The exposed linker accounts for the difficulty in purifying full-length collagenase. The C-terminal domain provides a structural model for haemopexin and its homologues. It controls the specificity of MMPs, affecting both substrate and inhibitor binding, although its role remains obscure. These structural results should aid the design of site-specific mutants which will reveal further details of the specificity mechanism.

No abstract

The binding properties of the newly described tissue inhibitor of metalloproteinases-4 (TIMP-4) to progelatinase A and to the COOH-terminal hemopexin-like domain (C domain) of the enzyme were examined. We present evidence for the first time of a specific, high affinity interaction between TIMP-4 and the C domain of human gelatinase A and show that TIMP-4 binds both progelatinase A and the C domain in a similar manner to that of TIMP-2. Saturable binding of recombinant C domain to TIMP-4 and to TIMP-2 but not to TIMP-1 was demonstrated using a microwell protein binding assay.

Collagens are the major structural proteins of connective tissues such as skin, bone, cartilage and tendon. Interstitial collagen types I, II and III are the most abundant, and the native triple helical structure of these molecules makes them highly resistant to proteolysis. However, collagenases of the matrix metalloproteinase (MMP) family [1] cleave native collagen types I, II and III at a specific site in all three chains of the triple helix, approximately three-quarters of the length from the N-terminus. The action of these collagenase enzymes is therefore critical for the initiation of collagenolysis. Once initiated, the cleaved helix unwinds at physiological temperatures and becomes susceptible to degradation by other, less-specific proteinases. MMP collagenases are active at neutral pH and play a highly important role in collagen degradation in vivo. The mammalian MMP collagenases currently include the 'classical' collagenases, MMP-1, and also the gelatinolytic enzyme, , and MMP-14 (MT1-MMP) [8], a member of the membrane-type subclass of MMPs.MMP-9 (also known as gelatinase B, 92 kDa gelatinase or 92 kDa type IV collagenase, EC 3.4.24.35) shares a close structural similarity with MMP-2 [9,10]. It was originally identified as a gelatinolytic enzyme produced by polymorphonuclear leukocytes [11] and subsequent studies have demonstrated secretion in the latent form (proMMP-9) by a variety of cell types. It has also been implicated in the pathogenesis of several human diseases, including arthritis [12][13][14][15]. Unlike other MMPs, MMP-9 and MMP-2 both contain three fibronectin type II repeats inserted into the catalytic Interstitial collagen types I, II and III are highly resistant to proteolytic attack, due to their triple helical structure, but can be cleaved by matrix metalloproteinase (MMP) collagenases at a specific site, approximately three-quarters of the length from the N-terminus of each chain. MMP-2 and -9 are closely related at the structural level, but MMP-2, and not MMP-9, has been previously described as a collagenase. This report investigates the ability of purified recombinant human MMP-9 produced in insect cells to degrade native collagen types I and III. Purified MMP-9 was able to cleave the soluble, monomeric forms of native collagen types I and III at 37°C and 25°C, respectively. Activity against collagens I and III was abolished by metalloproteinase inhibitors and was not present in the concentrated crude medium of mock-transfected cells, demonstrating that it was MMP-9-derived. Mutated, collagenase-resistant type I collagen was not digested by MMP-9, indicating that the three-quarters ⁄ one-quarter locus was the site of initial attack. Digestion of type III collagen generated a three-quarter fragment, as shown by comparison with MMP-1-mediated cleavage. These data demonstrate that MMP-9, like MMP-2, is able to cleave collagens I and III in their native form and in a manner that is characteristic of the unique collagenolytic activity of MMP collagenases.Abbreviations APMA, p-aminopheny...

YKL-40 is expressed in arthritic cartilage and produced in large amounts by cultured chondrocytes, but its exact role is unclear, and the identities of its physiological ligands remain unknown. Purification of YKL-40 from resorbing bovine nasal cartilage and chondrocyte monolayers demonstrated the existence of three isoforms, a major and minor form from resorbing cartilage and a third species from chondrocytes. Affinity chromatography experiments with purified YKL-40 demonstrated specific binding of all three forms to collagen types I, II, and III, thus identifying collagens as potential YKL-40 ligands. Binding to immobilized type I collagen was inhibited by soluble native ligand, but not heat-denatured ligand, confirming a specific interaction. Binding of the chondrocyte-derived species to type I collagen was also demonstrated by surface plasmon resonance analysis, and the dissociation rate constant was calculated (3.42 ؋ 10 ؊3 to 4.50 ؋ 10 ؊3 s ؊1 ). The chondrocyte-derived species was found to prevent collagenolytic cleavage of type I collagen and to stimulate the rate of type I collagen fibril formation in a concentration-dependent manner. By contrast, the cartilage major form had an inhibitory effect on type I collagen fibrillogenesis. Digestion with N-glycosidase F, endoglycosidase H and lectin blotting did not reveal any difference in the carbohydrate component of these two YKL-40 species, indicating that this does not account for the opposing effects on fibril formation rate.