Control of Promatrilysin (MMP7) Activation and Substrate-specific Activity by Sulfated Glycosaminoglycans

Abstract

Matrix metalloproteinases are maintained in an inactive state by a bond between the thiol of a conserved cysteine in the prodomain and a zinc atom in the catalytic domain. Once this bond is disrupted, MMPs become active proteinases and can act on a variety of extracellular protein substrates. In vivo, matrilysin (MMP7) activates pro-␣-defensins (procryptdins), but in vitro, processing of these peptides is slow, with about 50% conversion in 8 -12 h. Similarly, autolytic activation of promatrilysin in vitro can take up to 12-24 h for 50% conversion. These inefficient reactions suggest that natural cofactors enhance the activation and activity of matrilysin. We determined that highly sulfated glycosaminoglycans (GAG), such as heparin, chondroitin-4,6-sulfate (CS-E), and dermatan sulfate, markedly enhanced (>50-fold) the intermolecular autolytic activation of promatrilysin and the activity of fully active matrilysin to cleave specific physiologic substrates. In contrast, heparan sulfate and less sulfated forms of chondroitin sulfate did not augment matrilysin activation or activity. Chondroitin-2,6-sulfate (CS-D) also did not enhance matrilysin activity, suggesting that the presentation of sulfates is more important than the overall degree of sulfation. Surface plasmon resonance demonstrated that promatrilysin bound heparin (K D , 400 nM) and CS-E (K D , 630 nM). Active matrilysin bound heparin (K D , 150 nM) but less so to CS-E (K D , 60 M). Neither form bound heparan sulfate. These observations demonstrate that sulfated GAGs regulate matrilysin activation and its activity against specific substrates. Matrix metalloproteinases (MMPs)3 comprise a family of endopeptidases that act on a variety of extracellular proteins, such as chemokines, antimicrobial peptides, matrix components, and more, to effect numerous repair, immune, and disease processes (1-3). For many substrates, MMP cleavage results in gain-of-function processing, such as the activation of latent antimicrobial peptides (4, 5) and cytokines (1), or altered biologic activity, as with limited proteolysis of chemokines (6, 7) and shedding of cell surface proteins (8). Thus, the mechanisms controlling zymogen activation and proteinase activity against specific substrates would sit high in the hierarchy of events controlling many host response pathways. As for all proteinases, the activity of MMPs is regulated at four points: gene expression, compartmentalization (i.e. pericellular accumulation of enzyme), proenzyme (or zymogen) activation, and enzyme inactivation, and is further controlled by substrate availability, concentration, and affinity.ProMMPs are kept in a catalytically inactive state by the interaction between the thiol of the conserved prodomain cysteine and the zinc ion of the catalytic site. To become active, the thiol-Zn 2ϩ interaction, commonly called the "cysteine switch," must be disrupted (9), which can be mediated by proteolysis of the prodomain, post-translational modification of the thiol, allosteric interactions with other macromolecules...

Heparan Sulfate Chains of Syndecan-1 Regulate Ectodomain Shedding

“…We do not know if the heparan sulfates of syndecan bind to MMPs and inhibit their activity directly. It has been shown in some models that MMP-7 activity is enhanced by exogenous heparin, although it is not enhanced by heparan sulfate (38,39). However, in our model, we found that exogenous heparin had no effect on shedding, suggesting that it is unlikely that syndecans are binding to MMPs and directly inhibiting their activity.…”

A Role for Versican in the Development of Leiomyosarcoma

“…In the case of LMS, it may be that the expression of versican isoforms with abundant chondroitin sulfate chains (e.g. V0 and V1) by the LMS cells leads to a GAG-dependent localization and activation of cytokines, growth factors, and metalloproteinases (62,63) that promote a proliferative and metastatic microenvironment.…”

Mice Deficient in N-Acetylgalactosamine 4-Sulfate 6-O-Sulfotransferase Are Unable to Synthesize Chondroitin/Dermatan Sulfate containing N-Acetylgalactosamine 4,6-Bissulfate Residues and Exhibit Decreased Protease Activity in Bone Marrow-derived Mast Cells

“…substrates, procryptdins (20). Alcian Blue, which is used for detection of sulfated glycosaminoglycans, was reported to stain an outer rim around each granule of Paneth cells (53,67).…”