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Glycobiology. 2002 Aug;12(8):473-83.

Processing of N-linked carbohydrate chains in a patient with glucosidase I deficiency (CDG type IIb).

Author information

1
Institut für Physiologische Chemie, Universität Bonn, Bonn, Germany.

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.

PMID:
12145188
DOI:
10.1093/glycob/cwf050
[Indexed for MEDLINE]

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