Protein O‐Mannosylation in Metazoan Organisms

Vladislav M. Panin1, Lance Wells2

1 Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, 2 Complex Carbohydrate Research Center, Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 12.12
DOI:  10.1002/0471140864.ps1212s75
Online Posting Date:  February, 2014
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Abstract

Protein O‐mannosylation is a special type of glycosylation that plays prominent roles in metazoans, affecting development and physiology of the nervous system and muscles. A major biological effect of O‐mannosylation involves the regulation of α‐dystroglycan, a membrane glycoprotein mediating cell–extracellular matrix interactions. Genetic defects of O‐mannosylation result in the loss of ligand‐binding activity of α‐dystroglycan and cause congenital muscular dystrophies termed dystroglycanopathies. Recent progress in mass spectrometry and in vitro analyses has shed new light on the mechanism of α‐dystroglycan glycosylation; however, this mechanism is underlain by complex genetic and molecular elements that remain poorly understood. Protein O‐mannosylation is evolutionarily conserved in metazoans, yet the pathway is simplified and more amenable to genetic analyses in invertebrate organisms, indicating that genetically tractable in vivo models could facilitate research in this area. This unit describes recent methodological strategies for studying protein O‐mannosylation using in vitro and in vivo approaches. Curr. Protoc. Protein Sci. 75:12.12.1‐12.12.29. © 2014 by John Wiley & Sons, Inc.

Keywords: O‐glycosylation; O‐mannose; congenital muscular dystrophy; dystroglycan; Drosophila ; mass spectrometry

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: In Vitro Assay for O‐Mannosyltransferase Activity
  • Support Protocol 1: Expression and Purification of O‐Mannosyltransferase Substrates Using E. coli
  • Support Protocol 2: In Vivo Expression of Drosophila O‐Mannosyltransferases and Dystroglycan
  • Support Protocol 3: Purification of Membrane Microsomal Fractions with O‐Mannosyltransferase Activity
  • Basic Protocol 2: Analysis of Drosophila Dystroglycan O‐Mannosylation by Lectin Blotting
  • Alternate Protocol 1: Analysis of Drosophila Dystroglycan O‐Mannosylation by Glycosidase Treatment and Immunoblotting
  • Support Protocol 4: Purification of In Vivo−Expressed Drosophila Dystroglycan Using Affinity Beads
  • Basic Protocol 3: Release and Permethylation of O‐Linked Glycans for Mass Spectrometry
  • Support Protocol 5: Preparation of Column for Cation‐Exchange Chromatography
  • Support Protocol 6: Preparation of Anhydrous Base for Permethylation
  • Basic Protocol 4: Preparation of O‐Glycosylated Peptides for Mass Spectrometry
  • Basic Protocol 5: Glycopeptide Analysis by Neutral‐Loss Triggered MS n Approaches
  • Basic Protocol 6: Glycopeptide Analysis by HCD‐Triggered ETD Approaches
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: In Vitro Assay for O‐Mannosyltransferase Activity

  Materials
  • Dolichol phosphate‐activated [3H]mannose (Dol‐P‐[3H]Man) in methanol/chloroform (100 µCi/ml, 60 Ci/mmol; American Radiolabeled Chemicals)
  • Nitrogen stream
  • Assay buffer (see recipe)
  • Recombinant Dg‐GST protein expressed and purified from E. coli (see protocol 2)
  • Microsomal membrane fraction with POMT1 and POMT2 expression (see Support Protocols protocol 32 and protocol 43)
  • Bovine serum albumin (BSA, fraction V; Roche)
  • PBS containing 1% and 0.5% (v/v) Triton X‐100
  • GST affinity beads (glutathione agarose resin; Pierce, Thermo cat. no. 15160)
  • Sonicator
  • Scintillation counter with scintillation liquid and vials

Support Protocol 1: Expression and Purification of O‐Mannosyltransferase Substrates Using E. coli

  Materials
  • E. coli BL21(D3) cells (e.g., New England BioLabs, cat. no. C2527I) transformed with pET‐41‐Dg‐GST, e.g., by electroporation (Sharma and Schimke, )
  • LB medium ( )
  • Isopropyl β‐D‐1‐thiogalactopyranoside (IPTG)
  • Wash buffer (see recipe), ice cold
  • GST affinity beads (glutathione agarose resin; Pierce, Thermo cat. no. 15160)
  • Phosphate‐buffered saline (PBS; ) with 0.1% (w/v) phenylmethylsulfonyl fluoride (PMSF) added just before use
  • Elution buffer (see recipe)
  • Dialysis buffer (see recipe)
  • Shakers in 37° and 28°C incubators
  • 50‐ml Falcon tubes
  • Sonicator
  • 0.45‐µm low‐protein‐binding syringe filter (Millipore)
  • Glass fiber
  • Spectra/Por dialysis membrane (MWCO 15,000; Spectrum)
  • Centrifugal concentrators (MWCO 15,000; Millipore)
  • Additional reagents and equipment for SDS‐PAGE and Coomassie staining (unit )

