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• W1973198536 abstract "A minicollagen containing the COL1 and NC1 domains of chicken collagen XII has been produced in insect cells. Significant amounts of trimers contain a triple-helical domain in which the cysteines are not involved in inter- but in intrachain bonds. In reducing conditions, providing that the triple-helix is maintained, disulfide exchange between intra- and interchain bonding is observed, suggesting that the triple-helix forms first and that in favorable redox conditions interchain bonding occurs to stabilize the molecule. This hypothesis is verified by in vitro reassociation studies performed in the presence of reducing agents, demonstrating that the formation of interchain disulfide bonds is not a prerequisite to the trimeric association and triple-helical folding of the collagen XII molecule. Shortening the COL1 domain of minicollagen XII to its five C-terminal GXY triplets results in an absence of trimers. This can be explained by the presence of a collagenous domain that is too short to form a stable triple-helix. In contrast, the presence of five additional C-terminal triplets in COL1 allows the formation of triple-helical disulfide-bonded trimers, suggesting that the presence of a triple-helix is essential for the assembly of collagen XII. A minicollagen containing the COL1 and NC1 domains of chicken collagen XII has been produced in insect cells. Significant amounts of trimers contain a triple-helical domain in which the cysteines are not involved in inter- but in intrachain bonds. In reducing conditions, providing that the triple-helix is maintained, disulfide exchange between intra- and interchain bonding is observed, suggesting that the triple-helix forms first and that in favorable redox conditions interchain bonding occurs to stabilize the molecule. This hypothesis is verified by in vitro reassociation studies performed in the presence of reducing agents, demonstrating that the formation of interchain disulfide bonds is not a prerequisite to the trimeric association and triple-helical folding of the collagen XII molecule. Shortening the COL1 domain of minicollagen XII to its five C-terminal GXY triplets results in an absence of trimers. This can be explained by the presence of a collagenous domain that is too short to form a stable triple-helix. In contrast, the presence of five additional C-terminal triplets in COL1 allows the formation of triple-helical disulfide-bonded trimers, suggesting that the presence of a triple-helix is essential for the assembly of collagen XII. fibril-associated collagen with interrupted triple-helices base pair(s) dithiothreitol reduced glutathione oxidized glutathione 3-(N-maleimidylpropionyl)biocytin N-ethylmaleimide hydroxyproline polyacrylamide gel electrophoresis phenylmethylsulfonyl fluoride melting temperature polymerase chain reaction cytomegalovirus N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine Collagens are the most abundant proteins of the extracellular matrix. They consist of collagenous triple-helical domains, flanked by non-collagenous domains, that differ in their size and number from one collagen to another. Collagens can be homotrimers or heterotrimers, and their structure is stabilized by interchain disulfide bonds (1van der Rest M. Garrone R. Herbage D. Adv. Mol. Cell. Biol. 1993; 6: 1-67Crossref Scopus (36) Google Scholar, 2Prockop D.J. Kivirikko K.I. Annu. Rev. Biochem. 