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• W2029492079 abstract "HSP47 is a collagen-specific molecular chaperone that specifically recognizes and binds to the triple helical domain of various types of collagens. Here we report the cloning of the entire coding region of a novel collagen-like protein by yeast two-hybrid screening of a 17.5-day whole mouse embryo cDNA library using HSP47 as a bait. The cDNA of this protein and its deduced amino acid sequence are 2,690 bp and 438 amino acids long, respectively. The protein contains two clusters of Gly-X-Y collagenous repeats and three noncollagenous domains. Northern blot analysis showed that its mRNA is specifically expressed in the testis and ovary in adult tissues and that expression in these tissues is highest in the neonate. Biochemical characterization of this protein revealed that its proline residues are hydroxylated, it undergoes N-linked glycosylation, it forms trimers, and it is secreted in vitro. Immunohistochemical studies showed that the myoid cells and the pre-theca cells synthesized it in the testis and ovary, respectively, resulting in the accumulation of this protein in the extracellular spaces of these organs. These observations suggest that this protein is a new member of the collagen protein family. We thus designated this protein as type XXVI collagen. HSP47 is a collagen-specific molecular chaperone that specifically recognizes and binds to the triple helical domain of various types of collagens. Here we report the cloning of the entire coding region of a novel collagen-like protein by yeast two-hybrid screening of a 17.5-day whole mouse embryo cDNA library using HSP47 as a bait. The cDNA of this protein and its deduced amino acid sequence are 2,690 bp and 438 amino acids long, respectively. The protein contains two clusters of Gly-X-Y collagenous repeats and three noncollagenous domains. Northern blot analysis showed that its mRNA is specifically expressed in the testis and ovary in adult tissues and that expression in these tissues is highest in the neonate. Biochemical characterization of this protein revealed that its proline residues are hydroxylated, it undergoes N-linked glycosylation, it forms trimers, and it is secreted in vitro. Immunohistochemical studies showed that the myoid cells and the pre-theca cells synthesized it in the testis and ovary, respectively, resulting in the accumulation of this protein in the extracellular spaces of these organs. These observations suggest that this protein is a new member of the collagen protein family. We thus designated this protein as type XXVI collagen. endoplasmic reticulum cytomegalovirus collagenous domain 1–2 glutathione S-transferase human embryonic kidney 4-morpholinepropanesulfonic acid noncollagenous domain 1–3 phosphate-buffered saline peptideN-glycosidase F rapid amplification of cDNA ends Collagen is the most abundant protein in vertebrates. It is a major component of the extracellular matrix, which consists of specialized fibrils and networks around cells and in the interstitial spaces between the cells. Twenty-five types of collagen have now been identified (1Myllyharju J. Kivirikko K.J. Ann. Med. 2001; 33: 7-21Crossref PubMed Scopus (534) Google Scholar, 2Fitzgerald J. Bateman J.F. FEBS Lett. 2001; 505: 275-280Crossref PubMed Scopus (62) Google Scholar, 3Gordon M.K. Gerecke D.R. Hahn R.A. Bhatt P. Goyal M. Koch M. Invest. Ophthalmol. Visual Sci. 2000; 41: S752Google Scholar, 4Gordon M.K. Hahn R.A. Zhou P. Kistler A. Gerecke D.R. Koch M. Invest. Ophthalmol. Visual Sci. 2002; (in press)Google Scholar, 5Hashimoto T. Wakabayashi T. Watanabe A. Kowa H. Hosoda R. Nakamura A. Kanazawa I. Arai T. Takio K. Mann D.M. Iwatsubo T. EMBO J. 2002; 21: 1524-1534Crossref PubMed Scopus (171) Google Scholar). During the development of mammals, these collagens are expressed in various spatiotemporal patterns. The fibril-forming collagens are the most abundant of the collagens and include types I, II, and III (6Kadler K.E. Holmes D.F. Trotter J.A. Chapman J.A. Biochem. J. 1996; 316: 1-11Crossref PubMed Scopus (1056) Google Scholar). Other collagens known as the FACIT collagens (Fibril-associated Collagens withInterrupted Triple Helices), which include types IX, XII, XIV, XVI, and XIX, associate with the surface of the collagen fibrils and modify their interactive properties (7Koch M. Foley J.E. Hahn R. Zhou P. Burgeson R.E. Gerecke D.R. Gordon M.K. J. Biol. Chem. 2001; 276: 23120-23126Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 8Olsen B.R. Int. J. Biochem. Cell Biol. 1997; 29: 555-558Crossref PubMed Scopus (83) Google Scholar, 9Prockop D.J. Kivirikko K.I. Annu. Rev. Biochem. 