Enzymatic processing of β‐dystroglycan recombinant ectodomain by MMP‐9: Identification of the main cleavage site

Abstract

SummaryDystroglycan (DG) is a membrane receptor belonging to the complex of glycoproteins associated to dystrophin. DG is formed by two subunits, a-DG, a highly glycosylated extracellular matrix protein, and b-DG, a transmembrane protein. The two DG subunits interact through the C-terminal domain of a-DG and the N-terminal extracellular domain of b-DG in a noncovalent way. Such interaction is crucial to maintain the integrity of the plasma membrane. In some pathological conditions, the interaction between the two DG subunits may be disrupted by the proteolytic activity of gelatinases (i.e. MMP-9 and/or MMP-2) that removes a portion or the whole b-DG ectodomain producing a 30 kDa truncated form of b-DG. However, the molecular mechanism underlying this event is still unknown. In this study, we carried out proteolysis of the recombinant extracellular domain of b-DG, b-DG(654-750) with human MMP-9, characterizing the catalytic parameters of its cleavage. Furthermore, using a combined approach based on SDS-PAGE, MALDI-TOF and HPLC-ESI-IT mass spectrometry, we were able to identify one main MMP-9 cleavage site that is localized between the amino acids His-715 and Leu-716 of b-DG, and we analysed the proteolytic fragments of b-DG(654-750) produced by MMP-9 enzymatic activity.

Human matrix metalloproteinases: An ubiquitarian class of enzymes involved in several pathological processes

“…In turn, TGF-b induces MMP-2 gene transcription, creating a vicious cycle which definitely promotes tumor growth (Binker et al, 2011;Kim et al, 2007). MMP-2 activates a plethora of cytokines, growth factors and further inactivates adhesion molecules, as TNF-a, MCP-3 (McQuibban et al, 2000), IGF, b-dystroglycan and FGFR (Fowlkes et al, 1994;Bozzi et al, 2009), modulating tumor cells metabolism, immune responses, receptor turnover (Levi et al, 1996) and cell resistance to apoptotis (Gondi et al, 2009). As matter of the fact, glioma cells treated with anti-MMP-2 siRNA trigger apoptosis (Badiga et al, 2011).…”

“…Both these factors display positive properties for tumor growth and contribute to the epithelial-mesenchimal transition (Aresu et al, 2011;Ongusaha et al, 2004;Wu et al, 2011a). MMP-9 cleaves nexin-1 and adhesion molecules, such as the b-subunit of dystroglycan Bozzi et al, 2009), enhancing tumor detachment from the primary site and tumor invasiveness. Furthermore, MMP-9 degrades collagen IV and other basement membrane components and exposes cryptic binding sites for cell migration or releases hidden bio-active factors (Schenk and Quaranta, 2003;Stetler-Stevenson and Yu, 2001).…”

Enzymatic processing by MMP‐2 and MMP‐9 of wild‐type and mutated mouse β‐dystroglycan

“…This result is consistent with the notion that nonpolar amino acids are likely to better adapt in the S1 site of the MMP-2 catalytic pocket, and it further reflects a common feature of MMPs behavior (25,26). This proteolytic mechanism is quite different from what observed in a previous study for MMP-9, where the first cleavage event took place between the amino acids His-715 and Leu-716 of the b-DG ectodomain, leading to the formation of a very stable Cterminal fragment (17). The N-terminal fragment, which originated from that first cleavage, was further processed.…”

“…The activity of 100 nM MMP-2 and MMP-9 was investigated on some b-DG(654-750) mutants, namely (a) F700A and F718A, which displayed a reduced affinity toward the C-terminal portion of a-DG (18), and (b) L716A, which alters the MMP-9 main cleavage site (17). Figures 4A-4C show that the F700A and L716A mutants were degraded with the same apparent efficiency by MMP-2 and MMP-9 when compared with wild-type, indicating that the introduced mutations are not sufficient to modulate the enzymatic activity.…”

“…We have previously characterized the proteolytic digestion profile of the recombinant b-DG ectodomain, spanning the amino acid residues 654-750, b-DG(654-750), by MMP-9, for which a precise cleavage site has been identified, and by MMP-2, which cleaves the recombinant b-DG ectodomain with higher efficiency (17).…”