Ivan E. Collier

On the structure and chromosome location of the 72- and 92-kDa human type IV collagenase genes

The 72- and 92-kDa type IV collagenases are members of a group of secreted zinc metalloproteases. Two members of this family, collagenase and stromelysin, have previously been localized to the long arm of chromosome 11. Here we assign both of the two type IV collagenase genes to human chromosome 16. By sequencing, the 72-kDa gene is shown to consist of 13 exons, 3 more than have been reported for the other members of this gene family. The extra exons encode the amino acids of the fibronectin-like domain which has so far been found in only the 72- and 92-kDa type IV collagenase. The evolutionary relationship among the members of this gene family is discussed.

Diffusion of MMPs on the surface of collagen fibrils: the mobile cell surface-collagen substratum interface.

Remodeling of the extracellular matrix catalyzed by MMPs is central to morphogenetic phenomena during development and wound healing as well as in numerous pathologic conditions such as fibrosis and cancer. We have previously demonstrated that secreted MMP-2 is tethered to the cell surface and activated by MT1-MMP/TIMP-2-dependent mechanism. The resulting cell-surface collagenolytic complex (MT1-MMP)2/TIMP-2/MMP-2 can initiate (MT1-MMP) and complete (MMP-2) degradation of an underlying collagen fibril. The following question remained: What is the mechanism of substrate recognition involving the two structures of relatively restricted mobility, the cell surface enzymatic complex and a collagen fibril embedded in the ECM? Here we demonstrate that all the components of the complex are capable of processive movement on a surface of the collagen fibril. The mechanism of MT1-MMP movement is a biased diffusion with the bias component dependent on the proteolysis of its substrate, not adenosine triphosphate (ATP) hydrolysis. It is similar to that of the MMP-1 Brownian ratchet we described earlier. In addition, both MMP-2 and MMP-9 as well as their respective complexes with TIMP-1 and -2 are capable of Brownian diffusion on the surface of native collagen fibrils without noticeable dissociation while the dimerization of MMP-9 renders the enzyme immobile. Most instructive is the finding that the inactivation of the enzymatic activity of MT1-MMP has a detectable negative effect on the cell force developed in miniaturized 3D tissue constructs. We propose that the collagenolytic complex (MT1-MMP)2/TIMP-2/MMP-2 represents a Mobile Cell Surface – Collagen Substratum Interface. The biological implications of MT1-MMP acting as a molecular ratchet tethered to the cell surface in complex with MMP-2 suggest a new mechanism for the role of spatially regulated peri-cellular proteolysis in cell-matrix interactions.

Secreted proteases. Regulation of their activity and their possible role in metastasis.

Extracellular matrix metalloproteases are secreted by the resident cells of the tissue in a proenzyme form, and their extracellular activity is regulated at the level of gene expression, proenzyme activation, and interaction with inhibitors. To understand the molecular mechanisms that control the activity of ECM metalloproteases and their effect on the cellular phenotype, we have established cell lines in which the transcription of the protease genes is repressed. We also have undertaken a detailed study of the pathway of extracellular activation of interstitial procollagenase. Stable transfection of three human tumor cell lines--H-ras-transformed bronchial epithelial cells TBE-1, fibrosarcoma cells HT1080, and melanoma cells A2058--with the adenovirus E1A gene dramatically repressed the expression of the secreted proteases, type IV and interstitial collagenases, and urokinase-type plasminogen activator. Concomitantly, E1A-expressing cells showed reduced metastatic activity in vivo and reduced ability to traverse a reconstituted basement membrane in vitro. Monospecific anti-type IV collagenase antibody inhibited the invasive activity of parental tumor cell lines in the in vitro system, suggesting a possible causal relationship between the effect of E1A on the expression of secreted proteases and the reduced metastatic potential of the E1A-expressing transformants. We have also studied the mechanism of regulation of metalloprotease activity at the level of extracellular activation by investigating the cascade of proteolytic events that results in the activation of interstitial procollagenase. Cocultivation of the major cellular components of skin, dermal fibroblasts, and epidermal keratinocytes induces activation of interstitial procollagenase and prostromelysin in the presence of plasminogen. This activation occurs through a uPA-plasmin-dependent pathway in which plasmin catalyzes the first step in activation of both collagenase and stromelysin by amino-terminal processing. Activated stromelysin can in turn convert plasmin-activated collagenase into a fully active enzyme by removal of approximately 15 amino acid residues from the carboxyl end of the enzyme. This second step of activation results in a 5-8-fold further increase in specific activity of collagenase. This cascade of proteolytic events may constitute a major physiologic pathway of collagenase activation.

Substrate recognition by gelatinase A: the C-terminal domain facilitates surface diffusion.

An investigation of gelatinase A binding to gelatin produced results that are inconsistent with a traditional bimolecular Michaelis-Menten formalism but are effectively accounted for by a power law characteristic of fractal kinetics. The main reason for this inconsistency is that the bulk of the gelatinase A binding depends on its ability to diffuse laterally on the gelatin surface. Most interestingly, we show that the anomalous lateral diffusion and, consequently, the binding to gelatin is greatly facilitated by the C-terminal hemopexin-like domain of the enzyme whereas the specificity of binding resides with the fibronectin-like gelatin-binding domain.