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Glycosylation defects: To add sugar, reduce fat
Functional Glycomics (12 August 2010) | doi:10.1038/fg.2010.25Standfirst
SRD5A3 catalyzes a reduction step in the synthesis of precursors for N-glycosylation, and is mutated in congenital disorders of glycosylation.
Cantagrel, V. et al. Cell 142, 203–217 (2010) doi:10.1016/j.cell.2010.06.001
Congenital disorders of glycosylation (CDGs) are hereditary disorders caused by mutations that affect the maturation, transfer or processing of N-glycans. In characterizing a new CDG, Vincent Cantagrel et al. identify steroid 5α-reductase type 3 (SRD5A3) as a polyprenol reductase that has a conserved role in preparation of the lipid anchor required for N-glycan assembly.
Most eukaryotic secreted and plasma membrane proteins are posttranslationally modified with N-linked glycans, thus disruptions in glycosylation can cause wide-ranging and severe phenotypes. The glycan precursor is assembled on a lipid anchor in the ER membrane, transferred to the nascent protein, and modified in the Golgi. The identity of the lipid carrier — dolichol phosphate (Dol-P), generated by reduction of a precursor polyprenol followed by phosphorylation— has long been known, but the reductase enzyme required for its synthesis was unidentified.
Reporting in Cell, the authors describe a new CDG caused by mutations in the SRD5A3 gene, and show that the enzyme and its orthologs are likely to be the polyprenol reductases required for eukaryotic N-glycan synthesis.
The unexpected involvement of SRD5A3 in glycosylation was discovered in a genome-wide linkage analysis performed on a family with an apparent congenital disorder. The phenotype resembled that of known CDGs, and in screening tests the authors found that glycosylation was defective in the affected individuals. A mutation in SRD5A3 was found, and five independent mutations in the gene were discovered in a screen of patients with CDGs of unknown genetic cause. Fibroblasts from SRD5A3 deficient patients showed reduced synthesis of the lipid linked oligosaccharide Dol-PP-GlcNAc1/2, and this was rescued by exogenous Dol-P.
To find out whether the role of SRD5A3 is conserved, the authors investigated its yeast and mouse orthologs, DFG10 and Srd5a3. In yeast, underglycosylation of a secreted protein in DFG10-deficient cells was rescued by expression of SRD5A3, whereas mouse embryos with mutant Srd5a3 did not survive beyond day 12.5. Whole transcriptome analysis of these embryos revealed upregulation of genes involved in the unfolded protein response, consistent with the critical role of N-glycosylation for proper protein folding during development.
A defect in the metabolism of polyprenols was discovered in each of the tested organisms. Analysis by liquid chromatography-mass spectrometry showed that polyprenols were undetectable in samples of wildtype origin, but increased in mutant yeast cells and mouse embryos, and in plasma from human patients. By adding exogenous polyprenol to cultured human embryonic kidney cells, the authors showed that overexpression of SRD5A3 increased the conversion of polyprenols to dolichol.
This study shows that SRD5A3 reduces the nonsteroid lipids polyprenols, and is involved in N-glycosylation. Residual dolichol detected in the mutant organisms suggests that an alternative pathway for its biosynthesis might exist. Nevertheless, these data explain the pathogenesis of one type of CDG and help to define the complex mechanics of glycosylation.
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