Collagenous Sequence Governs the Trimeric Assembly of Collagen XII

A minicollagen containing the COL1 and NC1 domains of chicken collagen XII has been produced in insect cells. Significant amounts of trimers contain a triple-helical domain in which the cysteines are not involved in inter- but in intrachain bonds. In reducing conditions, providing that the triple-he...

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Bibliographic Details
Published in:The Journal of biological chemistry 2001-07, Vol.276 (30), p.27989-27998
Main Authors: Mazzorana, M, Cogne, S, Goldschmidt, D, Aubert-Foucher, E
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Biochemistry, Molecular Biology
Cell Line
Chickens
Chromatography, Gel
COL1 gene
Collagen - chemistry
Cysteine - chemistry
Dimerization
Disulfides
Electrophoresis, Polyacrylamide Gel
Genetic Vectors
Glutathione
HeLa Cells
Humans
Hydroxylation
Insecta
Life Sciences
Molecular Sequence Data
Oxidation-Reduction
Pepsin A - chemistry
Protein Conformation
Protein Folding
Protein Structure, Tertiary
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
Sequence Homology, Amino Acid
Transfection
Trypsin - pharmacology
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Summary:A minicollagen containing the COL1 and NC1 domains of chicken collagen XII has been produced in insect cells. Significant amounts of trimers contain a triple-helical 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 G X Y 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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M101633200