General methods for detection of enzyme reaction products of polypeptide N-acetylgalactosaminyltransferase, β1,3-glycosyltransferase, and β1,4-glycosyltransferases

Akira Togayachi; Takashi Sato; Tomomi Kubota; Hisashi Narimatsu

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Nishihara S, Angata K, Aoki-Kinoshita KF, et al., editors. Glycoscience Protocols (GlycoPODv2) [Internet]. Saitama (JP): Japan Consortium for Glycobiology and Glycotechnology; 2021-.

General methods for detection of enzyme reaction products of polypeptide N-acetylgalactosaminyltransferase, β1,3-glycosyltransferase, and β1,4-glycosyltransferases

Akira Togayachi, Ph.D.

Glycan & Life System Integration Center (GaLSIC), SOKA University

Email: pj.ca.akos @ihcayagot

Corresponding author.

Takashi Sato, Ph.D.

National Institute of Advanced Industrial Science and Technology (AIST)

Email: pj.og.tsia @otas-ihsakat

Tomomi Kubota, Ph.D.

National Institute of Advanced Industrial Science and Technology (AIST)

Email: pj.og.tsia@atobuk.mot

Hisashi Narimatsu, M.D. Ph.D.

National Institute of Advanced Industrial Science and Technology (AIST)

Email: moc.liamg@491kihsoyuy

Created: December 20, 2021; Last Revision: March 28, 2022.

Introduction

Due to the glycosyltransferase activity for acceptor substrates being assayed in the various reaction mixtures containing donor substrates as described in the previous chapters, a suitable system should be selected to separate the substrates and products. Glycosyltransferase reaction products are identified using several methods. This chapter reviews a general analyzing method for the enzyme assay of β1,3-glycosyltransferase family transferring sugars via a β1,3-linkage (1–21) and β1,4-glycosyltransferase family transferring sugars via a β1,4-linkage (22–28). Since it is a general method, it could be used for the analysis of other enzymes, such as pp-GalNAc-T (29, 30) enzyme, as well.

Protocol

This protocol describes general methods for detecting glycosyltransferase reaction products (31, 32).

Materials

1.: TSK-gel ODS-80TS column (4.6 × 300 mm; Tosoh, Tokyo, Japan)
2.: PALPAK type R column (4.6 × 250 mm; TAKARA BIO Inc., Shiga, Japan)
3.: C₁₈ reverse column (Waters, 5C18-AR, 4.6 × 250 mm)
4.: 20 mM of ammonium acetate buffer (pH 4.0)
5.: High-performance thin-layer chromatography (HPTLC / TLC) Kieselgel 60 TLC palate, 5641 (MERCK, Darmstadt, Germany)
6.: Sep-Pak Plus C₁₈ cartridge (Waters, Milford, MA)
7.: Liquid scintillation cocktail (GE Healthcare, Chicago, IL)

Instruments

1.: High-performance liquid chromatography (HPLC) system
2.: Fluorescence detector: JASCO FP-920 (JASCO, Tokyo, Japan)
3.: Mass spectrometer
4.: Liquid scintillation counter (Beckman Coulter, Brea, CA)
5.: Imaging analyzer (e.g., FLA-3000 Imaging Analyzer [Fujifilm, Tokyo, Japan])

Methods

1.

HPLC (for oligosaccharide) (Note 1): For various substrates, such as oligosaccharides, glycopeptides, glycoproteins, and glycolipids.

a.: The enzyme reaction is terminated by boiling for 3 min followed by dilution with water.
b.: After centrifugation of the reaction mixtures at 15,000 rpm for 5 min, 10 μL of each supernatant is subjected to HPLC analysis through a TSK-gel ODS-80TS column (4.6 × 300 mm; Tosoh) or a PALPAK type R column (4.6 × 250 mm; TAKARA BIO Inc).
c.: The reaction products are eluted with 20 mM of ammonium acetate buffer (pH 4.0) at a flow rate of 1.0 mL/min at 25°C.
d.: The substrate and product are monitored using a fluorescence spectrophotometer, JASCO FP-920 (JASCO). (The substrate and product are detectable with radioisotope, UV, or fluorescence.)

2.

HPLC (for [glyco]peptide) (Note 2)

a.: HPLC method using a reverse-phase column is described, as an example.
b.: The reaction mixture is filtrated with a 0.22-μm filter.
c.: The resulting solution is applied to a C18 reverse column (Waters, 5C18-AR, 4.6 × 250 mm) equilibrated with 0.05% TFA on the HPLC. The column temperature is 40°C. The flow rate is 1.0 mL/min.
d.: The substrate and the products are typically eluted with a 0%–50% linear gradient of acetonitrile in 0.05% TFA. Note that the elution conditions will be affected by the peptide sequence, the attached hydrophobic and/or fluorescent tag, and the number of attached saccharides.
e.: The elution pattern of absorbance at 220 nm or fluorescence is analyzed. Usually, GalNAc attachment will shorten the retention time in the reversed-phase chromatography. (Figure 1).
f.: The eluted peak fractions can be recovered to identify the number of GalNAc and the attachment sites (should be linked to another entry). Typically, the negative control without the donor substrate or the enzyme should be concomitantly analyzed.

