1

This document is a post-print version of a book chapter in Methods in molecular

biology (Clifton, N.J.). The final publication is available at link.springer.com:

http://link.springer.com/protocol/10.1007%2F978-1-62703-465-4_27

Glycosyltransferases in Chemo-enzymatic Synthesis of

Oligosaccharides.

Boris Tefsen1#

and Irma van Die1

1Department of Molecular Cell Biology and Immunology, VU University Medical

Center, van den Boechorststraat 7, 1081 BT, Amsterdam, The Netherlands

# Corresponding author: Department of Molecular Cell Biology and Immunology, VU

University Medical Center, van den Boechorststraat 7, 1081 BT, Amsterdam, The

Netherlands, +31204448150, b.tefsen@vumc.nl.

Running title: Glycosyltransferases in chemo-enzymatic synthesis

Summary

Many oligosaccharides are not commercially available, which limits studies focused

on elucidation of glycan functions; therefore chemo-enzymatic methods to synthesize

them can be very useful. Here, we describe the procedure to synthesize the Gal1-

3GalNAc1-4GlcNAc-R (Gal-LDN) moiety, containing the Gal1-3GalNAc

epitope found on the parasitic helminth Haemonchus contortus. An acceptor substrate

providing a terminal N-acetylglucosamine was prepared by coupling the fluorescent

2

hydrophobic aglycon, 2,6-diaminopyridine (DAP), to N,N’-diacetylchitobiose. By the

subsequent action of recombinant Caenorhabditis elegans 1,4-N-

acetylgalactosaminyltransferase the substrate was efficiently converted to GalNAc1-

4GlcNAc-R (LDN-R). Since no recombinant 1,3-galactosyltransferase has been

described that acts on terminal βGalNAc, we used bovine 1,3-galactosyltransferase

to obtain a partial conversion of LDN-R to the Gal-LDN antigen. This method can be

applied to synthesize any oligosaccharide, provided that specific glycosyltransferases

are available, or related enzymes that can be pushed to elongate the selected acceptor.

Key Words: neoglycoconjugate; 2,6-Diaminopyridine; galactosyltransferase, chemo-

enzymatic synthesis, Gal-LDN, helminth, chitobiose

1. Introduction

Glycan molecules linked to proteins or lipids play important roles in cellular

communication, adhesion and signalling and are key molecules in regulation of

immune responses. To establish the role of individual glycans in diverse aspects of

biology, the availability of oligosaccharides or neoglycoconjugates that carry defined

glycan antigens is of crucial importance. Neoglycoconjugates are attractive tools to

define anti-glycan responses in infection or immunization (1,2), or to define specific

carbohydrate recognition by lectin receptors that occur on many immune cells (3).

Neoglycoconjugates are also used in vaccines to elicit carbohydrate-specific

antibodies that can confer protection to infection, for example to Neisseria

meningitides and Streptococcus pneumoniae (4,5).

Helminth parasites express a variety of unusual glycan antigens that are highly

antigenic in infection (6). The study of the biological properties of particular helminth

3

glycan antigens highly depends on the availability of neoglycoconjugates, which in

contrast to many mammalian-type glycans are not commercially available. A

drawback for the enzymatic synthesis of unusual glycans such as helminth glycans is

that not many recombinant parasite-type glycosyltransferases are available.

Previously, we have synthesized LDNF antigen using 1,4-N-

acetylgalactosaminyltransferase from the albumen gland of the snail Lymnaea

stagnalis and partially purified 1,3-FucT from human milk (7) and also using the

1,4-N-acetylgalactosaminyltransferase from C. elegans in combination with

recombinant human 1,3-fucosyltransferase VI (8). Neoglycoconjugates containing

the LDNF-moiety can be applied when antigens directly isolated from helminths are

hard to obtain, as has been shown in the case of serodiagnosis of trichinellosis (9).

Recently, we showed that the Gal1-3GalNAc epitope is present on the sheep

parasite Haemonchus contortus and that IgG antibodies against Gal1-3GalNAc were

elicited during a vaccination trial with ES antigens (10). In this chapter, we describe a

chemo-enzymatic method to synthesize the neoglycoconjugate Gal1-3GalNAc1-

4GlcNAc conjugated to DAP (Gal-LDN-DAP) in nanomolar amounts.

