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

Abstract

SummaryDystroglycan (DG) is a membrane-associated protein complex formed by two noncovalently linked subunits, a-DG, a highly glycosylated extracellular protein, and b-DG, a transmembrane protein. The interface between the two DG subunits, which is crucial to maintain the integrity of the plasma membrane, involves the C-terminal domain of a-DG and the N-terminal extracellular domain of b-DG. It is well known that under both, physiological and pathological conditions, gelatinases (i.e. MMP-9 and/or MMP-2) can degrade DG, disrupting the connection between the extracellular matrix and the cytoskeleton. However, the molecular mechanisms and the exact cleavage sites underlying these events are still largely unknown. In a previous study, we have characterized the enzymatic digestion of the murine b-DG ectodomain by gelatinases, identifying a main cleavage site on the b-DG ectodomain produced by MMP-9. In this article, we have deepened the pattern of the b-DG ectodomain digestion by MMP-2 by using a combined approach based on SDS-PAGE, Orbitrap, and HPLC-ESI-IT mass spectrometry. Furthermore, we have characterized the kinetic parameters of the digestion of some b-DG ectodomain mutants by gelatinases.2012 IUBMB IUBMB Life, 64(12): 988-994, 2012

α-dystroglycan is a potential target of matrix metalloproteinase MMP-2

“…Further, in two recently published works we have described the gelatinases as the main players in degrading the β-DG ectodomain in vitro, though following a distinct pattern of digestion (Bozzi et al, 2009;Sbardella et al, 2012b). Because of the fairly complex pattern of domain-dependent degradation, likely different for the two gelatinases, in this paper we only focussed on the enzymatic activity of MMP-2; therefore, the enzymatic activity of MMP-9 both on the native α-DG and on the α-DG recombinant domains will be the subject of a following paper.…”

“…Accordingly, in our previous works, we found out that MMP-9 induces a main cleavage within the extracellular N-terminal domain of β-DG, producing a very stable C-terminal peptide (30 amino acids long) and an N-terminal region, which undergoes further proteolytic digestion (Bozzi et al, 2009). Conversely, the degradation by MMP-2 occurs through a complex pattern, which leads to several cleavages over the entire β-DG ectodomain (Sbardella et al, 2012b). As far as α-DG is concerned, although no direct experimental evidences are reported of its specific degradation by MMP-2 and MMP-9, it has been demonstrated a correlation between gelatinases over-expression and a reduction of both α-and β-DG levels in prostatic diseases (Hetzl et al, 2012).…”

“…Equal aliquots from the reaction mixture were collected at the indicated time-points and analyzed as reported elsewhere (Sbardella et al, 2012b).…”

“…However, they are both over-represented in flogistic and malignant conditions, where their contribution to the loss of cell adhesiveness and motility is of the utmost importance ( Van den Steen et al, 2002;Björklund and Koivunen, 2005;Sbardella et al, 2012a). Further, MMP-2 and MMP-9 mediate the shedding of the N-terminal domain of β-DG, thus exposing the α-DG to the extracellular environment (Bozzi et al, 2009;Sbardella et al, 2012b). Accordingly, in our previous works, we found out that MMP-9 induces a main cleavage within the extracellular N-terminal domain of β-DG, producing a very stable C-terminal peptide (30 amino acids long) and an N-terminal region, which undergoes further proteolytic digestion (Bozzi et al, 2009).…”

A role for dystroglycan in the pathophysiology of acute leukemic cells

“…Thus, we hypothesize that proteolysis of the -Dg ectodomain [38] or of -Dg's N-terminal portion [39][40][41] produced by Matrix MetalloProteinases (MMP) Furin andsecretase [42], respectively, may contribute to the pathophysiology of the AML, as these have been implicated in cancer progression in other systems [43,44]. Recently, it was established that hypoglycosylation severely alters -Dg's capability to bind to ECM partners, facilitating its degradation by MMP-2 or by other enzymes not identified to date [45]; additionally, in some patients affected by dystroglycanopathy, an important reduction of the Dg core protein was identified [46]. These events are in accordance with our results, in that we simultaneously observed a significant reduction in glycosylated -Dg forms, as well as a reduction of Dg core protein.…”