Support Protocol 2: In Vivo Expression of Drosophila O‐Mannosyltransferases and Dystroglycan

  Materials
  • Transgenic Drosophila strains carrying UAS constructs of interest, e.g., UAS‐RT, UAS‐TW, and UAS‐ExDg‐FLAG (available upon request from V.M.P.; Lyalin et al., ; Nakamura et al., )
  • 25% (w/v) sucrose, ice cold
  • Phosphate‐buffered saline (PBS; ), ice cold
  • 70% (v/v) ethanol, ice cold
  • Ethanol/dry ice or liquid N 2 bath (optional)
  • Microscope (e.g., Nikon SMZ645) with CO 2 diffusion pads (for fly sorting)
  • Glass dissection trays (e.g., Corning, cat. no. 7223‐34)
  • 1.5‐ml microcentrifuge tubes
  • Additional reagents and equipment for fly rearing (e.g., Roberts, ) and immunoblotting (unit )

Support Protocol 3: Purification of Membrane Microsomal Fractions with O‐Mannosyltransferase Activity

  Materials
  • Live Drosophila third‐instar larvae co‐expressing RT and TW (see protocol 3)
  • Homogenization buffer (see recipe)
  • Assay buffer (see recipe)
  • Glass Dounce homogenizer
  • 1.5‐ml microcentrifuge tubes
  • Sonicator (e.g., Branson Sonifier 150) with microtip
  • Beckman TL100 ultracentrifuge with TLA 100.3 rotor and 13 × 15−mm tubes
  • Bradford assay reagent (e.g., Sigma, cat. no. B6916)
  • Additional reagents and equipment for Bradford protein assay (unit )
NOTE: As protein O‐mannosyltransferase activity of microsomal membrane fractions is not stable in vitro at room temperature, care should be taken to maintain samples at 4°C during all purification steps.

Basic Protocol 2: Analysis of Drosophila Dystroglycan O‐Mannosylation by Lectin Blotting

  Materials
  • Purified ExDg‐FLAG protein on FLAG affinity beads (see protocol 7)
  • Protein N‐glycosidase F (PNGase F; New England Biolabs, cat. no. P0704S) with 10× G7 buffer [50 mM sodium phosphate, pH 7.5, 1% (v/v) NP‐40]
  • BSA Fraction V (Roche)
  • TBST (see recipe)
  • Biotinylated lectins (Vector Laboratories): e.g., concanavalin A (ConA), Vicia villosa agglutinin (VVA), and peanut agglutinin (PNA)
  • Inhibiting sugars for sugar competition controls (e.g., methyl α‐D‐mannopyranoside, GalNAc, or Gal for ConA, VVA, or PNA, respectively)
  • Chemiluminescent kit for biotin detection (e.g., Vectastain ABC, Vector Laboratories)
  • Instrumentation for chemiluminescent immunoblot analyses (e.g., ChemiDoc XRS system with Quantity One software; Bio‐Rad)
  • Additional reagents and equipment for SDS‐PAGE and immunoblotting (units & )

Alternate Protocol 1: Analysis of Drosophila Dystroglycan O‐Mannosylation by Glycosidase Treatment and Immunoblotting

  Additional Materials (also see protocol 5)
  • Glycosidase with specific activity (e.g., Jack bean α‐mannosidase; Sigma)
  • 10× G2 buffer (500 mM sodium citrate, pH 4.5; New England Biolabs)
  • Mouse anti‐FLAG M2 antibody (Sigma)
  • HRP‐conjugated anti‐mouse secondary antibody (e.g., Jackson ImmunoResearch Labs, cat. no. 11‐035‐003)
  • Pierce SuperSignal WestPico Chemiluminescent Substrate kit (Thermo Scientific)

Support Protocol 4: Purification of In Vivo−Expressed Drosophila Dystroglycan Using Affinity Beads

  Materials
  • Drosophila with transgenically expressed ExDg‐FLAG (see protocol 3)
  • Lysis buffer (see recipe)
  • Anti‐FLAG M2 affinity gel (Sigma, cat. no. A2220)
  • Glass Dounce homogenizer
NOTE: As protein O‐mannosyltransferase activity of microsomal membrane fractions is not stable in vitro at room temperature, care should be taken to maintain samples at 4°C during all purification steps.

