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• W2034709458 abstract "Tubulointerstitial nephritis antigen (TIN-ag) is a 58-kDa basement membrane glycoprotein that is recognized by human autoantibodies in certain forms of tubulointerstitial nephritis. To further characterize this macromolecule and isolate cDNAs encoding TIN-ag, amino acid sequences from tryptic peptides were used to design and synthesize primers in order to amplify a probe for screening a rabbit kidney cortex cDNA library. A cDNA encoding TIN-ag was cloned and sequenced. The predicted amino acid sequence deduced from this cDNA includes the chemically determined sequences of peptides derived from TIN-ag, supporting its authenticity. The predicted amino acid sequence also shows that the carboxyl-terminal region of the molecule exhibits a 30% homology with human preprocathepsin B, a member of the cysteine proteinase family of proteins. A domain in the amino-terminal region of TIN-ag contains an epidermal growth factor-like motif that shares homology with laminin A and S chains, α1 chain of type I collagen, von Willebrand's factor, and mucin, suggesting structural and perhaps functional similarities among these molecules. Immunoprecipitation of in vitro generated recombinant protein using a TIN-ag-specific monoclonal antibody (A8), confirms the identity of the isolated TIN-ag cDNA. In this report the cDNA and predicted amino acid sequences of TIN-ag are presented. Knowledge of the primary structure of TIN-ag will facilitate our understanding of the molecular structure of this novel basement membrane component and may provide clues toward understanding its functional role. Tubulointerstitial nephritis antigen (TIN-ag) is a 58-kDa basement membrane glycoprotein that is recognized by human autoantibodies in certain forms of tubulointerstitial nephritis. To further characterize this macromolecule and isolate cDNAs encoding TIN-ag, amino acid sequences from tryptic peptides were used to design and synthesize primers in order to amplify a probe for screening a rabbit kidney cortex cDNA library. A cDNA encoding TIN-ag was cloned and sequenced. The predicted amino acid sequence deduced from this cDNA includes the chemically determined sequences of peptides derived from TIN-ag, supporting its authenticity. The predicted amino acid sequence also shows that the carboxyl-terminal region of the molecule exhibits a 30% homology with human preprocathepsin B, a member of the cysteine proteinase family of proteins. A domain in the amino-terminal region of TIN-ag contains an epidermal growth factor-like motif that shares homology with laminin A and S chains, α1 chain of type I collagen, von Willebrand's factor, and mucin, suggesting structural and perhaps functional similarities among these molecules. Immunoprecipitation of in vitro generated recombinant protein using a TIN-ag-specific monoclonal antibody (A8), confirms the identity of the isolated TIN-ag cDNA. In this report the cDNA and predicted amino acid sequences of TIN-ag are presented. Knowledge of the primary structure of TIN-ag will facilitate our understanding of the molecular structure of this novel basement membrane component and may provide clues toward understanding its functional role. Immunologically mediated tubulointerstitial nephritis (TIN)1( 1The abbreviations used are: TINtubulointerstitial nephritisTIN-agtubulointerstitial nephritis antigenTBMtubular basement membranePBSphosphate-buffered salineIP bufferimmunoprecipitation buffer.) is characterized by the linear deposition of immunogammaglobulin (IgG) and complement along the renal tubular basement membrane (TBM). Antibodies to the TBM have been identified in patients with TIN, various types of glomerular nephropathies, and recipients of a renal allograft(1Andres G.A. Fakatsu A. Brentjens J.R. Price R.G. Hudson B.G. Renal Basement Membranes in Health and Disease. Academic Press, London1987: 354-431Google Scholar). Tubulointerstitial nephritis may be either acute or chronic. Acute tubulointerstitial nephritis is characterized by the presence of interstitial edema, leukocytic infiltrates, and patchy tubular necrosis, whereas chronic tubulointerstitial nephritis is characterized by a mononuclear infiltration accompanied by widespread tubular fibrosis and extensive tubular atrophy. Alterations of the TBM as a result of the