Cleavage of type II and III collagens with mammalian collagenase: site of cleavage and primary structure at the amino-terminal portion of the smaller fragment released from both collagens

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Abstract
Collagenase cleavage of human infant articular cartilage Type II and III collagens was studied using a highly purified preparation of rabbit tumor collagenase. Progress of the reactions in solution was followed by viscometry, and the results indicated that under the conditions employed Type III collagen molecules were cleaved at approximately 5 times the rate of Type II molecules. Cleavage products of the reactions were isolated in denatured form by agarose molecular sieve chromatography. The molecular weights and amino acid compostions of the products demonstrated that Type II and III molecules were cleaved at the characteristic 3/4, 1/4 locus, giving rise to a large fragment derived from the NH2-tetminal portion of the molecule and a smaller fragment representing the COOH-terminal region. The amino acid sequence at the NH2-terminal portion of the smaller fragment derived from Type II collagen was determined to be Ile-Ala-Gly-Gln-Arg, and the corresponding region from Type III collagen had the sequence Leu-Ala-Gly-Leu-Arg. These sequences for .alpha.1(II) and .alpha.1(III) chains adjacent to the site of collagenase cleavage along with previous data for .alpha.1(I) and .alpha.2 chains indicate that the minimum specific sequence required for collagenase cleavage is Gly-Ile-Ala or Gly-Leu-Ala. Inspection of the available sequence data for collagen .alpha. chains indicates that the latter sequences are found in a least 3 additional locations at which collagenase cleavage does not occur. Each of the sequences which are apparently not substrates for collagenase are followed by a Gly-X-Hyp sequence. A minimum of 5 residues in collagen .alpha. chains COOH-terminal to the cleavage site probably comprise the substrate recognition site.

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