A novel beta(1,6)-N-acetylglucosaminyltransferase V (GnT-VB)(1).
UDP-N-acetylglucosamine:alpha(1,6)-D-mannoside beta(1,6)-N-acetylglucosaminyltransferase (GnT-V, Mgat5) functions in the biosynthesis of N-linked glycans and is transcriptionally upregulated by oncogene signaling. We report here the cloning and characterization of a human cDNA encoding a distinct enzyme with related substrate specificity, termed GnT-VB, which is predicted to have 53% similarity to the original amino acid sequence of GnT-V(A). Transient expression of GnT-VB cDNA in COS7 cells yielded significant increases of activity toward GnT-VA acceptors, including synthetic saccharides and N-linked glycopeptides, with some differences in specificity. Unlike GnT-VA, GnT-VB required divalent cation for full activity. EST databases showed expression of a 6 bp (+) splice isoform of GnT-VB; when expressed, this enzyme showed significantly reduced activity. CHO Lec4 cells, which do not express GnT-VA or B activity, lack synthesis of the N-linked beta(1,6) branch, and do not bind L-phytohemagglutinin (L-PHA), were transfected with GnT-VB or GnT-VA; both then bound significant amounts of L-PHA, demonstrating that both enzymes synthesized N-linked beta(1,6) branched glycans in vivo. Real-time polymerase chain reaction results showed that GnT-VB mRNA was highly expressed in brain and testis, with lesser levels in other tissues, while human GnT-VA showed a more general expression, but with low levels in brain and no expression in skeletal muscle.
Kaneko
Mika
M
Complex Carbohydrate Research Center and Department of Biochemistry and Molecular Biology, University of Georgia, 30605, Athens, GA, USA
Alvarez-Manilla
Gerardo
G
Kamar
Maria
M
Lee
Intaek
I
Lee
Jin Kyu
JK
Troupe
Karolyn
K
Zhang
Wei jie
Wj
Osawa
Motoki
M
Pierce
Michael
M
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics* / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism; D009928:Organ Specificity; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014162:Transfection
D018076:DNA, Complementary; D007527:Isoenzymes; D009844:Oligosaccharides; D011994:Recombinant Proteins; D017351:N-Acetylglucosaminyltransferases; C052254:alpha-1,6-mannosylglycoprotein beta 1,6-N-acetylglucosaminyltransferase