Binding of Active Matrilysin to Cell Surface Cholesterol Sulfate Is Essential for Its Membrane-associated Proteolytic Action and Induction of Homotypic Cell Adhesion

Abstract

Regulation of cell surface molecules by matrix metalloproteinases (MMPs), as well as MMPs-catalyzed degradation of extracellular matrix, is important for tumor invasion and metastasis. Our previous study (Kioi, M., Yamamoto, K., Higashi, S., Koshikawa, N., Fujita, K., and Miyazaki, K. (2003) Oncogene 22, 8662-8670) demonstrated that active matrilysin specifically binds to the surface of colon cancer cells and induces notable cell aggregation due to processing of the cell membrane protein(s). Furthermore, these aggregated cells showed a dramatically enhanced metastatic potential. To elucidate the mechanism of matrilysin-induced cell aggregation, we attempted to identify the matrilysin-binding substance on the cell surface. Here, we demonstrate that cholesterol sulfate on the cell surface is a major matrilysin-binding substance. We found that active matrilysin bound to the cell membrane and cholesterol sulfate incorporated into liposomes with similar affinities. Treatment of colon cancer cells with ␤-cyclodextrin significantly reduced not only matrilysin binding to the cell surface but also matrilysin-dependent proteolysis and cell aggregation. Interestingly, replenishment of cholesterol sulfate, but not cholesterol, neutralized the effects of ␤-cyclodextrin. Taken together, it is likely that binding of matrilysin to cholesterol sulfate facilitates the matrilysin-catalyzed modulation of cell surface proteins, thus inducing the cancer cell aggregation. Matrix metalloproteinases (MMPs)2 are zinc-dependent endopeptidases that degrade components of the extracellular matrix and play essential roles in tissue remodeling in physiological and pathological processes such as morphogenesis, differentiation, angiogenesis, tissue remodeling, and tumor invasion (1, 2). The association of MMPs with cancer cell invasion and metastasis has suggested that these proteinases represent attractive targets for the development of novel anti-tumor therapies.Recently, it has been suggested that the biological functions of various cell surface proteins are proteolytically modulated by several MMPs (1, 3, 4), including MT-MMPs, gelatinase A (MMP-2), gelatinase B (MMP-9), stromelysin (MMP-3), and matrilysin (MMP-7). These metalloproteinases are thought to regulate cellular functions by activating, inactivating, or releasing membrane proteins. Such regulation of cell surface molecules as well as MMP-catalyzed degradation of the extracellular matrix is important for tumor invasion and metastasis.Matrilysin is the smallest member of the MMP family, which lacks the C-terminal hemopexin-like domain. In cancer tissues, various MMPs are overexpressed both in stromal and tumor cells. In contrast, matrilysin has been detected specifically in tumor cells but not in stromal cells (5, 6). Among many MMPs, matrilysin appears to be one of the most important MMPs in human colon cancers because it is almost always overexpressed in colon cancers. Expression of matrilysin is also correlated well with malignancy and metastasis of cancers, especially in liver me...

A novel nonsynonymous variant of matrix metalloproteinase-7 confers risk of liver cirrhosis

“…These include CD44 heparan sulfate proteoglycan (HSPG), cholesterol sulfate, and CD151. [18][19][20] Cholesterol sulfate is a component of lipid raft and CD151 is a transmembrane 4 superfamily protein that appears in a detergent-insoluble lipid-containing microdomain. To learn the possible docking mechanism of MMP-7, the behavior of the membrane-associated MMP-7(Asp-137) variant was analyzed in the presence of detergent.…”

“…A previous study reported that active MMP-7, but not proMMP-7, efficiently binds to cholesterol sulfate on the cell surface. 19 In contrast, CD151 anchors latent proMMP-7 onto the cell surface, but its affinity to interact with the active form of MMP-7 is relatively low. 20 Therefore, we hypothesized that association of the pro-form of MMP-7(Asp-137) variant to CD151 at the cell surface provides the opportunity for MMP-7 activation.…”

Control of matrix metalloproteinase catalytic activity

“…Rather, proteinases, such as MMPs, would likely be anchored to the cell membrane, thereby maintaining a locally high enzyme concentration and targeting their catalytic activity to specific substrates within the pericellular space. In addition to the membrane-bound MMPs, several examples of specific cell-MMP interactions have been reported, such as the binding of MMP-2 to the α v β 3 integrin (Brooks et al, 1996), MMP-1 to the α 2 β 1 integrin Stricker et al, 2001), MMP-9 to CD44 (Yu and Stamenkovic, 2000), and MMP-7 to surface proteoglycans (Yu and Woessner, 2000;Yu et al, 2002), cholesterol (Yamamoto et al, 2006), and CD151 (Shiomi et al, 2005). As suggested for CD44 (Yu and Stamenkovic, 2000) and the α 2 β 1 integrin ), these membrane anchors may act as accessory factors that mediate both pro-enzyme activation and binding of both substrate and proteinase, thereby increasing the probability of proteolysis (Fig.…”

Identification of Amino Acid Residues of the Matrix Metalloproteinase-2 Essential for Its Selective Inhibition by β-Amyloid Precursor Protein-derived Inhibitor

“…After a 5-h induction, E. coli cells were broken in 50 mM Tris-HCl, pH 8.0 containing 50 mM NaCl and 5 mM EDTA with a sonicator, and the resultant inclusion bodies were collected by centrifugation. Solubilization of the inclusion bodies with guanidine-HCl followed by refolding by rapid dilution method was performed as described previously (35,36). In the case of GST fusion proteins, the refolded proteins were incubated with thrombin to remove the N-terminal GST region.…”