tir domain containing proteins . we performed co - immunoprecipitation experiments with tlr14 and the tir domain containing receptors tlr2 and tlr4 in order to determine if tlr14 could interact with either or both receptors . we found that tlr14 interacts strongly with overexpressed tlr2 and tlr4 as shown in fig9 a . mutation of the conserved proline residue to a histidine in the tir domain of tlrs is known to abolish tir - tir interactions ( 22 ). accordingly , the interaction between tlr14 and either tlr2 or tlr4 was significant reduced with mutant ( p / h ) forms of the receptors were co - expressed with tlr14 . tlr14 was also found to interact with the universal tir - domain containing adapter myd88 as shown in fig9 b . this supports the notion that tlr14 is a tir domain containing protein . finally , we were able to detect an interaction between tlr2 and endogenous tlr14 as shown in fig1 . in order to test this , we transfected hek293 cells with flag - tagged tlr2 . cells were then lysed and incubated with anti - flag beads in order to immunprecipitate tlr2 and any interacting proteins . following western blotting , we were able to detect a band corresponding to tlr14 using the anti - tlr14 antibody . tlr14 is found at high levels in serum and may be produced as a soluble protein . we prepared cellular fractions in order to determine whether tlr14 is localised to the plasma membrane . surprisingly , tlr14 was found in the cytosolic fraction of cells ( fig1 a ). in addition , high levels of the protein were found in fetal calf serum ( fig1 b ) suggesting that the protein may be a soluble secreted protein . mass spectroscopic analysis revealed that the band present in fcs was the bovine homolog of human tlr14 ( data not shown ). preliminary experiments have also shown that the protein is secreted from u373 cells following stimulation with lps . the protein does not appear to be cleaved as the molecular weight corresponds to that of the full length protein . maximum secretion occurs at 6 hours . the invention is not limited to the embodiments hereinbefore described which may be varied in detail . 2 . qureshi , s . t . et al . j . exp . med . 189 , 615 - 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