Satoshi Fukumoto

Molecular Cloning of a Novel α2,3-Sialyltransferase (ST3Gal VI) That Sialylates Type II Lactosamine Structures on Glycoproteins and Glycolipids

A novel member of the human CMP-NeuAc:β-galactoside α2,3-sialyltransferase (ST) subfamily, designated ST3Gal VI, was identified based on BLAST analysis of expressed sequence tags, and a cDNA clone was isolated from a human melanoma line library. The sequence of ST3Gal VI encoded a type II membrane protein with 2 amino acids of cytoplasmic domain, 32 amino acids of transmembrane region, and a large catalytic domain with 297 amino acids; and showed homology to previously cloned ST3Gal III, ST3Gal IV, and ST3Gal V at 34, 38, and 33%, respectively. Extracts from L cells transfected with ST3Gal VI cDNA in a expression vector and a fusion protein with protein A showed an enzyme activity of α2,3-sialyltransferase toward Galβ1,4GlcNAc structure on glycoproteins and glycolipids. In contrast to ST3Gal III and ST3Gal IV, this enzyme exhibited restricted substrate specificity,i.e. it utilized Galβ1,4GlcNAc on glycoproteins, and neolactotetraosylceramide and neolactohexaosylceramide, but not lactotetraosylceramide, lactosylceramide, or asialo-GM1. Consequently, these data indicated that this enzyme is involved in the synthesis of sialyl-paragloboside, a precursor of sialyl-Lewis X determinant.

Molecular Basis for the Progeroid Variant of Ehlers-Danlos Syndrome IDENTIFICATION AND CHARACTERIZATION OF TWO MUTATIONS IN GALACTOSYLTRANSFERASE I GENE

Progeroid type Ehlers-Danlos (E-D) syndrome was reported to be caused by defects in galactosyltransferase I (EC2.4.1.133), which is involved in the synthesis of common linkage regions of proteoglycans. Recently, we isolated cDNA of the galactosyltransferase I (XGalT-1) (Okajima, T., Yoshida, K., Kondo, T., and Furukawa, K. (1999) J. Biol. Chem.274, 22915–22918). Therefore, we analyzed mutations in this gene of a patient with progeroid type E-D syndrome by reverse transcription polymerase chain reaction and direct sequencing. Two changes of G and T to A and C at 186 and 206, respectively, were detected. Then, we determined the genomic DNA sequences encompassing the A186D and L206P mutations, revealing that the unaffected parents and two siblings were heterozygous for either one of the two different mutations and normal, while the patient had both of two different mutant genes. Enzymatic functions of cDNA clones of XGalT-1 containing the individual mutations were examined, elucidating that L206P clone completely lost the activity, while A186D retained ∼50% or 10% of the activity when analyzed with extracts from cDNA transfectant cells or recombinant soluble enzymes, respectively. Moreover, L206P enzyme showed diffuse staining in the cytoplasm of transfectant cells, while the wild type or A186D clones showed Golgi pattern. These results indicated that the mutations in XGalT-1 were at least one of main molecular basis for progeroid type E-D syndrome.

Deformation of lipid droplets in fixed samples

Abstract. Nile red, Sudanxa0III, and oil redxa0O have been used to stain lipid droplets (LDs) for fluorescence microscopy. We noticed that LDs labeled by Nile red are different in appearance from those stained by the latter two dyes. To understand the cause of the difference, we used sequential labeling procedures (first LD stain–photography–quenching–second LD stain–photography), and examined the effect of several factors. Immunofluorescence labeling for adipose differentiation-related protein (ADRP), an LD marker, was also observed comparatively with the lipid stains. As a result, we found that ethanol and isopropanol used for Sudanxa0III and oil redxa0O staining, respectively, and glycerol used for mounting, cause fusion of adjacent LDs even in glutaraldehyde-fixed samples. By the same treatment, immunofluorescence labeling for ADRP was dislocated to the rim of large LDs that were formed as a result of the artifactual fusion. The result indicates that the LD structure can be better observed with Nile red than with Sudanxa0III or oil redxa0O.

