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

Abstract

Dystroglycan (DG) is a membrane receptor belonging to the complex of glycoproteins associated to dystrophin. DG is formed by two subunits, α-DG, a highly glycosylated extracellular matrix protein, and β-DG, a transmembrane protein. The two DG subunits interact through the C-terminal domain of α-DG and the N-terminal extracellular domain of β-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 β-DG ectodomain producing a 30 kDa truncated form of β-DG. However, the molecular mechanism underlying this event is still unknown. In this study, we carried out proteolysis of the recombinant extracellular domain of β-DG, β-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 β-DG, and we analysed the proteolytic fragments of β-DG(654-750) produced by MMP-9 enzymatic activity. © 2009 IUBMB.