1995; 64: 403-434Crossref PubMed Scopus (1365) Google Scholar).Collagens IX, XII, XIV, XVI, and XIX constitute the FACITs1 family that are grouped on the basis of similarities in their short C-terminal domain COL1, which is thought to be important for the association with fibrils (3Mayne R. Brewton R.G. Curr. Opin. Cell Biol. 1993; 5: 883-890Crossref PubMed Scopus (115) Google Scholar, 4Ricard-Blum S. Dublet D. van der Rest M. Sheterline P. Unconventional Collagens. Types VI, VII, VIII, IX, X, XIV, XVI and XIX. Protein Profile. Oxford University Press, NY2000Google Scholar). The association of collagen type IX, XII, and XIV molecules with quarter-staggered fibrils has been shown by immunofluorescence and electron microscopy (5Vaughan L. Mendler M. Huber S. Brückner P. Winterhalter K.H. Irwin M.I. Mayne R. J. Cell Biol. 1988; 106: 991-997Crossref PubMed Scopus (258) Google Scholar, 6Sugrue S.P. Gordon M.K. Seyer J. Dublet B. van der Rest M. Olsen B.R. J. Cell Biol. 1989; 109: 939-945Crossref PubMed Scopus (61) Google Scholar, 7Keene D.R. Lunstrum G.P. Morris N.P. Stoddard D.W. Burgeson R.E. J. Cell Biol. 1991; 113: 971-978Crossref PubMed Scopus (142) Google Scholar, 8Garrone R. Lethias C. Le Guellec D. Microsc. Res. Tech. 1997; 38: 407-412Crossref PubMed Scopus (38) Google Scholar, 9Niyibizi C. Visconti C.S. Kavalkovich K. Woo S.L. Matrix Biol. 1995; 14: 743-751Crossref PubMed Scopus (49) Google Scholar). The biological functions of the FACITs are not yet fully understood. Rapid and reversible production of collagen XII by fibroblasts submitted to mechanical stress has been observed (10Chiquet M. Matthisson M. Koch M. Tannheimer M. Chiquet- Ehrismann R. Biochem. Cell Biol. 1996; 74: 737-744Crossref PubMed Scopus (169) Google Scholar, 11Trachslin J. Koch M. Chiquet M. Exp. Cell. Res. 1999; 247: 320-328Crossref PubMed Scopus (78) Google Scholar). Collagens XII and XIV potentially facilitate the contraction of collagen gels by fibroblasts (12Nishiyama T. McDonough A.M. Bruns R.R. Burgeson R.E. J. Biol. Chem. 1994; 269: 28193-28199Abstract Full Text PDF PubMed Google Scholar), and they are known to interact with other components of the extracellular matrix (13Font B. Aubert-Foucher E. Goldschmidt D. Eichenberger D. van der Rest M. J. Biol. Chem. 1993; 268: 25015-25018Abstract Full Text PDF PubMed Google Scholar, 14Font B. Eichenberger D. Rosenberg L.M. van der Rest M. Matrix Biol. 1996; 15: 341-348Crossref PubMed Scopus (96) Google Scholar, 15Brown J.C. Mann K. Wiedemann H. Timpl R. J. Cell Biol. 1993; 120: 557-567Crossref PubMed Scopus (76) Google Scholar). This suggests that they contribute to the cohesion and the modulation of the mechanical properties of the extracellular matrix. Collagen XIV is also a potential reservoir for procollagen N-proteinase (16Colige A. Beschin A. Samyn B. Goebels Y. Van Beeumen J. Nusgens B.V. Lapiere C.M. J. Biol. Chem. 1995; 270: 16724-16730Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) and promotes the adhesion of different cells (17Ehnis T. Dieterich W. Bauer M. Lampe B. Schuppan D. Exp. Cell. Res. 1996; 229: 388-397Crossref PubMed Scopus (67) Google Scholar, 18Klein G. Kibler C. Schermutzki F. Brown J. Muller C.A. Timpl R. Matrix Biol. 1998; 16: 307-317Crossref PubMed Scopus (26) Google Scholar).The assembly of fibril-forming collagens (collagen types I, II, III, V, and XI) is a complex process in which three chains must fold into a triple-helix. This results from chain association, nucleation, and propagation of the folding occurring from the C to N termini in a zipper-like fashion (19Engel J. Prockop D.J. Annu. Rev. Biophys. Biophys. Chem. 1991; 20: 137-152Crossref PubMed Scopus (266) Google Scholar, 20McLaughlin S.H. Bulleid N.J. Matrix Biol. 1998; 16: 369-377Crossref PubMed Scopus (98) Google Scholar). The C-terminal non-triple-helical domains, called the C-propeptides, play an important role in the initial steps of chain association (21Bachinger H.P. Davis J.M. Int. J. Biol. Macromol. 