accumulation of anti-tubular basement membrane antibodies and complement may contribute to altered renal function and may eventually lead to end stage renal disease(2Colvin R.B. Fang L.S.T. Tisher C.C. Brenner B.M. Renal Pathology. J. B. Lippincott Company, Philadelphia1994: 728-734Google Scholar). The molecular origin of the tubular basement membrane macromolecule(s) that are reactive with anti-tubular basement membrane antibodies in patients with tubulointerstitial nephritis has been the subject of many investigations. Immunologically mediated forms of TIN have been described in various animal models typically induced following immunization with kidney TBM. These studies have led to the description of various nephritogenic antigens between 30 and 70 kDa(3Zanetti K. Wilson C.B. J. Immunol. 1983; 130: 2173-2179PubMed Google Scholar, 4Clayman M.D. Martinez-Hernandez A. Michaud L. Alper R. Mann R. Kefalides N.A. Neilson E.G. J. Exp. Med. 1985; 161: 290-305Crossref PubMed Scopus (63) Google Scholar, 5Nielson E.G. Sun M.J. Kelly C.J. Hines W.H. Haverty T.P. Clayman M.D. Cooke N.E. Proc. Natl Acad. Sci. U. S. A. 1991; 88: 2006-2010Crossref PubMed Scopus (34) Google Scholar, 6Graindorge P.P. Mahieu P.R. Kidney Int. 1978; 14: 594-606Abstract Full Text PDF PubMed Scopus (23) Google Scholar, 7Wakashin Y. Takei I. Ueda S. Mori Y. Lesato K. Wakashin M. Clin. Immunol. Immunopathol. 1981; 19: 360-371Crossref PubMed Scopus (23) Google Scholar, 8Yoshioka K. Morimoto Y. Iseki T. Maki S. J. Immunol. 1986; 136: 1654-1660PubMed Google Scholar). tubulointerstitial nephritis tubulointerstitial nephritis antigen tubular basement membrane phosphate-buffered saline immunoprecipitation buffer. This laboratory has focused on the identification and characterization of a 58-kDa novel basement membrane protein, tubulointerstitial nephritis antigen (TIN-ag), previously reported to be associated with anti-TBM TIN(9Butkowski R.J. Kleppel M.M. Katz A. Michael A.F. Fish A.J. Kidney Int. 1991; 40: 838-846Abstract Full Text PDF PubMed Scopus (27) Google Scholar). This macromolecule was eventually purified from rabbit TBM. Limited amino acid sequencing indicated that TIN-ag is a novel component of the basement membrane(10Butkowski R.J. Langeveld J.P. Wieslander J. Brentjens J.R. Andres G.A. J. Biol. Chem. 1990; 265: 21091-21098Abstract Full Text PDF PubMed Google Scholar). Immunofluorescence studies have revealed an interesting pattern of reactivity(9Butkowski R.J. Kleppel M.M. Katz A. Michael A.F. Fish A.J. Kidney Int. 1991; 40: 838-846Abstract Full Text PDF PubMed Scopus (27) Google Scholar). TIN-ag was detected primarily in the basement membrane underlying proximal tubular epithelial cells, to a lesser extent in Bowman's capsule and the basement membrane of the distal tubules, and was absent from the glomerular basement membrane and the mesangial matrix. In extrarenal tissues, TIN-ag was detected in part of the small intestine (ileum) and the corneal and epidermal basement membranes. Recently, in vitro solid phase binding studies revealed that TIN-ag reacted with both type IV collagen and laminin but not with heparin(11Kalfa T.K. Thull J.D. Butkowski R.J. Charonis A.S. J. Biol. Chem. 1994; 269: 1654-1659Abstract Full Text PDF PubMed Google Scholar). These reactions were found to be specific and saturable with an affinity in the micromolar range. Furthermore, TIN-ag exhibited a concentration-dependent inhibitory effect on the polymerization of laminin and on preformed laminin networks. This observation is of importance in view of the fact that laminin polymers have been shown to exist in vivo, and therefore the presence of TIN-ag in certain areas may profoundly effect the structure of the specific basement membranes. A fundamental understanding of the molecular properties of this potential nephritogenic antigen is essential to understand the normal physiological contribution of these macromolecules, and in delineating the pathophysiology of interstitial nephritis associated with basement membrane macromolecules. In this investigation the cDNA encoding TIN-ag was cloned from a rabbit kidney cortex library. The predicted amino acid se-quence of TIN-ag is presented and compared with other known sequences. A rabbit kidney cortex library cloned into λgt11 was screened for cDNAs encoding TIN-ag. A 500-base pair TIN-ag cDNA clone was polymerase