Corin, a Mosaic Transmembrane Serine Protease Encoded by a Novel cDNA from Human Heart

A novel cDNA has been identified from human heart that encodes an unusual mosaic serine protease, designated corin. Corin has a predicted structure of a type II transmembrane protein and contains two frizzled-like cysteine-rich motifs, seven low density lipoprotein receptor repeats, a macrophage scavenger receptor-like domain, and a trypsin-like protease domain in the extracellular region. Northern analysis showed that corin mRNA was highly expressed in the human heart. In mice, corin mRNA was detected by in situ hybridization in the cardiac myocytes of the embryonic heart as early as embryonic day (E) 9.5. By E11.5–13.5, corin mRNA was most abundant in the primary atrial septum and the trabecular ventricular compartment. Expression in the heart was maintained through the adult. In addition, mouse corin mRNA was also detected in the prehypertrophic chrondrocytes in developing bones. By fluorescentin situ hybridization analysis, the human corin gene was mapped to 4p12–13 where a congenital heart disease locus, total anomalous pulmonary venous return, had been previously localized. The unique domain structure and specific embryonic expression pattern suggest that corin may have a function in cell differentiation during development. The chromosomal localization of the human corin gene makes it an attractive candidate gene for total anomalous pulmonary venous return. A novel cDNA has been identified from human heart that encodes an unusual mosaic serine protease, designated corin. Corin has a predicted structure of a type II transmembrane protein and contains two frizzled-like cysteine-rich motifs, seven low density lipoprotein receptor repeats, a macrophage scavenger receptor-like domain, and a trypsin-like protease domain in the extracellular region. Northern analysis showed that corin mRNA was highly expressed in the human heart. In mice, corin mRNA was detected by in situ hybridization in the cardiac myocytes of the embryonic heart as early as embryonic day (E) 9.5. By E11.5–13.5, corin mRNA was most abundant in the primary atrial septum and the trabecular ventricular compartment. Expression in the heart was maintained through the adult. In addition, mouse corin mRNA was also detected in the prehypertrophic chrondrocytes in developing bones. By fluorescentin situ hybridization analysis, the human corin gene was mapped to 4p12–13 where a congenital heart disease locus, total anomalous pulmonary venous return, had been previously localized. The unique domain structure and specific embryonic expression pattern suggest that corin may have a function in cell differentiation during development. The chromosomal localization of the human corin gene makes it an attractive candidate gene for total anomalous pulmonary venous return. Serine proteases are essential for a variety of biological processes including food digestion, complement activation, and blood coagulation (1Neurath H. Science. 1984; 224: 350-357Crossref PubMed Scopus (399) Google Scholar, 2Huber R. Bode W. Acc. Chem. Res. 1978; 11: 114-122Crossref Scopus (610) Google Scholar, 3Davie E.W. Fujikawa K. Kisiel W. Biochemistry. 1991; 30: 10363-10370Crossref PubMed Scopus (1627) Google Scholar). In Drosophila, serine proteases are also involved in developmental pathways. For example, serine proteases encoded by the nudel, gastrulation defective, easter, and snake genes are key components of a proteolytic cascade that is critical for the establishment of the dorsal-ventral pattern in developing embryos (4Morisato D. Anderson K.V. Annu. Rev. Genet. 1995; 29: 371-399Crossref PubMed Scopus (315) Google Scholar, 5LeMosy E.K. Kemler D. Hashimoto C. Development. 1998; 125: 4045-4053PubMed Google Scholar, 6Konrad K.D. Goralski T.J. Mahowald A.P. Marsh J.