Experimental Design Considerations for In Vitro Non‐Natural Glycan Display via Metabolic Oligosaccharide Engineering

Elaine Tan1, Ruben T. Almaraz1, Hargun S. Khanna1, Jian Du1, Kevin J. Yarema1

1 Department of Biomedical Engineering, The Johns Hopkins University, Baltimore, Maryland
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch100059
Online Posting Date:  September, 2010
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Abstract

Metabolic oligosaccharide engineering (MOE) refers to a technique where non‐natural monosaccharide analogs are introduced into living biological systems. Once inside a cell, these compounds intercept a targeted biosynthetic glycosylation pathway and in turn are metabolically incorporated into cell‐surface‐displayed oligosaccharides where they can modulate a host of biological activities or be exploited as “tags” for bio‐orthogonal and chemoselective ligation reactions. Undertaking a MOE experiment can be a daunting task based on the growing repertoire of analogs now available and the ever increasing number of metabolic pathways that can be targeted; therefore, a major emphasis of this article is to describe a general approach for analog design and selection and then provide protocols to ensure safe and efficacious analog usage by cells. Once cell‐surface glycans have been successfully remodeled by MOE methodology, the stage is set for probing changes to the myriad cellular responses modulated by these versatile molecules. Curr. Protoc. Chem. Biol. 2:171‐194 © 2010 by John Wiley & Sons, Inc.

Keywords: sialic acid glycoengineering; glycosylation; ManNAc analogs; selectin‐based adhesion

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Incubation of Cells with Sugar Analogs
  • Support Protocol 1: Routine Growth and Maintenance of Jurkat Cells
  • Basic Protocol 2: Cell Viability Assays
  • Basic Protocol 3: Periodate‐Resorcinol Assay to Measure Analog Uptake by a Cell and Incorporation into Metabolic Pathways
  • Basic Protocol 4: Quantitation of Cell‐Surface Glycoconjugates
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Incubation of Cells with Sugar Analogs

  Materials
  • Stock solutions (10 mM in ethanol or DMSO) of sugar analogs shown in Figure [Ac 5ManNTGc (1), Ac 4ManNAc (3), Ac 4ManNProp (4), and Ac 4ManNGc (5)], or other analogs of choice; if a free hydroxyl monosaccharide (e.g., ManNAc (2) or ManNLev; Mahal et al., ) is used in any experiment, a 500 mM stock solution should be prepared in PBS or serum‐free culture medium and filter sterilized
  • Jurkat clone E6‐1 (ATCC, cat. no. TIB‐152; see protocol 2 for culture and harvesting)
  • Complete RPMI 1640 medium (see recipe)
  • Ethanol (200 proof; Pharmco)
  • 6‐, 12‐, or 24‐well tissue culture plates
  • 15‐ or 50‐ml polypropylene centrifuge tubes
  • Z2 Coulter particle count and size analyzer (Beckman Coulter) or hemacytometer for counting cells
  • Additional reagents and equipment for harvesting Jurkat cells ( protocol 2)

Support Protocol 1: Routine Growth and Maintenance of Jurkat Cells

  Materials
  • Jurkat clone E6‐1 (ATCC, cat. no. TIB‐152)
  • Complete RPMI 1640 medium (see recipe)
  • Z2 Coulter particle count and size analyzer (Beckman Coulter Inc) or hemacytometer for counting cells
  • 75‐cm2 tissue culture flask (Sarstedt, cat. no. 83.1813)
  • 37°C, 5% CO 2, humidified incubator
  • 15‐ or 50‐ml polypropylene centrifuge tubes

Basic Protocol 2: Cell Viability Assays

  Materials
  • Jurkat clone E6‐1 (ATCC, cat. no. TIB‐152; see protocol 2 for culture and harvesting)
  • Complete RPMI 1640 medium (see recipe)
  • Phosphate‐buffered saline (PBS), pH 7.4 (Invitrogen, cat. no. 10010‐049)
  • 10 mM stock solutions of analog: e.g., Ac 4ManNAc (3) plus any additional analogs required for user‐specific applications)
  • Ethanol (EtOH) (200 proof) for controls
  • 24‐well tissue culture plates
  • Z2 Coulter particle count and size analyzer (Beckman Coulter) or hemacytometer for counting cells

Basic Protocol 3: Periodate‐Resorcinol Assay to Measure Analog Uptake by a Cell and Incorporation into Metabolic Pathways

