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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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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Basic Protocol 1: Lectin Resistance Test

  • 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

  • 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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Literature Cited

Literature Cited
  Aguilan, J.T., Sundaram, S., Nieves, E., and Stanley, P. 2009. Mutational and functional analysis of Large in a novel CHO glycosylation mutant. Glycobiology 19:971‐986.
  Campbell, C. and Stanley, P. 1984. A dominant mutation to ricin resistance in Chinese hamster ovary cells induces UDP‐GlcNAc:Glycopeptide beta‐4‐N‐acetylglucosaminyltransferase III activity. J. Biol. Chem. 259:13370‐13378.
  Chaney, W. and Stanley, P. 1986. Lec1A Chinese hamster ovary cell mutants appear to arise from a structural alteration in N‐acetylglucosaminyltransferase I. J. Biol. Chem. 261:10551‐10557.
  Chaney, W., Sundaram, S., Friedman, N., and Stanley, P. 1989. The Lec4A CHO glycosylation mutant arises from miscompartmentalization of a Golgi glycosyltransferase. J. Cell Biol. 109:2089‐2096.
  Chen, J., Moloney, D.J., and Stanley, P. 2001. Fringe modulation of Jagged1‐induced Notch signaling requires the action of beta 4galactosyltransferase‐1. Proc. Natl. Acad. Sci. U.S.A. 98:13716‐13721.
  Chen, W. and Stanley, P. 2003. Five Lec1 CHO cell mutants have distinct Mgat1 gene mutations that encode truncated N‐acetylglucosaminyltransferase I. Glycobiology 13:43‐50.
  Chen, W., Unligil, U.M., Rini, J.M., and Stanley, P. 2001. Independent Lec1A CHO glycosylation mutants arise from point mutations in N‐acetylglucosaminyltransferase I that reduce affinity for both substrates. Molecular consequences based on the crystal structure of GlcNAc‐TI. Biochemistry 40:8765‐8772.
  Debeljak, N. and Sytkowski, A.J. 2012. Erythropoietin and erythropoiesis stimulating agents. Drug Test. Anal. 4:805‐812.
  Eckhardt, M., Gotza, B., and Gerardy‐Schahn, R. 1998. Mutants of the CMP‐sialic acid transporter causing the Lec2 phenotype. J. Biol. Chem. 273:20189‐20195.
  Esko, J.D. and Stanley, P. 2009. Glycosylation mutants of cultured cells. In Essentials of Glycobiology, 2nd ed. (A. Varki, R.D. Cummings, J.D. Esko, H.H. Freeze, P. Stanley, C.R. Bertozzi, G.W. Hart, and M.E. Etzler, eds.). Cold Spring Harbor, New York.
  Fukuda, M.N., Sasaki, H., Lopez, L., and Fukuda, M. 1989. Survival of recombinant erythropoietin in the circulation: The role of carbohydrates. Blood 73:84‐89.
  Grabowski, G.A., Barton, N.W., Pastores, G., Dambrosia, J.M., Banerjee, T.K., McKee, M.A., Parker, C., Schiffmann, R., Hill, S.C., and Brady, R.O. 1995. Enzyme therapy in type 1 Gaucher disease: Comparative efficacy of mannose‐terminated glucocerebrosidase from natural and recombinant sources. Ann. Intern. Med. 122:33‐39.
  Hammond, S., Kaplarevic, M., Borth, N., Betenbaugh, M.J., and Lee, K.H. 2012. Chinese hamster genome database: An online resource for the CHO community at http://www.CHOgenome.org. Biotechnol. Bioeng. 109:1353‐1356.
  Hou, X., Tashima, Y., and Stanley, P. 2012. Galactose differentially modulates lunatic and manic fringe effects on Delta1‐induced NOTCH signaling. J. Biol. Chem. 287:474‐483.
  Jefferis, R. 2013. Review of Glycosylation Engineering of Biopharmaceuticals: Methods and Protocols: A book edited by Alain Beck. mAbs 5:638‐640.
  Kanda, Y., Yamane‐Ohnuki, N., Sakai, N., Yamano, K., Nakano, R., Inoue, M., Misaka, H., Iida, S., Wakitani, M., Konno, Y., Yano, K., Shitara, K., Hosoi, S., and Satoh, M. 2006. Comparison of cell lines for stable production of fucose‐negative antibodies with enhanced ADCC. Biotechnol. Bioeng. 94:680‐688.
