Analysis of site-directed mutations in human pro-α2(I) collagen which block cleavage by the C-proteinase

Seung Taek Lee, Efrat Kessler, Daniel S. Greenspan

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

We have used site-directed mutagenesis to obtain human proα2(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 proα2(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.

Original languageEnglish
Pages (from-to)21992-21996
Number of pages5
JournalJournal of Biological Chemistry
Volume265
Issue number35
Publication statusPublished - 1990 Dec 15

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Collagen
Mutation
Viperidae
alanylaspartic acid
proteinase C
Mutagenesis
Site-Directed Mutagenesis
Transgenic Mice
Culture Media
Rats
Peptide Hydrolases
Substitution reactions
Complementary DNA
Cells
Amino Acids
Enzymes

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

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abstract = "We have used site-directed mutagenesis to obtain human proα2(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 proα2(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.",
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Analysis of site-directed mutations in human pro-α2(I) collagen which block cleavage by the C-proteinase. / Lee, Seung Taek; Kessler, Efrat; Greenspan, Daniel S.

In: Journal of Biological Chemistry, Vol. 265, No. 35, 15.12.1990, p. 21992-21996.

Research output: Contribution to journalArticle

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