Basic Protocol 3: Release and Permethylation of O‐Linked Glycans for Mass Spectrometry

  Materials
  • Sodium borohydride (NaBH 4)
  • 50 mM sodium hydroxide (NaOH)
  • Intact isolated proteins (or proteolytic peptides)
  • Glacial acetic acid (AcOH)
  • Anhydrous methanol (dry MeOH)
  • Cation‐exchange chromatography column (see protocol 9)
  • Anhydrous dimethyl sulfoxide (dry DMSO)
  • Anhydrous base (see protocol 10), prepared immediately before use
  • Iodomethane
  • Nanopure water
  • Dichloromethane (DCM)
  • Sonicator
  • 8‐ml glass screwtop tubes with Teflon‐lined caps
  • SpeedVac evaporator
  • Glass syringes and bulbs
  • Concentrator attached to N 2 gas for drying sample

Support Protocol 5: Preparation of Column for Cation‐Exchange Chromatography

  Materials
  • Cation‐exchange resin
  • Methanol (MeOH)
  • 1 M HCl
  • 5% (v/v) acetic acid (AcOH)
  • 8‐ml glass tubes
  • 4‐ and 20‐ml glass chromatography columns

Support Protocol 6: Preparation of Anhydrous Base for Permethylation

  Materials
  • 50% (w/w) NaOH solution
  • Anhydrous methanol (MeOH)
  • Anhydrous dimethyl sulfoxide (DMSO)
  • 100 × 13−mm glass screwcap tube
  • Glass and plastic pipets

Basic Protocol 4: Preparation of O‐Glycosylated Peptides for Mass Spectrometry

  Materials
  • Enriched glycoprotein sample
  • Glycosidase enzyme cocktail (e.g., Enzymatic CarboRelease Kit, QA‐Bio, cat. no. KE‐DG01)
  • 40 mM ammonium bicarbonate (NH 4HCO 3), pH 8.1
  • 8 M urea in 40 mM NH 4HCO 3, pH 8.1
  • Dithiothreitol (DTT)
  • 27.5 mM iodoacetamide
  • Trypsin, sequencing grade
  • Trifluoroacetic acid (TFA)
  • Triethylamine (TEA)
  • Sodium hydroxide (NaOH)
  • Ethanol
  • Nucleophile: dithiothreitol or biotin pentylamine
  • C18 reversed‐phase spin columns (The Nest Group)
  • SpeedVac evaporator
  • Microcentrifuge tubes
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Figures

