title: Peters plus syndrome mutations disrupt a noncanonical ER quality-control mechanism.
abstract: BACKGROUND
  O-fucose is added to cysteine-rich domains called thrombospondin type 1 repeats (TSRs) by protein O-fucosyltransferase 2 (POFUT2) and is elongated with glucose by β3-glucosyltransferase (B3GLCT). Mutations in B3GLCT result in Peters plus syndrome (PPS), an autosomal recessive disorder characterized by eye and other developmental defects. Although 49 putative targets are known, the function of the disaccharide and its role in PPS remain unexplored.
  
  
  RESULTS
  Here we show that while POFUT2 is required for secretion of all targets tested, B3GLCT only affects the secretion of a subset, consistent with the observation that B3GLCT mutant phenotypes in PPS patients are less severe than embryonic lethal phenotypes of Pofut2-null mice. O-glycosylation occurs cotranslationally, as TSRs fold. Mass spectral analysis reveals that TSRs from mature, secreted protein are stoichiometrically modified with the disaccharide, whereas TSRs from protein still folding in the ER are partially modified, suggesting that O-glycosylation marks folded TSRs and promotes ER exit. In vitro unfolding assays demonstrate that fucose and glucose stabilize folded TSRs in an additive manner. In vitro refolding assays under redox conditions showed that POFUT2 recognizes, glycosylates, and stabilizes the folded form of TSRs, resulting in a net acceleration of folding.
  
  
  CONCLUSIONS
  While known ER quality-control machinery rely on identifying and tagging unfolded proteins, we find that POFUT2 and B3GLCT mediate a noncanonical ER quality-control mechanism that recognizes folded TSRs and stabilizes them by glycosylation. Our findings provide a molecular basis for the defects observed in PPS and potential targets that contribute to the pathology.
authors.affiliation: Department of Biochemistry and Cell Biology, 450 Life Sciences Building, Stony Brook University, Stony Brook, NY 11794-5215, USA.
authors.forename: Deepika
authors.initials: D
authors.lastname: Vasudevan
authors.affiliation: Department of Biochemistry and Cell Biology, 450 Life Sciences Building, Stony Brook University, Stony Brook, NY 11794-5215, USA.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Takeuchi
authors.affiliation: Department of Biochemistry and Cell Biology, 450 Life Sciences Building, Stony Brook University, Stony Brook, NY 11794-5215, USA.
authors.forename: Sumreet Singh
authors.initials: SS
authors.lastname: Johar
authors.affiliation: Department of Internal Medicine, Division of Hematology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA.
authors.forename: Elaine
authors.initials: E
authors.lastname: Majerus
authors.affiliation: Department of Biochemistry and Cell Biology, 450 Life Sciences Building, Stony Brook University, Stony Brook, NY 11794-5215, USA. Electronic address: robert.haltiwanger@stonybrook.edu.
authors.forename: Robert S
authors.initials: RS
authors.lastname: Haltiwanger
mesh_terms: D015153:Blotting, Western; D002971:Cleft Lip / Q000235:genetics* / Q000378:metabolism; D003315:Cornea / Q000002:abnormalities* / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000235:genetics* / Q000502:physiology; D005643:Fucose / Q000378:metabolism; D005647:Fucosyltransferases / Q000378:metabolism*; D005700:Galactosyltransferases / Q000235:genetics / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D006130:Growth Disorders / Q000235:genetics* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D017880:Limb Deformities, Congenital / Q000235:genetics* / Q000378:metabolism; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction; D017510:Protein Folding; D034741:RNA, Small Interfering / Q000235:genetics; D019700:Thrombospondin 1 / Q000378:metabolism
chemical_list: D034741:RNA, Small Interfering; D019700:Thrombospondin 1; D005643:Fucose; C514116:B3GLCT protein, human; D005647:Fucosyltransferases; D005700:Galactosyltransferases; D005964:Glucosyltransferases; C495567:POFUT2 protein, human; D005947:Glucose