1995; 64: 403-434Crossref PubMed Scopus (1355) Google Scholar). In addition, nonfibrillar collagens, which include types XIII, XVII, and XXV, are reported to have transmembrane domains and appear to localize at the cell surface (5Hashimoto T. Wakabayashi T. Watanabe A. Kowa H. Hosoda R. Nakamura A. Kanazawa I. Arai T. Takio K. Mann D.M. Iwatsubo T. EMBO J. 2002; 21: 1524-1534Crossref PubMed Scopus (171) Google Scholar, 10Pihlajaniemi T. Rehn M. Prog. Nucleic Acids Res. Mol. Biol. 1995; 50: 225-262Crossref PubMed Scopus (78) Google Scholar, 11Snellman A., Tu, H. Vaisanen T. Kvist A.P. Huhtala P. Pihlajaniemi T. EMBO J. 2000; 19: 5051-5059Crossref PubMed Scopus (77) Google Scholar). Despite the differences among the collagens, all share in common a triple helical structure composed of three polypeptides consisting of Gly-X-Y repeats, where X is any amino acid, and Y is frequently proline or hydroxyproline. Each chain is a left-handed helix, and the three chains wind around each other in a right-handed superhelix (1Myllyharju J. Kivirikko K.J. Ann. Med. 2001; 33: 7-21Crossref PubMed Scopus (534) Google Scholar, 6Kadler K.E. Holmes D.F. Trotter J.A. Chapman J.A. Biochem. J. 1996; 316: 1-11Crossref PubMed Scopus (1056) Google Scholar). The procollagens are synthesized in and cotranslationally transported into the endoplasmic reticulum (ER).1 After translation is completed, the procollagens form a trimer at the C terminus. Only properly folded triple helical forms of procollagens are secreted by the cells. During collagen biosynthesis, the nascent procollagen chain is modified by unique enzymes that include prolyl-4 hydroxylase, which stabilize the triple helical structure. After being secreted by the cell, the N- and C-propeptides are cleaved by specific proteases, thereafter forming collagen bundles or basement membranes (1Myllyharju J. Kivirikko K.J. Ann. Med. 2001; 33: 7-21Crossref PubMed Scopus (534) Google Scholar, 6Kadler K.E. Holmes D.F. Trotter J.A. Chapman J.A. Biochem. J. 1996; 316: 1-11Crossref PubMed Scopus (1056) Google Scholar, 9Prockop D.J. Kivirikko K.I. Annu. Rev. Biochem. 1995; 64: 403-434Crossref PubMed Scopus (1355) Google Scholar,12Bachinger H.P. Davis J.M. Int. J. Biol. Macromol. 1991; 13: 152-156Crossref PubMed Scopus (75) Google Scholar, 13Brass A. Kadler K.E. Thomas J.T. Grant M.E. Boot-Handford R.P. FEBS Lett. 1992; 303: 126-128Crossref PubMed Scopus (66) Google Scholar, 14Doege K.J. Fessler J.H. J. Biol. Chem. 1986; 261: 8924-8935Abstract Full Text PDF PubMed Google Scholar, 15Bulleid N.J. Dalley J.A. Less J.F. EMBO J. 1997; 16: 6694-6701Crossref PubMed Scopus (85) Google Scholar). HSP47 is an ER resident stress protein that functions as a collagen-specific molecular chaperone. This protein associates with procollagen during its folding and/or post-translational modification in the ER. Recent studies have revealed that HSP47 plays a critical role in collagen biosynthesis (16Nagata K. Trends Biochem. Sci. 1996; 21: 22-26Abstract Full Text PDF PubMed Scopus (249) Google Scholar). Type I collagen secreted by cells from hsp47-null mice is abnormal in that its N- and C-propeptides are improperly cleaved, and the protease-sensitivity in its triple helix domain is altered (17Nagai N. Hosokawa M. Itohara S. Adachi E. Matsushita T. Hosokawa N. Nagata K. J. Cell Biol. 2000; 150: 1499-1505Crossref PubMed Scopus (283) Google Scholar). The tissues ofhsp47-null mice lack collagen fibrils and basement membranes, and the mice died before E11.5. HSP47 has been reported to bind to various types of collagen (at least types I–V) in vitro (18Nakai A. Satoh M. Hirayoshi K. Nagata K. J. Cell Biol. 1992; 117: 903-914Crossref PubMed Scopus (221) Google Scholar, 19Natsume T. Koide T. Hirayoshi K. Nagata K. J. Biol. Chem. 1994; 269: 31224-31228Abstract Full Text PDF PubMed Google Scholar, 20Satoh M. Hirayoshi K. Yokota S.-i. Hosokawa N. Nagata K. J. Cell Biol. 1996; 133: 469-483Crossref PubMed Scopus (188) Google Scholar). Furthermore, synthetic model peptides representing the collagenous triple helix domain and yeast two-hybrid screening revealed that HSP47 specifically binds to the helix-forming portions of procollagen (21Koide T. Asada S. Nagata K. J. Biol. Chem. 1999; 274: 34523-34526Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 22Tasab M. Batten M.R. Bulleid N.J. EMBO J. 2000; 19: 2204-2211Crossref PubMed Scopus (170) Google Scholar, 23Koide T. Aso A. Yorihuzi T. Nagata K. J. Biol. Chem. 2000; 275: 27957-27963Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 24Koide T. Takahara Y. Asada S. Nagata K. J. Biol. Chem. 2002; 277: 6178-6182Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Thus, HSP47 appears to play a critical role in collagen biosynthesis. To investigate the role