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Literature Cited

Literature Cited
   Abbott, K.L. , Matthews, R.T. , and Pierce, M. 2008. Receptor tyrosine phosphatase β (RPTPβ) activity and signaling are attenuated by glycosylation and subsequent cell surface galectin‐1 binding. J. Biol. Chem. 283:33026‐33035.
   Akasaka‐Manya, K. , Manya, H. , Nakajima, A. , Kawakita, M. , and Endo, T. 2006. Physical and functional association of human protein O‐mannosyltransferases 1 and 2. J. Biol. Chem. 281:19339‐19345.
   Anumula, K.R. and Taylor, P.B. 1992. A comprehensive procedure for preparation of partially methylated alditol acetates from glycoprotein carbohydrates. Anal. Biochem. 203:101‐108.
   Aoki, K. , Porterfield, M. , Lee, S.S. , Dong, B. , Nguyen, K. , McGlamry, K.H. , and Tiemeyer, M. 2008. The diversity of O‐linked glycans expressed during Drosophila melanogaster development reflects stage‐ and tissue‐specific requirements for cell signaling. J. Biol. Chem. 283:30385‐30400.
   Barone, R. , Aiello, C. , Race, V. , Morava, E. , Foulquier, F. , Riemersma, M. , Passarelli, C. , Concolino, D. , Carella, M. , Santorelli, F. , Vleugels, W. , Mercuri, E. , Garozzo, D. , Sturiale, L. , Messina, S. , Jaeken, J. , Fiumara, A. , Wevers, R.A. , Bertini, E. , Matthijs, G. , and Lefeber, D.J. 2012. DPM2‐CDG: A muscular dystrophy‐dystroglycanopathy syndrome with severe epilepsy. Ann. Neurol. 72:550‐558.
   Barresi, R. , Michele, D.E. , Kanagawa, M. , Harper, H.A. , Dovico, S.A. , Satz, J.S. , Moore, S.A. , Zhang, W. , Schachter, H. , Dumanski, J.P. , Cohn, R.D. , Nishino, I. , and Campbell, K.P. 2004. LARGE can functionally bypass α‐dystroglycan glycosylation defects in distinct congenital muscular dystrophies. Nat. Med. 10:696‐703.
   Beedle, A.M. , Turner, A.J. , Saito, Y. , Lueck, J.D. , Foltz, S.J. , Fortunato, M.J. , Nienaber, P.M. , and Campbell, K.P. 2012. Mouse fukutin deletion impairs dystroglycan processing and recapitulates muscular dystrophy. J. Clin. Invest. 122:3330‐3342.
   Beltran‐Valero de Bernabe, D. , Currier, S. , Steinbrecher, A. , Celli, J. , van Beusekom, E. , van der Zwaag, B. , Kayserili, H. , Merlini, L. , Chitayat, D. , Dobyns, W.B. , Cormand, B. , Lehesjoki, A.E. , Cruces, J. , Voit, T. , Walsh, C.A. , van Bokhoven, H. , and Brunner, H.G. 2002. Mutations in the O‐mannosyltransferase gene POMT1 give rise to the severe neuronal migration disorder Walker‐Warburg syndrome. Am. J. Hum. Genet. 71:1033‐1043.
   Bleckmann, C. , Geyer, H. , Lieberoth, A. , Splittstoesser, F. , Liu, Y. , Feizi, T. , Schachner, M. , Kleene, R. , Reinhold, V. , and Geyer, R. 2009. O‐Glycosylation pattern of CD24 from mouse brain. Biol. Chem. 390:627‐645.
   Brand, A.H. , Manoukian, A.S. , and Perrimon, N. 1994. Ectopic expression in Drosophila . Methods Cell Biol. 44:635‐654.
   Breloy, I. , Schwientek, T. , Gries, B. , Razawi, H. , Macht, M. , Albers, C. , and Hanisch, F.G. 2008. Initiation of mammalian O‐mannosylation in vivo is independent of a consensus sequence and controlled by peptide regions within and upstream of the α‐dystroglycan mucin domain. J. Biol. Chem. 283:18832‐18840.
   Brockington, M. , Blake, D.J. , Prandini, P. , Brown, S.C. , Torelli, S. , Benson, M.A. , Ponting, C.P. , Estournet, B. , Romero, N.B. , Mercuri, E. , Voit, T. , Sewry, C.A. , Guicheney, P. , and Muntoni, F. 2001a. Mutations in the fukutin‐related protein gene (FKRP) cause a form of congenital muscular dystrophy with secondary laminin α2 deficiency and abnormal glycosylation of α‐dystroglycan. Am. J. Hum. Genet. 69:1198‐1209.
   Brockington, M. , Yuva, Y. , Prandini, P. , Brown, S.C. , Torelli, S. , Benson, M.A. , Herrmann, R. , Anderson, L.V. , Bashir, R. , Burgunder, J.M. , Fallet, S. , Romero, N. , Fardeau, M. , Straub, V. , Storey, G. , Pollitt, C. , Richard, I. , Sewry, C.A. , Bushby, K. , Voit, T. , Blake, D.J. , and Muntoni, F. 2001b. Mutations in the fukutin‐related protein gene (FKRP) identify limb girdle muscular dystrophy 2I as a milder allelic variant of congenital muscular dystrophy MDC1C. Hum. Mol. Genet. 10:2851‐2859.
   Brockington, M. , Torelli, S. , Prandini, P. , Boito, C. , Dolatshad, N.F. , Longman, C. , Brown, S.C. , and Muntoni, F. 2005. Localization and functional analysis of the LARGE family of glycosyltransferases: Significance for muscular dystrophy. Hum. Mol. Genet. 14:657‐665.
