Abstract Alexander's disease, a leukodystrophy characterized by Rosenthal fibers (RFs) in the brain, is categorized into three subtypes: infantile, juvenile, and adult. Although most are sporadic, occasional familial Alexander's disease cases have been reported for each subtype. Hereditary adult‐onset Alexander's disease shows progressive spastic paresis, bulbar or pseudobulbar palsy, palatal myoclonus symptomatologically, and prominent atrophy of the medulla oblongata and upper spinal cord on magnetic resonance imaging. Recent identification of GFAP gene mutations in the sporadic infantile‐ and juvenile‐onset Alexander's disease prompted us to examine the GFAP gene in two Japanese hereditary adult‐onset Alexander's disease brothers with autopsy in one case. Both had spastic paresis without palatal myoclonus, and magnetic resonance imaging showed marked atrophy of the medulla oblongata and cervicothoracic cord. The autopsy showed severely involved shrunken pyramids, but scarce Rosenthal fibers (RFs). Moderate numbers of Rosenthal fibers (RFs) were observed in the stratum subcallosum and hippocampal fimbria. In both cases, we found a novel missense mutation of a G‐to‐T transition at nucleotide 841 in the GFAP gene that results in the substitution of arginine for leucine at amino acid residue 276 (R276L). This is the first report of identification of the causative mutation of the GFAP gene for neuropathologically proven hereditary adult‐onset Alexander's disease, suggesting a common molecular mechanism underlies the three Alexander's disease subtypes.
We describe an unusual case of a patient with Machado-Joseph disease (MJD) who showed autonomic dysfunctions in addition to cerebellar ataxia. The number of CAG repeat units in the expanded allele of the MJD1 gene of the patient is smaller (56 CAG repeat units) than all previously reported numbers of CAG repeat units in expanded alleles. Thus, the findings in this patient indicate that the clinical features of MJD cover a wider spectrum than previously thought.
Abstract We have previously reported that ganglioside GM3 was remarkably increased during monocytoid differentiation of human myelogenous leukemia cell line HL-60 cells and that neolacto series gangliosides (NeuAc-nLc) were enriched during granulocytoid differentiation. In addition, HL-60 was differentiated into monocytic lineage by exogenous GM3 and into granulocytoid by NeuAc-nLc. In the present report, the enzymatic bases of glycosphingolipid biosynthesis in HL-60 during differentiation induced by 12-O-tetradecanoylphorbol-13-acetate and all-trans-retinoic acid were investigated. The following results were of particular interest. (i) Lactosylceramide alpha 2-->3 sialyltransferase (GM3 synthase) was remarkably up-regulated during monocyte differentiation, while the GM3 synthase level did not change in granulocytic differentiation. (ii) By contrast, lactosylceramide beta 1-->3N-acetylglucosaminyltransferase (Lc3Cer synthase) was down-regulated during monocytic differentiation, while the activity of Lc3Cer synthase was found to increase in granulocytic differentiation. (iii) The activities of four downstream glycosyltransferases (for synthesis of NeuAc-nLc) were found to increase or to remain unchanged during monocytic and granulocytic differentiation. These results strongly suggested the following. The dramatic GM3 increase and the decrease of NeuAc-nLc during monocytic differentiation are the consequences of the up-regulation of GM3 synthase and the down-regulation of Lc3Cer synthase, although the downstream enzymes are ready to catalyze their enzyme reactions. The notable increase of NeuAc-nLc and the relative decrease of GM3 during granulocytic differentiation are the results of the unchanged level of GM3 synthase and the up-regulation of Lc3Cer synthase together with the activation of the downstream glycosyltransferases. These results suggest that these two key upstream glycosyltransferases, GM3 synthase and Lc3Cer synthase, play critical roles in regulating the glycosphingolipid biosynthesis in HL-60 cells during differentiation. This switching mechanism of these two glycosyltransferases, together with our previous findings, might be one of the most important parts of the determining system of differentiation direction in human myeloid cells into monocytic or granulocytic lineages.
