HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pihlajamaa, T. Articles by Ala-Kokko, L. Search for Related Content PubMed PubMed Citation Articles by Pihlajamaa, T. Articles by Ala-Kokko, L. Social Bookmarking                      What's this?

J Biol Chem, Vol. 274, Issue 32, 22464-22468, August 6, 1999

Characterization of Recombinant Human Type IX Collagen
ASSOCIATION OF  CHAINS INTO HOMOTRIMERIC AND HETEROTRIMERIC MOLECULES^*

Tero Pihlajamaa, Merja Perälä^§, Mirka M. Vuoristo, Minna Nokelainen, Michael Bodo^¶, Therese Schulthess, Eero Vuorio^§, Rupert Timpl^**, Jürgen Engel, and Leena Ala-Kokko

From the  Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, Kajaanintie 52A, FIN-90220 Oulu, Finland, the ^§ Department of Medical Biochemistry and Molecular Biology, University of Turku, 20520 Turku, Finland, ^¶ FibroGen Inc., South San Francisco, California 94080, the  Department of Biophysical Chemistry, Biozentrum of the University, CH 4056 Basel, Switzerland, and the ^** Max-Planck-Institut für Biochemie, D-8033 Martinsried, Federal Republic of Germany

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

As type IX collagen is a minor cartilage component, it is difficult to purify sufficient amounts of it from tissues or cultured cells to study its structure and function. Also, the conventional pepsin digestion used for fibrillar collagens cannot be utilized for purifying type IX collagen, because it contains several interruptions in its collagenous triple helix. A baculovirus expression system was used here to produce recombinant human type IX collagen by coinfecting insect cells with three viruses containing full-length cDNAs for the 1(IX), 2(IX), and 3(IX) collagen chains together with a double promoter virus for the  and  subunits of human prolyl 4-hydroxylase. Correctly folded recombinant type IX collagen was secreted, consisting of the three  chains in a 1:1:1 ratio and showing the expected biphasic thermal melting profile. When the individual  chains were expressed, disulfide-bonded homotrimers and homodimers of the  chains were observed. When the cells were coinfected with the viruses for all three  chains, heterotrimers of 1(IX), 2(IX), and 3(IX) were detected in cell culture medium, and the other possible combinations were less prominent. When any two of the  chains were co-expressed, in addition to the homodimers and homotrimers, only 1(IX) and 3(IX) chains were disulfide-bonded. The results thus suggest that the most favored molecular species is an 1(IX)2(IX)3(IX) heterotrimer, but the chains are also able to form disulfide-bonded heterotrimers of 1(IX) and 3(IX) chains and (1(IX))₃, (2(IX))₃, and (3(IX))₃ homotrimers.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Type IX collagen, which belongs to the group of fibril-associated collagens with interrupted triple helices, is a component of hyaline cartilage, intervertebral discs, and the vitreous body. The molecule is a heterotrimer consisting of three genetically distinct chains, 1(IX), 2(IX), and 3(IX) (1) and possesses three collagenous domains (COL1 to COL3, numbered from the C terminus) flanked by four noncollagenous domains (NC1 to NC4) (2, 3). Type IX collagen is also a proteoglycan, because a glycosaminoglycan side chain is covalently attached to the NC3 domain of the 2(IX) chain (4).

Hyaline cartilage contains mixed fibrils of types II, IX, and XI collagens, of which type II is the major component. Type XI collagen is an internal component of the fibril, whereas type IX collagen is located on the surface. Covalent lysine-derived cross-links between the central COL2 region of the 3(IX) chain and the C-telopeptide of type II collagen and between the N-terminal ends of the COL2 domains of all the type IX collagen  chains and the N-telopeptide of type II collagen stabilize the interaction between type II and IX collagens (5-8). The flexibility of the NC3 domain of type IX collagen allows the COL3 and NC4 domains to project from the fibril surface, possibly to mediate interactions between cartilage collagens and noncollagenous proteins (6, 9, 10).

The association of  chains in proper stoichiometry and register is a prerequisite for the formation of a stable collagen helix. The mechanism of chain selection and association has been studied most extensively in the fibrillar collagens, for which the crucial role of large C-terminal propeptides in chain selection and association has been demonstrated. These propeptides contain specific recognition sequences that direct the association of  chains in a collagen type-specific manner (11, 12). For example, the fibrillar collagenous polypeptides synthesized by chondrocytes, namely 1(II), 1(XI), and 2(XI), are found only as two trimeric molecules in vivo, (1(II))₃ and 1(XI)2(XI)1(II), despite the 10 theoretically possible combinations (13).

