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 alpha-subunit of the heterodimeric glucosidase II (GII). GIIalphas hydrolytic activity is regulated by the mannose binding activity of the mannose 6-phosphate receptor (MPR) homology domain (MRH domain) contained within GII beta-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 GIIbeta MRH domain and nascent glycoproteins bearing Man9-containing glycan chains. To better understand the role of GIIbetas MRH domain, we previously determined the solution structure of Schizosaccharomyces pombe GIIbeta-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 GIIbeta-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.