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6819-6824Crossref PubMed Scopus (61) Google Scholar). Genetic defects in these genes often lead to the disruption of the dorsal-ventral axis, resulting in embryonic lethality (7Anderson K.V. Schneider D.S. Morisato D. Jin Y. Ferguson E.L. Cold Spring Harbor Symp. Quant. Biol. 1992; 57: 409-417Crossref PubMed Google Scholar). Most serine proteases of the trypsin family are secreted proteins. Several members from this family have been identified that contain an integral transmembrane domain. Hepsin, for example, is a serine protease expressed on the surface of hepatocytes. Structurally, hepsin is a type II transmembrane protein with the transmembrane domain at its amino terminus and the protease domain at the carboxyl terminus exposed to the outside of the cell (8Tsuji A. Torres-Rosado A. Arai T. Le Beau M.M. Lemons R.S. Chou S.H. Kurachi K. J. Biol. Chem. 1991; 266: 16948-16953Abstract Full Text PDF PubMed Google Scholar). In tissue culture studies, hepsin was shown to contribute to hepatocyte growth (9Torres-Rosado A. O'Shea K.S. Tsuji A. Chou S.H. Kurachi K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7181-7185Crossref PubMed Scopus (90) Google Scholar). However, the physiological significance of the growth stimulating activity of hepsin remains unknown (10Wu Q. Yu D. Post J. Halks-Miller M. Sadler J.E. Morser J. J. Clin. Invest. 1998; 101: 321-326Crossref PubMed Scopus (76) Google Scholar). In Drosophila, Stubble-stubbloid protein, another transmembrane serine protease, shares structural similarities with hepsin (11Appel L.F. Prout M. Abu-Shumays R. Hammonds A. Garbe J.C. Fristrom D. Fristrom J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4937-4941Crossref PubMed Scopus (104) Google Scholar). Genetic studies demonstrated that Stubble-stubbloid is essential for epithelial morphogenesis and development of the fruit fly. Defects in theStubble-stubbloid gene cause malformation of legs, wings, and bristles. Most recently, other transmembrane serine proteases were isolated and cloned from human trachea and small intestine (12Yamaoka K. Masuda K. Ogawa H. Takagi K. Umemoto N. Yasuoka S. J. Biol. Chem. 1998; 273: 11895-11901Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar,13Paoloni-Giacobino A. Chen H. Peitsch M.C. Rossier C. Antonarakis S.E. Genomics. 1997; 44: 309-320Crossref PubMed Scopus (181) Google Scholar). The biological function of these newly discovered membrane-bound serine proteases has not yet been determined. In this study, we report the cloning of a cDNA from the human heart that encodes a novel transmembrane serine protease, designated corin. Corin has a predicted structure of a type II transmembrane protein containing two frizzled-like cysteine-rich motifs, seven LDL 1The abbreviations used are: LDL, low density lipoprotein; EST, expressed sequence tag; FISH, fluorescent in situ hybridization; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ORF, open reading frame; RT, reverse transcriptase; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; TAPVR, total anomalous pulmonary venous return; kb, kilobase pair; bp, base pair; E, embryonic day. receptor repeats, a macrophage scavenger receptor-like domain, and a trypsin-like protease domain in the extracellular region. In situ hybridization revealed that corin mRNA was expressed in the embryonic heart as early as E9.5, and the expression in the heart was maintained through the adult stage. In addition, corin mRNA was detected in prehypertrophic chrondrocytes of the developing bones. The unusual domain structures and specific expression pattern suggested that corin may have a function in cell differentiation during embryonic development. Human cancer cell lines, HEC-1-A (endometrium adenocarcinoma), U2-OS (osteosarcoma), SK-LMS-1 (vulva sarcoma), RL95-2 (endometrium carcinoma), and AN3-CA (endometrium adenocarcinoma) were obtained from the American Type Culture Collection (ATCC). Human heart cDNA libraries and human and mouse multiple tissue Northern blots were purchased from CLONTECH (Palo Alto, CA). Mouse tissue sections used for in situ hybridization were purchased from Novagen (Madison, WI). Tissue culture media and supplements were from Life Technologies Inc. All other chemicals were obtained from Sigma. An expressed sequence tag (EST) clone was found in a human heart cDNA library from the Incyte EST data base that shared significant sequence homology with trypsin, indicating that the EST may encode a novel serine protease gene. A 2.1-kb EcoRI-XhoI