Trimerization and Triple Helix Stabilization of the Collagen XIX NC2 Domain (2008)

The mechanisms of chain selection and assembly of fibril-associated collagens with interrupted triple helices (FACITs) must differ from that of fibrillar collagens, since they lack the characteristic C-propeptide. We analyzed two carboxyl-terminal noncollagenous domains, NC2 and NC1, of collagen XIX as potential trimerization units and found that NC2 forms a stable trimer and substantially stabilizes a collagen triple helix attached to either end. In contrast, the NC1 domain requires formation of an adjacent collagen triple helix to form interchain disulfide bridges. The NC2 domain of collagen XIX and probably of other FACITs is responsible for chain selection and trimerization. The mechanisms of chain selection and assembly of fibril-associated collagens with interrupted triple helices (FACITs) must differ from that of fibrillar collagens, since they lack the characteristic C-propeptide. We analyzed two carboxyl-terminal noncollagenous domains, NC2 and NC1, of collagen XIX as potential trimerization units and found that NC2 forms a stable trimer and substantially stabilizes a collagen triple helix attached to either end. In contrast, the NC1 domain requires formation of an adjacent collagen triple helix to form interchain disulfide bridges. The NC2 domain of collagen XIX and probably of other FACITs is responsible for chain selection and trimerization. The fibril-associated collagens with interrupted triple helices (FACIT 2The abbreviations used are: FACIT, fibril-associated collagen with interrupted triple helices; COL, collagenous; NC, noncollagenous; Fmoc, N-(9-fluorenyl)methoxycarbonyl; HPLC, high pressure liquid chromatography. collagens) include type IX, XII, XIV, XVI, XIX, XX, XXI, and XXII. Collagen IX is a heterotrimer composed of three different ;-chains, and all others are homotrimers whose ;-chains are characterized by short collagenous (COL) domains interrupted by several noncollagenous (NC) domains (1Myllyharju J. Kivirikko K.I. Trends Genet. 2004; 20: 33-43Abstract Full Text Full Text PDF PubMed Scopus (894) Google Scholar, 2Ricard-Blum S. Ruggiero F. Pathol. Biol. 2005; 53: 430-442Crossref PubMed Scopus (272) Google Scholar). Unlike the fibril-forming collagens, the FACITs have significantly shorter carboxyl-terminal NC domains (NC1 domains): 37 residues for collagen XIV, 20-25 residues for collagen IX, and even fewer than 20 residues for collagen types XII and XIX (based on human sequences). In contrast, the carboxyl-terminal NC domains of fibrillar collagens (C-propeptides) are of a different type and contain about 260 residues. The FACITs share a remarkable sequence homology at their COL1/NC1 junctions, each having two strictly conserved cysteine residues separated by four residues in their NC1 domain. Several studies suggest that the COL1 domain and the NC1 domain are involved in the mechanism of chain selection in the assembly of collagens XII and XIV (3Mazzorana M. Gruffat H. Sergeant A. van der Rest M. J. Biol. Chem. 1993; 268: 3029-3032Abstract Full Text PDF PubMed Google Scholar, 4Mazzorana M. Giry-Lozinguez C. van der Rest M. Matrix Biol. 1995; 14: 583-588Crossref PubMed Scopus (14) Google Scholar, 5Lesage A. Penin F. Geourjon C. Marion D. van der Rest M. Biochemistry. 