Trimerization of collagen IX α-chains does not require the presence of the COL1 and NC1 domains

Abstract

Collagen IX is a heterotrimer of three alpha-chains, which consists of three COL domains (collagenous domains) (COL1-COL3) and four NC domains (non-collagenous domains) (NC1-NC4), numbered from the C-terminus. Although collagen IX chains have been shown to associate via their C-terminal NC1 domains and form a triple helix starting from the COL1 domain, it is not known whether chain association can occur at other sites and whether other collagenous and non-collagenous regions are involved. To address this question, we prepared five constructs, two long variants (beginning at the NC4 domain) and three short variants (beginning at the COL2 domain), all ending at the NC2 domain (or NC2 replaced by NC1), to study association and selection of collagen IX alpha-chains. Both long variants were able to associate with NC1 or NC2 at the C-terminus and form various disulfide-bonded trimers, but the specificity of chain selection was diminished compared with full-length chains. Trimers of the long variant ending at NC2 were shown to be triple helical by CD. Short variants were not able to assemble into disulfide-bonded trimers even in the presence of both conserved cysteine residues from the COL1-NC1 junction. Our results demonstrate that collagen IX alpha-chains can associate in the absence of COL1 and NC1 domains to form a triple helix, but the COL2-NC2 region alone is not sufficient for trimerization. The results suggest that folding of collagen IX is a co-operative process involving multiple COL and NC domains and that the COL1-NC1 region is important for chain specificity.

Trimerization and Triple Helix Stabilization of the Collagen XIX NC2 Domain

“…The trimerization properties and stabilization of the collagen triple helix on either end of the NC2 domain suggests a universal role for this domain in chain selection, chain registration, and initiation of the triple helix formation in all FACIT collagens. This is somewhat in contradiction with studies on collagen IX, where others believe that the COL2-NC2 region alone is not sufficient for trimerization (7). Although this might reflect a different role of the NC2 domain in type IX collagen, a more detailed analysis is required.…”

“…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 (7). This suggests that folding and chain selection of collagen IX is a cooperative process involving multiple COL and NC domains (7). 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 (8), but no experimental evidence has been reported so far.…”

“…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 (3)(4)(5)(6). 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 (7). This suggests that folding and chain selection of collagen IX is a cooperative process involving multiple COL and NC domains (7).…”

The NC2 Domain of Collagen IX Provides Chain Selection and Heterotrimerization

“…On the other hand, 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, although the COL1-NC1 region might be important for chain specificity (16). Additionally, the authors reported that the COL2-NC2 region of collagen IX is not sufficient for trimerization (16).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.…”

“…On the other hand, 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, although the COL1-NC1 region might be important for chain specificity (16). Additionally, the authors reported that the COL2-NC2 region of collagen IX is not sufficient for trimerization (16).…”