This synthesis starts with the chemical modification of the commercially available

N,N’-diacetylchitobiose (chitobiose) acceptor with 2,6-diaminopyridine (DAP) to

provide a fluorescent hydrophobic linker, facilitating detection and purification of the

oligosaccharide products (Figure 1), and allowing subsequent coupling to a protein

(11,12). In principle, each oligosaccharide acceptor structure can be derivatized with

DAP, and after elongation by the subsequent action of appropriate

glycosyltransferases, the final DAP-derivatized oligosaccharides can easily be used

for different downstream applications (8,9,11).

4

Subsequently, an N-acetylgalactosamine residue is coupled to the DAP-

derivatized chitobiose acceptor using recombinant C. elegans 1,4-N-

acetylgalactosaminyltransferase (13) as a catalyst, resulting in a terminal GalNAc1-

4GlcNAc moiety, which commonly is referred to as LacDiNAc (LDN). No enzymatic

activity has been reported before that can catalyze the transfer of a galactose residue

in 1-3 linkage to LDN. It has been described that some glycosyltransferases can be

forced to act on certain acceptors that they normally would not act on (14). Indeed,

when we used bovine 1,3-galactosyltransferase (15,16) as a catalyst on the LDN

acceptor, it proved to be successful, resulting in Gal-LDN-DAP. Notably, a maximum

of ten percent of LDN-DAP was transformed to Gal-LDN-DAP in a single reaction,

reflecting that this is a sub-optimal reaction. The progress in synthesis of intermediate

structures was monitored using normal-phase HPLC (Figure 2). Using preparative

HPLC, the synthesized oligosaccharide was purified and the structural identity of

Gal-LDN-DAP was verified by ESI-MS (Figure 3).

5

Figure 1. Oligosaccharides used in this study

A schematic representation of the oligosaccharides mentioned in this chapter is

depicted. Filled square (N-acetylglucosamine), open square (N-acetylgalactosamine),

circle (galactose), rectangle (derivatized, open-chain N-acetylglucosamine), DAP

(2,6-diaminopyridine). In addition, the abbreviations used for these oligosaccharides

and their molecular masses are given.

2. Materials

Prepare all solutions using ultrapure MilliQ water.

2.1. Derivatization of chitobiose with 2,6-diaminopyridine (DAP)

1. Derivatization solution: (see Note 1) mix 1722 L DMSO (Sigma-Aldrich, St.

Louis, MO, USA) with 778 L Acetic acid (glacial 99,99 %, Sigma-Aldrich).

2. 82.5 mg 2,6-diaminopyridine (DAP, 98 %, Sigma-Aldrich) is dissolved in 1250

L of the Derivatization solution.

3. 137.4 mg of sodium cyanoborohydride (NaCNBH3, Sigma-Aldrich) is dissolved

in 1250 L of the Derivatization solution (heat in heatblock at 65°C until

completely dissolved).

6

4. N,N′-Diacetylchitobiose (chitobiose, Sigma).

5. 12 cc reversed-phase C18 Sep-Pak columns (Waters, Milford, MA, USA).

6. Preparative normal-phase column (Zorbax NH2 PrepHT, 250 x 21.2 mm, 7 m,

Agilent Technologies, Santa Clara, CA, USA).

7. Buffer A contains acetonitrile (LC-MS, Chromasolv, Riedel-de Haen, Seelze,

Germany).

8. Buffer B contains 50 mM ammoniumformate pH 4.4: dissolve 5 ml 10 M

Ammonium formate solution and 200 l Formic acid in 1 L water.

9. Äkta Explorer HPLC apparatus (Amersham Pharmacia Biotech; now merged

with GE Healthcare Bio-Sciences AB, Uppsala, Sweden).

10. Speedvac centrifuge.

11. 1.5-mL reaction tubes.

12. 15-mL tubes.

2.2 Production of recombinant C. elegans UDP-GalNAc:GlcNAc 1,4-N-

acetylgalactosaminyltransferase

1. Human Embryonic Kidney 293T (HEK293T) (ATCC CRL 1573) cells.

2. Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Life Technologies,

Paisley, UK).

3. 10 % Foetal Calf Serum (FCS, Gibco).

4. Pencillin-Streptomycin (Lonza, Basel, Switzerland).

5. HEPES (Gibco).

6. 10 mM MEM non essential amino acids (Gibco).

7. 100 mM MEM sodium pyruvate (Gibco).

