tailieunhanh - Báo cáo khoa học: Characterization of recombinant forms of the yeast Gas1 protein and identification of residues essential for glucanosyltransferase activity and folding

Gas1p is a glycosylphosphatidylinositol-anchored plasma membrane glycoprotein ofSaccharomyces cerevisiaeand is a representative of Family GH72 of glycosidases/transgly-cosidases, which also includes proteins from human fungal pathogens. Gas1p, Phr1-2p from Candida albicansand Gel1p fromAspergillus fumigatushave been shown to be b-(1,3)-glucanosyltransferases required for proper cell wall assembly andmorphogenesis. Gas1p is organized into three modules: a catalytic domain; a cys-richdomain; andahighly O-glycosylated serine-rich region. . | Eur. J. Biochem. 271 3635-3645 2004 FEBS 2004 doi Characterization of recombinant forms of the yeast Gas1 protein and identification of residues essential for glucanosyltransferase activity and folding Cristina Carotti1 Enrico Raani1 Oscar Palomares2 Thierrv Fontaine3 Gabriella Tedeschi4 Rosalia Rodríguez2 Jean Paul Latge3 Marina Vai5 and Laura Popolo1 1 Dipartimento di Scienze Biomolecolari e Biotecnologie Universita degli Studi di Milano Milano Italy 2Departamento de Bioquimica y Biologia Molecular Facultad de Ciencias Quimicas Universidad Complutense Madrid Spain 3Institut Pasteur Laboratoire des Aspergillus Paris France 4Dipartimento di Patologia Animale Igiene e Sanita Pubblica Veterinaria Universita degli Studi di Milano Milano Italy 5Dipartimento di Biotecnologie e Bioscienze Universita degli Studi di Milano-Bicocca Milano Italy Gas1p is a glycosylphosphatidylinositol-anchored plasma membrane glycoprotein of Saccharomyces cerevisiae and is a representative of Family GH72 of glycosidases transgly-cosidases which also includes proteins from human fungal pathogens. Gas1p Phr1-2p from Candida albicans and Gel1p from Aspergillus fumigatus have been shown to be b- 1 3 -glucanosyltransferases required for proper cell wall assembly and morphogenesis. Gas1p is organized into three modules a catalytic domain a cys-rich domain and a highly O-glycosylated serine-rich region. In order to provide an experimental system for the biochemical and structural analysis of Gas1p we expressed soluble forms in the meth-ylotrophic yeast Pichia pastoris. Here we report that 48 h after induction with methanol soluble Gas1p was produced at a yield of w 10 mg L-1 of medium and this value was unaffected by the further removal of the serine-rich region or by fusion to a 6 X His tag. Purified soluble Gas1 protein showed b- 1 3 -glucanosyltransferase activity that was abolished by replacement of the putative catalytic residues E161 and E262 with glutamine. .