  Lewis, N.E., Liu, X., Li, Y., Nagarajan, H., Yerganian, G., O'Brien, E., Bordbar, A., Roth, A.M., Rosenbloom, J., Bian, C., Xie, M., Chen, W., Li, N., Baycin‐Hizal, D., Latif, H., Forster, J., Betenbaugh, M.J., Famili, I., Xu, X., Wang, J., and Palsson, B.O. 2013. Genomic landscapes of Chinese hamster ovary cell lines as revealed by the Cricetulus griseus draft genome. Nat. Biotechnol. 31:759‐765.
  Lux, A. and Nimmerjahn, F. 2011. Impact of differential glycosylation on IgG activity. Adv. Exp. Med. Biol. 780:113‐124.
  Natsume, A., Wakitani, M., Yamane‐Ohnuki, N., Shoji‐Hosaka, E., Niwa, R., Uchida, K., Satoh, M., and Shitara, K. 2005. Fucose removal from complex‐type oligosaccharide enhances the antibody‐dependent cellular cytotoxicity of single‐gene‐encoded antibody comprising a single‐chain antibody linked the antibody constant region. J. Immunol. Methods 306:93‐103.
  Oelmann, S., Stanley, P., and Gerardy‐Schahn, R. 2001. Point mutations identified in lec8 Chinese hamster ovary glycosylation mutants that inactivate both the udp‐galactose and cmp‐sialic acid transporters. J. Biol. Chem. 276:26291‐26300.
  Ogorek, C., Jordan, I., Sandig, V., and von Horsten, H.H. 2012. Fucose‐targeted glycoengineering of pharmaceutical cell lines. Methods Mol. Biol. 907:507‐517.
  Olsnes, S. 2004. The history of ricin, abrin and related toxins. Toxicon 44:361‐370.
  Patnaik, S.K. and Stanley, P. 2006. Lectin‐resistant CHO glycosylation mutants. Methods Enzymol. 416:159‐182.
  Patnaik, S.K., Zhang, A., Shi, S., and Stanley, P. 2000. Alpha(1,3)fucosyltransferases expressed by the gain‐of‐function Chinese hamster ovary glycosylation mutants LEC12, LEC29, and LEC30. Arch. Biochem. Biophys. 375:322‐332.
  Patnaik, S.K., Potvin, B., and Stanley, P. 2004. LEC12 and LEC29 gain‐of‐function Chinese hamster ovary mutants reveal mechanisms for regulating VIM‐2 antigen synthesis and E‐selectin binding. J. Biol. Chem. 279:49716‐49726.
  Patnaik, S.K., Potvin, B., Carlsson, S., Sturm, D., Leffler, H., and Stanley, P. 2006. Complex N‐glycans are the major ligands for galectin‐1, ‐3, and ‐8 on Chinese hamster ovary cells. Glycobiology 16:305‐317.
  Phillips, M.L., Nudelman, E., Gaeta, F.C., Perez, M., Singhal, A.K., Hakomori, S., and Paulson, J.C. 1990. ELAM‐1 mediates cell adhesion by recognition of a carbohydrate ligand, sialyl‐Lex. Science 250:1130‐1132.
  Ravdin, J.I., Stanley, P., Murphy, C.F., and Petri, W.A. Jr. 1989. Characterization of cell surface carbohydrate receptors for Entamoeba histolytica adherence lectin. Infect. Immun. 57:2179‐2186.
  Ripka, J., Adamany, A., and Stanley, P. 1986. Two Chinese hamster ovary glycosylation mutants affected in the conversion of GDP‐mannose to GDP‐fucose. Arch. Biochem. Biophys. 249:533‐545.
  Shaaltiel, Y., Bartfeld, D., Hashmueli, S., Baum, G., Brill‐Almon, E., Galili, G., Dym, O., Boldin‐Adamsky, S.A., Silman, I., Sussman, J.L., Futerman, A.H., and Aviezer, D. 2007. Production of glucocerebrosidase with terminal mannose glycans for enzyme replacement therapy of Gaucher's disease using a plant cell system. Plant Biotechnol. J. 5:579‐590.
  Shinkawa, T., Nakamura, K., Yamane, N., Shoji‐Hosaka, E., Kanda, Y., Sakurada, M., Uchida, K., Anazawa, H., Satoh, M., Yamasaki, M., Hanai, N., and Shitara, K. 2003. The absence of fucose but not the presence of galactose or bisecting N‐acetylglucosamine of human IgG1 complex‐type oligosaccharides shows the critical role of enhancing antibody‐dependent cellular cytotoxicity. J. Biol. Chem. 278:3466‐3473.
