A unique biosynthetic pathway for gangliosides exists in Xenopus laevis oocytes

AbstractIt was previously reported that monosialosylgangliopentaosyl ceramide (Ga1NAc-GM1b) was a major ganglioside in Xenopus laevis oocytes. Here we determined biosynthetic pathways for the ganglioside by detailed measurements of glycosyltransferase activities. CMP-NeuAc:asialo-GM1 α2–3 sialyltransferase (α2–3 ST) and UDP-Ga1NAc:GM1b β1–4 N-acetylgalactosaminyltransferase (β1–4 Ga1NAcT) exhibited much higher activity than CMP-NeuAc:Ga1NAc-GA1 α2–3 ST and UDP-Ga1NAc:asialo-GM1 β1–4 Ga1NAcT, respectively. These observations indicated the existence of a unique biosynthetic pathway in the oocytes as follows; asialo-GM1 → GM1b → Ga1NAc-GM1b

Chromosome mapping of the GD3 synthase gene (SIAT8) in human and mouse.

GD3 synthase (CMP-NeuAc:NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1\u27Cer alpha 2,8-sialyltransferase) is a member of the sialyltransferase family, whose members are characterized by having the sialyl motif and a key regulatory enzyme that controls the ganglioside biosynthesis pathway. The chromosomal location of the GD3 synthase gene (SIAT8) was determined in human and mouse using fluorescence in situ hybridization and interspecific backcross analysis, respectively. The human GD3 synthase gene was mapped to p12.1-p11.2 of chromosome 12. The mouse homologue was mapped 2.8 cM distal to D6Mit52 and 4.3 cM proximal to D6Mit25; this region is syntenic to the short arm of human chromosome 12

Roles of HNK-1 carbohydrate epitope and its synthetic glucuronyltransferase genes on migration of rat neural crest cells

HNK-1 carbohydrate epitope is localized on the surface of avian neural crest cells (NCCs), and is necessary for their migration. However, it is still disputed whether the epitope works in similar ways in mammalian embryos. In this study, we found that HNK-1 carbohydrate epitope was specifically detected in some of the cranial ganglia, migrating trunk NCCs and some non-NCC derivatives in the rat embryo. Two genes encoding glucuronyltransferases that synthesize the HNK-1 epitope in vitro (GlcAT-P and GlcAT-D) were recently identified in the rat. Interestingly, the NCCs in the cranial ganglia expressed the GlcAT-D gene, whereas the migrating trunk NCCs expressed the GlcAT-P gene. To investigate in vivo functions of the GlcATs in the NCC migration further, we overexpressed GlcAT genes by electroporation in the cranial NCCs in cultured rat embryos. Transfection of both GlcAT genes resulted in efficient synthesis of the HNK-1 epitope in the NCCs. GlcAT-P overexpression increased distance of cranial NCC migration, whereas GlcAT-D overexpression did not show this effect. Our data suggest that the HNK-1 epitope synthesized by different GlcATs is involved in migration in the sublineages of the NCCs in the rat embryo, and that GlcAT-P and GlcAT-D mediate different effects on the NCC migration

