Expression and characterization of functional Glucosidase II  subunit

ALCULUMBRE, SOLANA G; STIGLIANO, ID; CARAMELO, JJ; PARODI, AJ; D'ALESSIO, C

Puerto Madryn, Chubut, Argentina

Congreso; XLVI Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2010

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Glucosidase II (GII) is a key player in glycoprotein biogenesis in the endoplasmic reticulum (ER). It is a heterodimer composed of a GIIalpha subunit that catalyzes the sequential removal of the two innermost Glc residues from the Glc3Man9GlcNAc2 glycan transferred to Asn residues in nascent proteins, and a GIIbeta regulatory subunit. GIIbeta is responsible for GIIalpha retention in the ER and for N-glycan in vivo recognition by GII through its mannose 6 phosphate receptor homologue domain (MRH). Direct binding of the complete isolated GIIbeta subunit to N-glycans has never been reported. Here we report the expression, purification and characterization of the Schizosaccharomyces pombe GIIbeta subunit. Circular dichroism spectra of the affinity purified subunit showed that the protein has a predominantly a helix secondary structure. Purified GIIbeta resulted active in a functional complementation test as mixing the microsomal fraction of a S. pombe mutant expressing only GIIa in the ER with the purified GIIbeta subunit restored the ability of the catalytic subunit to efficiently hydrolyze the physiological substrate G1M9. This is the first report of the expression of an active isolated GIIbeta subunit. The protein will be used to study its structure by NMR and to asses its binding specificity toward N-glycans.