Collectrin, a Collecting Duct-specific Transmembrane Glycoprotein, Is a Novel Homolog of ACE2 and Is Developmentally Regulated in Embryonic Kidneys

Abstract

Collectrin, a novel homolog of angiotensin-converting enzyme-related carboxypeptidase (ACE2), was identified during polymerase chain reaction-based cDNA subtraction and up-regulated in 5/6 ablated kidneys at hypertrophic phase. Collectrin, with 222 amino acids, has an apparent signal peptide and a transmembrane domain; the sequence is conserved in mouse, rat, and human and shares 81.9% identity. Human collectrin has 47.8% identity with non-catalytic extracellular, transmembrane, and cytosolic domains of ACE2; however, unlike ACE and ACE2, collectrin lacks active dipeptidyl carboxypeptidase catalytic domains. The collectrin mRNA transcripts are expressed exclusively in the kidney. In situ hybridization reveals its mRNA expression in renal collecting ducts, and immunohistochemistry shows that it is localized to the luminal surface and cytoplasm of collecting ducts. Immunoprecipitation studies, using [35S]methionine-labeled renal cortical and inner medullar collecting duct cells, i.e. M-1 and mIMCD-3, indicate that the protein size is ∼32 kDa. During the development of mouse kidney, mRNA signal is detectable at day 13 of gestation, and the protein product is observed in the ureteric bud branches. Its expression is progressively increased during later stages of the gestation extending into the neonatal periods and then is decreased in adult life. Up-regulated expression of collectrin in the hypertrophic kidneys after renal ablation and restricted spatio-temporal expression during development indicates a possible role(s)in the process of progressive renal failure and renal organogenesis.AF178085 AF178086 AF229179 Collectrin, a novel homolog of angiotensin-converting enzyme-related carboxypeptidase (ACE2), was identified during polymerase chain reaction-based cDNA subtraction and up-regulated in 5/6 ablated kidneys at hypertrophic phase. Collectrin, with 222 amino acids, has an apparent signal peptide and a transmembrane domain; the sequence is conserved in mouse, rat, and human and shares 81.9% identity. Human collectrin has 47.8% identity with non-catalytic extracellular, transmembrane, and cytosolic domains of ACE2; however, unlike ACE and ACE2, collectrin lacks active dipeptidyl carboxypeptidase catalytic domains. The collectrin mRNA transcripts are expressed exclusively in the kidney. In situ hybridization reveals its mRNA expression in renal collecting ducts, and immunohistochemistry shows that it is localized to the luminal surface and cytoplasm of collecting ducts. Immunoprecipitation studies, using [35S]methionine-labeled renal cortical and inner medullar collecting duct cells, i.e. M-1 and mIMCD-3, indicate that the protein size is ∼32 kDa. During the development of mouse kidney, mRNA signal is detectable at day 13 of gestation, and the protein product is observed in the ureteric bud branches. Its expression is progressively increased during later stages of the gestation extending into the neonatal periods and then is decreased in adult life. Up-regulated expression of collectrin in the hypertrophic kidneys after renal ablation and restricted spatio-temporal expression during development indicates a possible role(s)in the process of progressive renal failure and renal organogenesis. AF178085 AF178086 AF229179 polymerase chain reaction angiotensin-converting enzyme representational difference analysis of cDNA rapid amplification of 5′- and 3′-cDNA ends angiotensin II angiotensin II type 1 receptor renin-angiotensin system enzyme-linked immunosorbent assay glyceraldehyde-3-phosphate dehydrogenase base pair(s) Reduction of nephron number in various immunological and non-immunological renal diseases has been implicated in the development of subsequent glomerulosclerosis and interstitial scarring of the kidney that is followed by a decline in renal function. Such a reduction can be experimentally achieved by 5/6 renal ablations in rat, a model in which glomerular hyperfiltration in renal nephrons plays a central role in the development of glomerulosclerosis (1Striker G.E. He C.J. Liu Z.H. Yang D.C. Zalups R.K. Esposito C. Striker L.J. Lab. Invest. 1995; 73: 596-605PubMed Google Scholar). In Wistar-Kyoto and Harlan Sprague Dawley rats, partial ablation of renal mass initiates a cycle of progressive renal injury in the remnant kidney associated with glomerular and tubular hypertrophy, hyperfiltration, and systemic hypertension (2Hostetter T.H. Olson J.L. Rennke H.G. Venkatachalam M., A. Brenner B.M. Am. J. Pathol. 