The  - and  -subunits are required for expression of catalytic activity in the hetero-dimeric glucosidase II complex from human liver

Abstract

The alpha- and beta-subunits of the hetero-dimeric glucosidase II complex from human liver were cloned and expressed in COS-1 cells. The 4106 bp full-length cDNA for the alpha-subunit contained a 2835 bp ORF encoding a 107 kDa polypeptide. The 2095 bp cDNA for the beta-subunit encodes a approximately 60 kDa protein in a continuous 1605 bp ORF. The alpha- and beta-subunits each contain two potential Asn-Xaa-Thr/Ser acceptor sites, with only one site in the alpha-subunit (Asn97) being glycosylated. Additional lambda-clones were isolated for each subunit containing in-frame insertions/deletions within the coding region, indicating alternative splicing. Analysis of different human tissues revealed approximately 4.4 kb and approximately 2.4 kb transcripts for alpha- and beta-subunit, respectively, consistent with their full-length cDNA. Coexpression of the alpha- and beta-subunits in COS-1 cells resulted in >4-fold increase of glucosidase II activity. An inactive protein was obtained, however, after transfection with the alpha-subunit alone, showing that both subunits are essential for expression of active glucosidase II. The observation that the enzyme, previously purified from pig liver and lacking the beta-subunit, was catalytically active indicates that the beta-subunit is involved in alpha-subunit maturation rather than being required for enzymatic activity once the alpha-subunit has acquired its mature form. The alpha-subunit is expressed in COS-1 cells as an ER-located protein, whether inactive or part of a catalytically active complex. This suggests that ER-localization of the alpha-subunit, when associated with the dimeric enzyme complex, is mediated by the C-terminal HDEL-signal in the beta-subunit, whereas the apparently incompletely folded form of the inactive alpha-subunit could be retained in the ER by the putative "glycoprotein-specific quality control machinery. "

Endoplasmic Reticulum α-Glycosidases of Candida albicans Are Required for N Glycosylation, Cell Wall Integrity, and Normal Host-Fungus Interaction

“…Again, this sequence was typical of a type II membrane protein, having a predicted 2-amino-acid cytosolic tail and a 20-amino-acid transmembrane region. The glycosyl hydrolase family 31 members contain a short peptide segment of conserved amino acids (DGXWIDMNEXSXF), including a conserved Asp residue that is thought to be involved in catalysis (25,32,33,38,72). A similar peptide sequence ( 487 IHLWNDMNEPSVF 499 ) is present in CaRot2, indicating that this protein also belongs to the same protein family.…”

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

“…Gtb1p is a soluble glycoprotein localized in the ER lumen, where it interacts with Gls2p in a manner reminiscent of the heterodimeric structure of mammalian Glucosidase II (11,12). Interestingly, neither Gls2p nor Gtb1p contain any recognizable ER retention motifs, and yet both are evidently ER-localized.…”

“…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.…”

Cloning and Characterization of the Glucosidase II Alpha Subunit Gene of Trichoderma reesei : a Frameshift Mutation Results in the Aberrant Glycosylation Profile of the Hypercellulolytic Strain Rut-C30

“…Since then, glucosidase II has been partially or completely purified from a wide variety of both lower and higher eukaryotic organisms (2,6,7,11,20,38,78,80,88). Following some initial controversy, it is now generally accepted that the protein is a rather asymmetric nonglobular heterodimer consisting of a catalytic alpha subunit with a molecular mass of about 110 kDa (GII␣) and a beta subunit with a molecular mass of 60 kDa (GII␤) (6,15,65,85,86,88).…”

“…Recent data also indicate the involvement of GII␤ in the maturation of the glucosidase II heterodimer. While coexpression of the alpha and beta subunits increases total glucosidase II activity, expression of GII␣ alone results in an insoluble and inactive aggregate (65,85). However, once GII␣ is fully folded, the beta subunit is no longer necessary for activity (86).…”