chain reaction-amplified using degenerate primer sequences based upon previously reported tryptic peptides (10Butkowski R.J. Langeveld J.P. Wieslander J. Brentjens J.R. Andres G.A. J. Biol. Chem. 1990; 265: 21091-21098Abstract Full Text PDF PubMed Google Scholar) from oligo(dt)-primed rabbit kidney total RNA. The conditions for amplification were as follows: 94°C for 5 min; 37°C for 2 min (two cycles) followed by 35 cycles at 72°C for 3 min; 94°C for 1 min 20 s; 37°C for 2 min; and finally at 72°C for 7 min. This amplified fragment was sequenced, subsequently random prime-labeled with [α-32P]dCTP, and was then used to screen duplicate nitrocellulose filter lifts under high stringency conditions (6 × SSC, 0.5% SDS twice at room temperature for 30 min each; 0.1 × SSC, 0.1% SDS for 60 min at 65°C). Five positive clones of various lengths were identified and subcloned into Stratagene pBluescript II SK-EcoRI site. DNA sequencing of the putative TIN-ag clones was performed by the dideoxynucleotide chain termination method of Sanger(12Sanger F. Coulson A.R. Barrell B.G. Smith A.J. Roe B.A. J. Mol. Biol. 1980; 143: 161-178Crossref PubMed Scopus (2195) Google Scholar), using Life Sciences Sequenase version 2.0. Both strands of the TIN-ag final construct were sequenced using a combination of vector-specific primers (T3 and T7) and internally derived sequence-specific primers. When sequence-specific primers were employed, all junctional sequences were included in sequence analysis. The nucleotide sequence and predicted amino acid sequence of TIN-ag were subjected to a data base search against the GenBank nucleotide and protein sequence data bases (release 72.0). Alignment of TIN-ag predicted amino acid sequence was performed by DNASTAR version 2.0 software utilizing the Pearson and Lipman (13Pearson W.R. Lipman D.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2444-2447Crossref PubMed Scopus (9381) Google Scholar) method of sequence alignment to determine the regions of maximum homology. Ten μg of rabbit genomic DNA was digested at 37°C for 4 h with restriction enzymes HIND III and EcoRI for Southern analysis(14Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning. Laboratory, Cold Spring Harbor, NY1982Google Scholar). The samples were electrophoresed through a .8% agarose Tris Borate EDTA gel. Following electrophoresis, the gel was depurinated (5 min in .25 M HCl), rinsed with denaturation buffer (0.5 M NaOH, 1.5 M NaCl), and blotted onto a nylon membrane (Boehringer Mannheim) overnight in denaturation buffer. The membrane was cross-linked using a stratalinker (Stratagene) prehybridized at 42°C for 4 h, and then probed using an SphI/EcoRI cDNA fragment of TIN-ag, which was random primed with [32P]dCTP according to the manufacturer's protocol (Boehringer Mannheim random prime labeling kit for nucleic acids). The blot was washed for 30 min in 6 × SSC, 0.5% SDS for 30 min at room temperature and finally at 62°C in 0.1 × SSC, 0.1% SDS for 1 h. The Southern membrane was then exposed to x-ray film at −70°C for 48 h using an intensifying screen. The full-length TIN-ag construct was subcloned into pBluescript II SK- and transcribed using vector-specific RNA polymerase T7. Two micrograms of nonlinearized TIN-ag plasmid DNA was purified using the GeneClean kit (BIO 101, Inc.) and resuspended in 2 μl of diethyl pyrocarbonate-treated H2O. To this, 5 μl of transcription buffer (200 mM Tris-HCl (pH 8.0), 40 mM MgCl2, 10 mM spermidine, 250 mM NaCl) was added to the template. Next, 1.0 μl each of 10 mM rATP, rGTP, rCTP, rUTP, and 0.75 M dithiothreitol was added in addition to 40 units of RNasin RNase inhibitor (Promega) to the transcription reaction. After the addition of 10 units of T7 RNA polymerase, diethyl pyrocarbonate H2O water was added to a final volume of 25 μl. The reaction was incubated in a 37°C water bath for 30 min. DNA templates present in the transcription reaction were removed by incubation at 37°C with 10 units of RNase-free DNase/μg of DNA template for 15 min. TIN-ag RNA was extracted once with phenol:chloroform (v/v) (1:1) and precipitated with 0.1 volumes of 3 M NaOAc (pH 5.2) and 3 volumes of ethanol. The mRNA was centrifuged at 14,000 × g for 15 min; the RNA pellet was allowed to dry, and was resuspended in 3 μl of diethyl pyrocarbonate-treated H2O. The mRNA from the above transcription reaction