8. Optimem I (Gibco).

7

9. Lipofectamin 2000 (Invitrogen, Life Technologies, Paisley, UK).

10. Incubator at 37°C/5 % CO2.

11. PBS.

12. Trypsinization solution: 0.025 % trypsin (Gibco) and 0.01 % EDTA in PBS.

13. T75 culture flasks.

2.3 β1-4GalNActransferase reaction

1. GalNActransferase reaction buffer: mix an equivalent of 4 µL 1 M MnCl2 (final

concentration is 40 mM), 2.5 L 4 M Sodium cacodylate (C2H12AsNaO5), pH

7.0 (final concentration is 100 mM).

2. 100 mM UDP-GalNAc (final concentration is 20 mM).

3. Culture medium containing recombinant C. elegans UDP-GalNAc:GlcNAc

1,4-N-acetylgalactosaminyltransferase (1,4GalNAcT) (section 3.2. item 11).

2.4. 1-3Galactosyltransferase reaction

1. Buffer for Galactosyltransferase reaction: mix an equivalent of 8 µL 1 M MnCl2

(final concentration is 20 mM), 5 L 4 M Sodium cacodylate (C2H12AsNaO5)

pH 7.0 (final concentration is 50 mM), 80 L 0.5 % BSA (final concentration is

0.1 %). Add water to an end-volume of (an equivalent of) 400 L.

2. 50 mM UDP-galactose.

3. Recombinant bovine 1,3-galactosyltransferase (see Note 2).

8

Figure 2. Monitoring product formation of intermediate structures by HPLC

Enzymatic synthesis was monitored by analytical normal-phase HPLC. The relative

intensity of DAP-derivatized glycans is plotted against time of elution from the

column. The starting compound Chi-2-DAP is shown (A), which is converted with C.

elegans 1,4-GalNAcT into LDN-DAP (B). After isolation of LDN-DAP with

reversed-phase cartridges, around 10 % of this compound is converted to Gal-LDN-

DAP with bovine 1,3-GalT (C).

2.5. Monitoring of the formation and purification of DAP-derivatized glycans

1. 6cc reversed-phase C18 Sep-Pak columns (Waters).

2. Methanol.

3. Normal-phase LudgerSep N1 Amide column (250 x 4.6 mm, Ludger,

Oxfordshire, UK).

4. Surveyor HPLC (Thermo Fischer Scientific, Waltham, MA, USA).

5. Fluorimeter 470 (Waters).

6. Buffer A: Acetonitrile.

9

7. Buffer B: 50 mM Ammonium formate pH 4.4: dissolve 5 ml 10 M Ammonium

formate solution (Sigma-Aldrich) and 200 l Formic acid (Sigma-Aldrich) in 1

L water.

2.6. Electrospray Injection Mass Spectrometry

1. LCQ DecaXP ion trap mass spectrometer equipped with a nano-ES ionization

source (Thermo Fischer Scientific).

2. 10 L syringe.

3. Medium NanoEs spray capillary (Proxeon, Thermo Fischer Scientific).

4. 50 % acetonitrile.

3. Methods

3.1. Derivatization of chitobiose with 2,6-diaminopyridine (DAP)

1. Mix the derivatization solutions containing DAP and cyanoborohydride in a 1:1

ratio (see Note 3).

2. Dissolve dry, water-free pellets of chitobiose (0.4 mg, 1 mol) (see Note 4) in

10 L of DMSO, and subsequently add 500 L of the mixture described in

section 3.1. item 1.

3. Incubate the reaction mixture for 18 h at 65°C in a heat block.

4. Directly after incubation, apply the content of 2 tubes (containing unreacted

chitobiose, Chi-2-DAP and free DAP) to one 12 cc Sep-Pak C18 column that

was pre-activated with 5 column volumes of methanol and washed with 10

column volumes of water. Subsequently wash (see Note 5) the column with 10

column volumes of water and elute the Chi-2-DAP (see Note 6) with 5 column

volumes of methanol.

10

5. Pool the eluted Chi-2-DAP coming from two C18 columns in a 15-mL tube and

dry in a speedvac; add 0.5 mL water to the resulting pellet (contains

approximately 4 mol Chi-2-DAP and excess free DAP) and let his stand for 10

h at 4°C (see Note 7).

6. Apply 0.5 mL from the dissolved material on the preparative normal-phase

column. (see Note 8) Use a gradient that starts with 81 % acetonitrile (buffer A)

and 29 % 50 mM ammonium formate, pH 4.4 (buffer B) and ends after 17 min

with 30 % buffer A and 70 % buffer B on an Äkta Explorer with a column flow

of 10 mL/min. DAP-containing fractions are detected by measuring absorbance

at 235 nm.

