Enzymatic processing of beta-dystroglycan recombinant ectodomain by MMP-9: identification of the main cleavage site.
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.
Bozzi
Manuela
M
Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Largo Francesco Vito 1, Rome, Italy.
Inzitari
Rosanna
R
Sbardell
Diego
D
Monaco
Susanna
S
Pavoni
Ernesto
E
Gioia
Magda
M
Marini
Stefano
S
Morlacchi
Simona
S
Sciandra
Francesca
F
Castagnola
Massimo
M
Giardina
Bruno
B
Brancaccio
Andrea
A
Coletta
Massimo
M
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
D011994:Recombinant Proteins; D049030:Dystroglycans; D020780:Matrix Metalloproteinase 9