1991; 13: 152-156Crossref PubMed Scopus (75) Google Scholar, 22Brass A. Kadler K.E. Thomas J.T. Grant M.E. Boot-Handford R.P. FEBS Lett. 1992; 303: 126-128Crossref PubMed Scopus (67) Google Scholar, 23Doege K.J. Fessler J.H. J. Biol. Chem. 1986; 261: 8924-8935Abstract Full Text PDF PubMed Google Scholar, 24Bulleid N.J. Dalley J.A. Lees J.F. EMBO J. 1997; 16: 6694-6701Crossref PubMed Scopus (86) Google Scholar). However, in the case of FACITs, the C-propeptides are replaced by significantly shorter non-triple-helical domains, NC1. These small domains consist of just about 75 amino acids for collagen XII and less than 30 amino acids for collagen IX, whereas the C-propeptides of the fibrillar collagens contain some 260 amino acids. The NC1 domains of the FACITs do not share common sequences. In addition, structural variations in the NC1 domains of rat and mouse collagen XII, generated by tissue-specific alternative splicing, have been shown (25Kania A.M. Reichenberger E. Baur S.T. Karimbux N.Y. Taylor R.W. Olsen B.R. Nishimura I. J. Biol. Chem. 1999; 274: 22053-22059Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). In contrast, FACITs display remarkable similarities in their COL1 domains with respect to their similar size and to two corresponding imperfections within the triple-helix. At the junction of the COL1 and NC1 domains, two cysteines separated by four amino acids are responsible for interchain disulfide bonding. These data suggest that the COL1 domain and the junction may serve a common function in all FACITs.The critical role of the COL1 domain in the assembly of the collagen XII molecule has first been suggested by the necessity to stabilize this domain by the hydroxylation of prolyl residues to obtain disulfide-bonded trimers. Indeed, reduction or inhibition of prolyl 4-hydroxylase (EC 1.14.11.2) prevented the formation of disulfide-bonded trimers in HeLa and insect cells, which produced a chicken minicollagen XII consisting of the C-terminal domains COL1 and NC1 (26Mazzorana M. Gruffat H. Sergeant A. van der Rest M. J. Biol. Chem. 1993; 268: 3029-3032Abstract Full Text PDF PubMed Google Scholar, 27Mazzorana M. Snellman A. Kivirikko K.I. van der Rest M. Pihlajaniemi T. J. Biol. Chem. 1996; 271: 29003-29008Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). In addition, a deletion covering most of the NC1 domain of chicken collagen XII, except for seven residues of the COL1/NC1 junction, does not prevent the formation of disulfide-bonded trimers correctly folded into a triple-helix (28Mazzorana M. Giry-Lozinguez C. van der Rest M. Matrix Biol. 1995; 14: 583-588Crossref PubMed Scopus (14) Google Scholar). In this report, we analyze the respective roles of the COL1 domain and of the junction in the formation of the homotrimeric collagen XII molecule. We show that the formation of the COL1 triple-helix is the key step of the trimeric association of the α chains of minicollagen XII, which is then stabilized by interchain disulfide bonds.EXPERIMENTAL PROCEDURESMaterialsHiTrap Q, HiTrap SP, and Superose 6 and 12 (HR 10/30) columns were from Amersham Pharmacia Biotech. Chymotrypsin type I-S, reduced glutathione, Triton X-100 (peroxide-free), and trypsin type XIII were from Sigma Chemical Co. NADPH and yeast glutathione reductase (EC 1.6.4.2) were from Roche Molecular Biochemicals. Express Five medium, pluronic acid, Dulbecco's modified Eagle's medium, fetal calf serum, penicillin, and streptomycin were from Life Technologies, Inc.Construction of the Vectors and Generation of Recombinant VirusesGeneration of baculoviruses recombinant for the minigene coding for minicollagen XII (COL XII 23) has been described (27Mazzorana M. Snellman A. Kivirikko K.I. van der Rest M. Pihlajaniemi T. J. Biol. Chem. 1996; 271: 29003-29008Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Recombinant baculovirus transfer vector constructs coding for chicken