7. Collect the peak containing Chi-2-DAP (eluting at min 11.5) in a 15-mL tube

(see Note 9).

8. Freeze-dry the collected Chi-2-DAP and store at -20°C (see Note 10).

3.2. Production of recombinant C. elegans UDP-GalNAc:GlcNAc 1,4-N-

acetylgalactosaminyltransferase (1,4GalNAcT).

1. Culture adherent HEK293T cells in DMEM containing 10 mM HEPES, 100 M

MEM non essential amino acids, 1 mM MEM sodium pyruvate, 10 % FCS and

100 U/mL penicillin-streptomycin in a T75 flask at 37°C/5 % CO2 until the

bottom is completely covered by a monolayer of cells (~6x106 cells/flask).

2. Prepare a transfection mixture by mixing 600 L Optimem I containing 54 L

Lipofectamin 2000 with 600 L Optimem I containing 9 g DNA of plasmid

pCMV-SH-Ceβ4GalNAcT (13).

3. Incubate the transfection mixture for 30 min at room temperature.

4. Wash the HEK293T cells twice with 9 mL Optimem I.

11

5. Mix 4.8 mL Optimem I with the transfection mixture.

6. Add this mixture to the cells and incubate the cells for 5-6 h at 37°C/5 % CO2.

7. Add 6 ml DMEM and culture the cells overnight at 37°C/5 % CO2.

8. Wash the cells with 2 mL PBS.

9. Treat the cells with 1 mL Trypsinization solution and transfer to a new T75

flask.

10. Culture the cells in 9 mL DMEM for another 3 days.

11. Harvest medium containing the active enzyme daily until 7 days after

transfection.

12. Determine the activity of the enzyme by monitoring the incorporation of

radiolabeled UDP-GalNAc in time (13) (see Note 11).

13. Store at -20°C. (see Note 12).

3.3 Addition of a β1-4GalNAc residue to Chi-2-DAP to create LDN-DAP

1. Dissolve 1 mol dried chitobiose-DAP (final concentration is 10 mM) in 6.5 L

GalNActransferase reaction buffer.

2. Add 20 L 100 mM UDP-GalNAc (final concentration is 20 mM) and 73.5 µL

medium containing 1,4GalNAcTransferase to a final volume of 100 L.

3. Incubate the reaction mixture at room temperature (20-24°C) until the Chi-2-

DAP has been completely converted to the desired end-product, LDN-DAP.

3.4. Monitoring the formation of LDN-DAP and subsequent purification

1. Position a LudgerSep N1 column in the HPLC.

2. Equilibrate the column with 100 % ammonium formate for 10 min, followed by

10 min of 70 % acetonitrile and 30 % 50 mM ammonium formate.

12

3. Dissolve 0.5 L of the 100 L reaction mixture of section 3.3. item 1 in 100 L

80 % acetonitrile.

4. Inject 25 L of this dilution onto the column.

5. Run a 30-min gradient starting with 70 % acetonitrile and 30 % 50 mM

ammonium formate, pH 4.4 and ending in a 50:50 ratio at 22°C.

6. Detect the glycan-DAP peaks by fluorescence in a fluorimeter by excitation at

345 nm and measuring the emission at 400 nm with a 10 times gain of signal.

The Chi-2-DAP peak elutes in this system around minute 17.8, whereas the

LDN-DAP elutes around minute 19.8 (see Fig. 2).

7. Purify LDN-DAP when conversion is complete from the reaction mixture from

section 3.3. item 2 using a 6 cc C18 reversed-phase Sep-Pak column as

described in section 3.1. item 4.

8. Dry the eluted LDN-DAP in the speedvac and store it at -20°C.

3.5. Addition of an α1-3Galactose residue to LDN-DAP to create Gal-LDN-DAP

1. Dissolve 1 mol dried LDN-DAP (final concentration is 1 mM) in 262.5 µL

Galactosyltransferase buffer.

2. Add 377.5 L water, 30 L 50 mM UDP-galactose (final concentration is 1.5

mM) and 360 L 1,3-galactosyltransferase to a final volume of 1 mL (see

Note 13).

3. Incubate the mixture at 37°C until no further enhancement of the desired end-

product, Gal-LDN-DAP, is observed (see Note 14).

3.6. Monitoring the formation of Gal-LDN-DAP and subsequent purification

13

1. The formation of Gal-LDN-DAP is monitored in time as described in section

3.4.

