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

Abstract

Collagenase cleavage of human Type II and III collagens has been 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 five 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 compositions of the products demonstrated that Type II and III molecules had been cleaved at the characteristic three-quarter, one-quarter locus, giving rise to a large fragment derived from the NH2-terminal 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 was found to have the sequence Leu-Ala Gly-Leu-Arg. These sequences for alpha1(II) and alpha1(III) chains adjacent to the site of collagenase cleavage along with previous data for alpha1(I) and alpha2 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 at least three additional locations at which collagenase cleavage does not occur. Each of the sequences which are apparently not substrates for collagenase, however, are followed by a Gly-X-Hyp sequence. We suggest, then, that a minimum of five residues in collagen alpha chains COOH-terminal to the cleavage site comprise the substrate recognition site.

Characterization of the Amino-Terminal Segment in Type III Procollagen

“…The position of the upper band did not change by reduction (Fig. 2, slot 2) and the band was identified as ~l ( I 1 1 )~ with a molecular weight of 75000 [7]. The lower band was identified as the triple-stranded carboxy-terminal fragment [a1(III)B]3 which could be converted into a faster migrating component ~l ( I 1 1 )~ (molecular weight 25000) by reduction (Fig.…”

“…proved useful in establishing the location of interchain disulfide bonds as well as the location of precursor-specific peptides at either end of the a-chain [7,10,20,36,37]. Here interchain disulfide bonding in type 111 collagen and procollagen was examined by electrophoresis in dodecylsulfate comparing nonreduced with reduced samples (Fig.…”

“…More recently, a third type of collagen has been demonstrated in several tissues, including skin, blood vessels and internal organs [3 -51. These type I11 collagen molecules contain three identical a l(II1) chains, covalently linked by disulfide bonds located in their carboxy-terminal regions [6,7].These collagens are synthesized as precursor molecules, procollagens, which are larger than collagen because of additional peptides that occur at both the amino and carboxyl ends of the chains [8 -101. In the case of type I procollagen it is known that the precursor-specific region connected to the amino end of the al(1) chain has a molecular mass of about 15000 daltons while the carboxy-terminal precursor peptide has about 35 000 daltons.…”

“…More recently, a third type of collagen has been demonstrated in several tissues, including skin, blood vessels and internal organs [3 -51. These type I11 collagen molecules contain three identical a l(II1) chains, covalently linked by disulfide bonds located in their carboxy-terminal regions [6,7].…”

Proteinases in Human Polymorphonuclear Leukocytes

“…In particular, this structure is remarkably resistant to a variety of proteolytic enzymes, and the degradation of collagen in vivo may be initiated only by specific metalloproteinases, collagenases [I -31. The vertebrate collagenases degrade collagen only at a specific region located at three-quarters of the length of the molecule from the amino terminus; the cleavage of type I, 11, and I11 collagens has been shown to occur at Gly-Leu or Gly-Ile sequences [4,5]. Specific collagenolytic enzymes have been demonstrated in several human cells and tissues, including polymorphonuclear leukocytes, macrophages, and fibroblasts, as well as skin and synovium explants in culture [2,6].…”