was heated to 68°C for 1 min to eliminate any secondary structure formation in the mRNA. Two μl of nonradioactive amino acid mixture was added to the reaction to a final concentration of 50 μM. Twenty μl of rabbit reticulocyte lysate (Stratagene) was added, and after thorough mixing was allowed to proceed for 60 min at 30°C. Following the translation reaction, the contents of the tube were centrifuged and stored at −20°C. SDS-polyacrylamide gel electrophoresis was performed as described by Laemmli (15Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207211) Google Scholar) on 10% gels. Gels were run for 4-5 h at approximately 30 mA. Protein A-agarose was used to immunoprecipitate the immune complexes formed at 4°C from a lysate containing the in vitro translated product from the TIN-ag cDNA clone. Rabbit anti-mouse IgG was used as a bridging molecule between the protein A-agarose and the primary antibody. The following general procedure was carried out as follows in a 1.5-ml conical microfuge tube. Protein A-agarose beads were conjugated to rabbit anti-mouse IgG by incubation at room temperature for 1 h. Following this, the conjugated complex was washed 3 times in immunoprecipitation buffer (IP buffer) (50 mM Tris-HCl, 0.14 M NaCl, 1.0% Triton X-100, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2, 1 mM phenylmethylsulfonyl fluoride, 1 mMN-ethylmaleimide, 0.2% NaN3, pH 7.2). Normal mouse IgG was then conjugated to the complex, and the reaction was placed on a rotary platform at room temperature for 1 h. Following this incubation, the tubes were washed 3 times with IP buffer, and the samples were added to the tubes for preclearance of nonspecific binding proteins at 4°C overnight. Samples were washed 3 times with IP buffer, and supernatants were transferred to tubes containing the primary antibody (A8), which was raised against purified TIN-ag isolated from tubular basement membrane. This complex was conjugated to rabbit anti-mouse and protein A-agarose beads for immunoprecipitation of the desired in vitro expression product of TIN-ag at 4°C overnight. Samples were washed 4 times in IP wash buffer (50 mM Tris-HCl, 0.4 M NaCl, 1.0% Triton X-100, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2, 1 mM phenylmethylsulfonyl fluoride, 1 mMN-ethylmaleimide, 0.2% NaN3, pH 7.2) and centrifuged to collect pellet. Immunoprecipitation samples were resuspended in loading buffer and subjected to SDS-polyacrylamide gel electrophoresis in 10% polyacrylamide gels according to the methods of Laemmli et al.(15Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207211) Google Scholar). Protein was then electrophoretically transferred (0.2 A for 2 h) from the polyacrylamide gel to nylon membranes (Immobilon-P; Millipore, Bedford, MA) as described by Towbin et al.(16Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44923) Google Scholar), and unoccupied sites on the nitrocellulose were blocked by incubating with PBS containing 3% skim milk overnight at 4.0°C. The blocked membrane was incubated for 1 h at room temperature with a previously described TIN-ag monoclonal antibody (A8) diluted 1:50 in a solution of PBS and 3% skim milk. The membrane was then washed with PBS 4 times for 15 min and incubated for 30 min at room temperature with horseradish peroxidase-conjugated goat anti-mouse IgG antibody (UBI, Lake Placid, NY) at a 1:2000 dilution in PBS containing 0.05% Tween 20 (TPBS). After washing the membrane 2 times for 15 min and 4 times for 5 min each with TPBS, the bound secondary antibody was detected by enhanced chemiluminescence (Amersham Corp.) according to the manufacturer's protocol. To facilitate the isolation of cDNA sequences encoding rabbit TIN-ag, we took advantage of the known amino acid sequence of the amino terminus and an internal tryptic peptide(10Butkowski R.J. Langeveld J.P. Wieslander J. Brentjens J.R. Andres G.A. J. Biol. Chem. 1990; 265: 21091-21098Abstract Full Text PDF PubMed Google Scholar). To amplify a specific probe, degenerate primers corresponding to the known TIN-ag amino terminus (5′ WSY ATA TTY CAR GGI CAR TA 3′) and an internally derived amino acid sequence from the previously described TIN-ag tryptic T4-13 peptide (3′ GGI CTY TGI TGI CTR RAI GGI CT 5′) were used to amplify a cDNA from oligo(dt)-primed rabbit kidney total RNA (Fig. 1A). The reaction amplified a fragment