   Buysse, K. , Riemersma, M. , Powell, G. , van Reeuwijk, J. , Chitayat, D. , Roscioli, T. , Kamsteeg, E.J. , van den Elzen, C. , van Beusekom, E. , Blaser, S. , Babul‐Hirji, R. , Halliday, W. , Wright, G.J. , Stemple, D.L. , Lin, Y.Y. , Lefeber, D.J. , and van Bokhoven, H. 2013. Missense mutations in β‐1,3‐N‐acetylglucosaminyltransferase 1 (B3GNT1) cause Walker‐Warburg syndrome. Hum. Mol. Genet. 22:1746‐1754.
   Chiba, A. , Matsumura, K. , Yamada, H. , Inazu, T. , Shimizu, T. , Kusunoki, S. , Kanazawa, I. , Kobata, A. , and Endo, T. 1997. Structures of sialylated O‐linked oligosaccharides of bovine peripheral nerve α‐dystroglycan. The role of a novel O‐mannosyl‐type oligosaccharide in the binding of α‐dystroglycan with laminin. J. Biol. Chem. 272:2156‐2162.
   de Bernabe, D.B. , Inamori, K. , Yoshida‐Moriguchi, T. , Weydert, C.J. , Harper, H.A. , Willer, T. , Henry, M.D. , and Campbell, K.P. 2009. Loss of α‐dystroglycan laminin binding in epithelium‐derived cancers is caused by silencing of LARGE. J. Biol. Chem. 284:11279‐11284.
   Dobzhansky, T. 1946. Genetics of Natural Populations. Xiii. Recombination and variability in populations of Drosophila pseudoobscura . Genetics 31:269‐290.
   Endo, T. and Manya, H. 2006. O‐Mannosylation in mammalian cells. Methods Mol. Biol. 347:43‐56.
   Finne, J. , Krusius, T. , Margolis, R.K. , and Margolis, R.U. 1979. Novel mannitol‐containing oligosaccharides obtained by mild alkaline borohydride treatment of a chondroitin sulfate proteoglycan from brain. J. Biol. Chem. 254:10295‐10300.
   Fujimura, K. , Sawaki, H. , Sakai, T. , Hiruma, T. , Nakanishi, N. , Sato, T. , Ohkura, T. , and Narimatsu, H. 2005. LARGE2 facilitates the maturation of α‐dystroglycan more effectively than LARGE. Biochem. Biophys. Res. Commun. 329:1162‐1171.
   Greis, K.D. , Hayes, B.K. , Comer, F.I. , Kirk, M. , Barnes, S. , Lowary, T.L. , and Hart, G.W. 1996. Selective detection and site‐analysis of O‐GlcNAc‐modified glycopeptides by β‐elimination and tandem electrospray mass spectrometry. Anal. Biochem. 234:38‐49.
   Grewal, P.K. , McLaughlan, J.M. , Moore, C.J. , Browning, C.A. , and Hewitt, J.E. 2005. Characterization of the LARGE family of putative glycosyltransferases associated with dystroglycanopathies. Glycobiology 15:912‐923.
   Haines, N. , Seabrooke, S. , and Stewart, B.A. 2007. Dystroglycan and protein O‐mannosyltransferases 1 and 2 are required to maintain integrity of Drosophila larval muscles. Mol. Biol. Cell 18:4721‐4730.
   Harrison, R. , Hitchen, P.G. , Panico, M. , Morris, H.R. , Mekhaiel, D. , Pleass, R.J. , Dell, A. , Hewitt, J.E. , and Haslam, S.M. 2012. Glycoproteomic characterization of recombinant mouse α‐dystroglycan. Glycobiology 22:662‐675.
   Haslam, S.M. , North, S.J. , and Dell, A. 2006. Mass spectrometric analysis of N‐ and O‐glycosylation of tissues and cells. Curr. Opin. Struct. Biol. 16:584‐591.
   Hutzler, J. , Schmid, M. , Bernard, T. , Henrissat, B. , and Strahl, S. 2007. Membrane association is a determinant for substrate recognition by PMT4 protein O‐mannosyltransferases. Proc. Natl. Acad. Sci. U.S.A. 104:7827‐7832.
   Ichimiya, T. , Manya, H. , Ohmae, Y. , Yoshida, H. , Takahashi, K. , Ueda, R. , Endo, T. , and Nishihara, S. 2004. The twisted abdomen phenotype of Drosophila POMT1 and POMT2 mutants coincides with their heterophilic protein O‐mannosyltransferase activity. J. Biol. Chem. 279:42638‐42647.
   Inamori, K. , Yoshida‐Moriguchi, T. , Hara, Y. , Anderson, M.E. , Yu, L. , and Campbell, K.P. 2012. Dystroglycan function requires xylosyl‐ and glucuronyltransferase activities of LARGE. Science 335:93‐96.
   Inamori, K.I. , Hara, Y. , Willer, T. , Anderson, M.E. , Zhu, Z. , Yoshida‐Moriguchi, T. , and Campbell, K.P. 2013. Xylosyl‐ and glucuronyltransferase functions of LARGE in α‐dystroglycan modification are conserved in LARGE2. Glycobiology 23:295‐302.
   Jae, L.T. , Raaben, M. , Riemersma, M. , van Beusekom, E. , Blomen, V.A. , Velds, A. , Kerkhoven, R.M. , Carette, J.E. , Topaloglu, H. , Meinecke, P. , Wessels, M.W. , Lefeber, D.J. , Whelan, S.P. , van Bokhoven, H. , and Brummelkamp, T.R. 2013. Deciphering the glycosylome of dystroglycanopathies using haploid screens for lassa virus entry. Science 340:479‐483.