The factors responsible for chain selection and assembly in the case of the fibril-associated collagens with interrupted triple helices are likely to be different from those affecting the fibrillar collagens, because their C-terminal NC1 domains are much smaller and there is no appreciable homology with the C-propeptides of the fibrillar collagens. The chain assembly of type IX collagen has been studied both in vitro and in vivo. Labourdette and van der Rest (14) carried out chain association experiments in vitro using the polypeptide components of a pepsin-resistant low molecular weight fragment (15) isolated from bovine cartilage and showed that homotrimers can be formed, especially by the 1(IX) and 2(IX) chains, although the heterotrimer 123 was the predominant molecule formed when all three chains were present. Similar results were obtained using synthetic peptides containing the complete NC1 domains and five C-terminal Gly-X-Y tripeptide units of the COL1 domain (16). These peptides were able to associate into trimers stabilized by disulfide bonds, thus indicating a significant role of the NC1 domain in chain selection and association. Interestingly, generation of a mouse line harboring an inactivated Col9a1 gene (17) led to a functional knockout of all type IX collagen polypeptides, suggesting that homotrimers or heterotrimers of the 2(IX) and 3(IX) chains do not exist in vivo without the 1(IX) chain (18).

In the present work, we have used a baculovirus expression system to produce recombinant human type IX collagen in insect cells in order to study the structure and chain assembly of type IX collagen. We report here for the first time on the production of type IX collagen, which consists of three different  chains, in insect cells simultaneously with the tetrameric enzyme prolyl 4-hydroxylase, needed for the production of stable collagen.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Construction of Full-length cDNAs for Type IX Collagen  Chains and Generation of the Recombinant Viruses-- Total RNA was extracted from human fetal cartilage of several individuals by the guanidium isothiocyanate method, and about 1 µg of total RNA was reverse-transcribed using an oligo(dT) primer and Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.). Aliquots of cDNA were used for a single step amplification by polymerase chain reaction (Expand^TM long template polymerase chain reaction system; Roche Molecular Biochemicals) using oligonucleotide primers for the 5'- and 3'-ends of the three  chains. Specific oligonucleotide primers for the 1(IX) chain were designed based on the published sequences (19, 20). The oligonucleotide MH-18 (ACT CCC TTG CGG CCG CTT CTT CAT AGG), corresponding to the 5'-noncoding sequence of the 1(IX) cDNA, contained an engineered NotI cleavage site, and the oligonucleotide MH-19 (TCA TGC AGA CGG CCG TGC AGC AGT AAG), corresponding to the 3'-noncoding sequence, contained an engineered EagI cleavage site. For amplification of the 2(IX) cDNA, the specific oligonucleotides MH-22 (TCT GCC GTC GGT GCG GCC GCG GAC ACG C), corresponding to the 5'-noncoding sequence of the 2(IX) cDNA, and MH-23 (TCA TGC AGA CGG CCG TGC AGC AGT AAG), corresponding to the 3'-noncoding sequence, were designed based on published sequences (21, 22). The oligonucleotide MH-22 contained an engineered NotI cleavage site, and MH-23 contained an engineered EagI cleavage site. Specific oligonucleotides for the 5'- and 3'-ends of the 3(IX) cDNA were designed based on the published sequences (23). The oligonucleotides MH-29 (CCC GAC GCC GCA GTC TAG ACT CCG CCA CGC), corresponding to the 5'-noncoding sequence of the 3(IX) cDNA, and MH-30 (TCG GGC GTC CTT GTC TCT AGA TTC CTC ACG), corresponding to the 3'-noncoding sequence, contained engineered XbaI cleavage sites. The cDNAs were digested with the enzymes indicated above and ligated into the pVL1392 vector. The cDNAs were completely sequenced (Sequenase^TM reagent kit, Amersham Pharmacia Biotech) using cDNA-specific sequencing primers. The three recombinant constructs were co-transfected into Spodoptera frugiperda (Sf9; Invitrogen) insect cells with a modified Autographa californica nuclear polyhedrosis virus by means of the BaculoGold transfection kit (Pharmingen), and the resultant viral pools were collected, amplified, and plaque-purified (24).

