Consequences of disrupting the gene that encodes α‐glucosidase II in the N‐linked oligosaccharide biosynthesis pathway of Dictyostelium discoideum

Abstract

We have identified and disrupted the gene coding for alpha-glucosidase II in Dictyostelium discoideum. This enzyme is responsible for removing two alpha 1,3-linked glucose residues from N-linked oligosaccharides on newly synthesized glycoproteins. Mutagenesis by restriction enzyme-mediated integration (REMI) generated a clone, DG1033, which grows well but forms abnormal fruiting bodies with short, thick stalks. The strain lacks alpha-glucosidase II activity and makes incompletely processed N-linked oligosaccharides that are abnormally large and have fewer sulfate and phosphate esters. The morphological, enzymatic, and oligosaccharide profile phenotypes of the disruption mutant are all recapitulated by a targeted disruption of the normal gene. Furthermore, all of these defects are corrected in cells transformed with a normal, full-length copy of the gene. The phenotypic characteristics of DG1033 as well as chromosomal mapping of the disrupted gene indicate that it is the site of the previously characterized modA mutation. The Dictyostelium gene is highly homologous to alpha-glucosidase II genes in the human and the pig, C. elegans, and yeast. Although various cell lines have been reported to be defective in alpha-glucosidase II activity, disruption of the Dictyostelium gene gives the first example of a clear developmental phenotype associated with loss of this enzyme.

Endoplasmic Reticulum Glucosidase II Is Required for Pathogenicity ofUstilago maydis [W]

“…Glucosidases are proposed to be crucial for ER quality control of glycoprotein folding and exit from the ER (Helenius and Aebi, 2001). In all systems studied to date, GIIa is encoded by a unique gene (Trombetta et al, 1996;Freeze et al, 1997;Simons et al, 1998). In the available U. maydis sequence, we have detected one gene with significant similarity to gas1 whose product shares with Gas1 the catalytic site motif of family-31 glycosyl hydrolases (Henrissat, 1991) (Broad Institute, http://www.broad.mit.edu/seq/; contig 1.92, position 7939-4835).…”

“…The only other case where mutation in an a-glucosidase leads to a developmental defect is D. discoideum, where modA mutants are perfectly viable but produce stunted fruiting bodies with a short, thickened stalk. For this system, it has been speculated that correctly trimmed sugar chain structures in glycoproteins may be required for correct stalk assembly (Freeze et al, 1997). N-linked sugars of glycoproteins have been implicated in correct protein folding, secretion, and protein stability (Rademacher et al, 1988;Varki, 1993).…”

N‐glycomic profiling of a glucosidase II mutant of Dictyostelium discoideum by ‘‘off‐line’’ liquid chromatography and mass spectrometry

“…Inhibition or ablation of this enzyme is expected to prevent further processing by glycosidases and glycosyltransferases in the secretory pathway. Previous data on the Dictyostelium discoideum M31 glucosidase II mutant have indicated that radiolabelled N-glycans are increased in size (as judged by gel filtration) and have a decreased degree of sulphation and phosphorylation (as shown by anion-exchange chromatography) in comparison to the wild-type (15,24,47) . Our new mass spectrometric data confirm that the N-glycome of Dictyostelium is strongly affected in the M31 glucosidase II mutant; nevertheless, our study indicates that a remarkable degree of processing still takes place.…”

“…The Dictyostelium M31 mutant (16) was previously shown to be phenocopied by a specific mutation of the modA glucosidase II gene in the DG1033 strain as well as be rescued by transformation with the wild-type gene under control of an actin promoter (24) . However, the exact nature of the mutation in M31 was not known.…”

“…In this study, we apply our methodology to examination of the N-glycans of a glucosidase II mutant of D. discoideum; this M31 strain has been previously shown to have altered size and charge of its N-glycans, which are rescuable effects phenocopied by a specific knock-out of the gene encoding the glucosidase II catalytic α subunit (24) . Thereby we note a reduced glycomic complexity as compared to the wild-type, but also the presence of anionic oligosaccharides with unexpected backbone structures.…”