  Materials
  • Jurkat clone E6‐1 (ATCC, cat. no. TIB‐152; see protocol 2 for culture and harvesting)
  • Complete RPMI 1640 medium (see recipe)
  • 10 mM Ac 4ManNAc (3) stock solution plus additional sugar analogs of choice
  • Ethanol (EtOH) (200 proof; Pharmco) for controls
  • Phosphate‐buffered saline (PBS) pH 7.4 (Invitrogen, cat. no. 10010‐049)
  • 0.4 M periodic acid stock solution (see recipe)
  • 6% (w/v) resorcinol (Sigma, cat. no. 108‐46‐3; store at –20°C)
  • t‐butyl alcohol (2‐methyl‐propan‐2‐ol; Sigma, cat. no. 3972‐25‐6)
  • 10 mM sialic acid stock solution (see recipe)
  • 2.5 mM CuSO 4 (see recipe)
  • Concentrated HCl
  • Z2 Coulter particle count and size analyzer (Beckman Coulter) or hemacytometer for counting cells
  • 25‐cm2 tissue culture flasks
  • Heat block
  • 96‐well microtiter plates
  • Microplate reader (µQuant, Bio‐Tek Instruments)

Basic Protocol 4: Quantitation of Cell‐Surface Glycoconjugates

  Materials
  • Jurkat clone E6‐1 (ATCC, cat. no. TIB‐152; see protocol 2 for culture and harvesting)
  • Complete RPMI culture medium (see recipe)
  • 10 mM Ac 5ManNTGc (1) stock solution for thiol labeling experiments and/or Ac 4ManNLev (Kim et al., ) [or 1,3,4‐O‐Bu 3ManNLev (Aich et al., )) stock solution for ketone labeling]
  • Ethanol (EtOH; 200 proof)
  • Phosphate‐buffered saline (PBS) pH 7.4 (Invitrogen, cat. no. 10010‐049)
  • Tris(2,carboxyethyl)phosphine hydrochloride (TCEP; Sigma, cat. no. C4706)
  • MB solution (see recipe), freshly prepared
  • Biotin buffer (see recipe), freshly prepared
  • 5.0 mM biotin hydrazide stock solution (see recipe)
  • Avidin staining buffer (ASB; see recipe)
  • Fluorescein isothiocyanate (FITC)‐labeled avidin stock solution (see recipe)
  • Z2 Coulter particle count and size analyzer (Beckman Coulter Inc) or hemacytometer for counting cells
  • 6‐well tissue culture plates
  • Centrifuge
  • Tubes for flow cytometer (5‐ml polystyrene round‐bottom tubes, 12 × 75–mm; BD Falcon, BD Biosciences, cat. no. 352054)
  • Flow cytometer equipped with a 488‐nm argon laser (Becton Dickinson FACSCalibur, BD Biosciences)
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Figures

