Abstract
The mechanism of chain selection and trimerization of fibril-associated collagens with interrupted triple helices (FACITs) differs from that of fibrillar collagens that have special C-propeptides. We recently showed that the second carboxyl-terminal non-collagenous domain (NC2) of homotrimeric collagen XIX forms a stable trimer and substantially stabilizes a collagen triple helix attached to either end. We then hypothesized a general trimerizing role for the NC2 domain in other FACITs. Here we analyzed the NC2 domain of human heterotrimeric collagen IX, the only member of FACITs with all three chains encoded by distinct genes. Upon oxidative folding of equimolar amounts of the alpha1, alpha2, and alpha3 chains of NC2, a stable heterotrimer with a disulfide bridge between alpha1 and alpha3 chains is formed. Our experiments show that this heterotrimerization domain can stabilize a short triple helix attached at the carboxyl-terminal end and allows for the proper oxidation of the cystine knot of type III collagen after the short triple helix.
Highlights
fibrilassociated collagens with interrupted triple helices (FACITs) is able to form an ␣-helical coiled coil, bearing an ability to trimerize those collagens [7]
A recent study of full-length protein and several deletion mutants expressed in insect cells showed that COL1 and NC1 are not required for trimerization of collagen IX, the COL1-NC1 region might be important for chain specificity [16]
Design of Constructs—Constructs containing the NC2 regions of three human collagen IX chains (␣1, ␣2, or ␣3) either extended or not with a collagen triple helical sequence ending with the cystine knot of collagen III (Table 1) were cloned as part of a fusion molecule
Summary
FACITs is able to form an ␣-helical coiled coil, bearing an ability to trimerize those collagens [7]. Upon reduction and reassociation, followed by the formation of disulfide-bonded multimers, only a negligible amount of ␣1␣2␣3 was observed [14] Another in vitro study was focused on either NC1 sequences or NC1 sequences extended with short fragments of COL1 [15]. To explore the trimerization potential of the collagen IX, we have studied folding and stability of the NC2 domain alone as well as in junction with a collagenous sequence containing the type III collagen cystine knot. The cystine knot naturally found in type III collagen is located at the end of the collagenous domain and forms interchain disulfide bonds. The NC2 domain appeared to be an effective heterotrimerization domain that promotes chain selection and folding of the triple helix It must play an important postfolding role in stabilizing the triple helix. Protection of the collagen IX NC2 domain against MMP-3 cleavage can stabilize the integrity of cartilage and prevent onsets of cartilage diseases
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