NMR STRUCTURE OF THE LECTIN DOMAIN OF GLUCOSIDASE II AND CONFIRMATION OF THE MODEL OF GLYCAN BINDING

ORSI, R.; OLSON, LJ; ALCULUMBRE, SOLANA G; PETERSON, F.C; STIGLIANO, I.D.; PARODI AJ; DAHMS, NM; D'ALESSIO, C.

Mendoza

Congreso; XLVIII reunión anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2012

Glucosidase II (GII) removes the two innermost Glc residues from the glycan (Glc3Man9GlcNAc2) transferred to proteins during N-glycosylation. It also participates in cycles involving the lectin/chaperones calnexin and calreticulin as it removes the single Glc unit added to folding intermediates and misfolded glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase, a glycoprotein folding sensor. GII is a heterodimer whose alpha subunit (GIIα) bears the active site. We have previously shown that the C-terminus Mannose 6-Phosphate Receptor Homologous (MRH) domain present in GII beta subunit (GIIβ) enhances the deglucosylation activity of GII. Here we show that isolated GIIβ MRH domain competes with full length GIIβ for N-glycan binding and present its NMR structure in solution. We demonstrate that the amino acid W409, conserved in almost all GIIβ MRH domains from different species, is involved in the enhancement of GII activity by MRH as mutagenesis of that residue reduces activity of heterodimeric GII in vitro and delays N-glycan deglucosylation of ER glycoproteins in vivo without affecting neither GIIα-GIIβ interaction, nor GIIα active site. Our results show how the MRH domain of the beta subunit presents the glucose-containing arm of the N-glycan to the catalytic site of GII?s alpha subunit