title: Enzymatic processing of beta-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, alpha-DG, a highly glycosylated extracellular matrix protein, and beta-DG, a transmembrane protein. The two DG subunits interact through the C-terminal domain of alpha-DG and the N-terminal extracellular domain of beta-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 beta-DG ectodomain producing a 30 kDa truncated form of beta-DG. However, the molecular mechanism underlying this event is still unknown. In this study, we carried out proteolysis of the recombinant extracellular domain of beta-DG, beta-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 beta-DG, and we analysed the proteolytic fragments of beta-DG(654-750) produced by MMP-9 enzymatic activity.
authors: Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Largo Francesco Vito 1, Rome, Italy.
authors: Manuela
authors: M
authors: Bozzi
authors: Rosanna
authors: R
authors: Inzitari
authors: Diego
authors: D
authors: Sbardell
authors: Susanna
authors: S
authors: Monaco
authors: Ernesto
authors: E
authors: Pavoni
authors: Magda
authors: M
authors: Gioia
authors: Stefano
authors: S
authors: Marini
authors: Simona
authors: S
authors: Morlacchi
authors: Francesca
authors: F
authors: Sciandra
authors: Massimo
authors: M
authors: Castagnola
authors: Bruno
authors: B
authors: Giardina
authors: Andrea
authors: A
authors: Brancaccio
authors: Massimo
authors: M
authors: Coletta
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D049030:Dystroglycans / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D020780:Matrix Metalloproteinase 9 / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D011994:Recombinant Proteins; D049030:Dystroglycans; D020780:Matrix Metalloproteinase 9