高感度酵素抗体法を用いたRett症候群および他の神経疾患における髄液ガングリオシド分析

年齢2～10歳のRett症候群患者12名と,同年齢の髄液検査が必要であった他疾患患者19名,および正常対照例7名において,脳脊髄液中の5つの主要なガングリオシドGM1, GD1a, GD1b, GT1bおよびGQ1bを感度の高い酵素抗体法で測定した.我々は,この方法を用いて標準曲線を作成し,これらを測定できた.本法は,高感度な検査法であることを証明した.対象症例における脳脊髄液中の総ガングリオシド量は,脳脊髄液1ml当たり,正常対照例で100～900ng,神経疾患患者で25ngから最大5,000ngの値をとっていた.Rett症候群を含め,どの神経疾患でも特別なパターンは認められなかった.過去にRett症候群においては,あるガングリオシドが大脳および小脳で低下しているという所見の報告があったが,この所見と関連する結果は髄液では得られなかった.また,我々が,髄液における5つの主要なガングリオシドの値をRett症候群患者と正常および疾患対照例と比較した所では,そのデータからRett症候群を予測させるような低下パターンを見出すことはできなかった.但し,12例中5例でGD1aが, 4例でGT1bおよびGQ1bの合計値が正常対照例の下限値よりも低下していた.Rett症候群の独歩可能な患児と不可能な患児における髄液ガングリオシド値の比較検討では差がなく,また異った臨床ステージの患者間の比較検討でも差がなかった.しかしながら,髄膜炎の急性期,熱性けいれんの直後,またRett症候群のけいれんの多い児2名を含み,てんかん患児の一部で髄液中のガングリオシド,主として,GD1b,GT1b値が上昇していた.The cerebrospinal fluid (CSF) gangliosides GM1, GDla, GDlb, GTlb and GQlb were measured using a highly sensitive enzyme-immunostaining technique, in 12 patients with Rett syndrome (RS) ranging in age from 2y to 10y, in age-matched patients with various neurologic diseases (n=19) and in normal control subjects (n=7). The method used proved to be highly sensitive; a standard curve was established and we were able to obtain reliable assay results. Total CSF gangliosides varied widely among normal controls (100～900 ng/ml CSF) as well as in those with neurologic diseases (25 to 5,000 ng/ml CSF). No specific ganglioside pattern was found in any of the disease groups, including RS. Although previous reports have shown reductions in the levels of gangliosides in the RS brain, we found no evidence of a predictable and constant pattern of reduced levels of any of the five major CSF gangliotetraose series gangliosides in this syndrome; however, the amount of GDla was reduced in five of the 12 RS patients, and the amount of GQ1b+GT1b was reduced in four as compared with the minimum value obtained in the control group. Analysis of CSF ganglioside levels in RS patients revealed no differences between ambulant and non-ambulant patients, nor were there any differences among patients in different clinical stages. There was, however, a positive correlation between CSF ganglioside levels, mainly the b-pathway gangliosides, and the presence and frequency of seizures in some patients, including the one with febrile convulsion, those in the acute stage of meningitis, two of the RS patients and some of those with epileptic syndromes

Two Pathways for Importing GDP-fucose into the Endoplasmic Reticulum Lumen Function Redundantly in the O-Fucosylation of Notch in Drosophila*

Notch is a transmembrane receptor that shares homology with proteins containing epidermal growth factor-like repeats and mediates the cell-cell interactions necessary for many cell fate decisions. In Drosophila, O-fucosyltransferase 1 catalyzes the O-fucosylation of these epidermal growth factor-like repeats. This O-fucose elongates, resulting in an O-linked tetrasaccharide that regulates the signaling activities of Notch. Fucosyltransferases utilize GDP-fucose, which is synthesized in the cytosol, but fucosylation occurs in the lumen of the endoplasmic reticulum (ER) and Golgi. Therefore, GDP-fucose uptake into the ER and Golgi is essential for fucosylation. However, although GDP-fucose biosynthesis is well understood, the mechanisms and intracellular routes of GDP-fucose transportation remain unclear. Our previous study on the Drosophila Golgi GDP-fucose transporter (Gfr), which specifically localizes to the Golgi, suggested that another GDP-fucose transporter(s) exists in Drosophila. Here, we identified Efr (ER GDP-fucose transporter), a GDP-fucose transporter that localizes specifically to the ER. Efr is a multifunctional nucleotide sugar transporter involved in the biosynthesis of heparan sulfate-glycosaminoglycan chains and the O-fucosylation of Notch. Comparison of the fucosylation defects in the N-glycans in Gfr and Efr mutants revealed that Gfr and Efr made distinct contributions to this modification; Gfr but not Efr was crucial for the fucosylation of N-glycans. We also found that Gfr and Efr function redundantly in the O-fucosylation of Notch, although they had different localizations and nucleotide sugar transportation specificities. These results indicate that two pathways for the nucleotide sugar supply, involving two nucleotide sugar transporters with distinct characteristics and distributions, contribute to the O-fucosylation of Notch