insert from this EST clone was used to screen a human heart cDNA library (CLONTECH). Approximately, 5 × 106 lambda phage clones were screened, and two positive clones were isolated that contained inserts of 3.5 and 3.1 kb, respectively. The DNA sequences of these two clones were determined. Oligonucleotide primers were designed to clone further 5′ end cDNA sequences by 5′ rapid amplification of cDNA ends (RACE) using Marathon-ready human heart cDNA templates (CLONTECH). The PCR products from 5′ RACE were cloned into pCRII vector (Invitrogen, San Diego, CA) and sequenced. Oligonucleotide primers used in the 5′ RACE experiments were 5′-CAGTTGGTTTGAACAAGTGCAGGG-3′, 5′-TGCAAGGAGGGATACGCTCGCCTG-3′, 5′-AATCCCAAGAACAGACTCACAGCG-3′, 5′-CGGGTCACAGAGAGAGCTACCACC-3′, 5′-GGTCTCCTTCTTGACATGAATCTG-3′, 5′-CGGAGCCCCATGAAGTTAAACCA-3′, and 5′-AACAAAAGGATCCTTGGAGGTCGGACGAGT-3′. The final 5′ end sequence of human corin cDNA was derived from at least three independent clones. The full-length cDNA sequence was compiled using the Genetics Computer Group (GCG) software (version 9.1, Madison, WI). Northern blots containing poly(A)+ RNA samples (2 μg/lane) from multiple human and mouse tissues were purchased from CLONTECH. Human and mouse corin cDNA probes were labeled with 32PdCTP using a random primed DNA labeling kit (Roche Molecular Biochemicals). Northern hybridization was performed at 42 °C overnight in a solution containing 40% formamide, 5× Denhardt's solution, 6× SSC, 100 μg/ml salmon sperm DNA, and 0.1% SDS. Blots were washed with 0.2× SSC, 0.1% SDS at 60 °C and then exposed to Fuji imaging plates. As a control, the blots were reprobed with a human actin cDNA probe provided byCLONTECH. mRNA samples were isolated from Hec-1-A, U2-OS, SK-LMS-1, and AN3-CA cells using a commercial RNA preparation kit (Oligotex Direct mRNA Mini Kits, Qiagen). First strand cDNAs were synthesized using SuperScript II RNase− reverse transcriptase (Life Technologies Inc.). Human corin-specific oligonucleotide primers (sense primer, 5′-AACAAAAGGATCCTTGGAGGTCGGACGAGT-3′, and antisense primer, 5′-CGGAGCCCCATGA AGTTAATCCA-3′) were used to amplify a 630-bp fragment of corin cDNA between nucleotides 2475 and 3105. Oligonucleotide primers TFR1 (5′-GTCAATGTCCCAAACGTCACCAGA-3′) and TFR2 (5′-ATTTCGGGAATGCTGAGAAAACAGACAGA-3′), derived from the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, were used as an internal quantification control. PCR reactions were performed with a thermal cycler (Perkin-Elmer, model 480). PCR products were separated on 1% agarose gels and visualized by ethidium bromide staining. Mouse adult heart and embryonic tissue sections were deparaffinized in xylene, rehydrated, and fixed in 4% paraformaldehyde. The tissues were digested with proteinase K (20 μg/ml), then treated with triethanolamine/acetic anhydride, and dehydrated. An 800-bp mouse corin cDNA fragment from the coding region was cloned into pCRII (Invitrogen) in two orientations to yield plasmids pM11 and pM41. The plasmids were linearized byHindIII digestion. Sense and antisense probes were synthesized using T7 RNA polymerase (T7/SP6 transcription kit, Roche Molecular Biochemicals) and labeled with 33PUTP (Amersham Pharmacia Biotech). The hybridization was carried out as described (14Jen Y. Manova K. Benezra R. Dev. Dyn. 1997; 208: 92-106Crossref PubMed Scopus (185) Google Scholar). The slides were dehydrated and dipped in Kodak NTB-2 emulsion and exposed for 4 weeks in light-tight boxes at 4 °C. Photographic development was carried out in a Kodak D-19 developer. The slides were stained with hematoxylin/eosin and analyzed using both light- and dark-field optics of a Zeiss microscope. P1 phage clones containing the human corin gene were isolated by filter hybridization using a human corin cDNA as the probe. One clone was confirmed by DNA sequencing using a primer from human corin cDNA. The DNA fragment from this P1 phage was labeled with digoxigenin-dUTP. The labeled probe was combined with sheared human DNA and hybridized to metaphase chromosomes derived from PHA-stimulated peripheral blood lymphocytes in a solution containing 50% formamide, 10% dextran sulfate, and 2× SSC. Hybridization signals