Expression of Recombinant Type IX Collagen-- Sf9 or Trichoplusia ni (High Five; Invitrogen) insect cells were cultured in monolayers in TNM-FH medium (Sigma) supplemented with 10% fetal bovine serum (Bioclear), and High Five cells were also cultured in suspension in Sf-900 II SFM medium (Life Technologies, Inc.) supplemented with 5% fetal bovine serum at 27 °C. Prior to the infection, the insect cells were seeded at densities of 5-6 × 10⁵ cells/ml for the expression of recombinant proteins in monolayers and 1 × 10⁶ cells/ml for expression in suspension. The cells were co-infected with three viruses coding for the 1(IX), 2(IX), and 3(IX) chains and a double promoter virus 4PH coding for the - and -subunits of human prolyl 4-hydroxylase (25), with multiplicities of infection of 2:2:2:4, respectively. Ascorbate (80 µg/ml) was added daily to the culture medium.

Isolation of Recombinant Type IX Collagen from Insect Cells-- After 72 h of infection, the High Five cells were detached from the culture plates by pipetting and harvested by centrifugation at 1000 × g for 5 min. Those cultured in suspension were also harvested by centrifugation. Intracellular proteins were extracted from the cells by homogenization in 0.27 M NaCl, 0.2% Triton X-100, and 0.07 M Tris-HCl buffer, pH 7.4, as described earlier (26). The supernatant of the homogenate was stored at 4 °C, and the Triton-insoluble pellet was dissolved in 1% SDS at room temperature for 2 h, after which the insoluble remains were discarded. Alternatively, the cells were suspended and homogenized in 0.75 M NaCl, 0.5 M acetic acid, pH 2, on ice (7.5 × 10⁶ cells/ml) for 30 s using a glass-Teflon homogenizer. The homogenate was centrifuged at 12,000 × g for 20 min at 4 °C, and proteins were precipitated from the supernatant by increasing the NaCl concentration to 3 M and mixing at 4 °C for 12-16 h followed by centrifugation and dissolving of the pellet in 50 mM acetic acid (27). Homogenization was performed either without protease inhibitors or in the presence of 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µM E-64, 1 µM leupeptin, 1 µM pepstatin, and 75 nM aprotinin, separately or in various combinations.

Purification of Recombinant Protein from Culture Medium-- The presence of type IX collagen in the culture medium was assayed by dialyzing the medium against 50 mM acetic acid followed by SDS-PAGE¹ and Western blotting with the monoclonal antibody 95D1A.² To purify the type IX collagen, the proteins were precipitated from the culture medium by adding solid ammonium sulfate to a 25% saturation and placing the mixture on ice for 1 h. The precipitate was collected by centrifugation at 12,000 × g for 20 min at 4 °C and dissolved in 0.5 M urea, 0.2 M NaCl, and 0.05 M Tris-HCl buffer, pH 7.4, at 4 °C overnight to a concentration of about 1 mg/ml. The dissolved recombinant protein was then purified by gel filtration through Sephacryl S-300HR in the same buffer. Further purification was achieved by cation exchange on a CM-Sepharose fast flow column in a buffer of 2 M urea, 50 mM PIPES, and 20 mM NaCl at pH 6.5, eluting with an increasing NaCl concentration gradient (0.02-1 M NaCl).