minicollagen XII with shortened COL1 domains were generated by PCR overlap extension using pRc/CMV COL XII 23 as a template (28Mazzorana M. Giry-Lozinguez C. van der Rest M. Matrix Biol. 1995; 14: 583-588Crossref PubMed Scopus (14) Google Scholar). PVL-XII-PCR1 was generated using a 5′-oligonucleotide primer H1 (5′-GCAGTCCTCGAGGGCTCCACAGGATCACGAGG-3′) complementary to the sequence downstream of the second imperfection of the COL1 domain (an XhoI site has been introduced in this oligonucleotide to facilitate subsequent subcloning) and a 3′-oligonucleotide primer H2 (5′-AGGCACAGTCGAGGC-3′) complementary to the sequence of the pRc/CMV vector (Invitrogen) located 50 nucleotides downstream of theXbaI site of the polylinker. PCR yielded a 415-bp fragment (PCR1), which was cut by XhoI and XbaI and subcloned in BKS ENS 23 (28) digested by the same enzymes. The resulting plasmid BKS XII-PCR1 contains also the signal peptide of the α1 chain of human collagen I and two tagging sequences, each consisting of a short fragment of human c-Myc protein. BKS XII-PCR1 is digested by HindIII and XbaI, and the resulting fragment is then cloned in the mammalian expression vector pRc/CMV, giving rise to pRc/CMV XII-PCR1. Concurrently, BKS XII-PCR1 and the baculovirus transfer vector pVL 1392 (PharMingen) are linearized by HindIII and NotI, respectively, and filled with Klenow. Both are then digested with XbaI. TheHindIII-filled/XbaI fragment of BKS XII-PCR1 is then ligated to pVL 1392 containing a NotI-filled extremity and a cohesive XbaI extremity. pVL XII-PCR2 has been prepared in the same way using a 5′-oligonucleotide primer I (5′-GCAGTCCTCGAGGGAAATGCGGGTATTCG-3′) complementary to the sequence coding for the last 15 amino acids of COL1 and H2 as 3′-oligonucleotide primer. The PCR2 fragment was 363 bp in size. In the final baculovirus transfer vector, pVL1392, the minigenes of interest are inserted downstream of the polyhedrin promoter. In view of homologous recombination, each of the two recombinant pVL constructs has been co-transfected into Spodoptera frugiperdaSf9 cells together with wild type-purified DNA ofAutographa californica polyhedrosis virus. The resultant viral pool was collected, amplified, and plaque-purified to screen for recombinant viruses (29Summers M.D. Smith G.E. Texas Agric. Exp. Station Bull. 1987; 1555: 1-57Google Scholar). After several rounds of purification, the ensuing recombinant viruses, called XII-PCR1 and XII-PCR2, were checked by a PCR-based method (30Malitschek B. Schartl M. BioTechniques. 1991; 11: 177-178PubMed Google Scholar), amplified, and stored at 4 °C in the dark until use.Infection of Insect Cells with Recombinant BaculovirusesTrichoplusia ni Tn-SB1-4 cells (marketed by Invitrogen as High Five cells) were infected with COL XII 23, XII-PCR1, or XII-PCR2 viruses together with recombinant viruses encoding the α and β subunits of human prolyl 4-hydroxylase (31Vuori K. Pihlajaniemi T. Marttila M. Kivirikko K.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7467-7470Crossref PubMed Scopus (114) Google Scholar). These viruses were a generous gift from Prof. Taı̈na Pihlajaniemi, University of Oulu, Finland. Cells were cultured at 27 °C in serum-free medium containing 0.1% pluronic acid, penicillin, and streptomycin. For cells cultured in plates, 6 × 106 cells were seeded per 100-mm culture dish. For cells cultured in suspension in a spinner system (Cell Spin, Integra Biosciences), 100 ml of medium was inoculated with 108 cells in a 250-ml spinner flask to ensure proper aeration and stirred continuously at 40 rpm until the cells were adapted to suspension culture conditions. Cells were maintained at 106 cells/ml by removing the excess of cell suspension and replacing it with fresh culture medium. They were infected when