1981; 241: F85-93Google Scholar). The remaining viable renal mass undergoes the following phases: 1) hypertrophic phase that occurs after 2–4 weeks of ablation, 2) the quiescent phase that follows during the next 4–10 weeks with minimal histological alterations, and 3) the development of segmental glomerular sclerosis and tubulointerstitial fibrosis that ensue after 10 weeks (3Waldherr R. Gretz N. Contr. Nephrol. 1988; 60: 64-72Crossref PubMed Google Scholar). To investigate the molecular mechanism(s) and the genes that may be involved in the initiation of such a pathobiologic response, we employed PCR1-based subtractive hybridization method, i.e. representational difference analysis of cDNA (cDNA-RDA; Refs. 4Wada J. Kumar A. Ota K. Wallner E.I. Kanwar Y.S. Kidney Int. 1997; 51: 1629-1638Abstract Full Text PDF PubMed Scopus (35) Google Scholar and 5Wada J. Kanwar Y.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 144-149Crossref PubMed Scopus (61) Google Scholar) to screen the differentially expressed genes in remnant kidney at hypertrophic phase of 5/6 nephrectomized mice (6Zhang H. Wada J. Kanwar Y.S. Tsuchiyama Y. Hiragushi K. Hida K. Shikata K. Makino H. Kidney Int. 1999; 56: 549-558Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). In the process of cDNA subtraction, the cDNA fragment of a novel gene, NX-17, was isolated, and its mRNA expression was up-regulated in 5/6 ablated remnant kidney compared with control kidney (6Zhang H. Wada J. Kanwar Y.S. Tsuchiyama Y. Hiragushi K. Hida K. Shikata K. Makino H. Kidney Int. 1999; 56: 549-558Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). In the present study, we have reported cDNA cloning of the full coding sequence of NX-17. The gene product of NX-17 has been designated as collectrin, because it is a novel transmembrane glycoprotein specifically expressed in collecting tubules of the kidney. With homology searches, collectrin was determined to be a novel homolog of ACE-related carboxypeptidase (ACE2; Refs. 7Tipnis S.R. Hooper N.R. Hyde R. Karran E. Christie G. Turner A.J. J. Biol. Chem. 2000; 275: 33238-33243Abstract Full Text Full Text PDF PubMed Scopus (1572) Google Scholar, 8Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano M. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: e1-e9Crossref PubMed Google Scholar), which has been recently identified as the human homolog of ACE (8Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano M. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: e1-e9Crossref PubMed Google Scholar). ACE2, like ACE, is a membrane-associated and secreted enzyme expressed predominantly on endothelium, but unlike ACE it is highly restricted in humans to heart, kidney, and testis (8Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano M. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: e1-e9Crossref PubMed Google Scholar). ACE2 has a single carboxypeptidase active site and catalyzes the cleavage of angiotensin I (Ang I) to Ang1–9, whereas ACE has two active site domains and converts Ang I to angiotensin II (Ang II) (8Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano M. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: e1-e9Crossref PubMed Google Scholar). Collectrin shares 47.8% identity with C-terminal regions including non-catalytic extracellular, transmembrane, and cytosolic domains of ACE2, and this segment does not share homology with ACE. Although collectrin lacks catalytic domains, its highly restricted expression in collecting ducts and differential expression in embryonic and ablated kidneys suggest that it has a unique role in the pathobiology of collecting ducts that may be related to the organogenesis and organ failure of the kidney. Using cDNA-RDA, we previously described the isolation of differentially expressed genes in the 5/6 nephrectomized mouse remnant kidney (3Waldherr R. Gretz N. Contr. Nephrol. 1988; 60: 64-72Crossref PubMed Google Scholar, 4Wada J. Kumar A. Ota K. Wallner E.I. Kanwar Y.S. Kidney Int. 1997; 51: 1629-1638Abstract Full Text PDF PubMed Scopus (35) Google Scholar). Among the various novel genes,NX-17 (collectrin partial-length cDNA) had a ∼2-fold up-regulated mRNA expression in nephrectomized mouse with 5/6 ablated kidney (5Wada J. Kanwar Y.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 144-149Crossref PubMed Scopus (61) Google Scholar). Using this partial-length cDNA, the full coding sequence of collectrin cDNA from mouse, rat, and human was isolated with the Marathon cDNA amplification kit (CLONTECH, Palo Alto, CA). Total kidney RNAs from 8-week-old CD-1 (ICR) mice and 4-week-old Harlan Sprague Dawley rats (Charles River Co., Yokohama, Japan) were extracted by guanidinium isothiocyanate-CsCl ultracentrifugation (8Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano M. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: e1-e9Crossref PubMed Google Scholar, 9Yang Q. Dixit B. Wada J. Tian Y. Wallner E.I. Srivastva S.K. Kanwar Y.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9896-9901Crossref PubMed Scopus (48) Google Scholar, 10Wada J. Kumar A. Liu Z.Z. Ruoslahti E. Reichardt L. Marvaldi J. Kanwar Y.S. J. Cell Biol. 1996; 132: 1161-1176Crossref PubMed Scopus (62) Google Scholar, 11Wada J. Kanwar Y.S. J. Biol. Chem. 1997; 292: 6078-6086Abstract Full Text Full Text PDF Scopus (277) Google Scholar), and mRNAs were further isolated by FastTrack 2.0 Kit (Invitrogen, San Diego, CA). Human kidney poly(A)+ RNA was purchased fromCLONTECH. Double-stranded cDNAs were synthesized from 1.0 μg of mRNA and subjected to the rapid amplification of 5′- and 3′-cDNA ends (RACE) reactions using 5′-GCCCGTCTGGATTATTGTATTCGGTGTG-3′ (506–533 bp, S1) and 5′-CATGTCCAAGGGATCACAAGGGATGCC-3′ (669–695, AS1) as primers. The 5′- and 3′-RACE products were subcloned into the pCR2.1-TOPO vector (Invitrogen) and sequenced by automated DNA sequencing (ABI PRISM 310 Genetic Analyzer, Perkin-Elmer, Foster City, CA). At least four different clones were sequenced to ensure the fidelity ofTaq polymerase. Kyte and Doolittle hydrophobicity/hydrophilicity plot analyses (12Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (16899) Google Scholar) were performed using GENETYX-WIN (Software Development, Tokyo, Japan). An online homology search was performed using BLAST (National Library of Medicine). Subsequently, structural analyses were performed with online programs available on the ExPASy Molecular Biology and Network Protein Sequence Analysis servers. Multiple sequence alignment was performed using ClustalW and DIALIGN. The presence of the transmembrane domain was assessed by SOSUI and the cytoplasmic localization of collectrin by PSORT II. Antibodies were raised in rabbits against the synthetic peptide, GIRQRRRNNKGPPGV, the sequence of which was derived from the amino acid stretch between residues 165 and 179 of the intracellular domain of collectrin (Fig. 1 A). This segment of collectrin is highly conserved and identical between the mouse and rat. The cystine residue was added to the N terminus for conjugation of the peptide to keyhole limpet hemocyanin (KURABO, Osaka, Japan). To assess the specificity of the anti-collectrin antibody, ELISA (enzyme-linked immunosorbent assay) and competitive inhibition ELISA assays were performed as described previously (13Wada J. Ota K. Kumar A. Wallner E.I. Kanwar Y.S. J. Clin. Invest. 1997; 99: 2452-2461Crossref PubMed Scopus (254) Google Scholar). SV40 MES 13 (glomerular mesangial cells from an SV40 transgenic mouse, CRL-1927, ATCC), M-1 (kidney cortical collecting duct cells from an SV40 transgenic mouse, CRL-2038, ATCC) and mIMCD-3 (kidney inner medullar collecting duct cells, CRL-2123, ATCC) were grown in a mixture of Dulbecco's modified Eagle's medium, Ham's F12 medium, and 5% fetal bovine serum. For immunoprecipitation, the cells were grown to ∼70% confluence in a cell culture flask (∼5 × 106 cells) and labeled with [35S]methionine and [35S]cysteine (0.25 mCi/ml, Amersham Pharmacia Biotech) for 12 h. The cells were with culture and in 1 1 10 10 and by at for h. The were at × for at and the were with and protein Pharmacia Immunoprecipitation was performed by 10 of anti-collectrin to of and at for 1 h. The were further with of protein for 1 at on a The protein was at and the were with 1 of The were in a and and subjected to The were with a acid and and using a were using the Japan). analyses were performed using mRNAs isolated from various CD-1 mouse and including heart, kidney, and In RNAs were isolated in various stages of and adult kidneys from the CD-1 μg of poly(A)+ RNA isolated from adult were subjected to and were to the Pharmacia For and adult mouse μg of RNA were Human was purchased fromCLONTECH. this μg of poly(A)+ RNA from human heart, kidney, and were The were with mouse, rat, and human collectrin, and × 106 cDNA at in for h. were i.e. four in at followed by two at in The kidneys of CD-1 mice were after with in at for and in The of the CD-1 mouse at day 13 of gestation was with in and were in were and on residues of mouse collectrin cDNA 1) to a were synthesized and as and were as and In situ hybridization was performed using and in situ kit following the In the were with and with a The were with a for The were to the and at for the were by and and the were with and with CD-1 mouse adult kidneys were in and in were and The were with and with anti-collectrin for with was and for were with and stages of CD-1 mouse kidneys were and with anti-collectrin and The were with