   Jang‐Lee, J. , Curwen, R.S. , Ashton, P.D. , Tissot, B. , Mathieson, W. , Panico, M. , Dell, A. , Wilson, R.A. , and Haslam, S.M. 2007. Glycomics analysis of Schistosoma mansoni egg and cercarial secretions. Mol. Cell. Proteom. 6:1485‐1499.
   Jiang, X. , Han, G. , Feng, S. , Jiang, X. , Ye, M. , Yao, X. , and Zou, H. 2008. Automatic validation of phosphopeptide identifications by the MS2/MS3 target‐decoy search strategy. J. Proteome Res. 7:1640‐1649.
   Jiao, J. , Zhang, H. , and Reinhold, V.N. 2011. High performance IT‐MS sequencing of glycans (spatial resolution of ovalbumin isomers). Int. J. Mass Spectrom. 303:109‐117.
   Julenius, K. , Molgaard, A. , Gupta, R. , and Brunak, S. 2005. Prediction, conservation analysis, and structural characterization of mammalian mucin‐type O‐glycosylation sites. Glycobiology 15:153‐164.
   Kobata, A. 1979. Use of endo‐ and exoglycosidases for structural studies of glycoconjugates. Anal. Biochem. 100:1‐14.
   Kobayashi, K. , Nakahori, Y. , Miyake, M. , Matsumura, K. , Kondo‐Iida, E. , Nomura, Y. , Segawa, M. , Yoshioka, M. , Saito, K. , Osawa, M. , Hamano, K. , Sakakihara, Y. , Nonaka, I. , Nakagome, Y. , Kanazawa, I. , Nakamura, Y. , Tokunaga, K. , and Toda, T. 1998. An ancient retrotransposal insertion causes Fukuyama‐type congenital muscular dystrophy. Nature 394:388‐392.
   Krusius, T. , Finne, J. , Margolis, R.K. , and Margolis, R.U. 1986. Identification of an O‐glycosidic mannose‐linked sialylated tetrasaccharide and keratan sulfate oligosaccharides in the chondroitin sulfate proteoglycan of brain. J. Biol. Chem. 261:8237‐8242.
   Kunz, S. , Rojek, J.M. , Kanagawa, M. , Spiropoulou, C.F. , Barresi, R. , Campbell, K.P. , and Oldstone, M.B. 2005. Posttranslational modification of α‐dystroglycan, the cellular receptor for arenaviruses, by the glycosyltransferase LARGE is critical for virus binding. J. Virol. 79:14282‐14296.
   Lefeber, D.J. , Schonberger, J. , Morava, E. , Guillard, M. , Huyben, K.M. , Verrijp, K. , Grafakou, O. , Evangeliou, A. , Preijers, F.W. , Manta, P. , Yildiz, J. , Grunewald, S. , Spilioti, M. , van den Elzen, C. , Klein, D. , Hess, D. , Ashida, H. , Hofsteenge, J. , Maeda, Y. , van den Heuvel, L. , Lammens, M. , Lehle, L. , and Wevers, R.A. 2009. Deficiency of Dol‐P‐Man synthase subunit DPM3 bridges the congenital disorders of glycosylation with the dystroglycanopathies. Am. J. Hum. Genet. 85:76‐86.
   Lommel, M. and Strahl, S. 2009. Protein O‐mannosylation: Conserved from bacteria to humans. Glycobiology 19:816‐828.
   Longman, C. , Brockington, M. , Torelli, S. , Jimenez‐Mallebrera, C. , Kennedy, C. , Khalil, N. , Feng, L. , Saran, R.K. , Voit, T. , Merlini, L. , Sewry, C.A. , Brown, S.C. , and Muntoni, F. 2003. Mutations in the human LARGE gene cause MDC1D, a novel form of congenital muscular dystrophy with severe mental retardation and abnormal glycosylation of α‐dystroglycan. Hum. Mol. Genet. 12:2853‐2861.
   Lyalin, D. , Koles, K. , Roosendaal, S.D. , Repnikova, E. , Van Wechel, L. , and Panin, V.M. 2006. The twisted gene encodes Drosophila protein O‐mannosyltransferase 2 and genetically interacts with the rotated abdomen gene encoding Drosophila protein O‐mannosyltransferase 1. Genetics 172:343‐353.
   Manya, H. , Chiba, A. , Yoshida, A. , Wang, X. , Chiba, Y. , Jigami, Y. , Margolis, R.U. , and Endo, T. 2004. Demonstration of mammalian protein O‐mannosyltransferase activity: Coexpression of POMT1 and POMT2 required for enzymatic activity. Proc. Natl. Acad. Sci. U.S.A. 101:500‐505.
   Manya, H. , Suzuki, T. , Akasaka‐Manya, K. , Ishida, H.K. , Mizuno, M. , Suzuki, Y. , Inazu, T. , Dohmae, N. , and Endo, T. 2007. Regulation of mammalian protein O‐mannosylation: Preferential amino acid sequence for O‐mannose modification. J. Biol. Chem. 282:20200‐20206.
   Manya, H. , Bouchet, C. , Yanagisawa, A. , Vuillaumier‐Barrot, S. , Quijano‐Roy, S. , Suzuki, Y. , Maugenre, S. , Richard, P. , Inazu, T. , Merlini, L. , Romero, N.B. , Leturcq, F. , Bezier, I. , Topaloglu, H. , Estournet, B. , Seta, N. , Endo, T. , and Guicheney, P. 2008. Protein O‐mannosyltransferase activities in lymphoblasts from patients with α‐dystroglycanopathies. Neuromuscul. Disord. 18:45‐51.