they had reached 98% viability and 18- to 24-h doubling time. In both culture conditions, cells were infected at a multiplicity of infection 5:1:1 for the recombinant baculoviruses coding for any one of the minicollagens, the α subunit, and the β subunit of the prolyl 4-hydroxylase, respectively. Sodium ascorbate was added daily at 50 µg/ml immediately after the replacement of the viral inocula with fresh medium (7 ml) for cells cultured in plates or 1 h after inoculation of the viruses for the cells cultured in suspension. Cells and media were collected 48 h after infection. Cell fractions were used either immediately or stored as pellets at −80 °C after freezing in liquid nitrogen. Media were centrifuged at 12,000 × g for 15 min at 4 °C after addition of protease inhibitors (10 mm EDTA, 10 mm NEM, and 1 mm PMSF) and frozen at −80 °C.Transient Transfection of HeLa CellsCells (106) were plated in 100-mm culture dishes in Dulbecco's modified Eagle's medium supplemented with 5% heat-inactivated fetal calf serum, penicillin, and streptomycin. After cell adhesion and spreading, 5 µg of expression vector pRc/CMV XII-PCR1 and 10 µg of plasmid carrier DNA were used to transfect the cells using the calcium-phosphate procedure (32Kriegler M. Gene Transfer and Expression: A Laboratory Manual. Stockton Press, NY1990Crossref Google Scholar). After 16 h of contact with the DNA-calcium phosphate coprecipitate, cells were rinsed and sodium ascorbate (50 µg/ml) was added in 7 ml of fresh medium. After 24 h, medium and cells were collected for immunodetection experiments.SDS-PAGE and Western Blotting AnalysisThe cells cultured in plates were homogenized in ice-cold buffer (0.2 m NaCl, 0.1% Triton X-100, 50 mmTris-HCl, pH 7.4, containing 10 mm EDTA, 10 mmNEM, and 1 mm PMSF, 300 µl/106 cells). Aliquots of homogenates or cell extracts and of the corresponding conditioned media were precipitated with 10% trichloroacetic acid and analyzed by SDS-PAGE, followed by Coomassie Blue staining or Western blotting with antibody Myc-9E10.2 (designated here as 9E10). This antibody is a mouse monoclonal antibody directed against the tagging sequences derived from the human c-Myc protein present at the N terminus of minicollagen XII constructs.Purification of Minicollagen XII from Medium of Infected Insect Cells Cultured in SuspensionConditioned medium containing protease inhibitors was chromatographed on DEAE-cellulose. After rinsing of the gel with 50 mm NaCl, 10 mm Tris-HCl, pH 7.4, 5 mm EDTA, 5 mm NEM, elution of minicollagen XII was performed with the same buffer containing 0.5 m NaCl. Minicollagen XII was further purified with an anion exchange fast-protein liquid chromatography column (HiTrap Q, 5 ml) equilibrated in 10 mm Tris-HCl, pH 7.4, containing 0.1 mNaCl and 5 mm EDTA. A linear gradient of 0.1 to 0.7m NaCl in 50 min was applied at a flow rate of 1 ml/min. In this gradient, species that migrate as single bands with an apparent molecular mass corresponding to monomers in SDS-gels elute earlier than trimers. These fractions were separately injected to a Superose 6 fast-protein liquid chromatography column equilibrated in 0.15m NaCl, 10 mm Tris-HCl, 5 mm EDTA, pH 7.4, at 0.4 ml/min. This gel filtration allows preparation of two fractions corresponding to trimers and monomers, which elute after 12 and 16 ml, respectively.Pepsin Digestion of Minicollagen XIIMinicollagen XII (about 700 µg) purified from the medium of infected insect cells cultured in suspension as described above (except that the gel-filtration step was omitted) was adjusted to pH 3 with acetic acid and incubated in the presence of pepsin (20 µg) for 18 h at 4 °C. Fresh pepsin was added (30 µg), and after 90 min at 20 °C digested minicollagen XII was purified by gel filtration on Superose 