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.

Oligosaccharide Analysis in Urine by MALDI-TOF Mass Spectrometry for the Diagnosis of Lysosomal Storage Diseases

“…In addition, our assay was able to characterize specific patterns of subtypes of some oligosaccharidosis conditions and differentiate between the subtypes with severe early onset and mild clinical presentation. This technique can also be used to screen numerous other disorders characterized by urinary excretion of oligosaccharides, such as Pompe disease and CDG-IIb (congenital disorder of glycosylation IIb) (31 ). Compared to previous reports of FOS analysis in oligosaccharidosis, this study provides the most comprehensive profiling for each condition.…”

Structural and mechanistic insight into N-glycan processing by endo-α-mannosidase

“…Cells in which glucosidase II has been inhibited or subjected to genetic knockout retain 40-80% normal N-glycan processing function through the intervention of endo-α-mannosidase (8,35,36). During a castanospermine (CST)-imposed α-glucosidase I and II blockade, HepG2 cells produced N-linked glycoproteins with the normal glycan structure and resulted in concomitant release of free Glc 1-3 Man oligosaccharides (6).…”

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

“…Hex 9-10 HexNAc 2 (PMe) 2 , resulted in a fragment of m/z 997 (Hex 5 [PMe] 2 ) suggesting that both the B and C branches were modified with terminal methylphosphate residues ( Figure 6G). An alternative form of di-anionic glycan species is represented by Hex 8-10 HexNAc 2-3 PMeS structures; MS/MS showed the presence of m/z 255 (Hex 1 PMe) and 403 (Hex 2 S) and, in some cases, 497 (Hex 2 PMeS) fragments ( Figure 6E and H).…”

“…After transfer of a tetradecasaccharide containing three glucose, nine mannose and two N-acetylglucosamine residues (Glc 3 Man 9 GlcNAc 2 ) to protein, the three glucose residues are removed by α-glucosidases I and II, which are resident in the endoplasmic reticulum and have a neutral pH optimum (3,4) . Two human diseases are associated with defects in these enzymes: mutations of glucosidase I are associated with a congenital disorder of glycosylation (5) , whereas defects of the β-subunit of glucosidase II are found in some patients with polycystic liver disease (6) . Homologues of genes encoding glucosidase I and the α and β subunits of glucosidase II (7,8) are present in a range of organisms including the cellular slime mould (alternatively known as a social amoeba) Dictyostelium discoideum.…”