Michel van der Rest

Synteny between the loci for a novel FACIT-like collagen locus (D6S228E) and α1(IX) collagen (COL9A1) on 6q12–q14 in humans

A 1.8-kb cDNA encoding portion of a novel collagenous chain was isolated from a human rhabdomyosarcoma cell line by cross-hybridization using a chicken type V collagen probe. Sequence analysis suggests that this chain belongs to the recently discovered group of collagens, termed the FACIT class of macromolecules. This cDNA was used to locate the corresponding gene (D6S228E) to chromosome 6, notably at position 6q12-q14. Interestingly, within this region of human chromosome 6 residues the alpha 1 (IX) collagen gene (COL9A1), a member of the FACIT group.

Common topology within a non-collagenous domain of several different collagen types

The secondary structure of a conserved non-collagenous module in alpha 1(V), alpha 1(XI), alpha 1(IX), alpha 1(XII), alpha 1(XIV) and alpha 1(XVI) collagen chains and in proline- and arginine-rich protein was analyzed using different algorithms. The results predict that a common anti-parallel beta-sheet structure composed of nine consensus beta-strands is present in these non-collagenous modules. A model for the packing of these beta-sheets is proposed which suggests that the predicted beta-sheet structure may be involved in molecular recognition functions.

Analysis of the role of the COL1 domain and its adjacent cysteine-containing sequence in the chain assembly of type IX collagen

The mechanisms of chain selection and assembly of type IX collagen, a heterotrimer α1(IX)α2(IX)α3(IX), must differ from that of fibrillar collagens since it lacks the characteristic C‐propeptide of these latter molecules. We have tested the hypothesis that the information required for this process is contained within the C‐terminal triple helical disulfide‐bonded region (LMW). The reassociations of the purified LMW fragments of pepsinized bovine type IX collagen were followed by the formation of disulfide‐bonded multimers. Our data demonstrate that only three triple helical assemblies form readily, (α1)3, (α2)3, and α1α2α3. The information required for chain selection and assembly is thus, at least in part, contained in the studied fragments. Molecular stoichiometries different from the classical heterotrimer may thus also form under certain conditions.

Schmid's metaphyseal chondrodysplasia mutations interfere with folding of the C-terminal domain of human collagen X expressed in Escherichia coli.

Human collagen X contains a highly conserved 161-amino acid C-terminal non-triple helical domain that is homologous to the C-terminal domain of collagen VIII and to the C1q module of the human C1 enzyme. We have expressed this domain (residues 545–680) inEscherichia coli as a glutathione S-transferase fusion protein. The purified fusion protein trimerizes spontaneouslyin vitro, and after thrombin cleavage, the purified C-terminal domain trimer (46.2 kDa) is extremely stable and trypsin-resistant. Mutations within the C-terminal domain have been observed in patients with Schmid’s metaphyseal chondrodysplasia (SMCD). Some of these mutations (Y598D, G618V, W651X, or H669X; X is the stop codon) were constructed by site-directed mutagenesis. Each mutation had identical consequences regarding the fusion protein: 1) absence of trimeric formation, 2) copurification of the ∼60-kDa GroEL chaperone protein, and 3) sensitivity of the monomeric fusion protein to trypsin digestion. These results show that the C-terminal domain of collagen X is sufficient to produce a very stable and compact trimer in the absence of collagen Gly-X-Y repeats. Moreover, mutations causing SMCD interfere in this system with the correct folding of the C-terminal domain. The existence of a similar mechanism in chondrocytes might explain the relative homogeneity of phenotypes in SMCD despite the diversity of mutations.

Involvement of Prolyl 4-Hydroxylase in the Assembly of Trimeric Minicollagen XII STUDY IN A BACULOVIRUS EXPRESSION SYSTEM

We have shown previously that hydroxylation played a critical role in the trimer assembly and disulfide bonding of the three constituent α chains of a minicollagen composed of the extreme C-terminal collagenous (COL1) and noncollagenous (NC1) domains of type XII collagen in HeLa cells (Mazzorana, M., Gruffat, H., Sergeant, A., and van der Rest, M. (1993) J. Biol. Chem. 268, 3029-3032). We have further characterized the involvement of prolyl 4-hydroxylase in the assembly of the three α chains to form trimeric disulfide-bonded type XII minicollagen in an insect cell expression system. For this purpose, type XII minicollagen was produced in insect cells from baculovirus vectors, alone or together with wild-type human prolyl 4-hydroxylase or with the human enzyme mutated in the catalytic site of its α or β subunits or with the individual α or β subunits. When type XII minicollagen was produced alone, negligible amounts of disulfide-bonded trimers were found to be produced by the cells. However, coproduction of the collagen with the two subunits of the wild-type human enzyme dramatically increased the amount of disulfide-bonded trimeric type XII minicollagen molecules. In contrast, coproduction of the collagen with α subunits that had a mutation completely inactivating the human enzyme failed to enhance the trimer assembly. These results directly show that an active prolyl 4-hydroxylase is required for the assembly of disulfide-bonded trimers of type XII minicollagen.