Characterization of Recombinant Type IX Collagen-- The recombinant protein isolated was characterized by SDS-PAGE followed by staining with Coomassie Brilliant Blue or Western blotting with the monoclonal antibody 95D1A. The purified material was dialyzed against 50 mM acetic acid, hydrolyzed in 6 M HCl at 110 °C for 16 h and subjected to amino acid analysis in an Applied Biosystems 421 analyzer. The thermal stability of the material was determined by CD analysis at a fixed wavelength (221 nm), raising the temperature linearly at a rate of 60 °C/h (28). For N-terminal sequencing, purified recombinant type IX collagen was electrophoresed under reducing conditions and transferred to ProBlott polyvinylidene difluoride-type membrane, and the excised bands were subjected to Edman degradation with 477/120A liquid-phase protein/peptide sequencer (Applied Biosystems).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Expression of Recombinant Human Type IX Collagen in Insect Cells-- Three recombinant viruses, each coding for one of the three  chains of human type IX collagen, were generated and used to infect High Five cells together with a double promoter virus, 4PH (25), coding for the  and  subunits of human prolyl 4-hydroxylase. The cells were harvested after 72 h of culture and homogenized in a buffer containing Triton X-100, as used to isolate other recombinant collagens from insect cells (26, 29) and to extract type IX collagen from tissues. No recombinant protein could be detected in the Triton X-100 soluble protein fraction by Coomassie staining, but individual  chains were clearly detectable in the insoluble fraction (not shown). Selective salt precipitation, which had been used previously to extract type IX collagen from tissues (27), was therefore used to isolate intracellular type IX collagen. The amount of intracellular type IX collagen produced by the insect cells was estimated by comparison with known amounts of Coomassie-stained recombinant human type II collagen to be 4-8 mg/liter of culture (not shown). However, analyses of the isolated material by Western blotting with the 95D1A antibody repeatedly revealed that the material was partially degraded, an effect that was not significantly reduced by the use of various protease inhibitors (not shown).

Purification of Type IX Collagen from the Culture Medium-- The presence of type IX collagen in the culture medium was shown by SDS-PAGE and Western blotting of dialyzed medium using the 95D1A antibody (Fig. 1). In comparison with the intracellular material extracted by acid/NaCl and precipitated with 3 M NaCl, the recombinant type IX collagen present in the medium appeared to be less seriously degraded (Fig. 1), and therefore a protocol for isolating and purifying type IX collagen from the culture medium was designed. Addition of solid ammonium sulfate to a 25% saturation resulted in specific precipitation of the type IX collagen (Fig. 2), and analysis of the supernatant from this precipitation by SDS-PAGE followed by Coomassie staining or Western blotting showed that only a minor amount of type IX collagen had remained in solution (results not shown). Up to 10 mg of type IX collagen was obtained from 1 liter of culture medium. The precipitated type IX collagen was dissolved in a buffer containing 0.5 M urea to enhance the solubilization of the protein, and the solution was chromatographed on a Sephacryl S-300HR column in the same buffer. Analysis of the fractions containing most of the type IX collagen indicated removal of low molecular weight contaminants, e.g. the remaining bovine serum albumin (Fig. 3, lane 3). Further purification was achieved by cation exchange chromatography on a CM-Sepharose fast flow column (Fig. 3, lane 4).

View larger version (42K): [in this window] [in a new window]   Fig. 1.   Comparison of the intracellular and secreted recombinant human type IX collagen by SDS-PAGE and Western blotting. After 72 h of coinfection of High Five cells with the recombinant baculoviruses for the 1(IX), 2(IX), and 3(IX) chains and the 4PH virus, intracellular type IX collagen was extracted from the cells in 0.75 M NaCl/0.5 M acetic acid and precipitated in 3 M NaCl in the presence of protease inhibitors. The culture medium from the expression was dialyzed against acetic acid and analyzed for the presence of extracellular type IX collagen. Samples were electrophoresed by 8% SDS-PAGE under reducing conditions and analyzed by Western blotting with a monoclonal antibody recognizing collagenous structures. Lane 1, molecular weight markers; lane 2, intracellular type IX collagen; lane 3, extracellular type IX collagen.

View larger version (58K): [in this window] [in a new window]   Fig. 2.   Efficiency of ammonium sulfate in selective precipitation of secreted recombinant type IX collagen. After 72 h of coinfection of High Five cells with the recombinant baculoviruses for the 1(IX), 2(IX), and 3(IX) chains and the 4PH virus, the culture medium was clarified by centrifugation, and solid ammonium sulfate was added to achieve the concentrations indicated (percentage of saturation). After 1 h, the precipitates were collected by centrifugation, dissolved, subjected to 10% SDS-PAGE under reducing conditions, and analyzed by Coomassie staining. Lane 1, molecular weight markers; lanes 2-6, extracellular material precipitated in the concentrations of ammonium sulfate indicated (calculated as saturation percentages).

View larger version (37K): [in this window] [in a new window]   Fig. 3.   Analysis of the efficiency of gel filtration and cation exchange in the purification of recombinant human type IX collagen. Type IX collagen was precipitated from the culture medium by adding ammonium sulfate to 25% saturation, and the precipitate was collected, dissolved (lane 2), and subjected to chromatography on a Sephacryl S-300HR gel filtration column (lane 3), followed by cation exchange on a CM-Sepharose fast flow column (lane 4). Samples of the unpurified ammonium sulfate precipitate and the chromatographed material were subjected to 5% SDS-PAGE under nonreducing conditions in A and to 10% SDS-PAGE under reducing conditions in B, followed by Coomassie staining. Lane 1, molecular weight markers.

