Temperature-induced post-translational over-modification of type I procollagen. Effects of over-modification of the protein on the rate of cleavage by procollagen N-proteinase and on self-assembly of collagen into fibrils.
Previous observations suggested that incubating fibroblasts at elevated temperature caused over-modification of type I procollagen by post-translational enzymes because of a delay in folding of the collagen triple helix. Here, human skin fibroblasts were incubated at 40.5 instead of 37 degrees C, and the type I procollagen secreted into the medium was isolated. Analysis of the protein indicated that there was an increase of about 5 residues of hydroxylysine/alpha chain and about 1 residue of glycosylated hydroxylysine/alpha chain. Assays with procollagen N-proteinase indicated that the N-propeptide of the over-modified collagen was cleaved at a decreased rate, apparently because the over-modification altered the conformation-dependent cleavage site for the enzyme. Assays in a system for assembly of collagen into fibrils demonstrated that the over-modified protein had a higher critical concentration for self-assembly. Also, the fibrils formed from the over-modified collagen at 31 and 29 degrees C had smaller diameters than fibrils formed from normal type I collagen. The results provide direct evidence for earlier suggestions that post-translational over-modification of a fibrillar collagen can alter the morphology of the fibrils formed. The results also indicate that some of the biological consequences of the mutations in type I procollagen causing heritable disorders must be ascribed to the effects of post-translational over-modifications that frequently occur as secondary consequences of changes in the primary structure of the protein.
Torre-Blanco
A
A
Department of Biochemistry and Molecular Biology, Jefferson Institute of Molecular Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
Adachi
E
E
Hojima
Y
Y
Wootton
J A
JA
Minor
R R
RR
Prockop
D J
DJ
D000328:Adult; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006868:Hydrolysis; D006909:Hydroxyproline / Q000378:metabolism; D008297:Male; D008854:Microscopy, Electron; D011347:Procollagen / Q000378:metabolism*; D011348:Procollagen N-Endopeptidase / Q000378:metabolism*; D011487:Protein Conformation; D011492:Protein Hydrolysates / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D013696:Temperature
D011347:Procollagen; D011492:Protein Hydrolysates; C007978:hydrolysin; D003094:Collagen; D011348:Procollagen N-Endopeptidase; D006909:Hydroxyproline