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Literature Cited

Literature Cited
   Aich, U. and Yarema, K.J. 2008. Metabolic oligosaccharide engineering: Perspectives, applications, and future directions. In Glycosciences. 2nd ed. (B. Fraser‐Reid, K. Tatsuta, and J. Thiem, eds.) pp. 2136‐2190. Springer‐Verlag, Berlin, Heidelberg.
   Aich, U., Campbell, C.T., Elmouelhi, N., Weier, C.A., Sampathkumar, S.‐G., Choi, S.S., and Yarema, K.J. 2008. Regioisomeric SCFA attachment to hexosamines separates metabolic flux from cytotoxcity and MUC1 suppression. ACS Chem. Biol. 3:230‐240.
   Aich, U., Meledeo, M.A., Sampathkumar, S.‐G., Fu, J., Jones, M.B., Weier, C.A., Chung, S.Y., Tang, B.C., Yang, M., Hanes, J., and Yarema, K.J. 2010. Development of delivery methods for carbohydrate‐based drugs: controlled release of biologically‐active short chain fatty acid‐hexosamine analogs. Glycoconjug. J. 27:445‐459.
   Bond, M.R., Zhang, H., Vu, P.D., and Kohler, J.J. 2009. Photocrosslinking of glycoconjugates using metabolically incorporated diazirine‐containing sugars. Nat. Protoc. 4:1044‐1063.
   Brossmer, R. and Gross, H.J. 1994. Fluorescent and photoactivatable sialic acids. Meth. Enzymol. 247:177‐193.
   Büttner, B., Kannicht, C., Schmidt, C., Löster, K., Reutter, W., Lee, H.‐Y., Nöhring, S., and Horstkorte, R. 2002. Biochemical engineering of cell surface sialic acids stimulates axonal growth. J. Neurosci. 22:8869‐8875.
   Campbell, C.T., Sampathkumar, S.‐G., Weier, C., and Yarema, K.J. 2007. Metabolic oligosaccharide engineering: perspectives, applications, and future directions. Mol. Biosys. 3:187‐194.
   Chang, P.V., Prescher, J.A., Hangauer, M.J., and Bertozzi, C.R. 2007. Imaging cell surface glycans with bioorthogonal chemical reporters. J. Am. Chem. Soc. 129:8400‐8401.
   Chefalo, P., Pan, Y.‐B., Nagy, N., Harding, C., and Guo, Z.‐W. 2004. Preparation and immunological studies of protein conjugates of N‐acylneuraminic acids. Glycoconjug. J. 20:407‐414.
   Cohen, M., Joester, D., Geiger, B., and Addadi, L. 2004. Spatial and temporal sequence of events in cell adhesion: From molecular recognition to focal adhesion assembly. ChemBioChem 5:1393‐1399.
   Collins, B.E., Fralich, T.J., Itonori, S., Ichikawa, Y., and Schnaar, R.L. 2000. Conversion of cellular sialic acid expression from N‐acetyl‐ to N‐glycolylneuraminic acid using a synthetic precursor, N‐glycolylmannosamine pentaacetate: Inhibition of myelin‐associated glycoprotein binding to neural cells. Glycobiology 10:11‐20.
   Du, J. and Yarema, K.J. 2010. Carbohydrate engineered cells for regenerative medicine. Adv. Drug Deliv. Rev. 62:671‐682.
   Du, J., Meledeo, M.A., Wang, Z., Khanna, H.S., Paruchuri, V.D., and Yarema, K.J. 2009. Metabolic glycoengineering: Sialic acid and beyond. Glycobiology 19:1382‐1401.
   Elmouelhi, N., Aich, U., Paruchuri, V.D.P., Meledeo, M.A., Campbell, C.T., Wang, J.J., Srinivas, R., Khanna, H.S., and Yarema, K.J. 2009. Hexosamine template: A platform for modulating gene expression and for sugar‐based drug discovery. J. Med. Chem. 52:2515‐2530.
   Freitas, R.A. Jr. 1999. Nanomedicine, Volume I: Basic Capabilities. Landes Bioscience, Georgetown, Texas.
   Gagiannis, D., Gossrau, R., Reutter, W., Zimmermann‐Kordmann, M., and Horstkorte, R. 2007. Engineering the sialic acid in organs of mice using N‐propanoylmannosamine. Biochim. Biophys. Acta 1770:297‐306.
   Han, S., Collins, B.E., Bengtson, P., and Paulson, J.C. 2005. Homo‐multimeric complexes of CD22 revealed by in situ photoaffinity protein‐glycan crosslinking. Nat. Chem. Biol. 1:93‐97.
   Hang, H.C. and Bertozzi, C.R. 2001. Ketone isosteres of 2‐N‐acetamidosugars as substrates for metabolic cell surface engineering. J. Am. Chem. Soc. 123:1242‐1243.
   Horstkorte, R., Rau, K., Laabs, S., Danker, K., and Reutter, W. 2004. Biochemical engineering of the N‐acyl side chain of sialic acid leads to increased calcium influx from intracellular compartments and promotes differentiation of HL60 cells. FEBS Lett. 571:99‐102.
