Expression cloning of a human GT3 synthase. GD3 AND GT3 are synthesized by a single enzyme.
Gangliosides of the C series such as GT3 are polysialylated glycosphingolipids whose synthesis is developmentally regulated. Here we report the expression cDNA cloning and characterization of GT3 synthase that adds the second alpha-2,8-sialic acid to GD3, NeuNAcalpha2-->8NeuNAcalpha2-->3Galbeta1-->4Glc-->Cer, thus forming GT3, NeuNAcalpha2-->8NeuNAcalpha2-->8NeuNAc alpha2-->3Galbeta1--> 4Glc-->Cer. Unexpectedly, the cloned cDNA was found to be identical to the cDNA that encodes GD3 synthase. The newly identified enzyme was therefore named GD3/GT3 synthase (GD3/GT3ST). GD3/GT3ST synthesized GT3 most efficiently when GM3, NeuNAcalpha2-->3Galbeta1-->4Glc-->Cer, was incubated as an acceptor, indicating that GD3/GT3ST is a polysialyltransferase that can transfer more than one sialic acid residue via alpha-2,8 linkage to gangliosides. Moreover, a longer period of incubation of GD3 with GD3/GT3ST produced a significant amount of GT3 and higher polysialogangliosides. Among various cell lines expressing GD3/GT3ST, higher polysialogangliosides including GT3 were detected only in cell lines where the amount of GD3/GT3 mRNA is sufficiently high. The expression of GD3/GT3ST mRNA among human tissues is highly restricted to fetal and adult brains. The GD3/GT3ST gene was found to be located at chromosome 12, region p12. Taken together, these results indicate that C series polysialogangliosides are synthesized by a ganglioside-specific polysialyltransferase, GD3/GT3ST, that is specifically expressed in neural tissues.
Nakayama
J
J
Glycobiology Program, La Jolla Cancer Research Foundation, Cancer Research Center, La Jolla, California 92037, USA.
Fukuda
M N
MN
Hirabayashi
Y
Y
Kanamori
A
A
Sasaki
K
K
Nishi
T
T
Fukuda
M
M
D000328:Adult; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology*; D002240:Carbohydrate Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D002874:Chromosome Mapping; D002881:Chromosomes, Human, Pair 12; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D005260:Female; D005333:Fetus; D005732:Gangliosides / Q000096:biosynthesis*; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D007790:Lactosylceramides / Q000096:biosynthesis*; D008297:Male; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000138:chemical synthesis / Q000737:chemistry; D009928:Organ Specificity; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D012799:Sialyltransferases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D017931:DNA Primers; D018076:DNA, Complementary; D005732:Gangliosides; D007790:Lactosylceramides; D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C026226:ganglioside, GD3; C047989:trisialosyllactosylceramide; D012799:Sialyltransferases; C043662:alpha-N-acetylneuraminate alpha-2,8-sialyltransferase