Preparation of antisera to recombinant, soluble N-acetylglucosaminyltransferase V and its visualization in situ.
N-Acetylglucosaminyltransferase V (GlcNAc-T V) is a glycosyltransferase which transfers N-acetylglucosamine in beta(1,6) linkage to the alpha(1,6)-linked mannose residue of Asn-linked oligosaccharides. This enzyme is characterized by several unusual properties: GlcNAc-T V is the largest lumenal Golgi glycosyltransferase described thus far, and its multiple mRNA transcripts range from 4.5 to about 9.5 kb; GlcNAc-T V mRNA and activity are regulated by the src tyrosine kinase signalling pathway; in brain tissue, large levels of GlcNAc-T V mRNA are present, but only relatively low levels of catalytic activity can be detected; a lectin-resistant cell line, Lec4A, expresses active GlcNAc-T V which is mislocalized intracellularly. In addition, the cell surface oligosaccharide products of this enzyme have been hypothesized to regulate intercellular adhesion. In order to devise specific inhibitors of this enzyme it is necessary to understand its physical structure and how structural changes can influence its activity and localization. We have expressed milligram amounts of a soluble form of recombinant rat GlcNAc-T V, purified it from CHO cell-conditioned media, and used it to prepare specific antisera. This antisera binds selectively to GlcNAc-T V and has been used to visualize B-16 mouse melanoma cell GlcNAc-T V on immunoblots after SDS-PAGE. When the antisera was used in immunofluorescence microscopy experiments on permeabilized B-16 and baby hamster kidney cells, intense, specific staining was observed in intracellular structures which appear to correspond to the Golgi apparatus.
Chen
L
L
Department of Biochemistry and Molecular Biology and Complex Carbohydrate Research Center, University of Georgia, Athens 30602, USA.
Zhang
N
N
Adler
B
B
Browne
J
J
Freigen
N
N
Pierce
M
M
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002240:Carbohydrate Sequence; D006224:Cricetinae; D015151:Immunoblotting; D007700:Kinetics; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000032:analysis* / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis; D013010:Sonication
D011994:Recombinant Proteins; D017351:N-Acetylglucosaminyltransferases; C052254:alpha-1,6-mannosylglycoprotein beta 1,6-N-acetylglucosaminyltransferase