of HSP47 further, we searched for proteins that interact with HSP47 by performing yeast two-hybrid screening using HSP47 as a bait. This experiment identified a gene encoding a novel collagen-like protein that contains two collagenous domains consisting of Gly-X-Y repeats and three noncollagenous domains. We demonstrate here that this protein is secreted into the culture medium with modifications typically seen in other types of collagens, namely, N-linked glycosylation and hydroxylation of proline and/or lysine residues. This protein also forms a trimer and accumulates in the extracellular matrix of mouse tissues. These collagen-like features led us to designate this protein as type XXVI collagen. This protein is produced in the testis and ovary of adult mice but is also present at higher levels in the reproductive tissues of neonates, suggesting that it plays an important role in the development of the reproductive tissues. Restriction enzymes and other DNA-modifying enzymes were purchased from Takara Shuzo Co. Ltd. (Tokyo, Japan). DNA fragments were synthesized by Hokkaido System Science, (Sapporo, Japan). Chemicals were purchased from either Nacalai Tesque (Kyoto, Japan) or Wako Pure Chemical Co. (Osaka, Japan). DNA sequencing was carried out on an ABI PRISM 377A sequencer (PerkinElmer Life Sciences, Forester City, CA). ICR mice were purchased from Japan SLC, Inc. (Shizuoka, Japan). The ER retention sequence of mouse hsp47cDNA was deleted, and the remaining cDNA was used as bait in yeast two-hybrid screening. The cDNA was amplified by PCR and inserted into pAS2-1 (Clontech, Palo Alto, CA). Following the methods recommended by the manufacturer, a 1.8-kb cDNA fragment of type XXVI collagen was cloned from the cDNA library of 17.5-day whole mouse embryo (Clontech). A 2.7-kb type XXVI collagen cDNA was also cloned from a 15.5-day mouse embryo cDNA library (Invitrogen) using a specific probe prepared from the sequence data of the 1.8-kb cloned fragment. Marathon-ReadyTM cDNA from mouse testis (Clontech) was used as a template for 5′-RACE and resulted in the acquisition of 79 bp of upstream sequence. The program BLAST was used to predict the putative signal sequence (25Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (68368) Google Scholar). Freshly removed organs of mice of various ages were homogenized in TRIZOL Reagent (Invitrogen). Total RNA was extracted with TRIZOL according to the manufacturer's instructions. RNA samples were fractionated on a formaldehyde, MOPS, EDTA, 1% agarose gel and then transferred to nylon membranes and cross-linked by baking at 80 °C for 2 h. The filters were subsequently incubated with several radiolabeled probes. These included a 696-bp fragment amplified by PCR from type XXVI collagen cDNA, a 1.5-kb EcoRI-HindIII fragment of hsp47cDNA (26Takechi H. Hirayoshi K. Nakai A. Kudo H. Saga S. Nagata K. Eur. J. Biochem. 1992; 206: 323-329Crossref PubMed Scopus (93) Google Scholar), and a 1.3-kb PstI fragment of chickGAPDH cDNA (27Dugaiczyk A. Haron J.A. Stone E.M. Dennison O.E. Rothblum K.N. Schwartz R.J. Biochemistry. 1983; 22: 1605-1613Crossref PubMed Scopus (376) Google Scholar). Prehybridization and hybridization were performed using Perfect Hyb. (Toyobo, Tokyo) according to the manufacturer's instructions. Type XXVI collagen cDNA generated by PCR was cloned into the expression vector pCMV-Tag4 (Stratagene, La Jolla, CA) to construct type XXVI collagen tagged with FLAG at its C terminus. The same expression vector was also used to create the protein without the tag. Polyclonal antibodies were raised in rabbits against GST-fusion proteins corresponding to the region of either NC2 or NC3 of the type XXVI collagen following the standard protocols. Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The filters were blocked with 5% skim milk in PBS for 1 h at room temperature and then incubated with a 1:500 dilution of rabbit antiserum against type XXVI collagen (NC3) in PBS with 5% skim milk. After washing with PBS, the filters were incubated with a 1:1,000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG (Cappel, Aurora, OH) for 30 min at room temperature and washed with 0.1% Tween 20 in PBS. Signals were detected using the ECL system (Amersham Biosciences). COS-7 cells were transiently transfected with the plasmid encoding C-terminal FLAG-tagged type XXVI collagen and then cultured on coverslips for ∼12 h in the presence of 136 μg/ml ascorbate phosphate. The cells were then fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. After permeabilization with 0.2% Triton X-100 in PBS for 5 min, the coverslips were rinsed and then blocked with 10% nonimmune goat serum and 2.5% skim milk in PBS overnight at 4 °C. Thereafter, the cells were incubated with a mixture of the primary antibodies specific for FLAG (Stratagene) and calnexin (SPA-860, Stressgen, Victoria, B. C., Canada) which had both been diluted 1:1,000 in PBS containing 10% nonimmune goat serum and 2.5% skim milk. Goat anti-mouse IgG coupled to rhodamine and fluorescein isothiocyanate-conjugated goat anti-rabbit IgG were used as secondary antibodies at a dilution of 1:200. The coverslips were mounted, and the cells were observed using an Axiophoto 2 and photographed with Axiovision (Carl Zeiss Co., Ltd., Jena, Germany). HEK-293 cells were cultured in Dulbecco's modified Eagle's medium/medium F12 supplemented with 10% fetal bovine serum. Transfection of the cells with 1 μg of the plasmids encoding type XXVI collagen was performed using the FuGENE 6 transfection reagent (Roche Molecular Biochemicals) according to the protocol recommended by the manufacturer. Cells transfected with the expression vector pCMV-Tag4 were used as a negative control. HEK-293 cells were preincubated with medium lacking methionine for 30 min and labeled with 35S-Promix (AmershamBiosciences) for 30 min. During the subsequent chase period, the cells were incubated with medium containing excess methionine. For immunoprecipitation, cell lysates (1% Nonidet P-40, 150 mmNaCl, 50 mm Tris-HCl, pH 8.0, 5 mm EDTA) were mixed with the anti-type XXVI collagen antibody NC3, and the immune complexes were collected using protein A-Sepharose beads (AmershamBiosciences). Immunoprecipitates were separated on 10% or 8% SDS-PAGE under reducing or nonreducing conditions. After electrophoresis, the gels were dried and exposed to Fuji HR-HA 30 film (Fuji Photo Film Co. Ltd., Kanagawa, Japan). To inhibit the N-linked glycosylation and prolyl-4 hydroxylation of type XXVI collagen, cells were incubated with 5 μg/ml tunicamycin for 12 h and treated with 0.3 mm α,α′-dipyridyl for 30 min before cell labeling. To examine the release of N-glycans from type XXVI collagen molecules, the immunoprecipitates were treated with either endoglycosidase H as reported (28Amara J.F. Lederkremer G. Lodish H.F. J. Cell Biol. 1989; 109: 3315-3324Crossref PubMed Scopus (90) Google Scholar) or with PNGase F according to the manufacturer's instructions (New England Biolabs, Inc., Beverly, MA). Samples were then resolved by 10% SDS-PAGE under reducing conditions. The neonatal testes (5 days after birth) and ovaries (7 days after birth) were fixed in 4% paraformaldehyde in PBS, embedded in methyl methacrylate resin, and sectioned into 5-μm slices. The sections were then blocked for 1 h at room temperature and incubated overnight at 4 °C with a 1:100 dilution of anti-type XXVI collagen antibody NC2 or a 1:100 dilution of preimmune rabbit serum. Signals were detected with Tyramide Signal Amplification plus dinitrophenyl horseradish peroxidase system (MENTM, Invitrogen) according to the manufacturer's instructions. After detection of the signals, sections were counterstained with 1% methyl green (Muto Pure Chemicals Ltd., Tokyo) and mounted. Signals were observed using an Axiophoto 2 and photographed with Axiovision. In search of proteins interacting with HSP47, a collagen-specific molecular chaperone, we performed yeast two-hybrid screening using as bait HSP47 lacking its ER retention sequence, RDEL. We obtained a clone containing a 1,825-bp fragment that encoded a protein with two collagenous domains. Because this clone was thought to contain only part of the full-length cDNA, further cloning was performed on a 15.5-day whole mouse embryo cDNA library encompassing 5 × 106clones. The probe used was a digoxigenin-labeled oligonucleotide probe prepared from the sequence data of the previously cloned fragment. A clone containing the 2,690-bp full-length cDNA sequence was obtained (Fig. 1 A). 5′-RACE was also performed using a specific probe prepared from the cDNA sequence data to obtain the 79-bp upstream region of the cDNA (Fig.1 A). The full-length cDNA clone encodes a 438-amino acid protein (Fig. 1 A) that consists of two collagenous domains (COL1 and COL2) and three noncollagenous domains (NC1–NC3) (Fig.1 B). The protein also contains a putative ER-targeting signal sequence at its N terminus and two Asn-X-Ser/Thr sequences that are consensus motifs for Asn-linked glycosylation. The homology search using BLAST revealed that this protein is almost identical to the mouse emu2 protein and the putative human emu2 protein with the exception of 2 amino acids. Only the sequences of these two proteins have been reported previously, but studies to characterize them have, to our knowledge, not been performed. Apart from its two collagenous regions, this protein has no obvious sequence