  Song, Y., Aglipay, J.A., Bernstein, J.D., Goswami, S., and Stanley, P. 2010. The bisecting GlcNAc on N‐glycans inhibits growth factor signaling and retards mammary tumor progression. Cancer Res. 70:3361‐3371.
  Stahl, M., Uemura, K., Ge, C., Shi, S., Tashima, Y., and Stanley, P. 2008. Roles of Pofut1 and O‐fucose in mammalian Notch signaling. J. Biol. Chem. 283:13638‐13651.
  Stanley, P. 1983. Selection of lectin‐resistant mutants of animal cells. Methods Enzymol. 96:157‐184.
  Stanley, P. 1984. Glycosylation mutants of animal cells. Annu. Rev. Genet. 18:525‐552.
  Stanley, P. 1992. Glycosylation engineering. Glycobiology 2:99‐107.
  Stanley, P. and Chaney, W. 1985. Control of carbohydrate processing: The lec1A CHO mutation results in partial loss of N‐acetylglucosaminyltransferase I activity. Mol. Cell Biol. 5:1204‐1211.
  Stanley, P., Caillibot, V., and Siminovitch, L. 1975. Selection and characterization of eight phenotypically distinct lines of lectin‐resistant Chinese hamster ovary cell. Cell 6:121‐128.
  Steentoft, C., Vakhrushev, S.Y., Vester‐Christensen, M.B., Schjoldager, K.T., Kong, Y., Bennett, E.P., Mandel, U., Wandall, H., Levery, S.B., and Clausen, H. 2011. Mining the O‐glycoproteome using zinc‐finger nuclease‐glycoengineered SimpleCell lines. Nat. Methods 8:977‐982.
  Su, D., Zhao, H., and Xia, H. 2010. Glycosylation‐modified erythropoietin with improved half‐life and biological activity. Int. J. Hematol. 91:238‐244.
  Tekoah, Y., Tzaban, S., Kizhner, T., Hainrichson, M., Gantman, A., Golembo, M., Aviezer, D., and Shaaltiel, Y. 2013. Glycosylation and functionality of recombinant ss‐glucocerebrosidase from various production systems. Biosci. Rep. 33:e00071.
  Turan, S., Zehe, C., Kuehle, J., Qiao, J., and Bode, J. 2013. Recombinase‐mediated cassette exchange (RMCE)—a rapidly‐expanding toolbox for targeted genomic modifications. Gene 515:1‐27.
  Walsh, M.J., Dodd, J.E., and Hautbergue, G.M. 2013. Ribosome‐inactivating proteins: Potent poisons and molecular tools. Virulence 4:774‐784.
  Weinstein, J., Sundaram, S., Wang, X., Delgado, D., Basu, R., and Stanley, P. 1996. A point mutation causes mistargeting of Golgi GlcNAc‐TV in the Lec4A Chinese hamster ovary glycosylation mutant. J. Biol. Chem. 271:27462‐27469.
  Yang, H., Wang, H., Shivalila, C.S., Cheng, A.W., Shi, L., and Jaenisch, R. 2013. One‐step generation of mice carrying reporter and conditional alleles by CRISPR/Cas‐mediated genome engineering. Cell 154:1370‐1379.
  Zhang, A., Potvin, B., Zaiman, A., Chen, W., Kumar, R., Phillips, L., and Stanley, P. 1999. The gain‐of‐function Chinese hamster ovary mutant LEC11B expresses one of two Chinese hamster FUT6 genes due to the loss of a negative regulatory factor. J. Biol. Chem. 274:10439‐10450.
  Zhang, P., Tan, D.L., Heng, D., Wang, T., Mariati, Yang, Y., and Song, Z. 2010. A functional analysis of N‐glycosylation‐related genes on sialylation of recombinant erythropoietin in six commonly used mammalian cell lines. Metabol. Engin. 12:526‐536.
  Zhang, P., Chan, K.F., Haryadi, R., Bardor, M., and Song, Z. 2013. CHO glycosylation mutants as potential host cells to produce therapeutic proteins with enhanced efficacy. Adv. Biochem. Eng. Biotechnol. 131:63‐87.
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
  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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