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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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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