homology to any other collagen. However, as described below, this protein is also collagen-like in its secretion from the cells, the localization in the extracellular spaces, and the post-translational modifications it undergoes. We have thus designated this novel protein as type XXVI collagen. The 13 cysteine residues of the protein are found only in the NC1 domain, including in the putative signal sequence (Fig. 1 A, shown by circles), which is a unique feature of this collagen-like protein relative to the other types of collagens. There is also a possible furin protease cleavage site (RRRR, Fig.1 A, broken line) just before the COL1 domain, which is commonly observed in the transmembrane collagens such as types XIII and XXV. These latter transmembrane collagens are cleaved just before the COL1 domain. However, we did not observe that type XXVI collagen undergoes this cleavage because the fragment that would result from this cleavage was not detected in the murine tissues that express type XXVI collagen (data not shown). Furthermore, cells transfected with cDNA encoding type XXVI collagen did not release the cleaved fragment into the medium (data not shown). Type XXVI collagen also does not have transmembrane domains. We next examined the regional expression of this novel collagen-like gene in adult murine tissues. Northern blotting revealed that type XXVI collagen gene mRNA is markedly expressed in the testis and ovary (Fig.2 A) and weakly in the kidney. We next examined its expression level during the postnatal development of the testis and ovary (Fig. 2 B). In the testis, the type XXVI collagen gene is expressed at its highest levels 1 day after birth, after which the mRNA levels gradually decrease during germ cell development. In the ovary, the type XXVI collagen gene is highly expressed in the first 2 weeks after birth, after which expression also decreases. These expression patterns of the type XXVI collagen gene are very similar to that of hsp47 expression in both the testis and ovary (Fig. 2 B). That the expression of hsp47correlates spatiotemporally with the expression of various collagen genes has previously been reported by our group (16Nagata K. Trends Biochem. Sci. 1996; 21: 22-26Abstract Full Text PDF PubMed Scopus (249) Google Scholar, 17Nagai N. Hosokawa M. Itohara S. Adachi E. Matsushita T. Hosokawa N. Nagata K. J. Cell Biol. 2000; 150: 1499-1505Crossref PubMed Scopus (283) Google Scholar, 18Nakai A. Satoh M. Hirayoshi K. Nagata K. J. Cell Biol. 1992; 117: 903-914Crossref PubMed Scopus (221) Google Scholar, 19Natsume T. Koide T. Hirayoshi K. Nagata K. J. Biol. Chem. 1994; 269: 31224-31228Abstract Full Text PDF PubMed Google Scholar). We raised two antibodies against type XXVI collagen in rabbits using GST-NC2 and GST-NC3 fusion proteins as antigens (see Fig. 1 B). These antibodies were tested for specificity with an extract of cells transfected with an expression plasmid containing type XXVI collagen cDNA. Both antibodies recognized a single 60 kDa band (data not shown). Western blot analysis under reducing conditions showed that both the anti-NC3 antiserum (Fig.3) and the anti-NC2 antiserum (data not shown) recognize an ∼60 kDa band in extracts of neonatal mouse testes and ovaries (Fig. 3). The levels of this 60 kDa band gradually decreased after birth as the testis and ovary matured (Fig. 3). These expression patterns are consistent with those revealed by Northern blot analysis. In adult mice (8 weeks old), low levels of type XXVI collagen protein were observed in both the testis and ovary, but no protein was detected in the somatic tissues such as the brain, heart, intestine, and kidney (data not shown). The antibodies also faintly recognized a 70-kDa protein in both the testis and ovary (shown by * in Fig. 3). This band is thought to be an unspecific signal rather than a modified form of type XXVI collagen because its expression pattern did not coincide with that of the mRNA in Northern blotting, nor did immunoprecipitation with anti-type XXVI collagen antibody precipitate a 70-kDa protein (data not shown). Two-dimensional gel electrophoresis of postnatal day 7 testicular extract showed that type XXVI collagen has an isoelectric point of 6.5 (data not shown). COS-7 cells were transfected with a plasmid encoding type XXVI collagen tagged with the FLAG epitope at its C terminus to assess the intracellular localization of type XXVI collagen. Double staining with anti-FLAG monoclonal antibody and anti-calnexin polyclonal antibody revealed that type XXVI collagen colocalizes with calnexin, an ER marker, indicating that type XXVI collagen is targeted to the ER (Fig.4). To determine whether type XXVI collagen is secreted, HEK-293 cells