Rapid Assays for Lectin Toxicity and Binding Changes that Reflect Altered Glycosylation in Mammalian Cells

Pamela Stanley1, Subha Sundaram1

1 Department of Cell Biology, Albert Einstein College of Medicine, New York, New York
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch130206
Online Posting Date:  June, 2014
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Abstract

Glycosylation engineering is used to generate glycoproteins, glycolipids, or proteoglycans with a more defined complement of glycans on their glycoconjugates. For example, a mammalian cell glycosylation mutant lacking a specific glycosyltransferase generates glycoproteins, and/or glycolipids, and/or proteoglycans with truncated glycans missing the sugar transferred by that glycosyltransferase, as well as those sugars that would be added subsequently. In some cases, an alternative glycosyltransferase may then use the truncated glycans as acceptors, thereby generating a new or different glycan subset in the mutant cell. Another type of glycosylation mutant arises from gain‐of‐function mutations that, for example, activate a silent glycosyltransferase gene. In this case, glycoconjugates will have glycans with additional sugar(s) that are more elaborate than the glycans of wild type cells. Mutations in other genes that affect glycosylation, such as nucleotide sugar synthases or transporters, will alter the glycan complement in more general ways that usually affect several types of glycoconjugates. There are now many strategies for generating a precise mutation in a glycosylation gene in a mammalian cell. Large‐volume cultures of mammalian cells may also generate spontaneous mutants in glycosylation pathways. This article will focus on how to rapidly characterize mammalian cells with an altered glycosylation activity. The key reagents for the protocols described are plant lectins that bind mammalian glycans with varying avidities, depending on the specific structure of those glycans. Cells with altered glycosylation generally become resistant or hypersensitive to lectin toxicity, and have reduced or increased lectin or antibody binding. Here we describe rapid assays to compare the cytotoxicity of lectins in a lectin resistance test, and the binding of lectins or antibodies by flow cytometry in a glycan‐binding assay. Based on these tests, glycosylation changes expressed by a cell can be revealed, and glycosylation mutants classified into phenotypic groups that may reflect a loss‐of‐function or gain‐of‐function mutation in a specific gene involved in glycan synthesis. Curr. Protoc. Chem. Biol. 6:117‐133 © 2014 by John Wiley & Sons, Inc.

Keywords: glycosylation mutants; mammalian cells; engineer glycans; glycan binding; lectins; antibodies; CHO cells

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Lectin Resistance Test
  • Alternate Protocol 1: Lectin Survival Assay Based on Viability Determined using MTT
  • Basic Protocol 2: Lectin‐ or Antibody‐Binding Assay using Flow Cytometry
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Lectin Resistance Test

  Materials
  • Lectins from Vector Labs or EY Labs (see recipe)
  • Tissue culture medium (e.g., Dulbecco's modified Eagle's medium, DMEM) containing 10% fetal bovine serum (FBS) (see recipe)
  • Cells growing exponentially, preferably in suspension culture that can also grow in monolayer culture
  • Methylene blue solution (see recipe)
  • 96‐well flat‐bottom tissue culture dish with lid
  • Tissue culture hood
  • Repeater Plus Pipettor with Combitip (Eppendorf)
  • Clear‐capped, sterile plastic tissue culture tubes (5 ml) to make lectin dilutions
  • 37°C tissue culture incubator humidified, atmosphere 5% CO 2
  • Particle cell counter (Coulter) or hemacytometer
  • Inverted phase microscope
  • Two 2‐liter plastic beakers
  • Paper towels
  • Funnel
  • 500‐ml glass bottles
  • 3MM Whatman paper
  • Rubber bands
NOTE: If cells grow only in monolayer, use enzyme‐free cell dissociation buffer (Millipore) to make a suspension for cell counting. Trypsinization releases glycoproteins from the membrane. If cells grow only in suspension, use protocol 2Alternate Protocol or protocol 3.

Alternate Protocol 1: Lectin Survival Assay Based on Viability Determined using MTT

  Additional Materials
  • MTT reagent (see recipe)
  • Phosphate‐buffered saline (PBS) containing 1 mM CaCl 2 and 1 mM MgCl 2, pH 7.2 (PBS/CaMg; see recipe)
  • Dimethyl sulfoxide (DMSO)
  • Paper towels
  • ELISA plate reader

Basic Protocol 2: Lectin‐ or Antibody‐Binding Assay using Flow Cytometry

  Materials
  • Cells in suspension
  • Enzyme‐free cell dissociation buffer (Millipore)
  • Binding buffer (BB) based on Hanks' balanced salts solution (HBSS), pH 7.2, containing 2% (w/v) bovine serum albumin (Fraction V) and 0.1% sodium azide (see recipe)
  • Ice
  • Lectins conjugated to a fluorescent probe such as fluorescein or phycoerythrin from Vector Labs or EY Labs (see recipe)
  • Antibodies with or without a conjugated probe (commercially available)
  • 0.5 µg/ml 7‐actinomycin D (7‐AAD; see recipe)
  • Cell counter (Coulter) or hemacytometer
  • Low‐speed clinical centrifuge
  • 15‐ and 50‐ml conical centrifuge tubes
  • Vacuum aspirator
  • 1.5‐ml microcentrifuge tubes
  • Rotator for microcentrifuge tubes
  • Flow cytometer
  • Software to analyze the flow cytometry data (e.g., FlowJo, Tree Star)
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Figures

Videos

Literature Cited

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Key References
  Patnaik and Stanley, 2006. See above.
  This reference contains a more extensive description of the panel of CHO glycosylation mutants that have been characterized and can be used for glycosylation engineering.
Internet Resources
  http://www.chogenome.org/
  This site has the latest information on the sequencing of the CHO genome and transcriptome and is useful for glycosylation mutations that arise in CHO cells.
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