Structure of the MRH domain of glucosidase II, a key enzyme in glycoprotein folding quality control

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

Congreso; Glycobiology Gordon Research Conference; 2013

Glucosidase II (GII) is a key player in glycoprotein folding in the endoplasmic reticulum (ER). GII is responsible for removal of both inner glucoses from the Glc3Man9GlcNAc2 glycan transferred upon protein N-glycosylation and of the glucose residue added to folding intermediates by the ER lumen glycoprotein conformational sensor, the UDP-Glc:glycoprotein glucosyltransferase. GII is a heterodimeric protein composed of a catalytic GIIα subunit and a regulatory GIIβ subunit. GIIβ is involved in GIIa?s ER localization and mediates in vivo enhancement of N-glycan trimming by GII through its C-terminal mannose 6-phosphate receptor homology (MRH) domain. We determined the structure of a functional GIIβ MRH domain by NMR spectroscopy. The 94-residue construct adopts a β-barrel fold similar to that of other MRH domains, but its binding pocket is the most shallow known to date as it accommodates a single mannose residue. In addition, we identified a conserved residue (W409) present in GIIβ MRH domain but not in other MRHs that influences GII glucose trimming activity in vivo.  Differences with the binding pocket of the MRH domain of OS-9, an ER protein responsible for driving misfolded glycoproteins to proteasomal degradation, could explain the different N-glycan specificity of both proteins: maximal lectin activity of glucosidase II and OS-9 MRH domains is observed with fully mannosylated glycans in the former case and with ER mannosidase-trimmed glycans in the latter. A model of the MRH domain bound to Man9 glycan provides insight into how GIIb could enhance the catalytic activity of GII: binding of the mannose-containing C arm of the glycan to the binding pocket and of B arm to W409 would position the glucose-containing A arm to be cleaved by GIIα. This is the first report of the three-dimensional structure of an MRH domain present in a protein with enzymatic activity.