1996; 35: 9647-9660Crossref PubMed Scopus (32) Google Scholar, 6Mazzorana M. Cogne S. Goldschmidt D. Aubert-Foucher E. J. Biol. Chem. 2001; 276: 27989-27998Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Contrary to these studies, very recent studies on collagen IX show that three ;-chains can associate in the absence of the COL1 and NC1 domains to form a triple helix, although the COL2-NC2 region alone is not sufficient for trimerization (7Jáálinoja J. Ylóstalo J. Beckett W. Hulmes D.J.S. Ala-Kokko L. Biochem. J. 2008; 409: 545-554Crossref PubMed Scopus (6) Google Scholar). This suggests that folding and chain selection of collagen IX is a cooperative process involving multiple COL and NC domains (7Jáálinoja J. Ylóstalo J. Beckett W. Hulmes D.J.S. Ala-Kokko L. Biochem. J. 2008; 409: 545-554Crossref PubMed Scopus (6) Google Scholar). It has also been hypothesized that the NC2 domain of all FACIT collagens is able to form an ;-helical coiled-coil, thus bearing an ability to trimerize those collagens (8McAlinden A. Smith T.A. Sandell L.J. Ficheux D. Parry D.A.D. Hulmes D.J.S. J. Biol. Chem. 2003; 278: 42200-42207Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), but no experimental evidence has been reported so far. Collagen XIX was identified from independently isolated clones from a human rhabdomyosarcoma cell line (9Yoshioka H. Zhang H. Ramirez F. Mattei M.G. Moradi-Ameli M. van der Rest M. Gordon M.K. Genomics. 1992; 13: 884-886Crossref PubMed Scopus (44) Google Scholar, 10Myers J.C. Sun M.J. D'Ippolito J.A. Jabs E.W. Neilson E.G. Dion A.S. Gene (Amst.). 1993; 123: 211-217Crossref PubMed Scopus (32) Google Scholar). The type XIX chain is composed of a 268-residue, noncollagenous amino terminus, an 832-residue discontinuous collagenous region, and a 19-residue carboxyl-terminal peptide (10Myers J.C. Sun M.J. D'Ippolito J.A. Jabs E.W. Neilson E.G. Dion A.S. Gene (Amst.). 1993; 123: 211-217Crossref PubMed Scopus (32) Google Scholar, 11Inoguchi K. Yoshioka H. Khaleduzzaman M. Ninomiya Y. J. Biochem. (Tokyo). 1995; 117: 137-146Crossref PubMed Scopus (29) Google Scholar, 12Myers J.C. Yang H. D'Ippolito J.A. Presente A. Miller M.K. Dion A.S. J. Biol. Chem. 1994; 269: 18549-18557Abstract Full Text PDF PubMed Google Scholar). It is by far the least abundant collagen so far purified, with a composition of ∼10-6% of the dry weight of umbilical cord (13Myers J.C. Li D. Amenta P.S. Clark C.C. Nagaswami C. Weisel J.W. J. Biol. Chem. 2003; 278: 32047-32057Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Several features in the type XIX sequence place this collagen in the largest subclass of the nonfibrillar group, the FACIT collagens. These include a ∼250-residue thrombospondin module at the amino terminus, the position of two 2-amino acid interruptions in the collagenous subdomain closest to the carboxyl terminus, and a Cys-Xaa4-Cys motif situated at the junction of the collagenous region and carboxyl peptide (COL1/NC1 junction) (11Inoguchi K. Yoshioka H. Khaleduzzaman M. Ninomiya Y. J. Biochem. (Tokyo). 1995; 117: 137-146Crossref PubMed Scopus (29) Google Scholar, 12Myers J.C. Yang H. D'Ippolito J.A. Presente A. Miller M.K. Dion A.S. J. Biol. Chem. 1994; 269: 18549-18557Abstract Full Text PDF PubMed Google Scholar). Characterization of mice harboring null or structural mutations in the collagen XIX (Col19a1) gene has revealed the critical contribution of this matrix protein to muscle physiology and differentiation (14Sumiyoshi H. Mor N. Lee S.Y. Doty S. Henderson S. Tanaka S. Yoshioka H. Rattan S. Ramirez F. J. Cell Biol. 2004; 166: 591-600Crossref PubMed Scopus (48) Google Scholar). The phenotype includes smooth muscle motor dysfunction and a hypertensive sphincter. Mice without collagen XIX also display impaired smooth-to-skeletal muscle cell conversion in the abdominal segment of the esophagus (14Sumiyoshi H. Mor N. Lee S.Y. Doty S. Henderson S. Tanaka S. Yoshioka H. Rattan S. Ramirez F. J. Cell Biol. 2004; 166: 591-600Crossref PubMed Scopus (48) Google Scholar). Electron microscope images of protein purified from human umbilical cord revealed a sharply kinked and highly polymorphic collagenous region as well as higher order complexes (13Myers J.C. Li D. Amenta P.S. Clark C.C. Nagaswami C. Weisel J.W. J. Biol. Chem. 2003; 278: 