Overexpression of Ganglioside GM1 Results in the Dispersion of Platelet-derived Growth Factor Receptor from Glycolipid-enriched Microdomains and in the Suppression of Cell Growth Signals

To investigate the molecular mechanisms of gangliosides for the regulation of cell proliferation, Swiss 3T3 cells were transfected with GM2/GD2 synthase and GM1 synthase cDNAs, resulting in the establishment of GM1-expressing (GM1+) lines. Compared with the vector control (GM1−) cell lines, GM1+ cells exhibited reduced cell proliferation by stimulation with platelet-derived growth factor (PDGF). In accordance with the reduced cell growth, GM1+ cells showed earlier decreases in the phosphorylation levels of PDGF receptor and less activation of MAP kinases than GM1− cells. To analyze the effects of GM1 expression on the PDGF/PDGF receptor (PDGFR) signals, the glycolipid-enriched microdomain (GEM) was isolated and the following results were obtained. (i) PDGFR predominantly distributed in the non-GEM fraction in GM1+ cells, while it was present in both GEM and non-GEM fractions in GM1− cells. (ii) Activation of PDGFR as detected by anti-phosphotyrosine antibody occurred almost in parallel with existing amounts of PDGFR in each fraction. (iii) GM1 binds with PDGFR in GEM fractions. These findings suggested that GM1 regulates signals via PDGF/PDGFR by controlling the distribution of PDGFR in- and outside of GEM, and also interacting with PDGFR in the GEM fraction as a functional constituent of the microdomain.

Expression Cloning of Rat cDNA Encoding UDP-galactose:GD2 β1,3-galactosyltransferase That Determines the Expression of GD1b/GM1/GA1

Using an anti-GD1b monoclonal antibody, expression cloning of a cDNA for the β1,3-galactosyltransferase gene (EC 2.4.1.62) was performed. KF4C, mouse melanoma B16 transfected with polyoma T antigen gene, and GM2/GD2 synthase cDNA was used as a recipient cell line for the cDNA library transfection. A cDNA clone of GD3 synthase, pD3T-31 was co-transfected with a cDNA library prepared from rat brain RNA using the pcDNAI expression vector. The isolated cDNA clone pM1T-9 predicted a type II membrane protein with 4 amino acids of cytoplasmic domain, 21 amino acids of transmembrane region, and a large catalytic domain with 346 amino acids. Introduction of the cDNA clone into a mouse melanoma line B16 previously transfected with a GM2/GD2synthase gene resulted in the neo-synthesis of GM1. Co-transfection of the cell line with pM1T-9 and a GD3synthase cDNA resulted in the expression of GD1b as well as GM1. Moreover, introduction of pM1T-9 into L cell (lacking GM3 synthase), previously transfected with GM2/GD2 synthase gene, resulted in the definite expression of asialo-GM1. These results indicated that GD1b/GM1/GA1 synthases were identical, as previously suggested based on enzymological analysis. In Northern blots of the β1,3-galactosyltransferase gene with total RNA from various rat tissues, a 1.6-kilobase mRNA was strongly expressed in spleen, thymus, kidney, and testis. However, the expression level of the gene in the adult brain tissue was not especially high. On the other hand, this gene was expressed at high levels in the rat brain of embryonal day 12, and reached a peak at around birth, then fell to low level in the adult brain.

Heterogeneity in the expression pattern of two ganglioside synthase genes during mouse brain development

Abstract: Gangliosides are synthesized by sequential catalytic reaction of multiple glycosyltransferases. GM2/GD2 synthase and GD3 synthase are key enzymes for ganglioside synthesis, because their relative activities regulate the main profiles of ganglioside expression. Mouse GD3 synthase (EC 2.4.99.8) cDNA was cloned by eukaryotic expression cloning, and its mRNA expression as well as that of GM2/GD2 synthase gene during the development of the mouse CNS was analyzed by using northern blotting, reverse transcription‐polymerase chain reaction, and in situ hybridization. When brain tissue was analyzed as a whole mass, a typical pattern corresponding to the reported findings obtained by biochemical analyses was observed, i.e., high expression of GD3 synthase gene in the early stage and gradual increase of GM2/GD2 synthase gene expression in the late stage of the development. However, the results of in situ hybridization of these two genes revealed that the expression kinetics of these two genes were heterogeneous among various sites in the brain under development. These findings suggest that various expression patterns of the two genes reflect differences in the course of the development of individual sites, and also different ganglioside components are required in individual portions of the brain for development and maintenance of the function.