3.

TLC (for oligosaccharides, especially neutral glycolipids): The product is detectable using a radioisotope; a chemical reagent, such as orcinol and resorcinol; or fluorescence (Note 3).

a.: Reaction products are separated using TLC (HPTLC Kieselgel 60, 5641; MERCK) with mixtures of chloroform/methanol/water (60: 35: 8).
b.: Products and substrate are chemically developed with reagents, such as orcinol or resorcinol solution, or they are immunostained with lectin or antibody.

4.

Mass spectrometry (MS): For various substrates, oligosaccharides, glycopeptides, and glycolipids. However, it is necessary to isolate the products from the reaction mixture. For example, a product is separated using chromatography (Note 4).

5.

Scintillation counter (for [oligo]saccharide): For various products from radioisotope-labeled donor substrates. However, it is necessity to isolate the products from the reaction mixture. For example, use an acceptor substrate, which is labeled with hydrophobic residue, such as (poly)LacNAc-Bz (or Np), and the reacted product is isolated with a SepPak C₁₈ cartridge (Note 5).

a.: Radioactive products are separated from the free radioisotope (RI)-labeled donor substrates using a Sep-Pak Plus C₁₈ cartridge (Waters).
b.: The cartridge is activated by being washed with 1 mL of 100% methanol and then twice with 1 mL of water.
c.: The enzyme reaction is terminated by the addition of 100 μL of H₂O, and the reaction mixture is applied to the equilibrated cartridge and washed twice with 1 mL of water.
d.: The radioactive product is eluted with 1 mL of 100% methanol.
e.: The eluted solution is added to 5 mL of liquid scintillation cocktail (GE Biosciences).
f.: The radioactivity is measured using a liquid scintillation counter (Beckman Coulter).

6.

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) (for glycoproteins and [long] glycopeptides): It is required that the substrate/product is labeled with radioisotope or fluorescence (Note 6).

a.: The enzyme reaction is performed at 37°C for 1–24 h.
b.: After incubation at 37°C for 16 h, the enzyme reaction is terminated by treatment at 100°C for 3 min, and then, the reaction mixture is subjected to 10%–20% SDS–PAGE.
c.: The radioactive intensities of the bands obtained are measured using an imaging analyzer, such as FLA-3000 Imaging Analyzer (Fujifilm, Tokyo, Japan).
d.: Alternatively, they are blotted on polyvinylidene fluoride (PVDF) membrane and are immunostained with lectin or antibody.

Notes

1.: For example, this method for measuring glycosyltransferase activity has been used in the following studies: Refs. 5, 6, 8, and 28.
2.: For example, this method for measuring glycosyltransferase activity has been used in the following studies: Refs. 29 and 30.
3.: For example, this method for measuring glycosyltransferase activity has been used in the following study: Ref. 16.
4.: For example, this method for measuring glycosyltransferase activity has been used in the following studies: Refs. 5 and 27.
5.: For example, this method for measuring glycosyltransferase activity has been used in the following studies: Refs. 5, 6, 27, and 28.
6.: For example, this method for measuring glycosyltransferase activity has been used in the following studies: Refs. 6, 8, and 27.

References

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30.: Iwasaki H, Zhang Y, Tachibana K, Gotoh M, Kikuchi N, Kwon YD, Togayachi A, Kudo T, Kubota T, Narimatsu H. Initiation of O-glycan synthesis in IgA1 hinge region is determined by a single enzyme, UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 2. J Biol Chem. 2003 Feb 21;278(8):5613–5621. [PubMed: 12438318] [CrossRef]
31.: Togayachi A, Kubota T, Sato T, Narimatsu H. Enzyme assay of polypeptide N-acetylgalactosaminyltransferase, β1,3-glycosyltransferase, and β1,4-glycosyltransferases. [4] General methods for detection of enzyme reaction products. GlycoPOD, 2015. https://jcggdb.jp/GlycoPOD/protocolShow.action?nodeId=t243.
32.: Sato T. Enzyme assay of polypeptide N-acetylgalactosaminyltransferase, β1,3-glycosyltransferase, and β1,4-glycosyltransferases. [5] Recombinant glycosyltransferase production in HEK293T cells using GGENTRtr library. GlycoPOD, 2015. https://jcggdb.jp/GlycoPOD/protocolShow.action?nodeId=t244.

Footnotes

: The authors declare no competing or financial interests.

Figures

Figure 1:

[Example] High-performance liquid chromatography elution pattern of the reaction products by pp-GalNAc-T2 and T10 on the IgA hinge peptide (29, 30). The reaction time is 5 min and 30 min for the upper and the lower lines, respectively. The numbers with arrows represent the number of GalNAc incorporated. Note that the divided peaks with the same number indicate the different Ser/Thr modified. In many cases, pp-GalNAc-T reaction is a sequential multiple reaction. Further analysis to determine the number of GalNAc and their sites will be demanded, which could be identified by MS and peptide sequencer, respectively.

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