Collagenous Sequence Governs the Trimeric Assembly of Collagen XII

Abstract

A minicollagen containing the COL1 and NC1 domains of chicken collagen XII has been produced in insect cells. Significant amounts of trimers contain a triplehelical domain in which the cysteines are not involved in inter-but in intrachain bonds. In reducing conditions, providing that the triple-helix is maintained, disulfide exchange between intra-and interchain bonding is observed, suggesting that the triple-helix forms first and that in favorable redox conditions interchain bonding occurs to stabilize the molecule. This hypothesis is verified by in vitro reassociation studies performed in the presence of reducing agents, demonstrating that the formation of interchain disulfide bonds is not a prerequisite to the trimeric association and triple-helical folding of the collagen XII molecule. Shortening the COL1 domain of minicollagen XII to its five C-terminal GXY triplets results in an absence of trimers. This can be explained by the presence of a collagenous domain that is too short to form a stable triple-helix. In contrast, the presence of five additional C-terminal triplets in COL1 allows the formation of triple-helical disulfide-bonded trimers, suggesting that the presence of a triple-helix is essential for the assembly of collagen XII.Collagens are the most abundant proteins of the extracellular matrix. They consist of collagenous triple-helical domains, flanked by non-collagenous domains, that differ in their size and number from one collagen to another. Collagens can be homotrimers or heterotrimers, and their structure is stabilized by interchain disulfide bonds (1, 2).Collagens IX, XII, XIV, XVI, and XIX constitute the FACITs 1 family that are grouped on the basis of similarities in their short C-terminal domain COL1, which is thought to be important for the association with fibrils (3, 4). The association of collagen type IX, XII, and XIV molecules with quarter-staggered fibrils has been shown by immunofluorescence and electron microscopy (5-9). The biological functions of the FACITsare not yet fully understood. Rapid and reversible production of collagen XII by fibroblasts submitted to mechanical stress has been observed (10, 11). Collagens XII and XIV potentially facilitate the contraction of collagen gels by fibroblasts (12), and they are known to interact with other components of the extracellular matrix (13)(14)(15). This suggests that they contribute to the cohesion and the modulation of the mechanical properties of the extracellular matrix. Collagen XIV is also a potential reservoir for procollagen N-proteinase (16) and promotes the adhesion of different cells (17, 18). The assembly of fibril-forming collagens (collagen types I, II, III, V, and XI) is a complex process in which three chains must fold into a triple-helix. This results from chain association, nucleation, and propagation of the folding occurring from the C to N termini in a zipper-like fashion (19,20). The C-terminal non-triple-helical domains, called the C-propeptides, play an important role in the initial steps of chain associatio...

Trimerization and Triple Helix Stabilization of the Collagen XIX NC2 Domain

“…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 (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).…”

“…To analyze the importance of the triple helical sequence composition on the trimer formation, we substituted the native sequence with (GPO) 6 (Table 1, peptide (GPO) 6 NC1), which is known to form a stable triple helix. The yield of the correctly oxidized product of the (GPO) 6 NC1 peptide was even slightly higher (data not shown).…”

“…The presence of a sequence that can form a stable collagen triple helix is enough to initiate disulfide knot formation. The nature of the amino acid sequence in the triple helix is not important for disulfide knot formation; indeed, the natural sequence can be successfully substituted with the artificial (GPO) 6 sequence. Furthermore, the deletion of the carboxylterminal half of the NC1 domain sequence has no effect on the yield of the correctly oxidized trimers.…”

“…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.…”

The NC2 Domain of Collagen IX Provides Chain Selection and Heterotrimerization

“…The FACITs share a remarkable sequence homology at their COL1/NC1 junctions by having two strictly conserved cysteine residues separated by four residues in the NC1 domain. These cysteines form interchain disulfide bonds, a so-called cystine knot, but only after the triple helix is formed (3)(4)(5)(6). In other words, the NC1 domain cannot trimerize itself and requires exogenous alignment of three chains.…”