Structural and functional characterization of Glucosidase II N-glycan binding domain

DAHMS, NM; OLSON, LJ; ALCULUMBRE, S.G.; STIGLIANO, I.D.; PETERSON, F.C; CARAMELO, JJ; PARODI, A.J.; D'ALESSIO, C.

San Diego, CA

Congreso; American Society for Biochemistry and Molecular Biology (ASBMB) at Experimental Biology 2012; 2012

American Society for Biochemistry and Molecular Biology

Glucosidase II (GII) is a key player in glycoprotein biogenesis in the endoplasmic reticulum (ER). GII is composed of a catalytic GIIalpha subunit, which removes sequentially the two innermost Glc residues from the Glc3Man9GlcNAc2 glycan on nascent proteins, and a GIIbeta regulatory subunit. GIIbeta retains GIIalpha in the ER and mediates N-glycan in vivo recognition by GII through its C-terminal mannose 6-phosphate receptor homology (MRH) domain. Here we report the expression, purification and characterization of the Schizosaccharomyces pombe GIIbeta subunit and its MRH domain. Purified GIIbeta was active in a functional complementation test, as mixing the microsomal fraction of a S. pombe mutant expressing only GIIalpha in the ER with the purified GIIbeta subunit restored the ability of the catalytic subunit to efficiently hydrolyze the physiological substrate G1M9. MRH domain was unable to functionally complement isolated GIIalpha activity. However, an excess of purified MRH domain inhibited full length GIIbeta-mediated restoration of GIIalpha activity. These results show that the MRH domain cannot interact with GIIalpha, suggesting that GIIbeta G2B domain is involved in GIIalpha-GIIbeta interaction. The results also show that the MRH domain competes with GIIbeta for G1M9 binding, and thus is fully functional in binding N-glycans. The NMR structure of the b-strand-containing MRH domain was determined. (NIH-NIDDK, Mizutani Foundation for Glycoscience, CONICET)