Crystal Structure of Glucosidase II beta Mannose 6-Phosphate Receptor Homology (MRH) Lectin Domain

DAHMS, NM; PETERSON, F.C; D'ALESSIO, C; KIM J; OLSON, LJ

St. Petersburg, FL

Congreso; Annual Meeting of the Society for Glycobiology; 2013

In the endoplasmic reticulum (ER) of eukaryotic cells, N-glycan structures attached to proteins are modified as part of a quality control mechanism ensuring the correct folding of these secretory pathway proteins. In most eukaryotes, Glc3Man9GlcNAc2 is transferred from a dolichol pyrophosphate derivative to nascent polypeptides in the ER. Glucosidase I immediately removes the terminal glucose of the glycan revealing two inner glucose moieties that are in turn trimmed by the catalytic α-subunit of the heterodimeric glucosidase II (GII). GII?s hydrolytic activity is regulated by the mannose binding activity of the mannose 6-phosphate receptor (MPR) homology domain (MRH domain) contained within GII?s β-subunit. Removal of the middle glucose creates a monoglucosylated glycan that is recognized by lectin-chaperones, calnexin (CNX) and calreticulin (CRT), known to enhance polypeptide folding and ER retention of misfolded glycoproteins. Additional opportunities for folding are available through the reglucosylating activity of UDP-Glc:glycoprotein glucosyltransferase (UGGT) which recognizes non-native conformations and regenerates monoglucosylated glycoproteins capable of reassociating with CNX/CRT. GII also removes glucose added by UGGT irrespective of the protein?s folding status. The opposing activities of GII and UGGT allow for cycles of deglucosylation-reglucosylation to occur until either the glycoprotein acquires its native conformation or it is marked for degradation in a process known as ER-associated degradation (ERAD). MRH domains have long been investigated in the context of trafficking acid hydrolases to endosomal/lysosomal compartments and more recently have been studied in the resident ER proteins OS-9 and XTP3-B, which function in ERAD, and in the non-catalytic subunit of Golgi GlcNAc-phosphotransferase, which modifies acid hydrolases for their subsequent interaction with MPRs. Optimal GII deglucosylation activity requires a functional GIIβ MRH domain and nascent glycoproteins bearing Man9-containing glycan chains. To better understand the role of GIIβ?s MRH domain, we previously determined the solution structure of Schizosaccharomyces pombe GIIβ?s MRH domain that revealed the conserved MRH fold observed in the MPRs and OS-9. We now report a 1.6Å crystal structure of S. pombe GIIβ?s MRH domain in the presence of bound mannose and compare its shallow binding pocket with the phosphorylated mannose-specific and Mana1,6Mana1,6Man-specific MRH domain structures of MPRs and OS-9, respectively.