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Enzyme assay of O-fucose, fringe, and O-GlcNAc transferases modifying epidermal growth factor domains ~Purification of enzymes expressed in Drosophila S2 cells. O-fucosyltransferase assay
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Enzyme assay of O-fucose, fringe, and O-GlcNAc transferases modifying epidermal growth factor domains ~Purification of enzymes expressed in Drosophila S2 cells. O-fucosyltransferase assay

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

Enzyme assay of O-fucose, fringe, and O-GlcNAc transferases modifying epidermal growth factor domains ~Purification of enzymes expressed in Drosophila S2 cells. O-fucosyltransferase assay

Authors
Okajima, Tetsuya *
Department of Biochemistry II, Nagoya University Graduate School of Medicine

Furukawa, Koichi
Department of Biochemistry II, Nagoya University Graduate School of Medicine
*To whom correspondence should be addressed.
KeyWords
Reagents

Expression vectors (eg. pRmHa3-Ofut1 or pRmHa3-Ofut1:V5His)

serum-free media (eg. HyQ-CCM3; HyClone Laboratories Inc., South Logan, UT)

Cellfectin (Invitrogen/Life Technologies, Carlsbad, CA)

Anti-V5 antibody-conjugated agarose (Sigma-Aldrich, St. Louis, MO)

2x Assay buffer A (200 mM imidazole-HCl, pH 7.0, 100 mM MnCl2)

2x Assay buffer B (100 mM Hepes, pH 7.0, 100 mM MnCl2 )

0.1 mM GDP-[14C]fucose (20000 dpm/nmol)

EGF domain of clotting Factor VII (Obtained from Dr. Robert Haltiwanger - Stony Brook University)

Instruments

Ultrafree-4 Filter Units (Merck Millipore, Billerica, MA)

Ni-magnetic beads (Promega Corp., Fitchburg, WI)

DSC-18 SPE tubes (50 mg; Supelco/Sigma-Aldrich, Bellefonte, PA)

Methods
1.

Purification of enzymes expressed in Drosophila S2 cells

1) 

 S2 cells are plated in 10-cm plates (2x106 cells/mL, 10 mL/plate).

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2) 

 After 2-3 h, Ofut1 expression vectors are transfected into S2 cells using Cellfectin.

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3) 

 24 h after transfection, the medium is replaced with serum-free medium containing 0.7 mM CuSO4.

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4) 

 After 3-4 days of incubation the culture medium is collected.

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5) 

 (For the enzyme that dose not possess an epitope tag) The medium is concentrated 100-fold using Ultrafree Filter Units, followed by buffer exchange wih the appropriate buffer. In case of Ofut1, 100 mM imidazole-HCl, pH 7.0 or 50 mM Hepes, pH 7.0, were used.

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6) 

 (For the enzyme that possesses an epitope tag) The enzyme is purified on anti-V5 antibody-conjugated agarose. After washing with 1x Assay buffer, the beads are used as an enzyme source. Alternatively, OFUT1-V5His can be further purified using Ni-magnetic beads.

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2.

O-fucosyltransferase assay

1) 

 Before the reaction, GDP-[14C]fucose was dried under vacuum (Speed-Vac) and resuspended in water.

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2) 

 For the assay, 0.1 mM GDP-[14C]fucose (20000 dpm/nmol), 20 μM EGF factor VII substrate and the enzyme were mixed in the glycosylation buffer in a volume of 20 μL.

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3) 

 After incubation at 37˚C for 2 h, the reaction was stopped by addition of 1 mL of ice-cold 50 mM EDTA.

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4) 

 Discovery DSC-18 SPE tubes were conditioned by loading 1 mL of 80% acetonitrile, 0.052% TFA, and then equilibrated with 1 mL of H2O.

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5) 

 The sample was loaded to the tube and washed with 5 mL of H2O, and the labeled substrates were then eluted with 2 mL of 80% acetonitrile, 0.052% TFA.

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6) 

 Radioactivity in the eluate was measured using a liquid scintillation counter.

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Figure & Legends

Figure & Legends

Fig. 1. Assay for O-fucosyltransferase 1 activity

O-fucosyltransferase activity was measured using an EGF domain from Factor VII as an acceptor substrate and GDP-[14C]fucose as a donor substrate. As enzyme sources, wild-type OFUT1 with a V5His tag and its two derivatives carrying either the OFUT1R245A or OFUT1R245K mutation were used. Both mutants exhibit negligible enzyme activity. Below is a Western blot probed with anti-V5 antibody (Sigma), which confirms the expression of each OFUT1 construct.

This figure was originally published in "Experimental Glycoscience -Glycobiology" edited by Taniguchi N. et al. Springer Japan KK. 2008, pp.33-35 (Okajima T. Part1: Section I).

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