Analysis of the Purified Recombinant Human Type IX Collagen-- The results of amino acid analysis of the purified material corresponded well with calculated values for human type IX collagen (Table I), and using these values, a purity of over 90% was estimated for the recombinant type IX collagen. The melting behavior of the recombinant type IX collagen was analyzed by CD. The profile was biphasic, with about of the transition centering at T_m = 37.5 °C and about of the transition centering at T_m = 46.0 °C (Fig. 4).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   CD analysis of denaturation of the purified recombinant type IX collagen. Denaturation was monitored by the change of mean molar ellipticity in 0.05 M acetic acid at a fixed wavelength (221 nm). The estimated Tm values were 37.5 °C (about of transition) and 46.0 °C (about of transition).

The amino acid sequence at the N terminus of the 1(IX) chain, as identified by Edman degradation, was AVKRRPR, corresponding to the predicted signal peptide cleavage site (19). The sequences for the 2(IX) and 3(IX) chains could not be determined, apparently because of N-terminal blocking. The tendency of the N termini of these two chains to get blocked may be explained by possible presence of glutamine (22, 23) at the N terminus of the polypeptides (30).

Association of  Chains into Disulfide-bonded Molecules-- The formation of disulfide-bonded homomeric and heteromeric molecules by the  chains was studied by expressing each chain individually and in all possible combinations together with prolyl 4-hydroxylase. Analyses of samples of the culture media by SDS-PAGE followed by Western blotting with the 95D1A antibody showed that all three  chains of human type IX collagen appear to be capable of forming disulfide-bonded homodimers. Whereas 1(IX) chains showed clear homotrimer formation, the homotrimers of 2(IX) and 3(IX) chains were not readily detectable (Fig. 5). When all three chains are expressed simultaneously, the heterotrimer 1(IX)2(IX)3(IX) is the dominant molecular species. However, it is possible that also other disulfide-bonded trimers are formed, but in quantities that are below the detection limit. Interestingly, co-expression of any two  chains results in formation of detectable amounts of disulfide-bonded heterodimeric molecules only when the chains concerned are 1(IX) and 3(IX) (Fig. 5). Any other combination of two  chains appears to result only in the formation of disulfide-bonded homodimers (Fig. 5). Abundance of monomeric molecules seen in Fig. 5 is likely to reflect the release of the monomers into the culture medium due to cell lysis during the expression. Second, some of the monomers may originate from secreted trimeric molecules that were not fully disulfide-bonded. Also, the abundance of monomers is in part an artifact, because the efficiency of electroblotting is inversely related to the size of the blotted molecules.

View larger version (41K): [in this window] [in a new window]   Fig. 5.   Analysis of type IX collagen chain association. The recombinant  chains were expressed individually and in all possible combinations together with prolyl 4-hydroxylase in an adherent culture of High Five cells. Samples of the culture media were analyzed by SDS-PAGE under nonreducing conditions and Western blotting with the antibody 95D1A. Molecular species were identified by differences in electrophoretic mobility. Lane 1, prolyl 4-hydroxylase expressed alone; lane 2, 1(IX); lane 3, 2(IX); lane 4, 3(IX); lane 5, 1(IX) and 2(IX); lane 6, 1(IX) and 3(IX); lane 7, 2(IX) and 3(IX); lane 8, all three  chains co-expressed.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

As type IX collagen is a minor cartilage component that is covalently cross-linked to type II collagen fibrils and has interruptions in its triple helix, it has been difficult to isolate intact type IX collagen molecules from tissues. We have now produced and isolated intact heterotrimeric human type IX collagen for the first time using a baculovirus expression system. To obtain stable type IX collagen, a double promoter virus for the  and  subunits of prolyl 4-hydroxylase (25) was co-expressed with three viruses for the  chains of type IX collagen itself.