32047-32057Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The higher order complexes involve the aminoterminal domain, which is responsible for intermolecular disulfide linkages and contains a heparin-binding site (13Myers J.C. Li D. Amenta P.S. Clark C.C. Nagaswami C. Weisel J.W. J. Biol. Chem. 2003; 278: 32047-32057Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). To explore the trimerization abilities of collagen XIX at the biophysical level, we have studied the NC1 and NC2 domains in their isolated form as well as in conjunction with a triple helix. We show that the folding and the interchain disulfide bridge formation in the NC1 domain is driven by the triple helix formation; in other words, the triple helix formation in the COL1 domain is necessary for the folding of the NC1 domain. The artificial triple helical sequence (GPO)6, which forms a triple helix by itself, efficiently induces the folding of the NC1 domain. The NC2 domain alone is an effective trimerization domain. It forms a stable trimer and substantially stabilizes the triple helical domains attached to it. Synthesis and Purification of Reduced NC1-containing Peptides—Peptides with the NC1 domain were synthesized using the native sequence of mouse collagen XIX from the Swiss Protein Data base (Swiss-Prot number Q0VF58). The peptides were synthesized on an ABI433A peptide synthesizer (Applied Biosystems) with 0.25 mm Fmoc-Gly-PEG-PS resin, a 4-fold excess of Fmoc-amino acids, and O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate as activating agent. The Fmoc-amino acids carried the protection groups Cys-Trt, Hyp-t-Bu, Gln-Trt, His-Trt, Ser-t-Bu, Glu-O-t-Bu, Arg-Pbf, Asn-Trt, and Tyr-t-Bu. The peptides were cleaved off of the resin and deprotected for 4 h at room temperature with 90% trifluoroacetic acid, 5% thioanisole, 3% 1,2-ethanedithiol, 2% anisole. Subsequently, the peptides were precipitated in cold ether, redissolved in H2O, and lyophilized. The reduced peptides were then purified by reverse phase HPLC using a C18 column (50 × 250 mm, 10-15-;m particle size, 300-Á pores; Vydac, Hesperia, CA) with an acetonitrile/water gradient and 0.1% trifluoroacetic acid as an ion-pairing agent. Finally, the peptides were characterized by electrospray/quadrupole/time-of-flight mass spectrometry (Q-Tof micro, Waters Associates). Oxidation of NC1-containing Peptides and Separation of Oligomers—The lyophilized, reduced peptides were dissolved in degassed and N2-saturated 50 mm sodium phosphate buffer, pH 8.0, under N2 atmosphere and were kept at 4 °C for 24 h in order to allow triple helix formation prior to oxidation. Oxidation was induced by the addition of reduced (10 mm) and oxidized (1 mm) glutathione and exposure to atmospheric O2. The pH was raised to 8.3 with a saturated solution of Tris. Oxidation was carried out for 5-7 days, and the peptide mass was periodically analyzed by liquid chromatography/mass spectrometry. To separate covalently linked oligomers, the oxidized crude material was dissolved in deionized 8 m urea containing 0.1% trifluoroacetic acid to prevent disulfide exchange and applied to a sieve column (BioSep-SEC-S 2000, 600 × 20 mm; Phenomenex). Fractions were characterized by electrospray/quadrupole/time-of-flight mass spectrometry. Trimer-containing fractions were pooled and further purified by reverse phase HPLC using a C18 column with an acetonitrile/water gradient and 0.1% trifluoroacetic acid. Cloning, Expression, and Purification of NC2, (GPP)10NC2, and NC2(GPP)10—To facilitate expression and folding of short peptides containing the NC2 domain of human collagen XIX, they were cloned as part of fusion molecules with a His-tagged minifibritin that has a thrombin cleavage site (HT-mf-thr). Minifibritin is an obligatory trimer with both its amino and carboxyl termini exposed to solvent. The modified version of minifibritin (HT-mf-thr) has recently been used to successfully express, initiate folding, and stabilize a set of collagen fragments (15Bachmann A. Kiefhaber T. Boudko S. Engel J. Báchinger H.P. