HNK-1 (human natural killer-1) epitope, comprising HSO3-3GlcAβ1-3Galβ1-4GlcNAc-, is expressed in the nervous system and functions in learning and memory. Structural and functional characteristic of this glycan is based on the terminal sulfated glucuronic acid, and specific glucuronyltransferases (GlcAT-P and GlcAT-S) and sulfotransferase (HNK-1ST) are required for its biosynthesis. Here we describe how to clone and express these enzymes into mammalian cells. |
| Category | Glycosyltransferases & related proteins |
| Protocol Name | Cloning and transfection of glucuronyltransferase for HNK-1 epitope |
Authors
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Kizuka, Yasuhiko
Disease Glycomics Team, Advanced Science Institute, RIKEN
Oka, Shogo
*
Department of Biological Chemistry, Human Health Sciences, Graduate School of Medicine, Kyoto University
*To whom correspondence should be addressed.
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Reagents
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Trizol (Invitrogen/Life Technologies, Carlsbad, CA) |
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Standard kit for reverse transcription (superscriptIII in our case) |
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DNA polymerase, ligase and restriction enzymes |
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Cloning plasmid and expression plasmid |
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Transfection reagent (FUGENE6 in our case) |
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HNK-1 monoclonal antibody (mAb) (ATCC, Manassas, VA) |
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Instruments
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Standard devices for DNA recombination and western blotting |
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| Methods |
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1. |
Isolation and reverse transcription of total RNA |
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Excise a whole brain from mouse or rat. |
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Isolate total RNA using Trizol according to manufacturer’s protocol. |
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Reverse-transcribe the total RNA to first strand cDNA by standard method with random hexamer (we use superscriptIII). |
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2. |
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Using reverse-transcribed cDNA as a template, amplify cDNA of GlcAT-P, GlcAT-S or HNK-1ST by standard method (see Comment 1, 2). |
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Insert the amplified cDNA into cloning vector (we use Zero-Blunt TOPO plasmid, Invitrogen/Life Technologies, Carlsbad, CA). |
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Subclone the cDNA into expression vector (see Comment 3, 4). |
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3. |
Expression of HNK-1 epitope by transfection of GlcAT-P(S) and HNK-1ST |
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Choose cell lines which you want to investigate (see Comment 5). |
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For a 10-cm dish, we transfect 4 μg plasmid DNA using FUGENE6 transfection reagent. |
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Further analysis (western blotting (see Comment 6, 7) or immunostaining). |
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| Copyrights |
 Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses
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| Date of registration：2013-12-27 16:53:37 |
- Kizuka, Y., Matsui, T., Takematsu, H., Kozutsumi, Y., Kawasaki, T., and Oka, S. (2006) Physical and functional association of glucuronyltransferases and sulfotransferase involved in HNK-1 biosynthesis. J Biol Chem. 281, 13644-13651 [PMID : 16543228]
- Kizuka, Y., Tonoyama, Y., and Oka, S. (2009) Distinct transport and intracellular activities of two GlcAT-P isoforms. J Biol Chem. 284, 9247-9256 [PMID : 19181664]
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How to Cite this Work in an article:
Kizuka, Yasuhiko,
Oka, Shogo,
(2013). GlycoPOD https://jcggdb.jp/GlycoPOD.
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How to Cite this Work in Website:
Kizuka, Yasuhiko,
Oka, Shogo,
(2013).
Cloning and transfection of glucuronyltransferase for HNK-1 epitope.
Retrieved 18,4,2024 ,
from https://jcggdb.jp/GlycoPOD/protocolShow.action?nodeId=t81.
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Kizuka, Yasuhiko,
Oka, Shogo,
(2013).
<b>Cloning and transfection of glucuronyltransferase for HNK-1 epitope</b>.
Retrieved 4 18,2024 ,
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