Cloning and transfection of glucuronyltransferase for HNK-1 epitope
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.
Standard kit for reverse transcription (superscriptIII in our case)
DNA polymerase, ligase and restriction enzymes
Cloning plasmid and expression plasmid
Transfection reagent (FUGENE6 in our case)
HNK-1 monoclonal antibody (mAb) (ATCC)
M6749 mAb (optional)
Standard devices for DNA recombination and western blotting
Isolation and reverse transcription of total RNA
Excise a whole brain from mouse or rat.
Isolate total RNA using Trizol according to manufacturer’s protocol.
Reverse-transcribe the total RNA to first strand cDNA by standard method with random hexamer (we use superscriptIII).
Cloning of cDNA
Using reverse-transcribed cDNA as a template, amplify cDNA of GlcAT-P, GlcAT-S or HNK-1ST by standard method (see Comment 1, 2).
Insert the amplified cDNA into cloning vector (we use Zero-Blunt TOPO plasmid, Invitrogen).
Subclone the cDNA into expression vector (see Comment 3, 4).
Expression of HNK-1 epitope by transfection of GlcAT-P(S) and HNK-1ST
Choose cell lines which you want to investigate (see Comment 5).
For a 10-cm dish, we transfect 4 μg plasmid DNA using FUGENE6 transfection reagent.
Incubate for 24 h or longer.
Further analysis (western blotting (see Comment 6, 7) or immunostaining).
Kizuka Y, Matsui T, Takematsu H, Kozutsumi Y, Kawasaki T, 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, Oka S (2009) Distinct transport and intracellular activities of two GlcAT-P isoforms. J Biol Chem 284: 9247-9256. [PMID : 19181664]