6.Purification of XII-PCR1 and XII-PCR2 from Insect Cell ExtractsXII-PCR1Infected High Five cells were homogenized (3.4 × 106 cells/ml) in cold extraction buffer (0.2m NaCl, 0.1% Triton X-100, 50 mm Tris-HCl, pH 7.4, containing 10 mm EDTA, 10 mm NEM, and 1 mm PMSF). After 30-min incubation on ice, the homogenate was centrifuged at 12,000 × g for 15 min at 4 °C. The extract was diluted 2-fold with extraction buffer lacking NaCl and chromatographed on a DEAE-cellulose column equilibrated in 0.1m NaCl, 50 mm Tris-HCl, 5 mm EDTA, 5 mm NEM, pH 7.4. Elution was performed by raising the NaCl concentration to 0.5 m. XII-PCR1 was further purified by HiTrap Q chromatography (a 100-min linear gradient of 0.1 to 0.5m NaCl in 10 mm Tris-HCl, pH 7.4, at 1 ml/min) followed by gel filtration on Superose 6 performed as described for minicollagen XII.XII-PCR2Infected High Five cells were homogenized, and the extract was subjected to DEAE-cellulose chromatography as described for XII-PCR1. Monomers elute in the flow-through, whereas dimers elute with the 0.5 m NaCl-containing buffer. Monomers were further purified by HiTrap Q chromatography using a 0.1 to 0.3 mNaCl gradient. For the analysis of the XII-PCR2 hydroxylation state, an additional reverse phase chromatography on C18 was performed before trypsin digestion.Trypsin/Chymotrypsin DigestionsThe fractions (3–6 µg) equilibrated for 5 min at the digestion temperature were incubated 3 min at 21 °C unless otherwise indicated in the presence of a mixture of trypsin (1–1.5 µg) and chymotrypsin (3–4.5 µg).Purification of the COL1 Domain of Minicollagen XIIAn extract corresponding to 25 × 106 infected insect cells cultured in plates was adjusted to pH 2–2.5 with HCl before digestion with 1.5 mg of pepsin for 2 h at 20 °C. After centrifugation for 15 min at 12000 × g, the supernatant was neutralized with NaOH, filtered, and applied to a HiTrap Q column. The unbound fraction was dialyzed against 50 mm sodium acetate, pH 5.1, and batch-incubated with CM-cellulose. Pepsinized minicollagen was eluted with 0.15m NaCl, 20 mm Tris-HCl, pH 7.4, and chromatographed on Superose 6 as described for the full-length minicollagen XII. An aliquot of pepsinized minicollagen XII (0.4 mg) was further digested using a mixture of trypsin (20 µg) and chymotrypsin (50 µg) for 15 min at 25 °C and then 30 min at 20 °C. The digest was injected to a HiTrap sulfopropyl column, and the trimeric disulfide-bonded COL1 domain was eluted using a 0 to 0.25m NaCl gradient in Bis-Tris-HCl buffer, pH 6.7. Further purification was achieved by gel filtration on a Superose 12 column equilibrated in 0.1 m NaCl, 20 mm Tris-HCl, pH 7.4, at 0.4 ml/min.Labeling of Cysteine Residues with MPBIsolated T, Mt, and Mm forms of minicollagen XII (0.1 mg/ml) were reacted with 120 µm MPB for 35 min at 30 °C either directly or after incubation for 15 min at 45 °C in the presence of 5 mm DTT followed by removal of DTT by reverse phase chromatography. The MPB labeling was stopped by addition of reducing Laemmli sample buffer. Aliquots (0.1 µg) were analyzed by SDS-PAGE followed by electroblotting. MPB-modified proteins were detected after incubation with avidin-conjugated alkaline phosphatase (ExtrAvidin, Sigma) using 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium (Bio-Rad).Analysis of XII-PCR2 Hydroxylation StateXII-PCR2 (120 µg) in 0.15 m NaCl, 50 mm Tris-HCl, pH 8, was incubated 20 h at 37 °C in the presence of 10 µg of trypsin (sequencing grade, Promega). The digest was chromatographed on a C18 reverse phase column (Supelcosyl LC-318, 25 cm × 4.6 mm, 5 µm, Supelco) using an aqueous acetonitrile gradient (0–45%) over 60 min in 0.1% trifluoroacetic acid at 1.2 ml/min. An aliquot of each peak was tested by a standard enzyme-linked immunosorbent assay for its recognition by a monoclonal antibody (17-8H11) whose epitope is located within the 22 first residues of the NC1 domain of chicken collagen XII. 2M. Mazzorana, S. Cogne, D. Goldschmidt, and E. Aubert-Foucher, unpublished results. The major immunoreactive peaks were analyzed by electrospray mass spectrometry and N-terminal sequencing.Determination of GSSG Concentration in GSH SolutionsGSSG concentrations were determined by a coupled assay as glutathione reductase-dependent oxidation of NADPH (33Sies H. Summer K.H. Eur. J. Biochem. 