ABSTRACT BACKGROUND AND PURPOSE Magnetic resonance imaging (MRI) of autosomal recessive spastic ataxia of Charlevoix‐Saguenay (ARSACS) cases in Quebec and Europe was reported to show linear hypointensities in T2‐weighted and Fluid Attenuated Inversion Recovery (FLAIR) images of the pons. We attempted to clarify the characteristics of the brain MRI findings in ARSACS cases. METHODS Eight Japanese early‐onset ataxia patients with ARSACS confirmed molecularly were investigated. We performed neurological examination, SACS gene analysis, and MRI in the patients. RESULTS Hypointensity lesions in the middle cerebellar peduncles in addition to the pons were observed in T2‐weighted and FLAIR images in all eight cases. Although superior cerebellar atrophy was seen in all cases, this MRI finding might not be specific for ARSACS. Upper cervical cord and medulla oblongata atrophy was not observed in 3 of the 7 patients examined. CONCLUSION Not only pontine but also middle cerebellar peduncle hypointensity lesions observed in T2‐weighted and FLAIR images could be specific findings for ARSACS even in cases with variable clinical phenotypes.
Ganglioside GM3 is a major glycosphingolipid in the plasma membrane and is widely distributed in vertebrates. We describe here the isolation of a human cDNA whose protein product is responsible for the synthesis of GM3. The cloned cDNA spanned 2,359 base pairs, with an open reading frame encoding a protein of 362 amino acids with a predicted molecular mass of 41.7 kDa. The deduced primary structure shows features characteristic of the sialyltransferase family, including a type II transmembrane topology and the sialylmotifs L at the center and S at the C-terminal region. An amino acid substitution from aspartic acid to histidine was demonstrated at a position invariant in sialylmotif L of all the other sialyltransferases so far cloned. The best acceptor substrate for the gene product was lactosylceramide, and cells transfected with the cloned cDNA clearly exhibited de novo synthesis of GM3, with a measurable decrease in the precursor lactosylceramide. Despite the ubiquitous distribution of ganglioside GM3 in human tissues, a major 2.4-kilobase transcript of the gene was found in a tissue-specific manner, with predominant expression in brain, skeletal muscle, and testis, and very low expression in liver. Ganglioside GM3 is a major glycosphingolipid in the plasma membrane and is widely distributed in vertebrates. We describe here the isolation of a human cDNA whose protein product is responsible for the synthesis of GM3. The cloned cDNA spanned 2,359 base pairs, with an open reading frame encoding a protein of 362 amino acids with a predicted molecular mass of 41.7 kDa. The deduced primary structure shows features characteristic of the sialyltransferase family, including a type II transmembrane topology and the sialylmotifs L at the center and S at the C-terminal region. An amino acid substitution from aspartic acid to histidine was demonstrated at a position invariant in sialylmotif L of all the other sialyltransferases so far cloned. The best acceptor substrate for the gene product was lactosylceramide, and cells transfected with the cloned cDNA clearly exhibited de novo synthesis of GM3, with a measurable decrease in the precursor lactosylceramide. Despite the ubiquitous distribution of ganglioside GM3 in human tissues, a major 2.4-kilobase transcript of the gene was found in a tissue-specific manner, with predominant expression in brain, skeletal muscle, and testis, and very low expression in liver. NeuAcα2–3Galβ1–4Glcβ1–1′Cer GalNAcβ1–4(NeuAcα2–3)Galβ1–4Glcβ1–1′Cer Galβ1–3GalNAcβ1–4 (NeuAcα2–3)Galβ1–4Glcβ1–1′Cer (ganglioside nomenclature is based on that of Svennerholm (35Svennerholm L. J. Neurochem. 1963; 10: 613-623Crossref PubMed Scopus (1313) Google Scholar)) 12-O-tetradecanoylphorbol-13 acetate monoclonal antibody fluorescein isothiocyanate fluorescence-activated cell sorter base pairs phosphate-buffered saline. It is known that sialic acid-containing glycosphingolipids, gangliosides, have various important biological functions (1Hakomori S. Annu. Rev. Immunol. 