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

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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 [Ac₅ ManNTGc (1 ), Ac₄ ManNAc (3 ), Ac₄ ManNProp (4 ), and Ac₄ ManNGc (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% CO₂ , 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., Ac₄ ManNAc (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 Ac₄ ManNAc (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 CuSO₄ (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 Ac₅ ManNTGc (1 ) stock solution for thiol labeling experiments and/or Ac₄ ManNLev (Kim et al., ) [or 1,3,4‐O ‐Bu₃ ManNLev (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

Figure 1. Overview of metabolic oligosaccharide engineering (MOE). MOE was pioneered for the sialic acid biosynthetic pathway, which is one of the glycosylation pathways of a cell that can accommodate non‐natural analogs. Modifications in this pathway can be introduced as “R₁ ”‐modified forms of (A ) ManNAc, (B ) Neu5Ac, or (C ) CMP‐Neu5Ac. These metabolic intermediates intercept the sialic acid pathway at various points, are utilized in place of the natural pathway components, and ultimately appear on the cell surface as chemically altered form of Neu5Ac, the most common form of sialic acid found in human cells. Examples of “R₁ ” modifications are shown in Figure .

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Figure 2. Flow diagram depicting various steps involved in a typical MOE experiment. Procedures that are not described in this unit are denoted with a star; additional information on these steps is provided in the references cited in the main text.

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Figure 3. Analog design features. (A ) The “core” structures of ManNAc, GalNAc, fucose, and GlcNAc analogs used in MOE that are further derivatized with R₁ and R₂ groups. Categories of R₁ groups, which are carried through on to the cell surface and displayed as a modify glycan, include those with (B ) enlarged hydrocarbon groups, (C ) heteroatom (or other) containing functional groups, and (D ) direct fluorophores. (E ) Examples of R₂ groups include a proton (i.e., the ‐H found on hydroxyl groups) and 2‐ to 5‐carbon short chain fatty acids that are ester‐linked to the hydroxyl groups of the monosaccharide analog. Ac and Bu denote acetyl and n ‐butanoyl substitutions shown in Figure and referenced throughout the main text.

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Figure 4. Chemical structures of ManNAc analogs used in this unit.

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Literature Cited

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