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Enzyme assay of polypeptide N-acetylgalactosaminyltransferase, β1,3-glycosyltransferase, ang β1,4-glycosyltransferases. [4] General methods for detection of enzyme reaction products
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Enzyme assay of polypeptide N-acetylgalactosaminyltransferase, β1,3-glycosyltransferase, ang β1,4-glycosyltransferases. [4] General methods for detection of enzyme reaction products

Authors：
Introduction Protocol References Credit lines
Category
Glycosyltransferases & related proteins
Protocol Name

Enzyme assay of polypeptide N-acetylgalactosaminyltransferase, β1,3-glycosyltransferase, ang β1,4-glycosyltransferases. [4] General methods for detection of enzyme reaction products

Authors
 Togayachi, Akira
Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST)

Kubota, Tomomi
Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST)

Sato, Takashi
Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST)

Narimatsu, Hisashi
Research Center for Medical Glycoscience, National Instutute of Advanced Industrial Science and Technology (AIST)
KeyWords
Reagents
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TSK-gel ODS-80TS column (4.6 × 300 mm; Tosoh Corp., Tokyo, Japan)

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PALPAK type R column (4.6 × 250 mm; Takara Bio Inc., Otsu, Japan)

Instruments
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HPLC system
Methods
1.

HPLC for oligosaccharides

1) 

 The enzyme reaction is terminated by the boil for 3 min, followed by dilution with water.

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2) 

 After centrifugation of the reaction mixtures at 15,000 r.p.m. 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 Corp.) or a PALPAK type R column (4.6 × 250 mm; Takara Bio Inc.).

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3) 

 The reaction products are eluted with 20 mM ammonium acetate buffer (pH 4.0) at a flow rate of 1.0 mL/min at 25°C.

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4) 

 The substrate and product are monitored with a fluorescence spectrophotometer, JASCO FP-920 (JASCO Corporation, Tokyo, Japan). (The substrate and product are detectable with radioisotope, UV, or fluorescence.)

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2.

HPLC for glycopeptides

1) 

 HPLC method using a reverse-phase column is described, as an example.

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2) 

 The reaction mixture is filtrated with a 0.22 μm filter.

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3) 

 The resulting solution is applied into 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.

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4) 

 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 fluorescence tag and the number of attached saccharide.

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5) 

 The elution pattern of absorbance at 220 nm or fluorescence is analyzed. Usually, GalNAc attachment will shorten the retention time in the reverse phase chromatography. (Fig. 1.)

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6) 

 The eluted peak fractions can be recovered in order to identify the number of GalNAc and the attachment sites (see Protocol Title "Large-scale identification of N-glycosylated peptides using lectin-mediated affinity capture, glycosylation site-specific stable isotope tagging, and LC/MS" by Kaji, Hiroyuki). Typically, the negative control without the donor substrate or the enzyme should be concomitantly analyzed.

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3.

Thin layer chromatography (TLC) for oligosaccharides, especially glycolipids

The product is detectable with radioisotope, a chemical reagent such as orcinol and resorcinol, or fluorescence.

1) 

 Reaction products are separated by TLC (Glass HPTLC Silica gel 60 plate ; MERCK MILLIPORE, Germany) with mixtures of chloroform/methanol/water (60: 35: 8).

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2) 

 Products and substrate are chemically developed with reagents, such as orcinol or resorcinol solution. Or they are immunostained with lectin or antibody.

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4.

Scintillation counter for various products from radioisotope-labeled donor substrates.

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 (Np), and the reacted product is isolated with SepPak C18 cartridge.

1) 

 Radioactive products are separated from the free RI-labeled donor substrates using a Sep-Pak Plus C18 cartridge (Waters Corp., Milford, MA).

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2) 

 The cartridge is activated by being washed with 1 mL of 100% methanol and then twice with 1 mL of water.

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3) 

 The enzyme reaction is terminated by addition of 100 μL of H2O, and the reaction mixture is applied to the equilibrated cartridge and washed twice with 1 mL of water.

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4) 

 The radioactive product is eluted with 1 mL of 100% methanol.

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5) 

 The eluted solution is added to 5 mL of liquid scintillation cocktail (GE Healthcare, Little Chalfont, UK).

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6) 

 The radioactivity is measured with a liquid scintillation counter (Beckman Coulter, Inc. Brea CA).

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5.

SDS-PAGE for glycopeptides and glycoproteins

It is required that the substrate/product is labeled with radioisotope or fluorescence.

1) 

 The enzyme reaction is performed at 37°C for 1–24 h.

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2) 

 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% SDS-PAGE.

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3) 

 The radioactive intensities of the bands obtained are measured with an FLA-3000 Imaging Analyzer (Fujifilm, Tokyo, Japan).

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4) 

 Or they are blotted to PVDF membrane and are immunostained with lectin or antibody.

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Figure & Legends

Figure & Legends

 

Fig. 1.  [Example] HPLC elution pattern of the reaction products by pp-GalNAc-T2 and T10 on the IgA hinge peptide. The reaction time is 5 min and 30 min for upper and lower lines, respectively. The numbers with arrows represent the number of GalNAc incorporated. Note that the divided peaks with 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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