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13897-13902Crossref PubMed Scopus (45) Google Scholar, 16Boudko S.P. Engel J. J. Mol. Biol. 2004; 335: 1289-1297Crossref PubMed Scopus (41) Google Scholar). The plasmid pET23-HisMf (17Boudko S.P. Kuhn R.J. Rossmann M.G. J. Mol. Biol. 2007; 366: 1538-1544Crossref PubMed Scopus (28) Google Scholar) has multiple cloning sites just after the HT-mf-thr gene. The plasmid (clone ID 8322416, NCBI accession number BC113364), containing the full-length human collagen XIX cDNA was purchased from Open Biosystems. The NC2 domain was PCR-amplified using two oligonucleotides: 5′-TGCAGATCTCCGGCTGATGCAGTTTCATTTGAA-3′ (forward primer fNC2; the BglII site is underlined) and 5′-GTCAGTCGACTTAGCCATAAGCTTGGGCAGCCAACATT-3′ (reverse primer rNC2; the SalI site is underlined). Two other constructs for (GPP10)NC2 and NC2(GPP)10 were PCR-amplified in two steps. The DNA fragment coding (GPP10)NC2 was prepared as follows. A synthetic oligonucleotide coding for (GPP)10 (5′-GGTCCGCCTGGCCCACCGGGCCCGCCCGGTCCGCCGGGCCCGCCGGGTCCCCCAGGTCCGCCCGGCCCGCCGGGCCCTCCGGGTCCACCG-3′) was used as a template to amplify the (GPP)10 part, including a complementary end for the NC2 domain, using two oligonucleotides, 5′-TCAGGATCCGGTCCGCCTGGCCCACCGGGC-3′ (forward primer fGPP; the BamHI cloning site is underlined) and 5′-TTCAAATGAAACTGCATCAGCCGGAATCCCGGGTGGACCCGGAGGGCCCGGC-3′ (reversed primer; one half of it is complementary to the end of (GPP)10, and the other half is complementary to the beginning of the NC2 domain). The product of this PCR was purified and used together with the plasmid containing the NC2 domain sequence as a complex annealed template to PCR-amplify the (GPP10)NC2 fragment using two oligonucleotides: fGPP and rNC2. The same strategy was applied to amplify the fragment NC2(GPP)10, using the following primers: 5′-GTTGGCTGCCCAAGCTTATGGGCGGCCGGGTCCGCCTGGCCCACCGGGC-3′ (forward primer; one half of it is complementary to the end of NC2 domain, and the other half is complementary to the beginning of (GPP)10) and 5′-GTCAGTCGACTTACGGTGGACCCGGAGGGCCCGG-3′ (reverse primer rGPP; the SalI site is underlined) for the first step and fNC2 and rGPP for the The PCR-amplified fragments NC2, (GPP)10NC2, and NC2(GPP)10 were by and SalI and cloned the pET23-HisMf using the sites BamHI and so that all three fragments were a part of the fusion the the with the thrombin cleavage and the The DNA were by DNA The were at °C in the after mm) for Purification of the fusion by on a column and of the fragments NC2, (GPP)10NC2, and NC2(GPP)10 after thrombin cleavage were carried out as in the cleavage was at 4 °C for 24 h with thrombin in 50 mm buffer, pH containing mm The fragments two amino acid the native amino acid The peptides were then purified by phase HPLC using a C18 column with an acetonitrile/water gradient and 0.1% trifluoroacetic acid as an ion-pairing agent. The peptides at a which is highly Finally, the peptides were characterized by electrospray/quadrupole/time-of-flight mass spectrometry and amino acid were on a was at were carried out at 20 °C in an using with were on an with of The were for and to the after of the were at for the NC2 peptide to the ;-helical and at for and NC2(GPP)10 to the collagen triple helix were by amino acid of the were on a mechanism in which three by the NC2 domain, to a native collagen triple helix, The is as and are of and native and is the of and are the native and in the region and is the at the and that and are the of to the and the of the NC1 and NC2 of FACIT FACIT collagens human type have at least two collagenous