The role of the C-terminal domain of human collagenase-3 (MMP-13) in the activation of procollagenase-3, substrate specificity, and tissue inhibitor of metalloproteinase interaction.
Recombinant human procollagenase-3 and a C-terminal truncated form (Delta249-451 procollagenase-3) have been stably expressed in myeloma cells and purified. The truncated proenzyme could be processed by aminophenylmercuric acetate via a short-lived intermediate form (N-terminal Leu58) to the final active form (N-terminal Tyr85). The kinetics of activation were not affected by removal of the hemopexin-like C-terminal domain. The specific activities of both collagenase-3 and Delta249-451 collagenase-3 were found to be similar using two quenched fluorescent substrates, but Delta249-451 collagenase-3 failed to cleave native triple helical collagens (types I and II) into characteristic one- and three-quarter fragments. It was noted, however, that the beta1,2(I) chains of type I collagen were susceptible to Delta249-451 collagenase-3, which indicates that the catalytic domain displays telopeptidase activity, thereby generating alpha1,2(I) chains that are slightly shorter than those in native type I collagen. It can be concluded that the C-terminal domain is only essential for the triple helicase activity of collagenase-3. Binding of procollagenase-3 and active collagenase-3 to type I collagen is mediated by the C-terminal domain. Both collagenase-3 and Delta249-451 collagenase-3 hydrolyzed the large tenascin C isoform, fibronectin, recombinant fibronectin fragments, and type IV, IX, X, and XIV collagens; thus, these events were independent from C-terminal domain interactions. In contrast, the minor cartilage type XI collagen was resistant to cleavage. Kinetic analysis of the mechanism of inhibition of wild-type and Delta249-451 collagenase-3 by wild-type and mutant tissue inhibitors of metalloproteinase (TIMPs) revealed that the association rates for complex formation were influenced by both N- and C-terminal domain interactions. The C-terminal domain of wild-type collagenase-3 promoted increased association rates with the full-length inhibitors TIMP-1 and TIMP-3 and the hybrid N.TIMP-2/C.TIMP-1 by a factor of up to 33. In contrast, the association rates for complex formation with TIMP-2 and N.TIMP-1/C.TIMP-2 were only marginally affected by C-terminal domain interactions.
Knäuper
V
V
Department of Cell and Molecular Biology, Strangeways Research Laboratory, Worts' Causeway, Cambridge CB1 4RN, United Kingdom. vk@srl.mrc-lmb.cam.ac.uk
Cowell
S
S
Smith
B
B
López-Otin
C
C
O'Shea
M
M
Morris
H
H
Zardi
L
L
Murphy
G
G
D000595:Amino Acid Sequence; D003094:Collagen / Q000378:metabolism; D017364:Collagenases / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D053509:Matrix Metalloproteinase 13; D061965:Matrix Metalloproteinase Inhibitors; D008969:Molecular Sequence Data; D011506:Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D019716:Tissue Inhibitor of Metalloproteinase-2
D004792:Enzyme Precursors; D061965:Matrix Metalloproteinase Inhibitors; D011506:Proteins; D019716:Tissue Inhibitor of Metalloproteinase-2; D003094:Collagen; D017364:Collagenases; C507079:MMP13 protein, human; D053509:Matrix Metalloproteinase 13; C022440:procollagenase