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

DAHMS N; OLSON L; ALCULUMBRE S; STIGLIANO I; PETERSON F; CARAMELO J; PARODI A; DALESSIO C

San Diego

Congreso; Annual Meeting of the American Society of Biochemistry and Molecular Biology; 2012

American Society of 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 GIIa subunit, which removes sequentially the two innermost Glc residues from the Glc3Man9GlcNAc2 glycan on nascent proteins, and a GIIb regulatory subunit. GIIb retains GIIa 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 GIIb subunit and its MRH domain. Purified GIIb was 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 GIIb subunit restored the ability of the catalytic subunit to efficiently hydrolyze the physiological substrate G1M9. MRH domain was unable to functionally complement isolated GIIa activity. However, an excess of purified MRH domain inhibited full length GIIb-mediated restoration of GIIa activity. These results show that the MRH domain cannot interact with GIIa, suggesting that GIIb G2B domain is involved in GIIa-GIIb interaction. The results also show that the MRH domain competes with GIIb for G1M9 binding, and thus is fully functional in binding N-glycans. The NMR structure of the b-strand-containing MRH domain was determined.