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

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-α-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 μm). 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-Zn²⁺ 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, or other possible mechanisms (10). About one-third of proMMPs contains a furin-recognition sequence and are activated in the secretion pathway by furin proprotein convertase cleavage of the prodomain. However, with the possible exception of proMMP2 activation by MMP14, the physiologic activation mechanism of most MMPs is not known (10).Matrilysin (28 kDa zymogen, 19 kDa active enzyme) is expressed by mucosal epithelia and some macrophages and functions as a key effector of repair and immunity. Established functions of matrilysin include facilitating re-epithelialization (11, 12), cleaving Fas ligand to promote apoptosis (13, 14), shedding syndecan-1 to control neutrophil influx (15), and macrophage-mediated elastolysis (16).In mice, matrilysin activates pro-α-defensins (procryptdins), a family of structurally similar 3–4 kDa antimicrobial peptides found in the granules of Paneth cells at the base of the crypts of Lieberkühn (17). Because of the lack of mature cryptdins, matrilysin-null (Mmp7^−/−) mice have an impaired ability to battle enteric pathogens (4). Cryptdins are packaged as pro-proteins of 7–8 kDa and are cleaved at a conserved site by matrilysin within the secretion granules (4, 18). In resting Paneth cells, the steady-state levels of pro- and activated cryptdins are roughly equivalent. Upon stimulation, the balance of procryptdins is rapidly activated indicating efficient proteolysis by matrilysin within the secretory pathway (18, 19). However, in defined in vitro reactions containing just substrate and proteinase, activation of procryptdins by matrilysin is slow, with only 50% of the precursor cleaved in 8 h or longer (4). Furthermore, both pro- and active matrilysin are present in Paneth cells granules (4) indicating that this MMP is activated in vivo by prodomain cleavage. The inefficient cleavage of procryptdins in vitro, their rapid processing in vivo, and the presence of activated matrilysin in Paneth cell granules led us to hypothesize that other factors regulate both the activation of promatrilysin and its activity against physiologic substrates.Yu et al. (20, 21) reported that heparin increases matrilysin activity about 2–4-fold in a transferrin zymogram assay, and they reported that matrilysin colocalizes to heparan sulfate molecules in tissue. However, transferrin is not a physiologic substrate of this MMP, and it is not known how heparin and other glycosaminoglycans affect matrilysin activity against established substrates, such as procryptdins. Therefore, we assessed matrilysin activity in vitro in the presence of various glycosaminoglycans (GAGs), and we found that both zymogen activation and activity against specific substrates are markedly enhanced by highly sulfated molecules. Our findings suggest that specific GAGs function to control matrilysin proteolysis.