were transiently transfected with a plasmid encoding type XXVI collagen, and a pulse-chase experiment was performed in the presence of ascorbate, a cofactor of prolyl-4 hydroxylase, prolyl-3 hydroxylase, and lysyl hydroxylase (Fig.5). Cell extracts and the medium were immunoprecipitated with the NC3 antibody, and the precipitates were run on a SDS-polyacrylamide gel under reducing and nonreducing conditions. The transfected HEK-293 cells release type XXVI collagen that appears as a ∼60-kDa protein under reducing conditions (Fig. 5 A,lane 2). Under nonreducing conditions, two higher molecular size species of ∼120 and ∼200 kDa were observed in the medium (Fig. 5 B, lane 2), suggesting that type XXVI collagen forms dimer and/or trimer structures like the other collagens. However, type XXVI collagen is secreted into the culture medium more slowly than other collagen molecules (Fig. 5 A) (29Hosokawa N. Hohenadl C. Satoh M. Kuhn K. Nagata K. J. Biochem. 1998; 124: 654-662Crossref PubMed Scopus (29) Google Scholar). Notably, in the medium, the trimer form of type XXVI collagen predominates (Fig. 5 B, lanes 7 and 8), whereas the type XXVI collagen in the cell extracts is present as a mix of monomers, dimers, and trimers (Fig. 5 B, lanes 2–4). The type XXVI collagen proteins secreted into the medium have a slightly higher molecular size than the proteins within the cells (Fig. 5 A, compare lanes 2–4 andlanes 7 and 8), suggesting the protein is glycosylated just before it is secreted (see Figs.6 and7).Figure 6Effects of the addition of ascorbate, α,α′-dipyridyl, and tunicamycin on the hydroxylation, glycosylation, and secretion of type XXVI collagen. HEK-293 cells were transiently transfected with cDNA encoding type XXVI collagen and then cultured for 24 h in the presence or absence of 136 μg/ml ascorbate phosphate. The cells were then preincubated with either 0.3 mmα,α′-dipyridyl for 30 min or with 5 μg/ml tunicamycin for 12 h, after which the cells were labeled by culturing them with 0.1 mCi/ml [35S]methionine in medium lacking methionine for 3 h. The cell extract and the medium were then immunoprecipitated with anti-type XXVI collagen NC3 antibody. Immunoprecipitated samples were resolved by 8% SDS-PAGE under reducing (A) or nonreducing (B) conditions.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Characterization of N-linked glycosylation of secreted type XXVI collagen. HEK-293 cells transiently transfected with type XXVI collagen cDNA were cultured for 24 h in medium containing 136 μg/ml ascorbate phosphate. The cells were labeled by culturing them with 0.1 mCi/ml [35S]methionine in medium lacking methionine for 3 h. The cellular and medium fractions were immunoprecipitated with anti-type XXVI collagen antibody NC3. Each immunocomplex was then divided into equal aliquots and treated with either PNGase F or endoglycosidase H (Endo H). The samples were resolved by 8% SDS-PAGE under reducing conditions.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To assess whether collagen XXVI is subjected to prolyl and lysyl hydroxylation, the cells were labeled and treated with α,α′-dipyridyl, an iron-chelating agent that prevents prolyl and lysyl hydroxylation in the presence of ascorbate. The type XXVI collagen originating from α,α′-dipyridyl-treated cells migrated slightly faster under reducing conditions than the collagen from untreated cells (Fig. 6 A, compare lanes 2 and3). In the absence of ascorbate, treatment with α,α′-dipyridyl did not affect the migration of type XXVI collagen derived from the cell (Fig. 6, lanes 10 and 11). Furthermore, in the absence of ascorbate, the secretion of type XXVI collagen was negligible (Fig. 6, lane 14). Cells treated with α,α′-dipyridyl in the presence of ascorbate dramatically reduced their secretion of type XXVI collagen (Fig. 6, lane 7). Thus, type XXVI collagen is hydroxylated in the ER like other collagens, and only hydroxylated type XXVI collagen is secreted into the medium. When the cells were labeled in the presence of tunicamycin, an inhibitor of N-linked glycosylation, a 51-kDa form of type XXVI collagen was detected in the cell extract (Fig. 6 A,lane 4). This suggests that the two putativeN-linked glycosylation sites in type XXVI collagen are indeed both glycosylated. The secretion of type XXVI collagen was also prevented by the tunicamycin treatment (Fig 6 A, comparelanes 6 and 8). As shown in Figs. 5 and 6, secreted type XXVI collagen migrates slower than the cell-derived protein under reducing conditions (Fig.5 A, lanes 3 and 7, and Fig.6 A, lanes 2 and 6). To assess why the migration rate differs, we treated the cells with PNGase F and endoglycosidase H. Both enzymes released the N-glycans from the