were detected by fluorescent-labeled antidigoxigenin antibodies and counter-staining with 4,6-diaminoidino-2-phenylindole. A total of 80 metaphase cells were analyzed of which 74 cells exhibited specific labeling. A model of the corin protease domain (amino acids 802–1042) was built based on the structure of bovine chymotrypsinogen A at 1.8-Å resolution (15Wang D. Bode W. Huber R. J. Mol. Biol. 1985; 185: 595-624Crossref PubMed Scopus (209) Google Scholar,16Ponder J.W. Richards F.M. J. Mol. Biol. 1987; 193: 775-791Crossref PubMed Scopus (1350) Google Scholar), using the homology program (Insight II, 1995, MSI, San Diego, CA). Rotamers were used for non-identical side chain replacements (16Ponder J.W. Richards F.M. J. Mol. Biol. 1987; 193: 775-791Crossref PubMed Scopus (1350) Google Scholar). Coordinates for the loop insertions were extracted from the Brookhaven protein data bank (17Bernstein F.C. Koetzle T.F. Williams G.J. Meyer Jr., E.E. Brice M.D. Rodgers J.R. Kennard O. Shimanouchi T. Tasumi M. J. Mol. Biol. 1977; 112: 535-542Crossref PubMed Scopus (8178) Google Scholar). The model was refined by energy minimization using the AMBER force field (Discover 95.0), with a distance-dependent dielectric constant. The minimization used the steepest descents and conjugate gradient methods as follows: first for the loops only where insertions and deletions occurred, then side chains, and a final round of minimization keeping the Cα atoms fixed. The residues of corin (His843, Asp892, and Ser985) corresponding to the catalytic triad of the template structure were also held fixed. A computer search using the BLAST program identified an EST clone from a human heart library that shared significant homology with serine protease family members, such as trypsin. The EST clone was used to isolate the full-length cDNA of a novel gene, designated corin for its abundant expression in the heart. The sequence of the full-length corin cDNA, 4933 bp in length, is shown in Fig. 1. The size of the cDNA is consistent with the length of corin mRNA (∼5 kb) detected by Northern analysis (Fig. 4A). An ATG codon is located at position 95 that may represent the translation initiation site. The open reading frame (ORF) spans 3126 bp with a 5′-untranslated region of 94 nucleotides before the initiation codon. At the 3′ end, there is a 1.7-kb 3′-untranslated region after the stop codon at position 3221. A polyadenylylation signal of AATAAA is present 12 nucleotides before the poly(A)+ tail.Figure 4Northern analysis of corin mRNA expression. Human and mouse multiple tissue Northern blots were hybridized with human and mouse corin cDNA probes, respectively. In human tissues (A and B), corin mRNA was detected only in samples from heart. In mouse tissues (C), abundant expression of corin mRNA was detected in samples from heart. Weak signals were also detected in samples from testis and kidney.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The ORF of the human corin cDNA encodes a polypeptide of 1042 amino acids with a calculated mass of 116 kDa. At the amino terminus of the predicted corin protein, there is no discernible signal peptide sequence. Hydropathy plots using the GCG program identified a highly hydrophobic region between amino acids 46 and 66 (Fig. 2B). This hydrophobic sequence could serve as a potential transmembrane domain. There are positively charged amino acid residues immediately preceding the putative transmembrane segment, suggesting that corin is a type II transmembrane protein with the amino terminus present in the cytosol (18Hartmann E. Rapoport T.A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5786-5790Crossref PubMed Scopus (488) Google Scholar). Consistent with this hypothesis, there are 19 predicted N-linked glycosylation sites present in the extracellular domains of corin (Fig. 1). Analysis of the corin protein sequence showed that in the extracellular region there are two frizzled-like cysteine-rich domains, seven LDL receptor repeats, one macrophage scavenger receptor-like domain, and one trypsin-like serine protease domain (Fig. 2A). As shown in Fig. 2A, two frizzled-like cysteine-rich domains are located at amino acids 134–259 and 450–573, respectively. Amino acid sequences of these two domains share significant similarities with the extracellular cysteine-rich domain of the DrosophilaFrizzled protein, a seven-transmembrane receptor essential for polarity determination during the development of the fruit fly (19Vinson C.R. Conover S. Adler P.N. Nature. 