of about 500 base pairs and required both the amino-terminal primer and the T4-13 primer. The 500-base pair amplified product was gel-purified and cloned into Novagen PT7 Blue vector. Preliminary double-stranded sequence analysis using 5′ U-19 mer primer in association with vector-specific T7 primer revealed no homology with any known sequences in GenBankTM. Sequence data indicated the presence of both primer sequences, and the amino acid sequence of the regions adjacent to the primers corresponded to the previously determined peptide sequence, supporting the notion that sequences encoding the tryptic fragment have been isolated in the cDNA clone. The amplified product was used to screen a λgt11 rabbit kidney cortex library for additional clones. Five positive clones of various lengths were identified and subcloned into Stratagene pBluescript II SK-EcoRI site for further sequence analysis. A consensus sequence for both strands of the full-length TIN-ag final construct was determined using a combination of vector-specific primers (T3 and T7) and internally derived sequence-specific primers (Fig. 1B). Sequence analysis indicated the presence of a 1425-base pair open reading frame encoding a protein of 474 amino acids. The 5′-untranslated region of the cDNA contains 120 nucleotides. The 3′-untranslated region contains a canonical hexanucleotide polyadenylation signal located 200 nucleotides downstream of the stop codon(17Nevins J.R. Annu. Rev. Biochem. 1983; 52: 441-446Crossref PubMed Scopus (235) Google Scholar). The originally described tryptic peptide fragments are present in the deduced amino acid sequence, providing evidence that the cDNA that was cloned from the rabbit kidney cortex library corresponds to the protein species initially isolated from rabbit tubular basement membrane. The amino-terminal region of the protein (amino acids 1-18), containing a charged NH2-terminal, central hydrophobic, and polar COOH-terminal region, represents the putative signal peptide of the molecule(18Heijne G.V. J. Mol. Biol. 1985; 184: 99-105Crossref PubMed Scopus (1535) Google Scholar). This observed signal sequence is characteristic for molecules associated with the extracellular/secretory pathway. Twenty-nine amino acid residues are present between the observed signal peptide and the previously described NH2-terminal region (10Butkowski R.J. Langeveld J.P. Wieslander J. Brentjens J.R. Andres G.A. J. Biol. Chem. 1990; 265: 21091-21098Abstract Full Text PDF PubMed Google Scholar). It is possible that this region may represent a propeptide that is cleaved during the early stages following protein synthesis(19Cooper A.A. Stevens T.H. BioEssays. 1993; 15: 667-674Crossref PubMed Scopus (19) Google Scholar). Alternatively, isolation procedures may have contributed to the shortening of the amino-terminal portion of the molecule. The deduced amino acid sequence contains seven potential glycosylation sites (Asn-X-Ser/Thr)(20Elbein A.D. Annu. Rev. Biochem. 1987; 56: 497-534Crossref PubMed Google Scholar), shown in Fig. 2. Cysteine residues are clustered in two discrete regions: amino acids 55-152 and amino acids 238-349. No RGD sequence is indicated in the predicted amino acid sequence of TIN-ag. A relatively high content (3.0%) of tryptophan is also observed. A search for homologies to other known proteins revealed that TIN-ag shares sequence homology primarily with the cathepsin family of cysteine proteinases. Fig. 3 compares the carboxyl region of the TIN-ag sequence with human preprocathepsin B (HCB). Within this homologous region, positions 366-375 of TIN-ag correspond to a previously determined tryptic peptide sequence (T4-17) not used in primer design. The result provides additional supporting evidence that the cDNA represents the originally described TIN-ag. Alignment of conserved cysteines between the molecules is suggestive of structural and perhaps functional similarities between TIN-ag and the cathepsin family of cysteine proteinases(21Dufour E. Biochimie. 