   Jacobs, C.L., Goon, S., Yarema, K.J., Hinderlich, S., Hang, H.C., Chai, D.H., and Bertozzi, C.R. 2001. Substrate specificity of the sialic acid biosynthetic pathway. Biochemistry 40:12864‐12874.
   Jones, M.B., Teng, H., Rhee, J.K., Baskaran, G., Lahar, N., and Yarema, K.J. 2004. Characterization of the cellular uptake and metabolic conversion of acetylated N‐acetylmannosamine (ManNAc) analogs to sialic acids. Biotechnol. Bioeng. 85:394‐405.
   Jourdian, G.W., Dean, L., and Roseman, S. 1971. The sialic acids. XI. A periodate‐resorcinol method for the quantitative estimation of free sialic acids and their glycosides. J. Biol. Chem. 246:430‐435.
   Kayser, H., Zeitler, R., Kannicht, C., Grunow, D., Nuck, R., and Reutter, W. 1992. Biosynthesis of a nonphysiological sialic acid in different rat organs, using N‐propanoyl‐D‐hexosamines as precursors. J. Biol. Chem. 267:16934‐16938.
   Keppler, O.T., Horstkorte, R., Pawlita, M., Schmidt, C., and Reutter, W. 2001. Biochemical engineering of the N‐acyl side chain of sialic acid: Biological implications. Glycobiology 11:11R‐18R.
   Khidekel, N., Ficarro, S.B., Peters, E.C., and Hsieh‐Wilson, L.C. 2004. Exploring the O‐GlcNAc proteome: Direct identification of O‐GlcNAc‐modified proteins from the brain. Proc. Natl. Acad. Sci. U.S.A. 101:13132‐13137.
   Kim, E.J., Sampathkumar, S.‐G., Jones, M.B., Rhee, J.K., Baskaran, G., and Yarema, K.J. 2004. Characterization of the metabolic flux and apoptotic effects of O‐hydroxyl‐ and N‐acetylmannosamine (ManNAc) analogs in Jurkat (human T‐lymphoma‐derived) cells. J. Biol. Chem. 279:18342‐18352.
   King, E.J. and Garner, R.J. 1947. The colorimetric determination of glucose. J. Clin. Pathol. 1:30‐33.
   Kontou, M., Bauer, C., Reutter, W., and Horstkorte, R. 2008. Sialic acid metabolism is involved in the regulation of gene expression during neuronal differentiation of PC12 cells. Glycoconjug. J. 25:237‐244.
   Lee, J.H., Baker, T.J., Mahal, L.K., Zabner, J., Bertozzi, C.R., Wiemar, D.F., and Welsh, M.J. 1999. Engineering novel cell surface receptors for virus‐mediated gene transfer. J. Biol. Chem. 274:21878‐21884.
   Lemieux, G.A. and Bertozzi, C.R. 1998. Chemoselective ligation reactions with proteins, oligosaccharides and cells. Trends Biotechnol. 16:506‐513.
   Lemieux, G.A. and Bertozzi, C.R. 2001. Modulating cell surface immunoreactivity by metabolic induction of unnatural carbohydrate antigens. Chem. Biol. 8:265‐275.
   Luchansky, S.J., Argade, S., Hayes, B.K., and Bertozzi, C.R. 2004. Metabolic functionalization of recombinant glycoproteins. Biochemistry 43:12358‐12366.
   Mahal, L.K., Yarema, K.J., and Bertozzi, C.R. 1997. Engineering chemical reactivity on cell surfaces through oligosaccharide biosynthesis. Science 276:1125‐1128.
   Mahal, L.K., Yarema, K.J., Lemieux, G.A., and Bertozzi, C.R. 1999. Chemical approaches to glycobiology: Engineering cell surface sialic acids for tumor targeting. In Sialobiology and Other Novel Forms of Glycosylation Y. Inoue, Y.C. Lee, and F.A. Troy II, eds.) pp. 273‐280. Gakushin Publishing Company, Osaka, Japan.
   Nauman, D.A. and Bertozzi, C.R. 2001. Kinetic parameters for small‐molecule drug delivery by covalent cell surface targeting. Biochim. Biophys. Acta 1568:147‐154.
   Piller, V., Piller, F., and Fukuda, M. 1990. Biosynthesis of truncated O‐glycans in the T cell line Jurkat. Localization of O‐glycan initiation. J. Biol. Chem. 265:9264‐9271.
   Pilobello, K.T., Krishnamoorthy, L., Slawek, D., and Mahal, L.K. 2005. Development of a lectin microarray for the rapid analysis of protein glycopatterns. ChemBioChem 6:985‐989.
   Sampathkumar, S.‐G., Jones, M.B., Meledeo, M.A., Campbell, C.T., Choi, S.S., Hida, K., Gomutputra, P., Sheh, A., Gilmartin, T., Head, S.R., and Yarema, K.J. 2006a. Targeting glycosylation pathways and the cell cycle: Sugar‐dependent activity of butyrate‐carbohydrate cancer prodrugs. Chem. Biol. 13:1265‐1275.