1975; 57: 503-512Crossref PubMed Scopus (228) Google Scholar). Briefly, samples (5–200 µl) were assayed for GSSG in a final volume of 1 ml of a 20 mm Tris-HCl, 0.1 m NaCl buffer (pH 8) containing 0.2 mm NADPH by addition of 0.6 unit of yeast glutathione reductase (120 units/ml) and monitoring the absorbance decrease at 340 nm. NADPH consumption was quantified using an extinction coefficient of 6.34 mm−1cm−1.Other ProceduresSDS-PAGE analysis was performed according to Laemmli (34Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205964) Google Scholar) unless otherwise noted. Alternatively, the procedure of Schägger and von Jagow (35Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10434) Google Scholar) was used. Amino acid sequence analysis was performed by automated Edman degradation on an Applied Biosystems 473A protein sequencer using the trifluoroacetic acid conversion program provided by the manufacturer. Mass spectrometry analysis was performed on an electrospray apparatus API 165 Sciex (PerkinElmer Life Sciences) with an ion spray source.DISCUSSIONThe experiments reported here demonstrate that the COL1 domain is the key element in the assembly of minicollagen XII. The characterization of recombinant minicollagen XII produced in insect cells cultured in suspension shows the presence of a mixture of correctly folded trimers in which the cysteines of the COL1/NC1 junction are inter- or intrachain-linked. Their trimeric, triple-helical structure, with the three chains in register, has been demonstrated. The intrachain bonds rearrange to form interchain disulfide bonds under reducing conditions, providing that the triple-helix is maintained. In contrast, the disruption of the triple-helix prevents the intra- to interchain bond rearrangement.In vitro reassociation studies of monomers of minicollagen XII or of its COL1 domain show that the triple-helix forms to the same degree independent of whether the redox state of the cysteines is involved in intrachain disulfide bonds or totally reduced.These results show that interchain disulfide bond formation is not a prerequisite for the association of the α chains, nucleation, and folding of the collagen XII molecules in trimers with a triple-helical COL1 domain. They are similar to those previously described for the fibrillar collagen III (38Bulleid N.J. Wilson R. Lees J.F. Biochem. J. 1996; 317: 195-202Crossref PubMed Scopus (62) Google Scholar) and are in favor of a mechanism in which the collagenous part has first to fold into a triple-helical conformation before interchain disulfide bond formation occurs. Because the triple-helix formation is the rate-limiting step, when the redox conditions are too oxidative, the cysteines of monomers form intrachain disulfide bonds. In this respect, the COL1/NC1 junction of collagen XII produced in cells cultured in suspension shows the same behavior as a synthetic peptide containing the COL1/NC1 junction of collagen XIV, (GPO)3GYCDPSSCAG, in reassociation studies. Indeed, air oxidation of this peptide mainly leads to formation of an intrachain disulfide bond (36Lesage A. Penin F. Geourjon C. Marion D.,. van der Rest M. Biochemistry. 