with a of and with an with was performed the and kit Foster City, CA). CD-1 mouse kidney were and The were with at for a in a of for collectrin in was to the and for at The were then in and the was for at The were with and the signal was with the system The were with and using and cDNA the sequence of mouse collectrin was The mouse collectrin cDNA bp, and it a by 5′- and The initiation was at and the 222 amino acid residues with a molecular mass of (Fig. 1 A). The and the clones of and human in Using of collectrin and of human collectrin cDNAs were isolated by ClustalW alignment indicates that the protein of mouse, rat, and human collectrin was highly conserved the sequence (Fig. 1 and 81.9% identity at the and amino acid of mouse, rat, and human collectrin at and amino acid acid sequence alignment was performed using the ClustalW in a Multiple sequence alignment was performed using the ClustalW Kyte and Doolittle hydrophobicity/hydrophilicity plot analysis (12Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (16899) Google Scholar) that the protein had two domains (Fig. 1 The domain was as a signal sequence by H. J. S. G. Protein 1997; PubMed Scopus Google Scholar), and a cleavage site was between amino acid residues and The domain between residues and and it to have the of a transmembrane domain S. S. 1998; PubMed Scopus Google Scholar). collectrin to be a type an and a signal were not The domain amino acid and it two and (Fig. and using the the of J. Res. 1997; PubMed Scopus Google Scholar) and homology with the human angiotensin-converting enzyme homolog angiotensin-converting enzyme-related carboxypeptidase and protein The of ACE2, and and were derived from identical The full coding sequence of and ACE2 mRNA amino acids, whereas which was identified and in the a coding sequence with amino Multiple sequence alignment on B. 1999; PubMed Scopus Google Scholar), that human collectrin 47.8% identity with the C-terminal of ACE2 to non-catalytic extracellular, transmembrane, and cytosolic domains and The between ACE and ACE2 that the ACE2 protein a single catalytic domain to and shares identity with catalytic domains of the human angiotensin-converting enzyme Collectrin lacks carboxypeptidase active site domains and it does not share the domains of ACE. The sequence alignment of the ACE, ACE2, and collectrin is in B. human indicates that the human collectrin cDNA the localized to were on the and to residues of the human collectrin cDNA (Fig. The to of the human collectrin cDNA was not in of human in The ACE2 gene was on and the alignment between ACE2 mRNA and the that the and was from the collectrin of collectrin mRNA was by analyses using mouse, rat, and human collectrin cDNAs as the hybridization single of was observed exclusively in the kidney, and it was not in The size was in mouse, rat, and and were To investigate the mRNA localization of situ hybridization was performed using CD-1 mouse kidney (Fig. The of mRNA were observed in collecting tubules in the renal and and (Fig. the segment of collecting duct expressed collectrin in situ hybridization of the mouse at 13 of gestation, collectrin mRNA was not expressed in kidney not The localization of collectrin was further To the specificity of anti-collectrin antibody, using M-1 and mIMCD-3 collecting duct cell and SV40 MES 13 glomerular mesangial cell were single was observed in an protein derived from M-1 and mIMCD-3 collecting duct cells, and the was in SV40 MES 13 cells not a to the size of The specificity of anti-collectrin was further by competitive inhibition ELISA assay as described previously not (13Wada J. Ota K. Kumar A. Wallner E.I. Kanwar Y.S. J. Clin. Invest. 1997; 99: 2452-2461Crossref PubMed Scopus (254) Google Scholar). Collectrin was localized in the cytoplasm of collecting duct cells in the (Fig. and (Fig. the luminal surface of collecting ducts (Fig. and The cells in the renal expressed collectrin (Fig. The of that the of collectrin (Fig. with which is the for collecting ducts (Fig. To further the of collectrin, the was Collectrin was expressed in the cytoplasm of collecting in the (Fig. and and the of the luminal surface was indicate that collectrin is a gene and localized to the cells the renal collecting tubules and ducts. collectrin is specifically expressed on collecting ducts, its expression was in various stages of the CD-1 mouse kidney. the mRNA signal was detectable at 13 of gestation, and its expression was progressively increased during the later stages of gestation extending into the neonatal mRNA expression was then in the adult of the CD-1 mouse studies, collectrin was observed in the ureteric bud the of collecting ducts, at day 13 of gestation of the