Our initial attempts to purify intracellular type IX collagen failed because the protein was insoluble in a buffer containing Triton X-100 and was easily degraded after acid extraction and selective salt precipitation. Unlike the intracellular material, type IX collagen purified from the medium migrated as a single band of over 200 kDa in SDS-PAGE under nonreducing conditions, and reduction indicated that the material consisted of three  chains in a 1:1:1 ratio. It is likely that the three viruses for the  chains and the virus for prolyl 4-hydroxylase do not infect all the insect cells with equal efficiency, which in turn may lead to the intracellular accumulation of underhydroxylated and improperly folded molecules that are insoluble or susceptible to degradation.

Amino acid analysis of the secreted trimeric type IX collagen showed that the composition of the material was in agreement with composition expected based on the cDNA deduced amino acid sequence. An adequate degree of 4-hydroxylation of the Y-position prolines in the Gly-X-Y sequences is required to stabilize the collagen triple helix (31), but the extent of prolyl 4-hydroxylation in type IX collagen is currently not known. That in the recombinant type IX collagen was about 80% of the theoretical maximum. CD analysis indicated that this degree of hydroxylation is adequate, because the T_m values of the recombinant type IX collagen corresponded well to the values reported for type IX collagen isolated from tissues (32). The transition profile was biphasic because the COL3 domain has a higher thermal stability than the rest of the molecule (32). The results of CD analysis also indicate proper folding of the recombinant type IX collagen.

The results presented here show that all recombinant type IX collagen  chains are able to form disulfide-bonded homodimers, whereas only the 1(IX) chains show homotrimer formation. These findings support previous observations that the C-terminal fragments of 1(IX) chains exhibit the highest potential for homotrimer formation and 3(IX) chains have the lowest ability for self-association (16). Monomers and disulfide-bonded dimeric molecules were also detected in the culture medium. Because collagens are secreted as trimeric molecules, these species may originate from secreted trimeric molecules that were not fully disulfide-bonded. Alternatively, the monomers and dimers could represent immature molecules that are released into the culture medium as a result of cell lysis.

In a reassociation study using pepsin-resistant C-terminal low molecular weight fragments of bovine type IX collagen, Labourdette and van der Rest (14) found some formation of homomeric molecules in addition to the expected 123 heterotrimer upon mixing of fragments of all three  chains. Our results obtained using full-length  chains indicate that the heterotrimer is the predominant product formed. It is therefore possible that the C termini of the NC1 domains that are missing in the low molecular weight fragments enhance formation of the heterotrimer in preference to homotrimers. Because our results were obtained using secreted material, it is possible that homotrimeric molecules are also formed but remain mostly intracellular. It is also possible that homotrimeric molecules are present in the culture medium, but in very low quantities.

Two cysteine residues located at the COL1/NC1 junction in all three (IX) chains are involved in interchain disulfide bond formation. The functionality of these cysteines in connecting together any two of the  chains has been demonstrated by Labourdette and van der Rest (14), who were able to detect all possible dimeric combinations in minor quantities. In the present case, however, 13 was the only heterodimeric molecular structure detected. It is possible that the other two disulfide-bonded heterodimeric species were also formed but in quantities that were below the detection limit of the experimental system. Similarly, it is possible that in a reassociation study using synthetic NC1 domains (16), heterodimeric molecules were formed but remained undetected. Even though our results were obtained with a semiquantitative analysis, it appears that co-expression of the 1(IX) and 3(IX) chains results in formation of the heterodimer, which dominates the respective homodimers. The results thus suggest that in the association of full-length type IX collagen  chains the most favored trimeric form is an 1(IX)2(IX)3(IX) heterotrimer, although 1(IX) chains are capable of forming disulfide-bonded homotrimeric molecules.

	ACKNOWLEDGEMENT

We thank Helena Lindqvist for expert technical assistance.

	FOOTNOTES

^* This work was supported in part by the European Community project BIO-4-CT96-0537 (to L. A.-K. and R. T.) and grants from the Academy of Finland (to L. A.-K. and M. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 358-8-5375756; Fax: 358-8-5375811; E-mail: Leena.Ala-Kokko@oulu.fi.

² This antibody was generated using a collagenous fragment of recombinant human type XIII collagen as an antigen. It was found to recognize the collagen domains of various denatured collagen chains (A. Snellman and T. Pihlajaniemi, unpublished observations).

