Analysis of site-directed mutations in human pro-alpha 2(I) collagen which block cleavage by the C-proteinase.
We have used site-directed mutagenesis to obtain human pro alpha 2(I) cDNAs containing novel mutations designed to inhibit cleavage at the C-proteinase site. Deletion of six relatively conserved amino acids which surround the cleavage site did not interfere with assembly of the triple helix in transfected rat cells, but blocked cleavage of the constituent mutated chains by endogenous C-proteinase. Substitution for a conserved Asp, which forms part of the Ala-Asp bond cleaved by C-proteinase, also blocked cleavage by endogenous C-proteinase. The conserved Asp is, therefore, a necessary component of the C-proteinase cleavage site. Incubation in vitro with a purified mouse C-proteinase, confirmed both mutations to be resistant to cleavage by high concentrations of the physiologically relevant enzyme. Mutant pro alpha 2(I) chains, resistant to cleavage by C-proteinase in culture media, were processed in cell layers by a different protease which cleaved telopeptide domains. Naturally occurring mutations at the C-proteinase site have not been described in human patients. The mutations characterized here, further define the C-proteinase cleavage site and provide reagents which may be informative when introduced into transgenic mice.
Lee
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Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison 53706.
Kessler
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Greenspan
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D055395:Bone Morphogenetic Protein 1; D019485:Bone Morphogenetic Proteins; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003094:Collagen / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D004252:DNA Mutational Analysis; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008666:Metalloendopeptidases; D009841:Oligonucleotides / Q000737:chemistry; D011347:Procollagen / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013329:Structure-Activity Relationship; D014162:Transfection
D019485:Bone Morphogenetic Proteins; D009841:Oligonucleotides; D011347:Procollagen; D003094:Collagen; D004247:DNA; D010450:Endopeptidases; D008666:Metalloendopeptidases; C525705:BMP1 protein, human; C525707:Bmp1 protein, mouse; C525708:Bmp1 protein, rat; D055395:Bone Morphogenetic Protein 1