Molecular cloning and characterization of UDP-GlcNAc:lactosylceramide beta 1,3-N-acetylglucosaminyltransferase (beta 3Gn-T5), an essential enzyme for the expression of HNK-1 and Lewis X epitopes on glycolipids.
A new member of the UDP-N-acetylglucosamine:beta-galactose beta1,3-N-acetylglucosaminyltransferase (beta3Gn-T) family having the beta3Gn-T motifs was cloned from rat and human cDNA libraries and named beta3Gn-T5 based on its position in a phylogenetic tree. We concluded that beta3Gn-T5 is the most feasible candidate for lactotriaosylceramide (Lc(3)Cer) synthase, an important enzyme which plays a key role in the synthesis of lacto- or neolacto-series carbohydrate chains on glycolipids. beta3Gn-T5 exhibited strong activity to transfer GlcNAc to glycolipid substrates, such as lactosylceramide (LacCer) and neolactotetraosylceramide (nLc(4)Cer; paragloboside), resulting in the synthesis of Lc(3)Cer and neolactopentaosylceramide (nLc(5)Cer), respectively. A marked decrease in LacCer and increase in nLc(4)Cer was detected in Namalwa cells stably expressing beta3Gn-T5. This indicated that beta3Gn-T5 exerted activity to synthesize Lc(3)Cer and decrease LacCer, followed by conversion to nLc(4)Cer via endogenous galactosylation. The following four findings further supported that beta3Gn-T5 is Lc(3)Cer synthase. 1) The beta3Gn-T5 transcript levels in various cells were consistent with the activity levels of Lc(3)Cer synthase in those cells. 2) The beta3Gn-T5 transcript was presented in various tissues and cultured cells. 3) The beta3Gn-T5 expression was up-regulated by stimulation with retinoic acid and down-regulated with 12-O-tetradecanoylphorbol-13-acetate in HL-60 cells. 4) The changes in beta3Gn-T5 transcript levels during the rat brain development were determined. Points 2, 3, and 4 were consistent with the Lc(3)Cer synthase activity reported previously.
Togayachi
A
A
Division of Cell Biology, Institute of Life Science, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577, Japan.
Akashima
T
T
Ookubo
R
R
Kudo
T
T
Nishihara
S
S
Iwasaki
H
H
Natsume
A
A
Mio
H
H
Inokuchi
J
J
Irimura
T
T
Sasaki
K
K
Narimatsu
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D000939:Epitopes / Q000737:chemistry*; D006017:Glycolipids / Q000737:chemistry*; D006801:Humans; D016256:Lewis X Antigen / Q000737:chemistry*; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D000939:Epitopes; D006017:Glycolipids; D016256:Lewis X Antigen; D011994:Recombinant Proteins; D017351:N-Acetylglucosaminyltransferases; C067641:lactosylceramide 1,3-N-acetyl-beta-D-glucosaminyltransferase