Deficiency of the first mannosylation step in the N-glycosylation pathway causes congenital disorder of glycosylation type Ik

Abstract

Defects of N-linked glycosylation represent diseases with multiple organ involvements that are classified as congenital disorders of glycosylation (CDG). In recent years, several CDG types have been attributed to defects of dolichol-linked oligosaccharide assembly in the endoplasmic reticulum. The profiling of [3H]mannose-labeled lipid-linked oligosaccharides was instrumental in identifying most of these glycosylation disorders. However, this method is poorly suited for the identification of short lipid-linked oligosaccharide biosynthesis defects. To adequately resolve deficiencies affecting the first steps of lipid-linked oligosaccharide formation, we have used a non-radioactive procedure employing the fluorescence detection of 2-aminobenzamide-coupled oligosaccharides after HPLC separation. By applying this method, we have detected the accumulation of dolichylpyrophosphate-GlcNAc2 in a previously untyped CDG patient. The accumulation pattern suggested a deficiency of the ALG1 beta1,4 mannosyltransferase, which adds the first mannose residue to lipid-linked oligosaccharides. This was supported by the finding that this CDG patient was compound heterozygous for three mutations in the ALG1 gene, leading to the amino acid substitutions S150R and D429E on one allele and S258L on the other. The detrimental effect of these mutations on ALG1 protein function was demonstrated in a complementation assay using alg1 Saccharomyces cerevisiae yeast mutants. The ALG1 mannosyltransferase defect described here represents a novel type of CDG, which should be referred to as CDG-Ik.

Glycoprotein Maturation and the UPR

“…To separate 2AB-labeled oligosaccharides, we have adapted the procedure of Royle et al (Royle et al, 2002) using a three buffer solvent system (Grubenmann et al, 2004). We use a GlykoSep-N column maintained at 30 °C.…”

Drosophila, an emerging model for cardiac disease

“…In this case, based on a phenotypic analysis of a mutant in a yeast CDG homolog (Imbach et al, 1999) and its rescue by the human homolog, a screen was devised to identify other genes acting in the same glycosylation pathway in yeast. Subsequently, it was found that mutations in human homologs of these newly identified yeast genes caused CDG (Westphal et al, 2002;Imbach et al, 2000;Grubenmann et al, 2004). Thus, model genetic systems can be viewed as first pass in vivo filters or as bgenetic bioassaysQ to search for relevant genetic interactions among a potentially large set of genes.…”

Gain-of-glycosylation mutations

“…Furthermore, on the basis of in silico analysis, some CDG patients would be expected to carry loss of O-or N-glycosylation mutations in the corresponding glycosylation enzyme (G Vogt et al, unpublished). For example, a loss of N-glycosylation has been reported for one mutant enzyme involved in O-glycosylation [12]; nine examples have been reported in which a loss of O-glycosylation was observed in two mutant enzymes involved in N-glycosylation [13,14] and in seven mutant enzymes involved in O-glycosylation [15][16][17][18]. Intriguingly, pathogenic mutations involving gains of glycosylation also occur and, paradoxically, some of these mutations also affect glycosylation enzymes and cause CDGs (see below).…”