Characterization and regulation of MT1‐MMP cell surface‐associated activity

Abstract

Quantitative assessment of MT1‐MMP cell surface‐associated proteolytic activity remains undefined. Presently, MT1‐MMP was stably expressed and a cell‐based FRET assay developed to quantify activity toward synthetic collagen‐model triple‐helices. To estimate the importance of cell surface localization and specific structural domains on MT1‐MMP proteolysis, activity measurements were performed using a series of membrane‐anchored MT1‐MMP mutants and compared directly with those of soluble MT1‐MMP. MT1‐MMP activity (kcat/KM) on the cell surface was 4.8‐fold lower compared with soluble MT1‐MMP, with the effect largely manifested in kcat. Deletion of the MT1‐MMP cytoplasmic tail enhanced cell surface activity, with both kcat and KM values affected, while deletion of the hemopexin‐like domain negatively impacted KM and increased kcat. Overall, cell surface localization of MT1‐MMP restricts substrate binding and protein‐coupled motions (based on changes in both kcat and KM) for catalysis. Comparison of soluble and cell surface‐bound MT2‐MMP revealed 12.9‐fold lower activity on the cell surface. The cell‐based assay was utilized for small molecule and triple‐helical transition state analog MMP inhibitors, which were found to function similarly in solution and at the cell surface. These studies provide the first quantitative assessments of MT1‐MMP activity and inhibition in the native cellular environment of the enzyme.

Matrix Metalloproteinases Shape the Tumor Microenvironment in Cancer Progression

“…The collagenolytic activity of MMP-14 on the cell surface is enhanced predominantly by its hemopexin domain-dependent homodimerization [ 124 ]. It is regulated by interactions with integrins, CD44, chondroitin/heparin sulfate proteoglycans, tetraspanins, pericellular MMP-14-inhibiting proteins TIMP-2, -3, and -4, and RECK [ 158 , 170 , 171 , 172 ]. The O-glycosylation pattern also determines the lifespan of MMP-14 and, thus, the invasiveness of cancer cells [ 173 ].…”

Kallikrein-Related Peptidase 14 Activates Zymogens of Membrane Type Matrix Metalloproteinases (MT-MMPs)—A CleavEx Based Analysis

“…However, the appearance of a corresponding band in the zymogram analysis indicated ability to cleave gelatin and suggested that this specific auto-processing by initial KLK14-mediation may be responsible for shedding of the active catalytic domain of MMP15 from the cell surface ( Figure 4 C). Interestingly, the cell surface localization of MMP15 was essential for its ability to cleave collagen in cell-based assays, and it was required for exhibition of invasive cancer cell phenotypes [ 36 , 37 ], while soluble MMP15 was found to display a nearly 13-fold higher activity on triple-helical substrates compared to the cell-surface located form [ 38 ]. This suggests, that release of the mature MMP15 from the cell surface may be an important regulatory step, resulting in enhanced triple helical collagen I degradation.…”

Kallikrein-related peptidase 14 activates zymogens of membrane type matrix metalloproteinases (MT-MMPs) - a CleavEx library-based analysis

“…Interestingly, the cell surface localization of MMP15 was essential for its ability to cleave collagen in cell-based assays and it was required for exhibition of invasive cancer cell phenotypes [34], [35], while soluble MMP15 was found to display a nearly 13-fold higher activity on triple-helical substrates compared to the cell-surface located form [36]. This suggests, that release of the mature MMP15 from the cell surface may be an important regulatory step, resulting in enhanced triple helical collagen I degradation.…”