Identification and characterization of a heparin binding site within the NC1 domain of chicken collagen XIV

Collagen XIV is known to bind to the dermatan sulfate chain of decorin and to the heparan sulfate chain of perlecan. To study its possible interaction with glycosaminoglycans, the NC1 domain of chicken collagen XIV was overproduced in E. coli. Purified NC1*(6-119)* appears poorly organized (the asterisks indicate the presence of extension sequences), but V8-protease generated fragments containing the 84-108 basic sequence tend to fold into alpha-helix. These fragments interact specifically with heparin, which induces an alpha-helical fold with a maximum effect for equimolar heparin/peptide ratio. These data demonstrate the existence of a glycosaminoglycan binding site in NC1.

Trimeric assembly of collagen XII: effect of deletion of the C-terminal part of the molecule.

The fibril-associated-collagens-with-interrupted-triple-helices (FACITs) are devoid of large C-propeptides like those involved in the trimeric assembly of the fibrillar collagens. Under these conditions, the C-terminal non triple-helical domain (NC1) and the adjacent triple-helical domain (COL1) are likely to be responsible for the trimeric assembly of these collagen molecules. Using a recombinant minigene of one of the FACITs, collagen XII, we show that a deletion covering most of the NC1 domain, except the first seven residues containing a cysteine and constituting the main part of the conserved junction between the COL1 and NC1 domains, does not prevent the formation of trimeric disulfide-bonded assembly of truncated alpha chains. These results suggest that if the non triple-helical NC1 domain is involved in the initial events governing the trimeric assembly, it must be through its amino acid residues participating in the junction. Our data confirm also the results obtained in a previous paper (Mazzorana et al.: J. Biol. Chem. 268:3029-3032, 1993) showing that the formation of disulfide bonds is dependent on hydroxylation and suggesting that the folding of the triple helix (or a part of it) precedes the formation of the disulfide bonds.

Different splice variants of cartilage α1(XI) collagen chain undergo uniform amino-terminal processing

Collagen XI is found mainly as a component of cartilage fibrils. Among the different transcripts identified by RT-PCR for the alpha1 (XI) chain, the major tissue form has been reported to be the splicing product of exons I, III and V. In this study, two other splice isoforms of the alpha1(XI) chain were identified using N-terminal sequencing. Like the major alpha1(XI) chain, the fully processed isoforms begin at Gln254 within the N-terminal domain encoded by exon I. This sequence is followed by sequences encoded by exon IIA or III. An anti-peptide antibody allowed the identification of the exon IV encoded sequence within both isoforms. Therefore, these isoforms of the alpha1(XI) chain correspond to the splicing of exons I, IIA, III, IV and V or of exons I, III, IV and V, thus presenting larger acidic sequences than the major form. They could mediate strong ionic interactions within the cartilage matrix.

Complete primary structure of the chicken α1(V) collagen chain

Chicken alpha1(V) collagen cDNAs have been cloned by a variety of methods and positively identified. We present here the entire translated sequence of the chick polypeptide and compare selected regions to other collagen chains in the type V/XI family.

Mechanisms of collagen trimer assembly

It is generally accepted that the folding of collagen triple helical domains occur from the C-terminus toward the N-terminus by a “zipper” mechanism. The regions at the C-terminus of the triple helices must therefore play a critical role in the processes of chain recognition and assembly to get the proper stoichiometries and of chain registration to align the chains for the folding of the triple helix. Examination of these regions reveals a broad diversity of structures and suggests that different mechanisms of assembly are used in the various collagen and collagen-like molecules. We review here three different mechanisms that have recently come to light. The collectins, a group of serum proteins containing collagen-like triple helical domains, are assembled through hydrophobic interactions in a tripleα helix. Collagens VIII and X, C1q and several related proteins contain homologous C-terminal domains that are characterized by aβ-pleated sheet structure. They assemble through very strong hydrophobic interactions that probably involve an “aromatic zipper”. Collagens IX, XII and XIV fibril associated collagen with interrupted triple helices (FACITs), are assembled by a mechanism in which both the C-terminal triple helix and a very short cysteine-containing sequence are involved.