1989; 338: 263-264Crossref PubMed Scopus (302) Google Scholar). The frizzled-like cysteine-rich domains have also been found in other proteins, such as Dfz2 in Drosophila (20Bhanot P. Brink M. Samos C.H. Hsieh J.C. Wang Y. Macke J.P. Andrew D. Nathans J. Nusse R. Nature. 1996; 382: 225-230Crossref PubMed Scopus (1228) Google Scholar), Lin-17 inCaenorhabditis elegans (21Sawa H. Lobel L. Horvitz H.R. Genes Dev. 1996; 10: 2189-2197Crossref PubMed Scopus (167) Google Scholar), and FZ-1 in human (22Chan S.D. Karpf D.B. Fowlkes M.E. Hooks M. Bradley M.S. Vuong V. Bambino T. Liu M.Y. Arnaud C.D. Strewler G.J. Nissenson R.A. J. Biol. Chem. 1992; 267: 25202-25207Abstract Full Text PDF PubMed Google Scholar). The sequences of the two frizzled-like cysteine-rich domains in corin are closest to those in Lin-17 and FZ-1. As shown in Fig. 2C, all the 10 conserved cysteine residues are present in the frizzled-like cysteine-rich domains of corin. Between amino acids 268–415 and 579–690 (Fig. 2, A andD), there are seven cysteine-rich repeats homologous to the LDL receptor class A repeats (23Brown M.S. Herz J. Goldstein J.L. Nature. 1997; 388: 629-630Crossref PubMed Scopus (148) Google Scholar). Each repeat is about 36 amino acids long and contains six cysteine residues as well as a highly conserved cluster of negatively charged amino acids. In the LDL receptor, these cysteine-rich repeats bind calcium ions and play an essential role in endocytosis of the extracellular ligands (23Brown M.S. Herz J. Goldstein J.L. Nature. 1997; 388: 629-630Crossref PubMed Scopus (148) Google Scholar). Similar motifs have been found in the extracellular domain of other membrane receptors, such as LDL receptor-related protein (LRP1) (24Krieger M. Herz J. Annu. Rev. Biochem. 1994; 63: 601-637Crossref PubMed Scopus (1061) Google Scholar), megalin (also known as LRP2 or gp330) (25Kounnas M.Z. Chappell D.A. Strickland D.K. Argraves W.S. J. Biol. Chem. 1993; 268: 14176-14181Abstract Full Text PDF PubMed Google Scholar), complement proteins (26Catterall C.F. Lyons A. Sim R.B. Day A.J. Harris T.J. Biochem. J. 1987; 242: PubMed Scopus Google Scholar), Y. Q. Sadler J.E. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus (148) Google Scholar), proteins and S. Mahowald A.P. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar, Hashimoto C. 1995; Full Text PDF PubMed Scopus Google Scholar). In to the frizzled-like cysteine-rich domains and LDL receptor-like repeats, there is another cysteine-rich region between amino acids and in corin (Fig. 2, A This region contains amino acids and is homologous to the cysteine-rich found in the macrophage scavenger receptor A. M. H. S. H. H. H. H. Y. T. K. R. T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). This is also present in the receptor D.S. J. Biol. Chem. 1989; Full Text PDF PubMed Google Scholar, J.E. R.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: PubMed Scopus Google and the serine protease, Y. Q. Sadler J.E. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus (148) Google Scholar). At the carboxyl terminus of corin protein between amino acid residues and there is a trypsin-like serine protease domain (Fig. 2A). This protease domain is highly homologous to the catalytic domain of members of the trypsin For example, amino acid sequence between corin and Fujikawa K. E.W. Biochemistry. PubMed Scopus Google Scholar), K. E.W. Biochemistry. PubMed Scopus Google Scholar), and hepsin Kurachi K. E.W. Biochemistry. PubMed Scopus Google are and respectively. All essential of serine protease sequences are well conserved in corin and The residues of the catalytic triad are located at Asp892, and The amino acid residues the are located at and residues are predicted to bind the P1 suggesting that corin its after such as or In addition, a putative was found at suggesting that corin synthesized as an