Protein O-Glucosyltransferase 1 (POGLUT1) Promotes Mouse Gastrulation through Modification of the Apical Polarity Protein CRUMBS2.
Crumbs family proteins are apical transmembrane proteins with ancient roles in cell polarity. Mouse Crumbs2 mutants arrest at midgestation with abnormal neural plate morphology and a deficit of mesoderm caused by defects in gastrulation. We identified an ENU-induced mutation, wsnp, that phenocopies the Crumbs2 null phenotype. We show that wsnp is a null allele of Protein O-glucosyltransferase 1 (Poglut1), which encodes an enzyme previously shown to add O-glucose to EGF repeats in the extracellular domain of Drosophila and mammalian Notch, but the role of POGLUT1 in mammalian gastrulation has not been investigated. As predicted, we find that POGLUT1 is essential for Notch signaling in the early mouse embryo. However, the loss of mouse POGLUT1 causes an earlier and more dramatic phenotype than does the loss of activity of the Notch pathway, indicating that POGLUT1 has additional biologically relevant substrates. Using mass spectrometry, we show that POGLUT1 modifies EGF repeats in the extracellular domain of full-length mouse CRUMBS2. CRUMBS2 that lacks the O-glucose modification fails to be enriched on the apical plasma membrane and instead accumulates in the endoplasmic reticulum. The data demonstrate that CRUMBS2 is the target of POGLUT1 for the gastrulation epithelial-to-mesenchymal transitions (EMT) and that all activity of CRUMBS2 depends on modification by POGLUT1. Mutations in human POGLUT1 cause Dowling-Degos Disease, POGLUT1 is overexpressed in a variety of tumor cells, and mutations in the EGF repeats of human CRUMBS proteins are associated with human congenital nephrosis, retinitis pigmentosa and retinal degeneration, suggesting that O-glucosylation of CRUMBS proteins has broad roles in human health.
Developmental Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, New York, United States of America; Program in Biochemistry and Structural Biology, Cell and Developmental Biology, and Molecular Biology, Weill Cornell Graduate School of Medical Sciences, Cornell University, New York, New York, United States of America.
Nitya
N
Ramkumar
Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, United States of America.
Beth M
BM
Harvey
Developmental Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, New York, United States of America.
Jeffrey D
JD
Lee
Developmental Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, New York, United States of America.
Heather L
HL
Alcorn
The Hospital for Sick Children, Arthur and Sonia Labatt Brain Tumor Research Center and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.
Nancy F
NF
Silva-Gagliardi
The Hospital for Sick Children, Arthur and Sonia Labatt Brain Tumor Research Center and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.
C Jane
CJ
McGlade
Department of Genetics and Development, College of Physicians and Surgeons of Columbia University, New York, New York, United States of America.
Timothy H
TH
Bestor
Department of Neuromedical Genetics, Netherlands Institute for Neuroscience, Amsterdam, The Netherlands.
Jan
J
Wijnholds
Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, United States of America.
Robert S
RS
Haltiwanger
Developmental Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, New York, United States of America.
Kathryn V
KV
Anderson
D000818:Animals; D004622:Embryo, Mammalian; D047108:Embryonic Development; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism; D054262:Gastrulation / Q000235:genetics; D005964:Glucosyltransferases / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D011499:Protein Processing, Post-Translational / Q000235:genetics; D051881:Receptor, Notch1 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
C448939:CRB1 protein, human; C568885:Crb2 protein, mouse; D005136:Eye Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C497762:Notch1 protein, mouse; D051881:Receptor, Notch1; D005964:Glucosyltransferases; C510651:POGLUT1 protein, human