domains, COL1 and and two noncollagenous domains, NC1 and NC2 the NC2 domain is and no sequence homology either NC1 or NC2 domains, with the of the cysteine at the junction COL1 and The of the NC1 domain from to 37 residues and XIV, that the first cysteine in the conserved cysteine to The of NC2 domains, including one on both from 37 to 50 residues and Oxidation of the NC1 of covalently is as an of the folding of the NC1 domain. To the ability of the NC1 domain alone to form a covalently linked we oxidized the NC1 peptide spectrometry of revealed the of two to a and a were separated on a sieve column under Two were to the and the by mass spectrometry. trimer was acid of peptides used in this Open in a The peptide contains the segment of the collagenous domain and the full-length NC1 domain. The of this helix to form a triple helix by at 4 since no was by The at 4 °C and as in the with the NC1 the of the peptide at °C in the of to as in 5Lesage A. Penin F. Geourjon C. Marion D. van der Rest M. Biochemistry. 1996; 35: 9647-9660Crossref PubMed Scopus (32) Google were In addition to and a of a trimer was The purified form the formation of a stable collagen triple helix This to the that the collagen triple helix formation a for the folding and of the NC1 domain. the addition of two to the beginning of the peptide the of the NC1 domain The reduced peptide collagen triple helix formation and the about 90% of the oxidized product of biophysical of the NC1-containing of Open in a To the of the triple helical sequence composition on the trimer we the native sequence with peptide which is to form a stable triple helix. The of the oxidized product of the peptide was even higher not the folding of the NC1 domain not on the composition of the triple helical The sequence of the NC1 domain is amino acids To its on folding, we synthesized a the residues of the NC1 domain are The of the oxidized trimer was the same as for the The of the triple helix in the oxidized form of the two peptides a of °C the part of the NC1 domain for of the triple helix, either a of the part with the triple helix or by the of the disulfide of the NC2 an of the NC2 domain, and The far that the NC2 domain has an ;-helical at 20 The of the NC2 domain was by revealed a mass of which is with a mass of the chain is at that the NC2 domain forms a highly stable that requires a of for The of is by the of the reverse temperature of the NC2 domain in a with m the the ;-helical was stable to of biophysical of the Open in a of the NC2 domain. were in 50 mm sodium phosphate buffer, pH 8.0, with m m m 4 m and m using mm peptide and a The was at with a of and are for the with m to the of the The NC2 the chain peptide with the sequence is able to form a collagen triple helix of The temperature for is a and is about °C at in S. Boudko S. K. T. Engel J. Báchinger H.P. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). It has been that the of (GPP)10 can by fusion to an obligatory stable trimer S. D. T. J. Y. A. Báchinger H.P. Engel J. J. Mol. Biol. 2001; PubMed Scopus Google Scholar) or by of all three S. Boudko S. K. T. Engel J. Báchinger H.P. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar, D. S. C. L. 2003; PubMed Scopus Google Scholar). of the (GPP)10 triple helix by trimerization is from both and carboxyl-terminal although the is highly S. Boudko S. K. T. Engel J. Báchinger H.P. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). of both and NC2(GPP)10 are in and significantly from the of the isolated NC2 domain of the NC2 domain from either or NC2(GPP)10 and of the the of the collagen triple helical in both peptides The (GPP)10 part in the peptide has higher of triple helix then in at at which the collagen triple helix can show a of the triple helix in both The are and °C for and NC2(GPP)10, The of the triple helical is the of the NC2 domain the by the NC2 domain. The in the also with the in the of the triple helical and are to a of for collagen S.P. Engel J. J. Mol. Biol. 2004; 335: 1289-1297Crossref PubMed Scopus (41) Google Scholar, J. Báchinger H.P. Matrix Biol. PubMed Scopus (45) Google Scholar, Y. Protein Sci. 2004; 13: PubMed Scopus Google Scholar). of the collagen triple helices in both peptides as a since three linked by the NC2 domain. A of the that the of and are for the NC2(GPP)10 peptide with the peptide and on the peptides containing the NC1 domain of collagen XIX that a stable triple helical region is for the disulfide formation in the junction region the COL1 and NC1 The full-length NC1 domain together with the residues of the COL1 domain is not of trimerization and disulfide The of a sequence that can form a stable collagen triple helix is to initiate disulfide The of the amino acid sequence in the triple helix is not for disulfide formation; the sequence can successfully with the artificial the of the carboxyl-terminal half of the NC1 domain sequence has no on the of the oxidized The same features are in the collagen carboxyl-terminal disulfide S.P. Engel J. J. Mol. Biol. 2004; 335: 1289-1297Crossref PubMed Scopus (41) Google Scholar, Báchinger H.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of the disulfide significantly the temperature of the collagen triple helix. This is very for all FACIT collagens, since the carboxyl-terminal collagenous region COL1 has a number of interruptions that significantly the triple helix. The of the disulfide just at the end of the collagen domain as a the carboxyl-terminal end of the collagen triple helix from or The NC2 domain alone forms a highly stable trimer with a higher than °C under This is the first that the trimerization abilities of the NC2 domain were The NC2 domain has an ;-helical and a as by (8McAlinden A. Smith T.A. Sandell L.J. Ficheux D. Parry D.A.D. Hulmes D.J.S. J. Biol. Chem. 2003; 278: 42200-42207Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). are that can form a of different including and of J. Trends Cell Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). contain a characteristic sequence whose are a to The in a is by at the a and with residues at the and A. Trends Biochem. Sci. 1996; Full Text PDF PubMed Scopus Google Scholar). The in the NC2 domains of FACIT collagens have interruptions and of and residues (8McAlinden A. Smith T.A. Sandell L.J. Ficheux D. Parry D.A.D. Hulmes D.J.S. J. Biol. Chem. 2003; 278: 42200-42207Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The NC2 domain is two adjacent collagenous domains, and its the of triple the a to form a are a of and ;-helical having four or with Y. J. W. M. 14: Full Text Full Text PDF PubMed Scopus Google Scholar, J. Y. C. M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, J. Y. C. M. Biochemistry. PubMed Scopus Google Scholar), although of the collagen of NC2 residues on a ;-helical of on one that with two other to form a The trimerization and of the collagen triple helix on either end of the NC2 domain suggests a for this domain in chain chain and of the triple helix formation in all FACIT collagens. This is in with studies on collagen IX, others that the COL2-NC2 region alone is not sufficient for trimerization (7Jáálinoja J. Ylóstalo J. Beckett W. Hulmes D.J.S. Ala-Kokko L. Biochem. J. 2008; 409: 545-554Crossref PubMed Scopus (6) Google Scholar). this a different of the NC2 domain in type IX a is In studies, we the NC2 domain of type IX collagen is responsible for chain selection of the FACIT no mutations are in NC2 domains of it to residues are for folding, and for chain To the of the NC2 domain, the of an by or is We for to the NC1 domain of mouse collagen XIX and and for peptide peptide mass amino acid and DNA