CD-1 mouse and During subsequent stages of gestation, collectrin was localized in the collecting ducts, and expression was increased during fetal and neonatal (Fig. In adult the of collectrin was decreased (Fig. and localization of collectrin in various stages of mouse kidney. study, collectrin is observed in the ureteric bud at day 13 of gestation of the CD-1 mouse and of collectrin is increased in day of gestation and it is expressed in the collecting ducts and the expression of collectrin is during i.e. and and and and Collectrin is expressed in the collecting ducts in the and cells the In 8-week-old adult kidneys and of collectrin is in and in and In this isolation and of a novel type transmembrane designated as collectrin, is analysis indicates that collectrin has a homology with ACE2, which is the human homolog of ACE and was identified from sequencing of a human failure cDNA (8Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano M. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: e1-e9Crossref PubMed Google Scholar). renal and of ACE of two of catalytic domains, whereas the of catalytic domain Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The ACE2 protein has active site domain and shares identity with ACE. Although ACE is expressed in the ACE2 is restricted to heart, kidney, and testis (8Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano M. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: e1-e9Crossref PubMed Google Scholar). In to ACE and ACE2, collectrin lacks the catalytic domains, it shares 47.8% identity with the non-catalytic extracellular, transmembrane, and intracellular domains of ACE2 and collectrin expression in kidney. ACE2 is present the and in tubular cells, whereas collectrin is present in collecting duct of human collectrin cDNA in the human indicate that it is localized to is localized in the that is to the collectrin and localization of ACE, ACE2, and collectrin that genes from a The catalytic of ACE2 has been the C-terminal from Ang I and In ACE the and converts Ang I to Ang II 8Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano M. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: e1-e9Crossref PubMed Google Scholar). sequence alignment indicates that the catalytic domain is and for domain have not as and in have that the ACE Ang II receptor and the of and various of The that the systemic renin-angiotensin system is not in of renal has to the that a plays in the of renal In this it is to that renal tubular cells are of Ang II and Ang II type 1 receptor that are implicated in various kidney In rats with and Ang II are up-regulated and implicated in the of fibrosis R.E. A. J.L. Am. J. Pathol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), as is the in Wistar-Kyoto rats, ACE has been reported to be up-regulated in tubules R. K. B. J. J. 1999; PubMed Scopus Google Scholar). Ang II is involved in renal cell and the of receptor G. Nephrol. 1997; Google Scholar), Ang II may be for the in this it is that up-regulated expression of the gene, as observed in tubular cell may to a to the of in Kidney Int. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). reported in the suggest that in the tubular cells, plays a role in the of interstitial The of is to the of renal in various kidney collectrin, which is to ACE2 and up-regulated in the hypertrophic phase of the ablated kidney in 5/6 nephrectomized rats, be that may be to the renal In to ACE and ACE2, collectrin does not dipeptidyl carboxypeptidase domains, and it may a role in hypertrophic phase kidneys to be mechanism(s) To such a role in the of renal of the of collectrin be the of the of structural and in the kidney, the expression of genes in fetal and to related to renal and development B. A. H. A. H. J. M. Kidney Int. Full Text PDF PubMed Scopus Google A. L. J. L. R. Am. 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Ang II and its receptor are up-regulated at the renal at and related to of the to compared with embryonic Y. S. H. A. J. Clin. Invest. 1998; PubMed Scopus Google Scholar). The that collectrin the and is expressed in collecting ducts and of renal that collectrin may have a role in the development of collecting ducts and renal collectrin has homology with ACE2, a novel of collectrin may be involved in the related to the the during the The of collectrin, a novel homolog of ACE2, to the and may the of novel of the in the of renal collecting ducts and At the that can be collectrin is catalytic domains and are its its up-regulated expression in the hypertrophic kidneys after renal ablation and embryonic and kidneys the possible role of collectrin in the process of progressive renal failure and renal organogenesis.