Structure of the Lectin Mannose 6-Phosphate Receptor Homology (MRH) Domain of Glucosidase II, an Enzyme That Regulates Glycoprotein Folding Quality Control in the Endoplasmic Reticulum

Abstract

Background: Glucosidase II is an endoplasmic reticulum enzyme involved in quality control of glycoprotein folding. Results: The structure of the lectin domain of GII was determined by NMR spectroscopy. Conclusion: GII lectin domain structure contains a unique Trp residue critical for GII activity. Significance: GII␤ MRH domain structure is the first determined of an MRH domain present in a protein with enzymatic activity.

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

“…Mutation of W409 decreased the efficiency of GII’s removal of glucose residues from G2M9 and G1M9 both in vivo and in vitro , although not to the extent observed for GII harboring a mutation of either glutamate or tyrosine in the primary mannose binding pocket. 18,33 However, contrary to the mutations within the primary mannose binding pocket, we observed that the W409A mutant was still able to bind Man α 1,2Man as monitored by 15 N– 1 H HSQC and surface plasmon resonance analyses using the lysosomal enzyme acid α -glucosidase. To further investigate the mechanism by which GII β binds glycans to enhance the catalytic activity of GII, structural studies of wild-type and mutant forms of the MRH domain of GII β subunit were performed and evaluated by in vitro and in vivo GII activity assays.…”

“…These constructs (pREP1-GII β Y372A and pREP1-GII β Y372F) were then electroporated into S. pombe competent Δ GIIβ cells. The pREP1-GII β W409A construct used in this study was described by Olson et al, 33 and the wild-type and MRH binding pocket mutant construct used in this study (pREP1-GII β and pREP1-GII β Y439F, respectively) were described by Stigliano et al (please note that the numbering of this last mutant was changed from Y462F used in ref 18 as throughout this work the numbering corresponds to the mature protein sequence instead of starting at the initial Met).…”

“…Briefly, GII activity in Δ GIIβ mutants transformed with either wild-type or mutant GII β subunits was assayed using 5 mM pNPG as the substrate in 0.1 M HEPES buffer (pH 7.2) for 20 min at 37 °C, and the absorbance at 405 nm was measured, or with [ 14 C-glucose]-Glc 1 Man 9 GlcNAc (obtained as described in ref 33) in 40 mM sodium phosphate buffer (pH 7.2) for 15 min at 30 °C. In the latter case, glucosidase activity was determined as the percentage of radioactive glucose released with respect to the total radioactivity.…”

“…Data were processed using NMRPipe 42 and visualized with XEASY. 43 Complete 1 H, 15 N, and 13 C resonance assignments for the W409A [0.7 mM protein, 10 mM deuterated imidazole (pH 7.1), and 150 mM NaCl] mutant of GII β MRH were determined by the transfer of assignments from the previously published wild-type structure collected under the same sample conditions 33 and confirmed using three-dimensional HNCO, HCCH total correlation spectroscopy and 13 C (aromatic)-edited NOESY-HSQC spectra. The structure was calculated using distance constraints derived from three-dimensional 15 N-edited NOESY-HSQC and 13 C-edited NOESY-HSQC spectra.…”

“…33 This structure revealed a tryptophan residue, W409 (Figure 2C), located close to the primary mannose binding pocket (which includes the four essential glutamine, arginine, glutamate, and tyrosine residues) that is conserved in GII β among all species. Mutation of W409 decreased the efficiency of GII’s removal of glucose residues from G2M9 and G1M9 both in vivo and in vitro , although not to the extent observed for GII harboring a mutation of either glutamate or tyrosine in the primary mannose binding pocket.…”