   Manya, H. , Akasaka‐Manya, K. , Nakajima, A. , Kawakita, M. , and Endo, T. 2010. Role of N‐glycans in maintaining the activity of protein O‐mannosyltransferases POMT1 and POMT2. J. Biochem. 147:337‐344.
   Manzini, M.C. , Tambunan, D.E. , Hill, R.S. , Yu, T.W. , Maynard, T.M. , Heinzen, E.L. , Shianna, K.V. , Stevens, C.R. , Partlow, J.N. , Barry, B.J. , Rodriguez, J. , Gupta, V.A. , Al‐Qudah, A.K. , Eyaid, W.M. , Friedman, J.M. , Salih, M.A. , Clark, R. , Moroni, I. , Mora, M. , Beggs, A.H. , Gabriel, S.B. , and Walsh, C.A. 2012. Exome sequencing and functional validation in zebrafish identify GTDC2 mutations as a cause of Walker‐Warburg syndrome. Am. J. Hum. Genet. 91:541‐547.
   Martinez, T. , Pace, D. , Brady, L. , Gerhart, M. , and Balland, A. 2007. Characterization of a novel modification on IgG2 light chain. Evidence for the presence of O‐linked mannosylation. J. Chromatogr. A 1156:183‐187.
   Mikesh, L.M. , Ueberheide, B. , Chi, A. , Coon, J.J. , Syka, J.E. , Shabanowitz, J. , and Hunt, D.F. 2006. The utility of ETD mass spectrometry in proteomic analysis. Biochim. Biophys. Acta 1764:1811‐1822.
   Muntoni, F. , Torelli, S. , Wells, D.J. , and Brown, S.C. 2011. Muscular dystrophies due to glycosylation defects: Diagnosis and therapeutic strategies. Curr. Opin. Neurol. 24:437‐442.
   Nakamura, N. , Lyalin, D. , and Panin, V.M. 2010a. Protein O‐mannosylation in animal development and physiology: From human disorders to Drosophila phenotypes. Semin. Cell Dev. Biol. 21:622‐630.
   Nakamura, N. , Stalnaker, S.H. , Lyalin, D. , Lavrova, O. , Wells, L. , and Panin, V.M. 2010b. Drosophila dystroglycan is a target of O‐mannosyltransferase activity of two protein O‐mannosyltransferases, rotated abdomen and twisted. Glycobiology 20:381‐394.
   OMIM. 2012. Online Mendelian Inheritance in Man. McKusick‐Nathans Institute of Genetic Medicine, Johns Hopkins University, Baltimore, MD), URL: http://omim.org/.
   Pacharra, S. , Hanisch, F.G. , and Breloy, I. 2012. Neurofascin 186 is O‐mannosylated within and outside of the mucin domain. J. Proteome Res. 11:3955‐3964.
   Rademaker, G.J. , Pergantis, S.A. , Blok‐Tip, L. , Langridge, J.I. , Kleen, A. , and Thomas‐Oates, J.E. 1998. Mass spectrometric determination of the sites of O‐glycan attachment with low picomolar sensitivity. Anal. Biochem. 257:149‐160.
   Roberts, D.B. 1998. Drosophila: A Practical Approach, 2nd ed. Oxford University Press, New York.
   Sharma, R.C. and Schimke, R.T. 1996. Preparation of electro‐competent E. coli using salt‐free growth medium. BioTechniques 20:42‐44.
   Shcherbata, H.R. , Yatsenko, A.S. , Patterson, L. , Sood, V.D. , Nudel, U. , Yaffe, D. , Baker, D. , and Ruohola‐Baker, H. 2007. Dissecting muscle and neuronal disorders in a Drosophila model of muscular dystrophy. EMBO J. 26:481‐493.
   Spradling, A.C. and Rubin, G.M. 1982. Transposition of cloned P elements into Drosophila germ line chromosomes. Science 218:341‐347.
   Stalnaker, S.H. , Hashmi, S. , Lim, J.M. , Aoki, K. , Porterfield, M. , Gutierrez‐Sanchez, G. , Wheeler, J. , Ervasti, J.M. , Bergmann, C. , Tiemeyer, M. , and Wells, L. 2010. Site mapping and characterization of O‐glycan structures on α‐dystroglycan isolated from rabbit skeletal muscle. J. Biol. Chem. 285:24882‐24891.
   Stalnaker, S.H. , Aoki, K. , Lim, J.M. , Porterfield, M. , Liu, M. , Satz, J.S. , Buskirk, S. , Xiong, Y. , Zhang, P. , Campbell, K.P. , Hu, H. , Live, D. , Tiemeyer, M. , and Wells, L. 2011a. Glycomic analyses of mouse models of congenital muscular dystrophy. J. Biol. Chem. 286:21180‐21190.
   Stalnaker, S.H. , Stuart, R. , and Wells, L. 2011b. Mammalian O‐mannosylation: Unsolved questions of structure/function. Curr. Opin. Struct. Biol. 21:603‐609.
   Tran, D.T. , Lim, J.M. , Liu, M. , Stalnaker, S.H. , Wells, L. , Ten Hagen, K.G. , and Live, D. 2012. Glycosylation of α‐dystroglycan: O‐Mannosylation influences the subsequent addition of GalNAc by UDP‐GalNAc polypeptide N‐acetylgalactosaminyltransferases. J. Biol. Chem. 287:20967‐20974.