cell-derived type XXVI collagen (Fig. 7, lanes 3 and5) but the N-glycans from the secreted type XXVI collagen were released by treatment with PNGase F only (Fig. 7,lane 9), not endoglycosidase H (Fig. 7, lane 11). Thus, the N-linked glycans on type XXVI collagen are processed to the complex type when the protein is in the Golgi apparatus awaiting secretion. To determine the localization of type XXVI collagen in the testis and ovary, we performed immunohistochemical analysis using the NC2 antibody. In the 5-day-old testis, only myoid cells were stained (Fig. 8 B). Other somatic cells such as Leydig or Sertoli cells were not stained nor were germ cells such as spermatogonia. In the 7-day-old ovary, although the primary follicles were not stained, pre-theca cells surrounding primary follicles and the extracellular matrix region of medulla and cortex were recognized by the antibody (Fig. 8 D). This observation confirms that type XXVI collagen is secreted in vivo. In both the testis and ovary, no signal was detected after staining with preimmune serum (Fig. 8, A and B). Thus, like other collagen molecules, type XXVI collagen accumulates in the extracellular matrix. This suggests that this novel collagen may be involved in the early development of testis and ovary as an extracellular matrix component. We performed yeast two-hybrid screening using HSP47 as a bait to identify proteins that interact with HSP47. This resulted in the cloning and characterization of a novel protein. This protein has a signal sequence at its N terminus, two collagenous domains, and three noncollagenous domains. Collagen-like features of this protein are indicated by several observations: first, it possesses two clusters of Gly-X-Y repeats; second, it forms a trimer and is secreted into the culture medium; third, it is hydroxylated andN-linked glycosylated; and fourth, immunohistochemical analysis of the testis and ovary indicates its presence in the extracellular matrix region. These features indicate that this protein can be considered as a new member of collagen. Thus, we designated this protein as type XXVI collagen because 25 types of collagens have been reported to date (1Myllyharju J. Kivirikko K.J. Ann. Med. 2001; 33: 7-21Crossref PubMed Scopus (534) Google Scholar, 2Fitzgerald J. Bateman J.F. FEBS Lett. 2001; 505: 275-280Crossref PubMed Scopus (62) Google Scholar, 3Gordon M.K. Gerecke D.R. Hahn R.A. Bhatt P. Goyal M. Koch M. Invest. Ophthalmol. Visual Sci. 2000; 41: S752Google Scholar, 4Gordon M.K. Hahn R.A. Zhou P. Kistler A. Gerecke D.R. Koch M. Invest. Ophthalmol. Visual Sci. 2002; (in press)Google Scholar, 5Hashimoto T. Wakabayashi T. Watanabe A. Kowa H. Hosoda R. Nakamura A. Kanazawa I. Arai T. Takio K. Mann D.M. Iwatsubo T. EMBO J. 2002; 21: 1524-1534Crossref PubMed Scopus (171) Google Scholar). In adult mice, type XXVI collagen mRNA is found mainly in the testis and ovary and slightly in the kidney. During early testis and ovary development, type XXVI collagen is highly expressed in myoid cells and pre-theca cells, respectively. That HSP47 is indispensable in collagen biosynthesis has been demonstrated by the analysis of hsp47 −/− mice (17Nagai N. Hosokawa M. Itohara S. Adachi E. Matsushita T. Hosokawa N. Nagata K. J. Cell Biol. 2000; 150: 1499-1505Crossref PubMed Scopus (283) Google Scholar). The null mutation of hsp47 is lethal during embryonic development, and it has been shown that the embryos lack collagen fibrils and basement membranes in their tissues. This suggests that in these mice, the molecular maturation of collagen is impaired. This notion is supported by the improper cleavage of the type I collagen N- and C-propeptides and the enhanced sensitivity of secreted type I collagen to proteases. Recently, it was shown that Gly-X-Arg triplets in the collagen triple helix are dominant binding sites for HSP47 (24Koide T. Takahara Y. Asada S. Nagata K. J. Biol. Chem. 2002; 277: 6178-6182Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Type XXVI collagen also possesses two Gly-X-Y clusters, both of which contain Gly-X-Arg triplets. Thus, HSP47 may also interact with this novel collagen through these motifs. Further supporting the importance in development of interactions between HSP47 and collagen is that although collagen XXVI is expressed in the adult testis and ovary, this expression is higher in the neonatal tissues. Furthermore, we also detected high levels of type XXVI collagen expression during embryogenesis. 