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Enzyme assay of polypeptide N-acetylgalactosaminyltransferase, β1,3-glycosyltransferase, and β1,4-glycosyltransferases. [2] β1,3-glycosyltransferase family
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Enzyme assay of polypeptide N-acetylgalactosaminyltransferase, β1,3-glycosyltransferase, and β1,4-glycosyltransferases. [2] β1,3-glycosyltransferase family

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

Enzyme assay of polypeptide N-acetylgalactosaminyltransferase, β1,3-glycosyltransferase, and β1,4-glycosyltransferases. [2] β1,3-glycosyltransferase family

Authors
 Togayachi, Akira
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)
*To whom correspondence should be addressed.
KeyWords
Reagents
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Donor substrates (see Note 1):

[Substrates]

・UDP-glucose (Glc)

・UDP-N-acetylglucosamine (GlcNAc)

・UDP-galactose (Gal)

・UDP-N-acetylgalactosamine (GalNAc)

(Sigma Aldrich, Calbiochem, GE Healthcare Biosciences Corp etc.)

[Radiolabeled chemicals]

・UDP-[14C]glucose (Glc)

・UDP-[14C]N-acetylglucosamine (GlcNAc)

・UDP-[14C]galactose (Gal)

・UDP-[3H] N-acetylgalactosamine (GalNAc)

(American Radiolabeled Chemicals, Inc.)

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Acceptor substrates (see Note 2):

Monosaccharides/Oligosaccharides

・Gal-β-pNP (para-nitrophenyl)

・GlcNAc-β-Bz (benzyl)

・GalNAc-α-pNP

・GalNAc-β-Bz

・Fuc-α-pNp

・Galβ1–3(GlcNAcβ1–6)GalNAc(core2)-pNP

・GlcNAcβ1–3GalNAc(core3)-pNP

・GlcNAcβ1–6GalNAc(core6)-pNP

・lactose (Galβ1– 4Glc)

・Lactoside-Bz

・N-acetyllactosamine (LN, Galβ1– 4GlcNAc)

・2LN: (Galβ1– 4GlcNAcβ1–3Galβ1– 4GlcNAc

・3LN: (Galβ1– 4GlcNAcβ1– 3)2Galβ1–4GlcNAc

・4LN: (Galβ1–4GlcNAcβ1–3)3Galβ1–4GlcNAc

・5LN: (Galβ1– 4GlcNAcβ1–3)4Galβ1– 4GlcNAc

・lacto-N-neotetraose (LNnT; Galβ1– 4GlcNAcβ1–3Galβ1– 4Glc)

・agalacto-LNnT (agalacto-LNnT)

・lacto-N-tetraose (LNT; Galβ1–3GlcNAcβ1–3Galβ1–4Glc)

・lacto-N-fucosylpentaose (LNFP) III (Galβ1– 4(Fucα1–3)GlcNAcβ1–3Galβ1– 4Glc),

・LNFP-II (Galβ1– 3(Fucα1– 4)GlcNAcβ1–3Gal1– 4Glc)

・LNFP-V (Galβ1–3GlcNAcβ1– 3Galβ1– 4(Fucα1–3)Glc)

・lacto-N-difucosylhexaose II (Galβ1–3(Fucα1–4)GlcNAcβ1–3Galβ1– 4(Fucα1–3)Glc)

Other acceptor substrates

gycopeptides

・e.g., FITC-conjugated glycopeptides: FITC-AHGVT(-GalNAc) SAPDTR

glycoproteins

・e.g., bovine submaxillary gland mucin (BSM), fetuin, asialofetuin, EGF and TSR domains, or etc.

glycolipids

・LacCer: lactosylceramide, Galβ1– 4Glcβ1–1Cer

・Lc3Cer: lactotriaosylceramide, GlcNAcβ1–3Galβ1– 4Glcβ1–1Cer

・nLc4Cer: neolactotetraosylceramide (paragloboside), Galβ1–4GlcNAcβ1–3Galβ1– 4Glcβ1–1Cer

・nLc5Cer: GlcNAcβ1–3Galβ1– 4GlcNAcβ1–3Galβ1– 4Glcβ1–1Cer

・nLc6Cer: Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4Glcβ1– 1Cer

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Reagents for reaction:

・Cell lysate (enzyme source) or membrane fraction of cells/tissues is prepared in a general method. (Concentration should be optimized for your samples.) (see Note 3)

・Triton X-100

・1 μM/10 nmol of each acceptor mixture (Concentration should be optimized for your samples.)

・50 mM donor substrate (e.g., UDP-GlcNAc, UDP-Gal, UDP-GalNAc, and UDP-Glc, (see Note 4)

・10 mM MnCl2

・5 mM CDP-choline (Enzyme reaction occurs in the presence or absence of CDP-choline. It is better to avoid degradation of donor substrates.)