2. The LDN-DAP elutes around minute 19.8 and Gal-LDN-DAP around minute

22.8 (see Fig. 2).

3. When the reaction from section 3.5. item 3 is stopped, purify the mixture of

LDN-DAP and Gal-LDN-DAP with a 6 cc Sep-Pak as described in section 3.1.

item 4.

4. Dissolve the pellet containing this mixture in water and apply it to the

preparative column, similarly as described for the purification of Chi-2-DAP in

section 3.1. item 6.

5. Collect the fractions where Gal-LDN-DAP elutes (see Notes 15 and 16). LDN-

DAP starts to elute around minute 14.5 and Gal-LDN-DAP elutes around

minute 15.5.

6. Dry the eluted DAP-derivatized glycans in the speedvac and store at -20°C.

3.7. Confirmation of the formation of Gal-LDN-DAP by mass spectrometry

1. Characterize product formation by ESI–MS on an LCQ DecaXP ion trap mass

spectrometer equipped with a nano-ES ionization source.

2. Take 0.5 L of a collected fraction from the Surveyor run, containing Gal-LDN-

DAP, and dilute in 50 L 50 % acetonitrile.

3. Inject a few L of this sample into a medium NanoEs spray capillary (Proxeon)

using the 10 L syringe.

4. Mount the capillary in front of the ion-trap and set the capillary temperature to

200°C.

14

5. Take spectra in the positive ion mode with a spray voltage of 1.0 kV and a

capillary voltage of 40.1 V and subject to tandem MS to confirm the synthesis

of Gal-LDN-DAP (see Fig. 3).

Figure 3. Tandem mass spectrum of Gal-LDN-DAP

Characterization of Gal-LDN–DAP by tandem mass spectrometry with ESI-MS.

Oligosaccharide products were characterized in the positive-ion mode. The expected

molecular mass of the protonated Gal-LDN–DAP ion (m/z = 883.11) was found, and

by fragmentation using tandem MS/MS the structure was confirmed. Schematic

figures of the oligosaccharides representing the found fragments are depicted above

the peaks. The asterisks indicate ions that are similar to the structures shown, but that

lack a water molecule (-18 Da).

4. Notes

1. These solutions should be made fresh on the day they will be used.

15

2. Initially, we could obtain the recombinant bovine 1,3-galactosyltransferase

from Sigma, but this product was discontinued. At that point, we kindly

received the enzyme from Dr. M.M. Palcic (16).

3. The less-toxic compound picoline borate was tested instead of

cyanoborohydride for the coupling of DAP to chitobiose, but it inhibited

subsequent enzymatic reactions and was therefore discarded.

4. We originally started the whole procedure described here using N,N’,N’’-

triacetylchitotriose as the starting acceptor to have a greater distance between

the immunogenic epitope of the neoglycoconjugate and the carrier protein.

However, degradation of this compound by a -N-acetylhexosaminidase present

in Fetal Calf serum made this strategy impossible.

5. This step removes free chitobiose and salts.

6. Free DAP molecules will also be eluted from the column.

7. This step can be shortened, but then the amount of dissolved Chi-2-DAP will be

less.

8. This step separates free DAP molecules from DAP-derivatized glycans (see

figure 2 in (8)).

9. Characterization by ESI-MS of the intermediate DAP-derivatized glycans, Chi-

2-DAP and LDN-DAP is described in (8).

10. No loss of material was observed after 3 months of storage at -20°C.

11. The enzyme-containing medium showed an activity of approximately 200

nmol/mL/h using acceptor p-nitrophenyl-N-acetyl-1-thio--D-glucosaminide.

12. The enzyme was stored at -20°C for several months without loss of activity.

13. Galactosyltransferase activity of the bovine 1,3galactosyltransferase was

determined essentially as described previously (17).

16

14. This was after approximately 72 h. Longer incubations never yielded more than

approximately 10 % of LDN-DAP to be elongated with a galactose molecule.

15. Gal-LDN-DAP purified in this way, can still contain between 5 and 10 % of the

starting material LDN-DAP.

16. The elutions containing LDN-DAP can be collected, dried and re-used starting

from step 3.5. item 2.

Acknowledgements

This work was supported by the Dutch Technology Foundation (STW). We kindly

thank Dr. M.M. Palcic (Carlsberg Laboratory, Denmark) for providing the

recombinant bovine 1,3-galactosyltransferase.

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