   van Reeuwijk, J. , Janssen, M. , van den Elzen, C. , Beltran‐Valero de Bernabe, D. , Sabatelli, P. , Merlini, L. , Boon, M. , Scheffer, H. , Brockington, M. , Muntoni, F. , Huynen, M.A. , Verrips, A. , Walsh, C.A. , Barth, P.G. , Brunner, H.G. , and van Bokhoven, H. 2005. POMT2 mutations cause α‐dystroglycan hypoglycosylation and Walker‐Warburg syndrome. J. Med. Genet. 42:907‐912.
   Vester‐Christensen, M.B. , Halim, A. , Joshi, H.J. , Steentoft, C. , Bennett, E.P. , Levery, S.B. , Vakrushev, S.Y. , and Clausen, H. 2013. Mining the O‐mannose glycoproteome reveals cadherins as major O‐mannosylated glycoproteins. Proc. Natl. Acad. Sci. U.S.A. 110:21018‐20123.
   Vosseller, K. , Hansen, K.C. , Chalkley, R.J. , Trinidad, J.C. , Wells, L. , Hart, G.W. , and Burlingame, A.L. 2005. Quantitative analysis of both protein expression and serine/threonine post‐translational modifications through stable isotope labeling with dithiothreitol. Proteomics 5:388‐398.
   Wada, Y. , Dell, A. , Haslam, S.M. , Tissot, B. , Canis, K. , Azadi, P. , Backstrom, M. , Costello, C.E. , Hansson, G.C. , Hiki, Y. , Ishihara, M. , Ito, H. , Kakehi, K. , Karlsson, N. , Hayes, C.E. , Kato, K. , Kawasaki, N. , Khoo, K.H. , Kobayashi, K. , Kolarich, D. , Kondo, A. , Lebrilla, C. , Nakano, M. , Narimatsu, H. , Novak, J. , Novotny, M.V. , Ohno, E. , Packer, N.H. , Palaima, E. , Renfrow, M.B. , Tajiri, M. , Thomsson, K.A. , Yagi, H. , Yu, S.Y. , and Taniguchi, N. 2010. Comparison of methods for profiling O‐glycosylation: Human Proteome Organisation Human Disease Glycomics/Proteome Initiative multi‐institutional study of IgA1. Mol. Cell. Proteom. 9:719‐727.
   Wairkar, Y.P. , Fradkin, L.G. , Noordermeer, J.N. , and DiAntonio, A. 2008. Synaptic defects in a Drosophila model of congenital muscular dystrophy. J. Neurosci. 28:3781‐3789.
   Wells, L. , Vosseller, K. , Cole, R.N. , Cronshaw, J.M. , Matunis, M.J. , and Hart, G.W. 2002. Mapping sites of O‐GlcNAc modification using affinity tags for serine and threonine post‐translational modifications. Mol. Cell. Proteom. 1:791‐804.
   Willer, T. , Lee, H. , Lommel, M. , Yoshida‐Moriguchi, T. , de Bernabe, D.B. , Venzke, D. , Cirak, S. , Schachter, H. , Vajsar, J. , Voit, T. , Muntoni, F. , Loder, A.S. , Dobyns, W.B. , Winder, T.L. , Strahl, S. , Mathews, K.D. , Nelson, S.F. , Moore, S.A. , and Campbell, K.P. 2012. ISPD loss‐of‐function mutations disrupt dystroglycan O‐mannosylation and cause Walker‐Warburg syndrome. Nat. Genet. 44:575‐580.
   Wing, D.R. , Rademacher, T.W. , Schmitz, B. , Schachner, M. , and Dwek, R.A. 1992. Comparative glycosylation in neural adhesion molecules. Biochem. Soc. Trans. 20:386‐390.
   Winterhalter, P.R. , Lommel, M. , Ruppert, T. , and Strahl, S. 2013. O‐Glycosylation of the non‐canonical T‐cahderin from rabbit skeletal muscle by single mannose residues. FEBS Lett. 587:3715‐3721.
   Yoshida, A. , Kobayashi, K. , Manya, H. , Taniguchi, K. , Kano, H. , Mizuno, M. , Inazu, T. , Mitsuhashi, H. , Takahashi, S. , Takeuchi, M. , Herrmann, R. , Straub, V. , Talim, B. , Voit, T. , Topaloglu, H. , Toda, T. , and Endo, T. 2001. Muscular dystrophy and neuronal migration disorder caused by mutations in a glycosyltransferase, POMGnT1. Dev. Cell 1:717‐724.
   Yoshida‐Moriguchi, T. , Yu, L. , Stalnaker, S.H. , Davis, S. , Kunz, S. , Madson, M. , Oldstone, M.B. , Schachter, H. , Wells, L. , and Campbell, K.P. 2010. O‐Mannosyl phosphorylation of α‐dystroglycan is required for laminin binding. Science 327:88‐92.
   Yoshida‐Morguchi, T. , Willer, T. , Anderson, M.E. , Venzke, D. , Whyte, T. , Muntoni, F. , Lee, H. , Nelson, S.F. , Yu, L. , and Campbell, K.P. 2013. SGK196 is a glycosylation‐specific O‐mannose kinase required for dystroglycan function. Science 341:896‐899.
   Yuen, C.T. , Chai, W. , Loveless, R.W. , Lawson, A.M. , Margolis, R.U. , and Feizi, T. 1997. Brain contains HNK‐1 immunoreactive O‐glycans of the sulfoglucuronyl lactosamine series that terminate in 2‐linked or 2,6‐linked hexose (mannose). J. Biol. Chem. 272:8924‐8931.
   Zhao, P. , Viner, R. , Teo, C.F. , Boons, G.J. , Horn, D. , and Wells, L. 2011. Combining high‐energy C‐trap dissociation and electron transfer dissociation for protein O‐GlcNAc modification site assignment. J. Proteome Res. 10:4088‐4104.
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