Hydrolysis of a Broad Spectrum of Extracellular Matrix Proteins by Human Macrophage Elastase

Abstract

Macrophage elastase (ME) was originally named when metal-dependent elastolytic activity was detected in conditioned media of murine macrophages. Subsequent cDNA cloning of the mouse and human enzyme demonstrated that ME is a distinct member of the matrix metalloproteinase family. To date, the catalytic parameters that describe the hydrolysis of elastin by ME have not been quantified and its activity against other matrix proteins have not been described. In this report, we have examined the action of purified recombinant human ME (rHME), produced in Escherichia coli, on elastin and other extracellular matrix proteins. On a molar basis, rHME is approximately 30% as active as human leukocyte elastase in solubilizing elastin. rHME also efficiently degrades ␣ 1 -antitrypsin (␣ 1 -AT), the primary physiological inhibitor of human leukocyte elastase. In addition, rHME efficiently degrades fibronectin, laminin, entactin, type IV collagen, chondroitan sulfate, and heparan sulfate. These results suggest that HME may be required for macrophages to penetrate basement membranes and remodel injured tissue during inflammation. Moreover, abnormal expression of HME may contribute to destructive processes such as pulmonary emphysema and vascular aneurysm formation. To further understand the specificity of HME, the initial cleavage sites in ␣ 1 -AT have been determined. In addition, the hydrolysis of a series of synthetic peptides with different P 1 residues has been determined. rHME can accept large and small amino acids at the P 1 site, but has a preference for leucine.Macrophage elastase (MMP-12) shares many properties with other members of the matrix metalloproteinase (MMP) 1 gene family, yet it is unique in several ways. Like other MMPs, metalloelastase requires zinc for catalytic activity, is inhibited by the tissue inhibitors of metalloproteinases (TIMPs), and has common structural domains with other MMPs (1, 2). Human macrophage elastase (HME) is most closely related to collagenase-1 (MMP-1) and stromelysin-1 (MMP-3), being 49% identical to each at the amino acid level (3). Moreover, the gene for macrophage elastase, composed of a common 10-exon, 9-intron structure, is on human chromosome 11q22.2/22.3 with at least six other MMPs (4). Despite these similarities, HME possesses certain distinct biological and biochemical properties. Expression appears to be largely restricted to tissue macrophages (4). Upon activation, it not only cleaves its 8-kDa N-terminal domain, but also has a unique propensity to autolytically release its 23-kDa C-terminal domain resulting in a mature active 22-kDa proteinase (3)(4)(5).Macrophage elastase shares its elastolytic activity (6, 7) with only a few MMPs, including the gelatinases (MMP-2 and MMP-9) and, to a lesser extent, matrilysin (8, 9). However, despite this characteristic activity, the relative capacity of metalloelastase (human or mouse) to degrade elastin has never been quantified. In addition, the catalytic capacity of metalloelastase against other extracellular matrix components has...

The cell biology of leukocyte-mediated proteolysis

“…This process may be facilitated by the lack of inhibitors of cathepsin D in mammalian tissues. Elastin, fibronectin, laminin, collagen IV, proteoglycan, inactivation of ␣ 1 -proteinase inhibitor [104] Classification by preferred substrate(s)…”

“…In vitro studies have demonstrated that reactive oxygen species released by activated leukocytes can inactivate serpins (␣ 1 -proteinase inhibitor and SLPI) as well as ␣ 2 -macroglobulin [98][99][100][101]. Serpins are also susceptible to proteolytic inactivation by several classes of proteinases: (1) several MMPs (MMP-1, MMP-3, MMP-7, MMP-8, MMP-9, and MMP-12) can inactivate ␣ 1 -proteinase inhibitor, ␣ 1 -antichymotrypsin, and also ␣ 2 -macroglobulin [25,26,[102][103][104]; (2) the serine proteinase, HLE, can inactivate its cognate inhibitor (␣ 1 -proteinase inhibitor) [105] and PAI-1 [106]; (3) cathepsin B (a cysteine proteinase) can inactivate ␣ 1 -proteinase inhibitor [107]; and (4) several bacterial proteinases can inactivate ␣ 1 -proteinase inhibitor and SLPI [108]. It is also noteworthy that serine proteinases can proteolytically inactivate TIMPs [109].…”

Matrix metalloproteinase 12 silencing: A therapeutic approach to treat pathological lung tissue remodeling?

“…As suggested by its trivial name (metalloelastase), MMP-12 is clearly the most active MMP against elastin [38] although it can cleave many of the other components of the extracellular matrix such as fibronectin, fibrillin-1, laminin, entactin, type IV collagen fragments, chondroitin sulfate and heparan sulfate proteoglycans and vitronectin [35,39,40]. However, MMP-12 can not significantly degrade fibrillar collagen or gelatin [39]. In vivo, MMP-12 has the ability to activate other MMPs such as pro-MMP-2 and pro-MMP-3, which, in turn, can activate pro-MMP-1 and pro-MMP-9.…”

Overexpression of Human Matrix Metalloproteinase-12 Enhances the Development of Inflammatory Arthritis in Transgenic Rabbits

“…7 Although the major substrate for MMP-12 is elastin, MMP-12 is able to degrade a broad spectrum of substrates such as type IV collagen, fibronectin, laminin, vitronectin, proteoglycans (PGs), chondroitin sulfate, myelin basic protein, ␣-1-anti-trypsin, and plasminogen. 8,9 Moreover, human and mouse MMP-12 undergo self-activation through autolytic processing, and recombinant rabbit MMP-12 has been shown to activate other MMPs such as MMP-2 and MMP-3. 5 Importantly, MMP-12 expression of macrophages is highly regulated by inflammatory mediators such as GM-CSF, MCP-1, 10,11 and CD40 ligands, 12 suggesting that in many inflammatory processes, once MMP-12 is up-regulated, there is a cascade of activation of other MMPs that leads to extracellular matrix degradation.…”