2K. Sato, N. Hosokawa, and K. Nagata, unpublished results. Thus, type XXVI collagen together with HSP47 may be important not only during testis and ovary development but also in embryogenesis. This is similar to type XIX collagen, which was reported to be ubiquitously expressed during embryogenesis, whereas in adulthood its expression was restricted to just a few tissues (30Sumiyoshi H. Inoguchi K. Khaleduzzaman M. Ninomiya Y. Yoshioka H. J. Biol. Chem. 1997; 272: 17104-17111Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The roles that type XXVI collagen and HSP47 play during embryogenesis are currently being investigated. In the case of type I collagen, three α-chains are associated in the C-propeptide region and form intermolecular disulfide bonds (1Myllyharju J. Kivirikko K.J. Ann. Med. 2001; 33: 7-21Crossref PubMed Scopus (534) Google Scholar, 6Kadler K.E. Holmes D.F. Trotter J.A. Chapman J.A. Biochem. J. 1996; 316: 1-11Crossref PubMed Scopus (1056) Google Scholar). In contrast, type XXVI collagen bears its 13 cysteine residues only in the N-terminal portion of its first noncollagenous region (NC1), including in its signal sequences. The electrophoresis of cell-derived and secreted XXVI collagen revealed that this protein forms multimers by establishing intermolecular disulfide bonds. Notably, the trimer form was the predominantly secreted form. Thus, trimers of type XXVI collagen must arise from intermolecular disulfide bonds between the NC1 regions. This is a unique feature of this protein relative to the other collagens. When extracts of the neonatal testis or of cells transfected with type XXVI collagen cDNA were immunoprecipitated with anti-type XXVI collagen antibody, only one band was detected. This suggests that the type XXVI collagen trimer probably consists of three identical α-chains (COL26A1). However, it remains possible that the trimer may actually be a heterotrimer consisting of different α-chains with identical molecular sizes. The studies to discriminate between these two possibilities are now in progress. HEK-293 cells transfected with type XXVI collagen cDNA release a protein that is ∼60 kDa in reducing SDS-PAGE. This is a slightly higher molecular size than is predicted from the actual amino acid length (438 amino acids). The additional mass appears to come from post-translational modifications of the protein that include glycosylation and hydroxylation of proline residues within the collagenous domain. Pulse-chase experiments revealed that both modifications are required for the secretion of the protein. Furthermore, the N-glycans that are added are further modified to more complex forms in the Golgi apparatus before the protein is secreted. Type XXVI collagen possesses RRRR sequences just before the first collagenous (COL1) region. These sequences are also found in types XIII and XXV collagens and were cleaved by furin convertase (5Hashimoto T. Wakabayashi T. Watanabe A. Kowa H. Hosoda R. Nakamura A. Kanazawa I. Arai T. Takio K. Mann D.M. Iwatsubo T. EMBO J. 2002; 21: 1524-1534Crossref PubMed Scopus (171) Google Scholar, 11Snellman A., Tu, H. Vaisanen T. Kvist A.P. Huhtala P. Pihlajaniemi T. EMBO J. 2000; 19: 5051-5059Crossref PubMed Scopus (77) Google Scholar). However, Western blotting of testis and ovary did not detect a short peptide fragment of type XXVI collagen resulting from cleavage by furin convertase (data not shown). The role this site plays in type XXVI collagen function is thus unclear at present. It also appears that type XXVI collagen is not a FACIT collagen. The FACIT collagens reported to date all have a thrombospondin N terminus-like domain and various numbers of von Willebrand factor-like A-domains (31Tuckwell D. Matrix Biol. 2002; 21: 63-66Crossref PubMed Scopus (30) Google Scholar). Type XXVI collagen does not bear such domains. In summary, we have identified a new collagen that interacts with HSP47 and is secreted into the extracellular matrix. It may be important in the adult reproductive organs as well as during the development of these organs. It bears many collagen-like features, including the trimeric form it assumes in its secreted state and its post-translational modifications. It also has some unusual features, including the fact that the disulfide bonds that form the trimer are made in an N-terminal noncollagenous domain. Further studies to investigate the functions of this novel collagen in development and adulthood are currently being conducted. We thank Yuko Tadokoro and Dr. Hiromitsu Tanaka (Osaka University) for excellent technical assistance and critical suggestions. We also thank Dr. Takehiko Koji (Nagasaki University) for technical assistance on histochemical analysis." @default.
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• W2029492079 title "Type XXVI Collagen, a New Member of the Collagen Family, Is Specifically Expressed in the Testis and Ovary" @default.
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