and that another trypsin-like was for its In the protease domain, there are 12 cysteine of these cysteine residues predicted by with other well serine such as trypsin and First three of cysteine residues present in all members of the trypsin are located at and of cysteine residues are present at the and two of cysteine residues are found in a of serine such as and Fujikawa K. E.W. Biochemistry. PubMed Scopus Google Scholar). The of and after the at the catalytic domain of corin to the of by a there is one of cysteine and present in corin. residues at these two were not found in other serine proteases in A search of data showed that a serine protease from the had two cysteine residues at the corresponding A model of the corin protease domain was built based on the structure of bovine chymotrypsinogen A (Fig. on this corin where the atoms of these two cysteine residues were held fixed during energy the between the atoms of side is about after The model that these two are to a two in the of the protease domain (Fig. expression of the corin gene in human Northern hybridization was performed using human corin cDNA As shown in Fig. an was detected only in the heart not in other tissues including and the heart is of cardiac Northern analysis was performed to the of corin mRNA in other human corin mRNA was detected in the heart not in small and (Fig. corin mRNA expression in mice, the full-length mouse corin cDNA was cloned by a Mouse corin cDNA shared sequence with human corin cDNA not Northern analysis was performed with RNA samples from mouse As shown in Fig. a of was detected in samples derived from the heart. In to Northern analysis with human low of corin mRNA were also detected in samples derived from the and In situ hybridization was performed to the and expression of corin In adult (Fig. corin mRNA was detected in cardiac myocytes of both and The of expression to in the the embryonic corin mRNA was first detected at in both and of the developing heart (Fig. Between and corin mRNA was highly expressed in the atrial and in the that in the (Fig. and By corin mRNA in the heart was in primary atrial (Fig. Weak signals to present in developing and not in the and (Fig. The expression of corin mRNA in the heart was maintained in the embryonic of corin mRNA in the developing heart. Tissue sections were from mouse embryos at day (A and B), andD), and and and stained with hematoxylin/eosin and Corin mRNA expression was detected by in situ hybridization in developing heart by and as The expression was in the primary atrial septum and the trabecular ventricular by By corin mRNA was detected in most cardiac myocytes in both and used in and are as E, Large Image Figure ViewerDownload Hi-res image Download (PPT) In to the corin mRNA was also detected in other mouse tissues situ For example, corin mRNA was present in the of and in the developing In the (Fig. corin mRNA expression was most abundant in the cells to the of the In the developing at corin mRNA was highly expressed in the cells in the not in the of the (Fig. This was consistent with the of Northern analysis in which a corin was found in RNA samples from mouse (Fig. in situ hybridization also identified corin mRNA in such as the in the the in the and the long in the (Fig. and Fig. the expression of corin mRNA in of in the of an By the of corin mRNA expression in the was as the not indicating that corin may play a role in the differentiation of This was by the expression of corin mRNA in developing Fig. and showed an early developing that of three of cells as follows: at the prehypertrophic to the and at the both Corin mRNA was found in the prehypertrophic (Fig. Hybridization signals were also present in (Fig. Fig. and showed a long in a that was at a developmental stage. The was by tissues containing cells and expression pattern of corin mRNA was found in the of the prehypertrophic and in the that corin expression was with a specific of A of human cancer cell were by Northern and for the of corin In most cell lines, such as and corin mRNA was not However, corin mRNA was found in cell derived from or As shown in Fig. corin mRNA was detected by in cell and cell SK-LMS-1, as well as in cell The