・0.4% Triton CF-54 (In case of hydrophobic substrates being used, i.e., glycolipids)

・150 mM sodium cacodylate buffer (pH 7.2) (see Note 5)

Instruments
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HPLC system
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Thermoregulated bath (Incubator)
Methods
1.

Preparation of enzyme source

Each enzyme is purified from the culture medium using anti-FLAG M2 agarose affinity gel (Sigma-Aldrich, St. Louis, MO), since recombinant enzyme is fused with FLAG peptide-tag (Sigma-Aldrich).

1) 

 A culture medium (10–50 mL) is mixed with anti-FLAG M2 agarose affinity gel and rotated slowly at 4°C overnight.

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2) 
 
The gel is then washed 2–5 times with 50 mM Tris buffered saline (TBS; 50 mM Tris-HCl, pH 7.4, and 150 mM NaCl) and finally suspended in 100 μL of TBS.
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3) 

 Enzymatic reactions of all glycosyltransferases are carried out using the suspension as an enzyme source.

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2.

Reaction mixture

1) 

 Glycosyltransferases are reacted in 20 μL of a basic reaction mixture containing following components.

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2) 

 The reaction mixture is incubated at 37°C for designated periods (usually 16 h).

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3) 

 The mixture is heated at 100°C for 3 min to terminate the reaction.

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4) 

 The mixture is centrifuged at 15,000 rpm for 5 min at 4°C and the supernatant is recovered.

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5) 

 The substrate and product are analyzed by the methods as described in section [IV] "General methods for detection of enzyme reaction products". As necessary, the product should be purified with any method (i.e., SepPak C18 column for hydrophobic-labeled substrates, HPLC for any substrates).

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Notes

1.  Donor Substrate: For example, UDP-GlcNAc is used as a donor acceptor for b3GnT assay. UDP-Gal and UDP-GalNAc are useful donor substrates for β3GalT and β3GalNAcT assay, respectively. In case radiolabeled chemicals are used, UDP-[14C]glucose (Glc), UDP-[14C]N-acetylglucosamine (GlcNAc), UDP-[14C]galactose (Gal), UDP-[3H] N-acetylgalactosamine (GalNAc), UDP-[14C]glucuronic acid (GlcA), GDP-[14C]mannose (Man) and GDP-[14C]fucose (Fuc) (American Radiolabeled Chemicals, Inc., are available and utilized as donor substrates.

 

2. The various acceptor substrates, such as monosaccharides, oligosaccharides, glycolipids, glycopeptides, and glycoproteins, were purchased from Calbiochem (San Diego, CA), Toronto Research Chemicals Inc. (Toronto, Canada), Seikagaku Corp. (Tokyo, Japan), Takara Bio Inc. (Otsu, Japan), Glycotech (Gaithersburg, MD) or Sigma-Aldrich(St. Louis, MO) etc. Oligosaccharides are fluorescently labeled with 2-aminobenzamide- (2AB) or pyridylamino- (PA) group and used as good acceptors for analysis using radioisotope (scintillation counter), High performance liquid chromatography (HPLC), and MS. Oligosaccharides are fluorescently labeled with nitrophenol (para-Np, ortho-Np) or benzene (Bz) and used as good acceptors for analysis using radioisotope (scintillation counter) and MS.

[Example: polylactosamine synthase (β3GnTs)]

For acceptor substrates: carbohydrate structures including terminal Gal residues on non-reducing end of their structures. For major polylactosamine synthase, β3GnT2 activity, a longer acceptor substrate, such as polylactosamine structure is recommended (at least 2-repeated lactosamine structure and over 3-repeated lactosamine structure is desirable).

 

[Exampe: type 1 structure synthase (β3GalTs)]

Pyridylaminated agalacto-LNnT (Agalacto-LNnT-PA) as acceptor substrate is prepared, and used for assaying β3Gal-T activity. The LNnT-PA is digested with 20 milliunits/mL streptococcal β-galactosidase (Seikagaku Corp., Tokyo, Japan) to remove the galactose residue at the non- reducing end.

 

3. For example, cells/tissues are solubilized in a 20 mM HEPES buffer (pH 7.2) at 2%. The total protein (10–20 mg) in the cell homogenates is used for the enzyme reaction.

 

4. For using a radioactive donor, add 2.5 mM of a radioactive donor substrate for reaction.

 

5. Reaction is optimized in various buffer systems i.e., MES, HEPES, Tris-HCl and Na-cacodylate buffer at different pH values, approximately pH 7.4. We use 150 mM sodium cacodylate buffer (pH 7.2).

Figure & Legends

Figure & Legends

 

 

Fig. 1. Table; β1,3-Glycosyltransferases and their enzymatic activity

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