Crystal Structure and Functional Analyses of the Lectin Domain of Glucosidase II: Insights into Oligomannose Recognition

Abstract

N-Glycans are modified as part of a quality control mechanism during glycoprotein folding in the endoplasmic reticulum (ER). Glucosidase II (GII) plays a critical role by generating monoglucosylated glycans that are recognized by lectin chaperones, calnexin and calreticulin. To understand how the hydrolytic activity of GIIα is enhanced by the mannose 6-phosphate receptor (MPR) homology domain (MRH domain) of its β subunit, we now report a 1.6 Å resolution crystal structure of the MRH domain of GIIβ bound to mannose. A comparison of ligand-bound and unbound structures reveals no major difference in their overall fold, but rather a repositioning of side chains throughout the binding pocket, including Y372. Mutation of Y372 inhibits GII activity, demonstrating an important role for Y372 in regulating GII activity. Comparison of the MRH domains of GIIβ, MPRs, and the ER lectin OS-9 identified conserved residues that are critical for the structural integrity and architecture of the carbohydrate binding pocket. As shown by nuclear magnetic resonance spectroscopy, mutations of the primary binding pocket residues and adjacent W409, all of which inhibit the activity of GII both in vitro and in vivo, do not cause a significant change in the overall fold of the GIIβ MRH domain but impact locally the stability of the binding pocket. W409 does not directly contact mannose; rather, its indole ring is stabilized by binding into a hydrophobic pocket of an adjacent crystallographic neighbor. This suggests that W409 interacts with a hydrophobic region of the GIIβ or GIIα subunit to modulate its effect on GII activity.

Structures of mammalian ER α-glucosidase II capture the binding modes of broad-spectrum iminosugar antivirals

“…The differences in the activity of the enzyme against glycoproteins carrying a variable number of N-linked glycans have led to the suggestion that more than one glycan is needed for α-GluII to allow entry of a glycoprotein into the calnexin/calreticulin cycle (29). In this case, the mannose 6-phosphate receptor homology (M6PRH) C-terminal domain of the β-subunit would bind a terminal α(1,2)-linked mannose residue on one N-linked glycan (30,31), helping the recruitment of the other N-linked glycan to the active site (29,32). The shape and dimensions of our Mmα-GluII SAXS envelope and Mmα-GluII Tryps crystal structure are compatible with a role for the M6PRH C-terminal domain of the β-subunit assisting glucose hydrolysis by binding a second glycan on glycoproteins carrying more than one glycan (29,32).…”

“…In this case, the mannose 6-phosphate receptor homology (M6PRH) C-terminal domain of the β-subunit would bind a terminal α(1,2)-linked mannose residue on one N-linked glycan (30,31), helping the recruitment of the other N-linked glycan to the active site (29,32). The shape and dimensions of our Mmα-GluII SAXS envelope and Mmα-GluII Tryps crystal structure are compatible with a role for the M6PRH C-terminal domain of the β-subunit assisting glucose hydrolysis by binding a second glycan on glycoproteins carrying more than one glycan (29,32). In the absence of the β-subunit, α-GluII activity is reduced in vivo (27) and in vitro (33) to about 5-10% of wt levels and the recombinant α-subunit is unstable unless associated with the β-subunit (4, 28).…”

“…It is therefore likely that the two substrates are inserted in the active site with the N-linked glycan bound to α-GluII in different orientations (34). These observations, in turn, have led to models in which a single Glc 1 Man x GlcNAc 2 glycan is simultaneously bound to the α-subunit active site and the M6PRH C-terminal domain of the β-subunit, which would assist the reorientation of the glycan needed for the second cleavage (29,32). Our docking of the Mmα-GluII Tryps crystal structure into the Mmα-GluII SAXS envelope shows that, in the absence of a major conformational change, the distance between the active site of the α-subunit and the closest portion of the β-subunit is comparable to the length of a single N-glycan in extended conformation.…”

Interaction mode between catalytic and regulatory subunits in glucosidase II involved in ER glycoprotein quality control

“…Using this microorganism, we have successfully determined crystal structures of enzymes involved in ER quality control, including GIIα . It has been confirmed that closely related species, such as Aspergillus oryzae and Schizosaccharomyces pombe, have enzymatically active GII with the same substrate specificity as that of the mammalian counterparts . These results encouraged us to perform a structural study of GIIβ derived from C. thermophilum .…”

“…Mutations in GANAB and PRKCSH encoding GIIα and GIIβ cause autosomal‐dominant polycystic kidney and liver diseases (ADPKD and ADPLD) . To date, the 3D structures of GIIα and the MRH domain of GIIβ have been determined . However, structural information of GIIβ G B as well as its mode of interaction with GIIα has not been elucidated.…”