	ABBREVIATIONS

The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; T_m, midpoint temperature of thermal transition; PIPES, 1,4-piperazinediethanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				M. Fresquet, T. A. Jowitt, J. Ylostalo, P. Coffey, R. S. Meadows, L. Ala-Kokko, D. J. Thornton, and M. D. Briggs Structural and Functional Characterization of Recombinant Matrilin-3 A-domain and Implications for Human Genetic Bone Diseases J. Biol. Chem., November 30, 2007; 282(48): 34634 - 34643. [Abstract] [Full Text] [PDF]

				B. Budde, K. Blumbach, J. Ylostalo, F. Zaucke, H. W. A. Ehlen, R. Wagener, L. Ala-Kokko, M. Paulsson, P. Bruckner, and S. Grassel Altered Integration of Matrilin-3 into Cartilage Extracellular Matrix in the Absence of Collagen IX Mol. Cell. Biol., December 1, 2005; 25(23): 10465 - 10478. [Abstract] [Full Text] [PDF]

				J. Kapyla, J. Jaalinoja, M. Tulla, J. Ylostalo, L. Nissinen, T. Viitasalo, P. Vehvilainen, V. Marjomaki, P. Nykvist, A.-M. Saamanen, et al. The Fibril-associated Collagen IX Provides a Novel Mechanism for Cell Adhesion to Cartilaginous Matrix J. Biol. Chem., December 3, 2004; 279(49): 51677 - 51687. [Abstract] [Full Text] [PDF]

				T. Pihlajamaa, H. Lankinen, J. Ylostalo, L. Valmu, J. Jaalinoja, F. Zaucke, L. Spitznagel, S. Gosling, A. Puustinen, M. Morgelin, et al. Characterization of Recombinant Amino-terminal NC4 Domain of Human Collagen IX: INTERACTION WITH GLYCOSAMINOGLYCANS AND CARTILAGE OLIGOMERIC MATRIX PROTEIN J. Biol. Chem., June 4, 2004; 279(23): 24265 - 24273. [Abstract] [Full Text] [PDF]

				J. Karppinen, E. Paakko, P. Paassilta, J. Lohiniva, M. Kurunlahti, O. Tervonen, P. Nieminen, H. H. H. Goring, A. Malmivaara, H. Vanharanta, et al. Radiologic Phenotypes in Lumbar MR Imaging for a Gene Defect in the COL9A3 Gene of Type IX Collagen Radiology, April 1, 2003; 227(1): 143 - 148. [Abstract] [Full Text] [PDF]

				P. Paassilta, J. Lohiniva, H. H. H. Goring, M. Perala, S. S. Raina, J. Karppinen, M. Hakala, T. Palm, H. Kroger, I. Kaitila, et al. Identification of a Novel Common Genetic Risk Factor for Lumbar Disk Disease JAMA, April 11, 2001; 285(14): 1843 - 1849. [Abstract] [Full Text] [PDF]

				A. Snellman, M.-R. Keranen, P. O. Hagg, A. Lamberg, J. K. Hiltunen, K. I. Kivirikko, and T. Pihlajaniemi Type XIII Collagen Forms Homotrimers with Three Triple Helical Collagenous Domains and Its Association into Disulfide-bonded Trimers Is Enhanced by Prolyl 4-Hydroxylase J. Biol. Chem., March 17, 2000; 275(12): 8936 - 8944. [Abstract] [Full Text] [PDF]

				P. Paassilta, T. Pihlajamaa, S. Annunen, R. G. Brewton, B. M. Wood, C. C. Johnson, J. Liu, Y. Gong, M. L. Warman, D. J. Prockop, et al. Complete Sequence of the 23-Kilobase Human COL9A3 Gene. DETECTION OF GLY-X-Y TRIPLET DELETIONS THAT REPRESENT NEUTRAL VARIANTS J. Biol. Chem., August 6, 1999; 274(32): 22469 - 22475. [Abstract] [Full Text] [PDF]

				J. Thur, K. Rosenberg, D. P. Nitsche, T. Pihlajamaa, L. Ala-Kokko, D. Heinegard, M. Paulsson, and P. Maurer Mutations in Cartilage Oligomeric Matrix Protein Causing Pseudoachondroplasia and Multiple Epiphyseal Dysplasia Affect Binding of Calcium and Collagen I, II, and IX J. Biol. Chem., February 23, 2001; 276(9): 6083 - 6092. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pihlajamaa, T. Articles by Ala-Kokko, L. Search for Related Content PubMed <