Generation of collagenase-resistant collagen by site-directed mutagenesis of murine pro alpha 1(I) collagen gene.
Collagenase (matrix metalloproteinase 1) cleaves type I, II, and III collagen helices at a specific site between Gly-Ile or Gly-Leu bonds (residues 775 and 776, P1-P1'). To understand the mechanism of collagen processing, mutations around the cleavage site have been introduced into the cloned murine pro alpha 1(I) collagen (Col1a1) gene. These mutant constructs have been transfected into homozygous Mov13 fibroblasts that do not express the endogenous Col1a1 gene due to a retroviral insertion. Secreted triple-helical type I collagens containing substitutions of Pro for Ile (position 776) (P1') were not cleaved by human rheumatoid synovial collagenase, whereas those containing substitutions of Met for Ile (position 776) were cleaved. Type I collagens containing double substitutions of Pro for Gln-774 (P2) and Ala-777 (P2') were not cleaved regardless of whether they contained the wild-type residue Ile at position 776 or the substitution of Met for Ile at position 776. The wild-type alpha 2(I) chains derived from the endogenous Col1a2 gene were also resistant to enzyme digestion when they were complexed with the mutant alpha 1(I) chains, indicating that the presence of normal alpha 1(I) sequences is critical for cleavage of the alpha 2(I) chains in the type I heterotrimer.
Wu
H
H
Department of Biology, Massachusetts Institute of Technology, Cambridge 02142.
Byrne
M H
MH
Stacey
A
A
Goldring
M B
MB
Birkhead
J R
JR
Jaenisch
R
R
Krane
S M
SM
D000595:Amino Acid Sequence; D000818:Animals; D001172:Arthritis, Rheumatoid / Q000201:enzymology; D001483:Base Sequence; D002478:Cells, Cultured; D002999:Clone Cells; D003094:Collagen / Q000378:metabolism*; D005347:Fibroblasts / Q000201:enzymology; D005796:Genes; D006801:Humans; D046911:Macromolecular Substances; D051379:Mice; D003012:Microbial Collagenase / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010449:Peptide Mapping; D011347:Procollagen / Q000235:genetics*; D011487:Protein Conformation; D011489:Protein Denaturation; D015183:Restriction Mapping; D013379:Substrate Specificity; D013582:Synovial Fluid / Q000201:enzymology; D014162:Transfection
D046911:Macromolecular Substances; D011347:Procollagen; D003094:Collagen; D003012:Microbial Collagenase