Epidermal growth factor (EGF) domains are posttranslationally modified with unique O-linked glycans. Here we describe methods to detect enzyme activity mediating the modification of O-GlcNAc and O-fucose glycans of EGF domains.
1. Purification of enzymes expressed in Drosophila S2 cells
To detect enzyme activity of Drosophila O-fucosyltransferase (Ofut1) and its mammalian homologue (Pofut1), they were expressed as a secreted form. The constructs were transfected into S2 cells and the culture medium was recovered and used as an enzyme source.
2. O-fucosyltransferase assay
The EGF domain from Factor VII and GDP-[14C]fucose were used as the acceptor substrate and the donor substrate, respectively. The enzyme products were purified by reverse-phase liquid chromatography; the incorporated radioactivity was measured using a liquid scintillation counter.
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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
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Okajima, Tetsuya
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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.
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KeyWords |
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Reagents
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Expression vectors (eg. pRmHa3-Ofut1 or pRmHa3-Ofut1:V5His) |
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serum-free media (eg. HyQ-CCM3; HyClone Laboratories Inc., South Logan, UT) |
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Cellfectin (Invitrogen/Life Technologies, Carlsbad, CA) |
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Anti-V5 antibody-conjugated agarose (Sigma-Aldrich, St. Louis, MO) |
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2x Assay buffer A (200 mM imidazole-HCl, pH 7.0, 100 mM MnCl2) |
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2x Assay buffer B (100 mM Hepes, pH 7.0, 100 mM MnCl2 ) |
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0.1 mM GDP-[14C]fucose (20000 dpm/nmol) |
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EGF domain of clotting Factor VII (Obtained from Dr. Robert Haltiwanger - Stony Brook University) |
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Instruments
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Ultrafree-4 Filter Units (Merck Millipore, Billerica, MA) |
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Ni-magnetic beads (Promega Corp., Fitchburg, WI) |
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DSC-18 SPE tubes (50 mg; Supelco/Sigma-Aldrich, Bellefonte, PA) |
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Methods |
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Purification of enzymes expressed in Drosophila S2 cells
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1) |
S2 cells are plated in 10-cm plates (2x106 cells/mL, 10 mL/plate). |
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After 2-3 h, Ofut1 expression vectors are transfected into S2 cells using Cellfectin. |
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24 h after transfection, the medium is replaced with serum-free medium containing 0.7 mM CuSO4. |
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After 3-4 days of incubation the culture medium is collected. |
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(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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(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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O-fucosyltransferase assay
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Before the reaction, GDP-[14C]fucose was dried under vacuum (Speed-Vac) and resuspended in water. |
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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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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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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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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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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). |
Copyrights |
Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses
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Date of registration:2014-05-27 13:20:00 |
- Okajima, T., and Irvine, K.D. (2002) Regulation of Notch signaling by O-linked fucose. Cell. 111, 893-904 [PMID : 12526814]
- Okajima, T., Xu, A., Lei, L., and Irvine, K.D. (2005) Chaperone activity of Protein O-fucosyltransferase I within the endoplasmic reticulum promotes folding of the Notch receptor. Science. 307, 1599-1603 [PMID : 15692013]
- Okajima, T., and Matsuda, T. (2006) Roles of O-fucosyltransferase 1 and O-linked fucose in notch receptor function. Methods Enzymol. 417, 111-126 [PMID : 17132501]
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Okajima, Tetsuya,
Furukawa, Koichi,
(2014). GlycoPOD https://jcggdb.jp/GlycoPOD.
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Okajima, Tetsuya,
Furukawa, Koichi,
(2014).
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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Furukawa, Koichi,
(2014).
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