title: Processing of N-linked carbohydrate chains in a patient with glucosidase I deficiency (CDG type IIb).
abstract: Recently, we reported a novel congenital disorder of glycosylation (CDG-IIb) caused by severe deficiency of the glucosidase I. The enzyme cleaves the alpha1,2-glucose residue from the asparagine-linked Glc(3)-Man(9)-GlcNAc(2) precursor, which is crucial for oligosaccharide maturation. The patient suffering from this disease was compound-heterozygous for two mutations in the glucosidase I gene, a T-->C transition in the paternal allele and a G-->C transition in the maternal allele. This gives rise in the glucosidase I polypeptide to the substitution of Arg486 by Thr and Phe652 by Leu, respectively. Kinetic studies using detergent extracts from cultured fibroblasts showed that the glucosidase I activity in the patient's cells was < 1% of the control level, with intermediate values in the parental cells. No significant differences in the activities of other processing enzymes, including oligosaccharyltransferase, glucosidase II, and Man(9)-mannosidase, were observed. By contrast, the patient's fibroblasts displayed a two- to threefold higher endo-alpha1,2-mannosidase activity, associated with an increased level of enzyme-specific mRNA-transcripts. This points to the lack of glucosidase I activity being compensated for, to some extent, by increase in the activity of the pathway involving endo-alpha1,2-mannosidase; this would also explain the marked urinary excretion of Glc(3)-Man. Comparative analysis of [(3)H]mannose-labeled N-glycoproteins showed that, despite the dramatically reduced glucosidase I activity, the bulk of the N-linked carbohydrate chains (>80%) in the patient's fibroblasts appeared to have been processed correctly, with only approximately 16% of the N-glycans being arrested at the Glc(3)-Man(9-7)-GlcNAc(2) stage. These structural and enzymatic data provide a reasonable basis for the observation that the sialotransferrin pattern, which frequently depends on the type of glycosylation disorder, appears to be normal in the patient. The human glucosidase I gene contains four exons separated by three introns with exon-4 encoding for the large 64-kDa catalytic domain of the enzyme. The two base mutations giving rise to substitution of Arg486 by Thr and Phe652 by Leu both reside in exon-4, consistent with their deleterious effect on enzyme activity. Incorporation of either mutation into wild-type glucosidase I resulted in the overexpression of enzyme mutants in COS 1 cells displaying no measurable catalytic activity. The Phe652Leu but not the Arg486Thr protein mutant showed a weak binding to a glucosidase I-specific affinity resin, indicating that the two amino acids affect polypeptide folding and active site formation differently.
authors.affiliation: Institut für Physiologische Chemie, Universität Bonn, Bonn, Germany.
authors.forename: Christof
authors.initials: C
authors.lastname: Völker
authors.forename: Claudine M
authors.initials: CM
authors.lastname: De Praeter
authors.forename: Birgit
authors.initials: B
authors.lastname: Hardt
authors.forename: Willi
authors.initials: W
authors.lastname: Breuer
authors.forename: Burga
authors.initials: B
authors.lastname: Kalz-Füller
authors.forename: Rudy N
authors.initials: RN
authors.lastname: Van Coster
authors.forename: Ernst
authors.initials: E
authors.lastname: Bause
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002236:Carbohydrate Conformation; D002239:Carbohydrate Metabolism, Inborn Errors / Q000201:enzymology* / Q000235:genetics* / Q000652:urine; D002240:Carbohydrate Sequence; D002478:Cells, Cultured; D005260:Female; D005347:Fibroblasts / Q000201:enzymology; D006031:Glycosylation; D006579:Heterozygote; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D008361:Mannosidases / Q000378:metabolism; D020125:Mutation, Missense; D010375:Pedigree; D017354:Point Mutation; D011134:Polysaccharides / Q000737:chemistry / Q000235:genetics; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000032:analysis; D000520:alpha-Glucosidases / Q000172:deficiency* / Q000235:genetics* / Q000378:metabolism
chemical_list: D011134:Polysaccharides; D012333:RNA, Messenger; D008361:Mannosidases; C042059:glucosidase I; D000520:alpha-Glucosidases