   Sampathkumar, S.‐G., Jones, M.B., and Yarema, K.J. 2006b. Metabolic expression of thiol‐derivatized sialic acids on the cell surface and their quantitative estimation by flow cytometry. Nat. Protoc. 1:1840‐1851.
   Sampathkumar, S.‐G., Li, A.V., Jones, M.B., Sun, Z., and Yarema, K.J. 2006c. Metabolic installation of thiols into sialic acid modulates adhesion and stem cell biology. Nat. Chem. Biol. 2:149‐152.
   Sampathkumar, S.‐G., Li, A.V., and Yarema, K.J. 2006d. Synthesis of non‐natural ManNAc analogs for the expression of thiols on cell surface sialic acids. Nat. Protoc. 1:2377‐2385.
   Sawa, M., Hsu, T.‐L., Itoh, T., Sugiyama, M., Hanson, S.R., Vogt, P.K., and Wong, C.‐H. 2006. Glycoproteomic probes for fluorescent imaging of fucosylated glycans in vivo. Proc. Natl. Acad. Sci. U.S.A. 103:12371‐12376.
   Saxon, E. and Bertozzi, C.R. 2000. Cell surface engineering by a modified Staudinger reaction. Science 287:2007‐2010.
   Schilling, B., Goon, S., Samuels, N.M., Gaucher, S.P., Leary, J.A., Bertozzi, C.R., and Gibson, B.W. 2001. Biosynthesis of sialylated lipooligosaccharides in Haemophilus ducreyi is dependent on exogenous sialic acid and not mannosamine: Incorporation studies using N‐acylmannosamine analogs, N‐glycolylneuraminic acid, and 13C‐labeled N‐acetylneuraminic acid. Biochemistry 40:12666‐12677.
   Schmidt, C., Stehling, P., Schnitzer, J., Reutter, W., and Horstkorte, R. 1998. Biochemical engineering of neural cell surfaces by the synthetic N‐propanoyl‐substituted neuraminic acid precursor. J. Biol. Chem. 273:19146‐19152.
   Sussich, F. and Cesaro, A. 2000. The kinetics of periodate oxidation of carbohydrates: A calorimetric approach. Carbohydr. Res. 329:87‐95.
   Tanaka, Y. and Kohler, J.J. 2008. Photoactivatable crosslinking sugars for capturing glycoprotein interactions. J. Am. Chem. Soc. 130:3278‐3279.
   Tiziani, S., Sussich, F., and Cesàro, A. 2003. The kinetics of periodate oxidation of carbohydrates 2. Polymeric substrates. Carbohydr. Res. 338:1083‐1095.
   Varki, A. 1993. Biological roles of oligosaccharides: All of the theories are correct. Glycobiology 3:97‐130.
   Villavicencio‐Lorini, P., Laabs, S., Danker, K., Reutter, W., and Horstkorte, R. 2002. Biochemical engineering of the acyl side chain of sialic acids stimulates integrin‐dependent adhesion of HL60 cells to fibronectin. J. Mol. Med. 80:671‐677.
   Viswanathan, K., Lawrence, S., Hinderlich, S., Yarema, K.J., Lee, Y.C., and Betenbaugh, M. 2003. Engineering sialic acid synthetic ability into insect cells: Identifying metabolic bottlenecks and devising strategies to overcome them. Biochemistry 42:15215‐15225.
   Walborg, E.F. Jr. and Christensson, L. 1965. A colorimetric method for the quantitative determination of monosaccharides. Anal. Biochem. 13:186‐193.
   Wang, Z., Du, J., Che, P.‐L., Meledeo, M.A., and Yarema, K.J. 2009. Hexosamine analogs: From metabolic glycoengineering to drug discovery. Curr. Opin. Chem. Biol. 13:565‐572.
   Weinbaum, S., Tarbell, J.M., and Damiano, E.R. 2007. The structure and function of the endothelial glycocalyx layer. Annu. Rev. Biomed. Eng. 9:121‐167.
   Yarema, K.J. 2002. A metabolic substrate‐based approach to engineering new chemical reactivity into cellular sialoglycoconjugates. In Cell Engineering 3. Glycosylation (M. Al‐Rubeai, ed.) pp. 171‐196. Kluwer Academic Publishers, Dordrecht, The Netherlands.
   Yarema, K.J., Mahal, L.K., Bruehl, R.E., Rodriguez, E.C., and Bertozzi, C.R. 1998. Metabolic delivery of ketone groups to sialic acid residues: Application to cell surface glycoform engineering. J. Biol. Chem. 273:31168‐31179.
   Zanghi, J.A., Mendoza, T.P., Knop, R.H., and Miller, W.M. 1998a. Ammonia decreases NCAM polysialylation in Chinese hamster ovary and small cell lung cancer cells. J. Cell. Physiol. 177:248‐263.
   Zanghi, J.A., Mendoza, T.P., Schmelzer, A.E., Knop, R.H., and Miller, W.M. 1998b. Role of nucleotide sugar pools in the inhibition of NCAM polysialylation by ammonia. Biotechnol. Prog. 14:834‐844.
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