The heterodimeric structure of glucosidase II is required for its activity, solubility, and localization in vivo

Abstract

Glucosidase II is an ER heterodimeric enzyme that cleaves sequentially the two innermost alpha-1,3-linked glucose residues from N-linked oligosaccharides on nascent glycoproteins. This processing allows the binding and release of monoglucosylated (Glc(1)Man(9)GlcNAc(2)) glycoproteins with calnexin and calreticulin, the lectin-like chaperones of the endoplasmic reticulum. We have isolated two cDNA isoforms of the human alpha subunit (alpha1 and alpha2) differing by a 66 bp stretch, and a cDNA for the corresponding beta subunit. The alpha1 and alpha2 forms have distinct mobilities on SDS-PAGE and are expressed in most of the cell lines we have tested, but were absent from the glucosidase II-deficient cell line PHA(R) 2.7. Using COS7 cells, the coexpression of the beta subunit with the catalytic alpha subunit was found to be essential for enzymatic activity, solubilization, and/or stability, and ER retention of the alpha/beta complex. Transfected cell extracts expressing either alpha1 or alpha2 forms with the beta subunit showed similar activities, while mutating( )the nucleophile (D542N) predicted from the glycoside hydrolase Family 31 active site consensus sequence abolished enzymatic activity. In order to compare the kinetic parameters of both alpha1/beta and alpha2/beta forms of human glucosidase II the protein was expressed with the baculovirus expression system. Expression of the human alpha or beta subunit alone led to the formation of active human/insect heteroenzymes, demonstrating functional complementation by the endogenous insect glucosidase II subunits. The activity of both forms of recombinant human glucosidase II was examined with a p-nitrophenyl alpha-D-glucopyranoside substrate, and a two binding site kinetic model for this substrate was shown. The K(M1-2) values and apparent K(i1-2 )for deoxynojirimycin and castanospermine were determined and found to be identical for both isoforms suggesting they have similar catalysis and inhibition characteristics. The substrate specificities of both isoforms using the physiological oligosaccharides were assessed and found to be similar.

Yeast GTB1 Encodes a Subunit of Glucosidase II Required for Glycoprotein Processing in the Endoplasmic Reticulum

“…Gls2p Localization Is Unaffected in ⌬gtb1 Cells-It has been suggested that GII␤ may contribute to the stability and/or ER retention of GII␣ in mammalian cells (11,12). We therefore further examined the expression of Gls2p in ⌬gtb1 cells.…”

Genetic and molecular interactions of the Erv41p-Erv46p complex involved in transport between the endoplasmic reticulum and Golgi complex

“…Mammalian glucosidase II consists of a catalytic ␣ subunit and a 58 kDa ␤ subunit that localizes the enzyme to the ER (Trombetta et al, 1996;Pelletier et al, 2000). Although a ␤ subunit has recently been identified in yeast as well, it is not required for localization of Rot2p (Wilkinson et al, 2006).…”

Cell Biology of the Endoplasmic Reticulum and the Golgi Apparatus through Proteomics

“…Briefly, newly synthesized N-linked glycoproteins are glycosylated with 14 sugars donated from a dolichol-linked lipid via oligosaccharyl transferase (Das and Heath 1980). Following the sequential removal of the terminal glucose by glucosidase I (Hettkamp et al 1984;Bause et al 1989), the glycoprotein may then be recognized by the protein malectin (Schallus et al 2008;Chen et al 2011;Galli et al 2011), potentially for presentation to glucosidase II for removal of the second glucose residue (Burns and Touster 1982;Reitman et al 1982;Pelletier et al 2000). The resulting mono-glucosylated glycoprotein is then a client for association with calnexin or calreticulin (Hammond et al 1994;Zapun et al 1997;Schrag et al 2003).…”