1996; 35: 9647-9660Crossref PubMed Scopus (32) Google Scholar). This could explain why, in the case of minicollagen XII produced in insect cells cultured in suspension, significant amounts of non-disulfide-bonded trimers are observed. However, when the redox conditions are less oxidative (cells cultured in plates), interchain disulfide bonding occurs as soon as the triple-helix has formed, because very few non-disulfide-bonded trimers are observed.3 In vitro reassociation data also show that the COL1 domain and the COL1/NC1 junction contain all the information necessary for trimerization of minicollagen XII and nucleation of its triple-helix. These results confirm those previously obtained in HeLa cells transfected with a minigene of collagen XII coding only for the COL1 domain and the COL1/NC1 junction, which showed that triple-helical disulfide-bonded trimers were produced (28Mazzorana M. Giry-Lozinguez C. van der Rest M. Matrix Biol. 1995; 14: 583-588Crossref PubMed Scopus (14) Google Scholar).To determine the respective roles of COL1 and of the junction in assembly, two shortened minicollagens XII, XII-PCR1 and XII-PCR2, constituted of the complete NC1 domain and of a COL1 domain reduced to its one-third or one-sixth C-terminal extremity, respectively, have been produced in insect cells. We show that the presence of one-third of the COL1 domain is sufficient to promote the formation of interchain disulfide-bonded trimers in which the ten remaining GXY triplets of COL1 have folded into a triple-helix. This result is also true at 37 °C, as shown by transfection experiments in HeLa cells. In contrast, XII-PCR2 does not form trimers neither disulfide-bonded nor non-disulfide-bonded. Because XII-PCR2 still contains the five C-terminal GXY triplets of COL1, a difference in reactivity of the cysteines of the junction due to a different environment is unlikely. Neither the hydroxylation state of the two prolyl residues in the Y position of the two remaining GPP triplets nor the redox state of the cysteines is responsible for the inability of the α chains of XII-PCR2 to associate into trimers, because we have shown that the two prolyl residues are hydroxylated and that the cysteines are mainly reduced. These results show that the COL1/NC1 junction is not sufficient to promote the association of the three chains, and that COL1, or a part of it, is required to ensure trimerization. A similar conclusion has emerged from studies using synthetic peptides corresponding to the NC1 sequences of collagen IX (39Mechling D.E. Gambee J.E. Morris N.P. Sakai L.Y. Keene D.R. Mayne R. Bächinger H.P. J. Biol. Chem. 1996; 271: 13781-13785Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) or to the sequence of the COL1/NC1 junction of collagen XIV (36Lesage A. Penin F. Geourjon C. Marion D.,. van der Rest M. Biochemistry. 1996; 35: 9647-9660Crossref PubMed Scopus (32) Google Scholar). The sequence of COL1 is crucial for the trimeric assembly of minicollagen XII. Taking into account the natural tendency of the GXY triplets to fold into a triple-helix, it is tempting to speculate that the nucleation of the helix is the driving force for the assembly process. In this case, the collagenous part of XII-PCR2 is probably too short to form a stable triple-helix. Indeed, the thermal stability of triple-helices has been reported for (GPO)npeptides of various lengths and the melting temper" @default.
• W1973198536 created "2016-06-24" @default.
• W1973198536 creator A5021793143 @default.
• W1973198536 creator A5037390780 @default.
• W1973198536 creator A5043367245 @default.
• W1973198536 creator A5085042381 @default.
• W1973198536 date "2001-07-01" @default.
• W1973198536 modified "2023-10-15" @default.
• W1973198536 title "Collagenous Sequence Governs the Trimeric Assembly of Collagen XII" @default.
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