Glycobiology, 2002, Vol. 12, No. 8 473-483
© 2002 Oxford University Press
Processing of N-linked carbohydrate chains in a patient with glucosidase I deficiency (CDG type IIb)
2 Institut für Physiologische Chemie, Universität Bonn, Bonn, Germany; 3 Department of Pediatrics, Division of Neonatal Intensive Care, Gent University Hospital, Gent; and 4 Department of Pediatrics, Division of Neurology and Metabolic Diseases, Gent University Hospital, Gent
Recently, we reported a novel congenital disorder of glycosylation (CDG-IIb) caused by severe deficiency of the glucosidase I. The enzyme cleaves the 
1,2-glucose residue from the asparagine-linked Glc3-Man9-GlcNAc2 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 GC 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 Man9-mannosidase, were observed. By contrast, the patient’s fibroblasts displayed a two- to threefold higher endo-1,2-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-1,2-mannosidase; this would also explain the marked urinary excretion of Glc3-Man. Comparative analysis of [3H]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 
16% of the N-glycans being arrested at the Glc3-Man9–7-GlcNAc2 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.
1 To whom correspondence should be addressed; E-mail: bause@institut.physiochem.uni-bonn.de
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