Abstract
In this study, we test the hypothesis that the carboxyl noncollagenous (NC1) domain of collagen X is sufficient to direct multimer formation without a triple helix. Two peptides containing the NC1 domain of avian collagen X have been synthesized using a bacterial expression system and their properties characterized. One peptide consists only of the NC1 domain, and the other is a chimeric molecule with a noncollagenous A domain of matrilin-1 fused to the N terminus of NC1. The NC1 peptide alone forms a 45-kDa trimer under native conditions, suggesting that NC1 contains all the information for trimerization without any triple helical residues. This trimeric association is highly thermostable without intermolecular disulfide bonds. This indicates that the NC1 domain contributes to the remarkable structural stability of collagen X. Chemical cross-linking of the NC1 trimer results in a series of varying sized multimers, the smallest of which is a trimer. Therefore the NC1 trimer is sufficient to form higher order multimers. The chimeric A-NC1 peptide forms a homotrimer by itself, and a series of heterotrimers with the NC1 peptide via the NC1 domain. Thus the NC1(X) domain directs multimer formation, even in a noncollagenous molecule.
Highlights
In this study, we test the hypothesis that the carboxyl noncollagenous (NC1) domain of collagen X is sufficient to direct multimer formation without a triple helix
Type X collagen is essential for the structural organization of the matrix preceding calcification [3, 4], occurring in two forms: one is a pericellular mat [5], which probably represents a multimeric form of type X collagen itself [6]; the other is in association with type II collagen containing fibrils where it interacts with proteoglycans [7, 8]
The first peptide, NC1, consists of the C-terminal 162 amino acid residues of ␣1(X), and comprises the entire NC1 domain (Fig. 2A). This peptide is devoid of any triple helical (Gly-X-Y) residues, and can be used to test whether the NC1 domain alone is sufficient to form a trimer and higher order multimers
Summary
We test the hypothesis that the carboxyl noncollagenous (NC1) domain of collagen X is sufficient to direct multimer formation without a triple helix. The NC1 peptide alone forms a 45-kDa trimer under native conditions, suggesting that NC1 contains all the information for trimerization without any triple helical residues. This trimeric association is highly thermostable without intermolecular disulfide bonds. The NC1 domain of type X collagen is highly conserved among species [15], with that of the human and avian molecules sharing 77.7% identity at the amino acid level. All the residues within the NC1 domain mutated in the SMCD patients are conserved between human and avian ␣1(X) These data, when taken together, suggest that the NC1 domain of type X from different species plays a similar or identical roles, such as in the assembly of type X collagen
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