1988; 70: 1335-1342Crossref PubMed Google Scholar). It is known that three distantly located domains contribute to the conformation of the active site of cysteine proteinases(22Kamphuis I.G. Drenth J. Baker E.N. J. Mol. Biol. 1985; 262: 14448-14453Google Scholar). In two out of the three domains TIN-ag contains sequences that are identical to those of the human preprocathepsin B molecule, as well as all the other members of the cysteine proteinase family. In the third region, a cysteine residue considered important for the catalytic activity and present in all members of the cysteine proteinase family is substituted by a serine residue in TIN-ag at position 241. Analysis of a domain in the amino terminus of the molecule reveals an epidermal growth factor-like repeat that is found within several classes of extracellular matrix molecules. As shown in Fig. 4, alignment of extracellular matrix molecules such as laminin A and S chains, von Willebrand's factor, mucin, and the α1 chain of type I collagen with that of amino acids 118-152 of TIN-ag, revealed a highly conserved domain with respect to the position of cysteines, again suggesting putative structural similarities between this domain and other domains of various extracellular matrix molecules. To verify the presence of TIN-ag gene sequences corresponding to the isolated cDNA sequence, Southern blot analysis was conducted. Rabbit genomic DNA, digested with the restriction enzymes HindIII (lane 1) and EcoRI (lane 2) revealed a simple endonuclease restriction fragment pattern indicative of a single gene (Fig. 5). These results demonstrate the presence of gene sequences that correspond to the isolated cDNA sequence. In vitro transcription and translation of the TIN-ag cDNA was carried out to verify the identity of the gene product encoded by the isolated TIN-ag cDNA. The in vitro translated product was immunoprecipitated with monoclonal antibody A8, (Fig. 6). Electrophoresis, under nonreducing conditions, of immunoprecipitated protein samples revealed a band with a mobility of 52 kDa only in the lane in which the in vitro reaction was carried out (lane 3). The electrophoretic mobility of the recombinant product is consistent with the predicted molecular weight of the translated construct. The observed difference in the electrophoretic mobility between the tissue-isolated TIN-ag and the recombinant product may be attributed to various factors such as lack of carbohydrate additions and differential disulfide bond formation. The immunoprecipitation data provided evidence that the recombinant product encodes the protein species that was originally characterized as being associated with anti-TBM TIN. In this report we provide the full coding sequence of the TIN-ag cDNA. The cDNA encodes a protein with a predicted molecular weight of 54.5 kDa that shares 30% homology with the cysteine proteinase family of molecules. Several chemically determined tryptic peptide sequences were present within the deduced amino acid sequence, providing evidence that the cDNA corresponds to the protein originally isolated from TBM. Southern blot analysis of genomic DNA reveals the presence of gene sequences that correspond to the isolated cDNA. A full-length construct derived from the isolated cDNAs was used to generate a recombinant protein. Immunoprecipitation of the in vitro generated recombinant product supports the hypothesis that the TIN-ag cDNA encodes a product associated with anti-TBM TIN. The structural data provided in this report, in combination with previously reported functional characteristics of TIN-ag suggest that TIN-ag may represent a macromolecule of importance in renal basement membrane biology. Collectively, the results of our investigation are suggestive of TIN-ag being a member of two distinct protein superfamilies(23Ohno S. Emori Y. Imajoh S. Kawasaki H. Kisaragi M. Suzuki K. Nature. 1984; 312: 566-569Crossref PubMed Scopus (253) Google Scholar). The NH2-terminal region of the molecule contains a highly conserved epidermal growth factor-like repeat common to several classes of extracellular matrix adhesive glycoproteins, whereas the COOH-terminal region shares extensive homology with the cathepsin family of cysteine proteinases. The spacing of the two putative domains may indicate that TIN-ag arose by the fusion of genes of extracellular adhesion glycoproteins and of cysteine proteinases, although the possibility of convergent evolution cannot be excluded. The identification of TBM components reactive with antisera from patients with anti-tubular basement membrane nephritis has yielded much information regarding the heterogeneous nature of this biologically active barrier. Clayman et al.