N-linked glycosylation is the most frequent modification of secreted and membrane-bound proteins in eukaryotic cells, disruption of which is the basis of the congenital disorders of glycosylation (CDGs). We describe a new type of CDG caused by mutations in the steroid 5α-reductase type 3 (SRD5A3) gene. Patients have mental retardation and ophthalmologic and cerebellar defects. We found that SRD5A3 is necessary for the reduction of the alpha-isoprene unit of polyprenols to form dolichols, required for synthesis of dolichol-linked monosaccharides, and the oligosaccharide precursor used for N-glycosylation. The presence of residual dolichol in cells depleted for this enzyme suggests the existence of an unexpected alternative pathway for dolichol de novo biosynthesis. Our results thus suggest that SRD5A3 is likely to be the long-sought polyprenol reductase and reveal the genetic basis of one of the earliest steps in protein N-linked glycosylation.
Bibliographical noteFunding Information:
We thank G.R. Fink, A. Jansen, F. Karst, K. Gable, T.M. Dunn, and R. Kolodner for providing yeast strains and helpful advice. We are grateful to J.H. Lin for valuable discussion. We thank D. Matern and the Mayo Clinic for providing complete results of patients' transferrin analysis and also C. Sault for additional clinical results from family CVH-385. We thank the University of California, San Francisco, microscopy core (P30 NS047101 and DK80506) and the Biomedical Genomics Core for help in imaging and microarray data analysis. A. de Rooij and K. Huyben are gratefully acknowledged for technical assistance. H.H.F. is a Sanford Research Professor. He and B.N. are supported by the Rocket Fund, R01 DK55615, and the Sanford Children's Health Research Center. Financial support from Euroglycanet (LSHM-CT2005-512131) to R.W. and Metakids and the Netherlands Brain Foundation to D.L. are kindly acknowledged. L.L. was supported by grants from the Deutsche Forschungsgemeinschaft and the Körber-Stiftung. The mass-spectrometry facility in the Department of Biochemistry of the Duke University Medical Center and Z.G. are supported by the LIPID MAPS Large Scale Collaborative Grant GM-069338 from the National Institutes of Health. V.C. is supported by a fellowship from Fondation pour la Recherche Médicale, and J.G.G. is an Investigator of the Howard Hughes Medical Institute. L.A.-G. ascertained and phenotyped family CVH-385 and MR3 and proposed the collaboration to identify the defective gene. W.B.D. reviewed brain imaging and suggested CDG defects, and H.F. suggested SRD5A3 as the polyprenol reductase. D.S., A.P.D.B., and H.v.B. coordinated homozygosity mapping. J.L.S. identified the SRD5A3 mutation, and S.L.B. performed linkage analysis and initiated generation of the gene trap mouse line. E.M. performed CDG Ix phenotype assessment on the remaining patients. D.J.L. performed biochemical diagnosis, functional studies, and dolichol analysis. R.A.W. coordinated biochemical analysis and homozygosity mapping. L.L. suggested a defect in dolichol biosynthesis and performed dolichol rescue experiments. E.S. helped with dolichol standards. D.B.-V., M.A., P.B., and J.S.C. contributed patients. Mass-spectrometry analysis was performed by Z.G. under the guidance of C.R.H.R. B.G.N. performed lipid extraction and patient LLO analysis. S.H. and B.R.A. provided technical assistance, discussions, and control samples. H.H. provided assistance with standard yeast techniques. V.C. performed all other experiments. J.G.G. directed the project. V.C. and J.G.G. wrote the manuscript, with help from the other authors.