O-Glycosylation Regulates Autolysis of Cellular Membrane Type-1 Matrix Metalloproteinase (MT1-MMP)

Abstract

MT1-MMP is a key enzyme in cancer cell invasion and metastasis. The activity of cellular MT1-MMP is regulated by furin-like proprotein convertases, TIMPs, shedding, autoproteolysis, dimerization, exocytosis, endocytosis, and recycling. Our data demonstrate that, in addition to these already known mechanisms, MT1-MMP is regulated by O-glycosylation of its hinge region. Insignificant autolytic degradation is characteristic for naturally expressed, glycosylated, MT1-MMP. In turn, extensive autolytic degradation, which leads to the inactivation of the protease and the generation of its C-terminal membrane-tethered degraded species, is a feature of overexpressed MT1-MMP. We have determined that incomplete glycosylation stimulates extensive autocatalytic degradation and self-inactivation of MT1-MMP. Self-proteolysis commences during the secretory process of MT1-MMP through the cell compartment to the plasma membrane. The strongly negatively charged sialic acid is the most important functional moiety of the glycopart of MT1-MMP. We hypothesize that sialic acid of the O-glycosylation cassette restricts the access of the catalytic domain to the hinge region and to the autolytic cleavage site and protects MT1-MMP from autolysis. Overall, our results point out that there is a delicate balance between glycosylation and self-proteolysis of MT1-MMP in cancer cells and that when this balance is upset the catalytically potent MT1-MMP pool is self-proteolyzed.Membrane-tethered MT1-MMP, 2 the most abundant member of the membrane-type (MT) matrix metalloproteinase subfamily, is distinguished from soluble MMPs by a short transmembrane domain and a cytoplasmic tail (1, 2). MT1-MMP functions in cancer cells as an important mediator of proteolytic events on the cell surface (3, 4), and it is directly engaged in the cleavage of cell surface receptors and the pericellular proteolysis of the extracellular matrix components (5-7). MT1-MMP expression is associated with a variety of pathophysiological conditions and especially with cell locomotion, tumor growth, and metastasis (4, 5, 8 -11).MT1-MMP and related membrane-tethered MMPs are regulated, both as proteinases and as membrane proteins, at the transcriptional and post-transcriptional levels by multifaceted coordinated mechanisms (12-22). The trafficking and the internalization of MT1-MMP have been identified as two additional mechanisms that regulate its biological functions. Both clathrin-coated pits and caveolae are involved in the internalization of 19,[23][24][25][26][27][28].To exercise its proteolytic activity, MT1-MMP requires the proteolytic removal of its N-terminal prodomain sequence (29). Because the prodomain part of MT1-MMP has the furin-cleavage motif, furin and related proprotein convertases (PCs) are the physiological activators of latent MT1-MMP (13, 30 -32). Proteolytic processing by the PCs leads to the activation of the latent MT1-MMP zymogen, which occurs primarily in the trans-Golgi network during the secretory passage of MT1-MMP (30, 33). The levels of expressio...

Dynamic Interdomain Interactions Contribute to the Inhibition of Matrix Metalloproteinases by Tissue Inhibitors of Metalloproteinases

“…Immediately following the biotinylation procedure, cells were incubated for 25 min at 37°C in serum-free DMEM supplemented with 1% insulin-transferrin-selenium to allow the internalization of biotin-labeled MT1-MMP (41,42). To remove the residual cell surface biotin, cells were incubated for 25 min on ice in Sorensen phosphate buffer containing membrane-impermeable MESNA (150 mM).…”

“…Cells were next lysed using 50 mM N-octyl-␤-D-glucopyranoside in Tris-buffered saline supplemented with 1 mM PMSF, 1 mM CaCl 2 , 1 mM MgCl 2 , 10 mM EDTA, and proteinase inhibitor mixture set III (N-octyl-␤-D-glucopyranoside buffer). The biotin-labeled plasma membrane proteins were pooled down using streptavidin beads (42). The precipitates were dissolved in SDS sample buffer (0.125 M Tris-HCl, pH 6.8, 20% glycerol, 2% SDS, 0.005% Bromphenol Blue) with 100 mM DTT and analyzed by Western blotting with the MT1-MMP antibody followed by the secondary HRP-conjugated antibody and a SuperSignal West Dura Extended Duration Substrate kit.…”

“…To inactivate cellular MT1-MMP and block its self-degradation, prior to immunostaining procedures the cells were cultured in the presence of GM6001 (42). The cells were then fixed and permeabilized or left untreated.…”

Contribution of sialidase NEU1 to suppression of metastasis of human colon cancer cells through desialylation of integrin β4

“…It has been described that MT1-MMP activity could be modulated by O-glycosylation (Wu et al, 2004) and that enhancement of its movement to the cell surface might occur during the enzyme activation (Remacle et al, 2006). The MT1-MMP-catalysed activation of matrix metalloprotease-2 was then compared between the NEU1/pp11-and mock-transfected cells by gelatin zymography, and furthermore, the amount of biotinylated MT1-MMP was observed by immunoblotting with anti-MT1-MMP antibody.…”

The Inactive 44-kDa Processed Form of Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) Enhances Proteolytic Activity via Regulation of Endocytosis of Active MT1-MMP

“…Expression of constitutively active Rac1 in HT1080 cells also promotes MT1-MMP processing (30). In addition, high levels of enzyme expression (36,48,(52)(53)(54) and low levels of TIMP-2 relative to MT1-MMP (6,21) are associated with enhanced MT1-MMP processing. Finally, agents such as conA and cytochalasin D, which are known to inhibit clathrin-dependent endocytosis (55,56), promote MT1-MMP processing, which indicates that the rate of MT1-MMP internalization influences processing.…”