MOLECULAR CLONING OF BRAIN-SPECIFIC GD1ALPHA SYNTHASE (ST6GALNAC V) CONTAINING CAG/GLUTAMINE REPEATS

A novel member of the mouse CMP-NeuAc: β-N-acetylgalactosaminide α2,6-sialyltransferase (ST6GalNAc) subfamily, designated ST6GalNAc V, was identified by BLAST analysis of expressed sequence tags. The sequence of the longest cDNA clone of ST6GalNAc V encoded a type II membrane protein with 8 amino acids comprising the cytoplasmic domain, 21 amino acids comprising the transmembrane region, and 306 amino acids comprising the catalytic domain. The predicted amino acid sequence showed homology to the previously cloned ST6GalNAc III and IV, with common amino acid sequences in sialyl motifs L and S among these three enzymes. Eleven CAG repeats were found in the stem region. A fusion protein with protein A and extracts from L cells transfected with ST6GalNAc V in a expression vector showed enzyme activity of α2,6-sialyltransferase almost exclusively for GM1b, but not toward glycoproteins. Sialidase treatment and thin layer chromatography immunostaining revealed that the product was GD1α. Northern blotting revealed that three transcripts of the gene were expressed specifically in brain tissues. It is concluded that this enzyme is involved in the synthesis of GD1α in the nervous tissues, and the CAG repeats may have implications in neurodegenerative diseases.

Expression cloning of mouse cDNA of CMP-NeuAc:Lactosylceramide alpha2,3-sialyltransferase, an enzyme that initiates the synthesis of gangliosides.

Expression cloning of a cDNA for the α2,3-sialyltransferase (GM3 synthase) (EC 2.4.99.-) gene was performed using a GM3-lacking mouse fibroblast line L cell and anti-GM3 monoclonal antibody. Plasmids from a cDNA library generated with poly(A)+ RNA of a mouse fibrosarcoma line CMS5j and pdl3027 (polyoma T antigen) were co-transfected into L cells. The isolated cDNA clone pM3T-7 predicted a type II membrane protein with 13 amino acids of cytoplasmic domain, 17 amino acids of transmembrane region, and a large catalytic domain with 329 amino acids. Introduction of the cDNA clone into L cells resulted in the neo-synthesis of GM3 and high activity of α2,3-sialyltransferase. Among glycosphingolipids, only lactosylceramide showed significant activity as an acceptor, indicating that this gene product is a sialyltransferase specific for the synthesis of GM3. An amino acid sequence deduced from the cloned cDNA showed the typical sialyl motif with common features among α2,3-sialyltransferases. Among various mouse tissues, brain, liver, and testis showed relatively high expression of a 2.3-kilobase mRNA, whereas all tissues, more or less, expressed this gene.

Novel functions of complex carbohydrates elucidated by the mutant mice of glycosyltransferase genes

Complex carbohydrates consist of carbohydrate moieties and protein or lipid portions, resulting in the formation of glycoproteins, proteoglycans or glycosphingolipids. The polymorphic carbohydrate structures are believed to contain profound biological implications which are important in cell-cell or cell-extracellular matrix interactions. A number of studies to delineate the roles of carbohydrates have been performed, and demonstrated definite changes in their profiles, cellular phenotypic changes or, sometimes, morphological and functional changes in tissues after modification of their structures. Recent successes in the isolation of glycosyltransferase genes and their modification enzyme genes has enabled clearer demonstrations of the roles of complex carbohydrates. In particular, genetic modification of glycosyltransferase genes in mice can elucidate the biological significances of their products in vivo. Here, we summarize recent advances in the understanding of the roles of complex carbohydrates provided from studies of gene knock-out mice of glycosyltransferase and modification enzyme genes focusing on novel functions which had not been expected.

Attenuation of Interleukin 2 Signal in the Spleen Cells of Complex Ganglioside-lacking Mice

T cell development and function in complex ganglioside-lacking (GM2/GD2 synthase gene-disrupted) mice were analyzed. GM1, asialo-GM1, and GD1b were representative gangliosides expressed on T cells of the wild type mice and completely deleted on those of the mutant mice. The sizes and cell numbers of the mutant mice spleen and thymus were significantly reduced. Spleen cells from the mutant mice showed clearly reduced proliferation compared with the wild type when stimulated by interleukin 2 (IL-2) but not when treated with concanavalin A or anti-CD3 cross-linking. Expression levels of IL-2 receptor α, β, and γ were almost equivalent, and up-regulation of α chain after T cell activation was also similar between the mutant and wild type mice. Activation of JAK1, JAK3, and SAT5 after IL-2 treatment was reduced, and c-fos expression was delayed and reduced in the mutant spleen cells, suggesting that the IL-2 signal was attenuated in the mutant mice probably due to the modulation of IL-2 receptors by the lack of complex gangliosides.