(4Clayman M.D. Martinez-Hernandez A. Michaud L. Alper R. Mann R. Kefalides N.A. Neilson E.G. J. Exp. Med. 1985; 161: 290-305Crossref PubMed Scopus (63) Google Scholar) identified a 48-kDa nephritogenic antigen, 3M-1, isolated from rabbit tubular basement membrane capable of inducing anti-TBM disease in rodents. Nielson et al.(5Nielson E.G. Sun M.J. Kelly C.J. Hines W.H. Haverty T.P. Clayman M.D. Cooke N.E. Proc. Natl Acad. Sci. U. S. A. 1991; 88: 2006-2010Crossref PubMed Scopus (34) Google Scholar) further characterized the human 3M-1 glycoprotein and defined it as a major nephritogenic antigen in anti-TBM disease. Yoshioka (8Yoshioka K. Morimoto Y. Iseki T. Maki S. J. Immunol. 1986; 136: 1654-1660PubMed Google Scholar) has also described nephritogenic antigens of 54 and 48 kDa isolated from human TBM through immunoprecipitation with human patient antisera. Interestingly, the same patient antisera used to characterize TIN-ag is also capable of immunoprecipitating the 3M-1 glycoprotein described by Clayman et al.(4Clayman M.D. Martinez-Hernandez A. Michaud L. Alper R. Mann R. Kefalides N.A. Neilson E.G. J. Exp. Med. 1985; 161: 290-305Crossref PubMed Scopus (63) Google Scholar) and Nielson et al.(5Nielson E.G. Sun M.J. Kelly C.J. Hines W.H. Haverty T.P. Clayman M.D. Cooke N.E. Proc. Natl Acad. Sci. U. S. A. 1991; 88: 2006-2010Crossref PubMed Scopus (34) Google Scholar) as well as the nephritogenic antigens reported by Yoshioka et al.(8Yoshioka K. Morimoto Y. Iseki T. Maki S. J. Immunol. 1986; 136: 1654-1660PubMed Google Scholar). This observation suggests the presence of multiple anti-TBM antibodies present in the patient antisera, but it also may indicate that the antigens have similar nephritogenic domains, are structurally related, or are conferred similar post-translational modifications. This latter interpretation is not entirely consistent with the data presented on the characterization of the 3M-1 glycoprotein by Neilson et al.(5Nielson E.G. Sun M.J. Kelly C.J. Hines W.H. Haverty T.P. Clayman M.D. Cooke N.E. Proc. Natl Acad. Sci. U. S. A. 1991; 88: 2006-2010Crossref PubMed Scopus (34) Google Scholar), since peptides thought to represent the major nephritogenic domain of 3M-1 show no similarities to the predicted amino acid sequence of our TIN-ag. Furthermore, biochemical analysis of 3M-1 reveals little similarity to TIN-ag in amino acid composition. These two observations suggest that the two antigens differ structurally and biochemically. Although immunofluorescence data using monoclonal antibodies against the nephritogenic antigen described by Yoshioka et al.(8Yoshioka K. Morimoto Y. Iseki T. Maki S. J. Immunol. 1986; 136: 1654-1660PubMed Google Scholar) show similar tissue distribution as TIN-ag, the reported NH2-terminal sequence (24Yoshioka K. Hino S. Takemura T. Miyasato H. Honda E. Maki S. Clin. Exp. Immunol. 1992; 90: 319-325Crossref PubMed Scopus (11) Google Scholar) is not found in the predicted sequence of TIN-ag, suggesting that these two nephritogenic antigens represent different macromolecules. The possibility cannot be excluded, however, that alternative processive forms of the same nephritogenic antigen are present in the TBM. Basement membranes represent highly specialized barriers that are present between the basal surface of polarized cells and the interstitial tissue. They are thought to contribute significantly to cellular phenotype, vascular permeability, and overall tissue architecture. Their macromolecular constituents are involved in these physiological processess. Knowledge of the TIN-ag amino acid sequence may provide valuable insight into the possible physiological role of this macromolecule in normal tissue and facilitate an understanding of diseases that are associated with the basement membrane. Furthermore, because the highest degree of immunohistochemical staining for TIN-ag is in the basement membranes beneath epithelial cells involved in the active transport of many metabolites including proteins, peptide molecules, and electrolytes, it is tempting to speculate that TIN-ag plays a crucial role in the creation and maintenance of these physiological environments." @default.
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• W2034709458 title "Identification of a cDNA Encoding Tubulointerstitial Nephritis Antigen" @default.
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