1984; 2: 103-126Crossref PubMed Scopus (541) Google Scholar, 2Hakomori S. J. Biol. Chem. 1990; 265: 18713-18716Abstract Full Text PDF PubMed Google Scholar), and their functions as well as their biosynthesis are currently clarified. In vertebrates, almost all the ganglio-series gangliosides are synthesized from a common precursor, ganglioside GM3,1 which has the simplest structure among the major gangliosides. GM3itself is known to participate in induction of differentiation (3Nojiri H. Takaku F. Tetsuka T. Motoyoshi K. Miura Y. Saito M. Blood. 1984; 64: 534-541Crossref PubMed Google Scholar, 4Nojiri H. Takaku F. Terui Y. Miura Y. Saito M. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 782-786Crossref PubMed Scopus (218) Google Scholar), modulation of proliferation (2Hakomori S. J. Biol. Chem. 1990; 265: 18713-18716Abstract Full Text PDF PubMed Google Scholar, 5Bremer E. Hakomori S. Bowen-Pope D.F. Raines E. Ross R. J. Biol. Chem. 1984; 259: 6818-6825Abstract Full Text PDF PubMed Google Scholar), maintenance of fibroblast morphology (6Meivar-Levy I. Sabanay H. Bershadsky A.D. Futerman A.H. J. Biol. Chem. 1997; 272: 1558-1564Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), signal transduction (7Hakomori S. Yamamura S. Handa K. Ann. N. Y. Acad. Sci. 1998; 845: 1-10Crossref PubMed Scopus (123) Google Scholar), and integrin-mediated cell adhesion (8Kojima N. Hakomori S.-I. Glycobiology. 1991; 1: 623-630Crossref PubMed Scopus (58) Google Scholar). Molecular cloning of genes whose protein products catalyze the transfer of sialic acid through an α-2,3 linkage has been reported (14Sasaki K. Watanabe E. Kawashima K. Sekine S. Dohi T. Oshima M. Hanai N. Nishi T. Hasegawa M. J. Biol. Chem. 1993; 268: 22782-22787Abstract Full Text PDF PubMed Google Scholar, 15Kitagawa H. Paulson J.C. Biochem. Biophys. Res. Commun. 1993; 194: 375-382Crossref PubMed Scopus (124) Google Scholar, 16Kitagawa H. Paulson J.C. J. Biol. Chem. 1994; 269: 17872-17878Abstract Full Text PDF PubMed Google Scholar, 17Giordanengo V. Bannwarth S. Laffont C. Van Miegem V. HarduinLepers A. Delannoy P. Lefebvre J.-C. Eur. J. Biochem. 1997; 247: 558-566Crossref PubMed Scopus (44) Google Scholar), and these protein products had the same acceptor substrates,i.e. the oligosaccharides of O- and N-linked glycoproteins and glycolipids. However, none of the gene products so far reported is known to be involved in the synthesis of ganglioside GM3. We demonstrated previously that the level of GM3 synthase activity was dramatically enhanced during the monocytic differentiation of HL-60 cells (21Nakamura M. Tsunoda A. Sakoe K. Gu J. Nishikawa A. Taniguchi N. Saito M. J. Biol. Chem. 1992; 267: 23507-23514Abstract Full Text PDF PubMed Google Scholar). Using a cDNA library prepared from the differentiated HL-60 cells, we have isolated a cDNA encoding the GM3 synthase by a modified expression cloning method. In the present study, we demonstrate that the GM3 synthase shows some features clearly distinct from those of all the other sialyltransferases so far cloned, although it possesses several features common to members of the sialyltransferase family. The human myelogenous leukemia cell line HL-60, and the mouse lung carcinoma cell line 3LL-J5 (22Inokuchi J. Jimbo M. Kumamoto Y. Shimeno H. Nagamatsu A. Clin. Exp. Metastasis. 1993; 11: 27-36Crossref PubMed Scopus (13) Google Scholar), which completely lacks acidic glycosphingolipids, and its derivative cell lines, 3LL-HK46, which stably expresses the polyoma virus large tumor antigen, and 3LL-ST28, which is a line permanently transfected with human GD3 synthase cDNA, were cultured in RPMI 1640 medium containing 10% fetal calf serum. HL-60 cells were inoculated at 2–3 × 105 cells/ml and treated with 24 nm TPA for 48 h, and poly(A)+ RNA was isolated using a Fast Track mRNA isolation kit (Invitrogen). After reverse transcription, followed by double-strand synthesis and blunt-ending of the termini, cDNA was ligated with BstXI adapter (Invitrogen) and cloned into pCEV18, which is a derivative of pCEV7 (24Ito N. Yonehara S. Schreurs J. Gorman D.M. Maruyama K. Ishii A. Yahara I. Arai K. Miyajima A. Science. 1990; 247: 324-327Crossref PubMed Scopus (291) Google Scholar). The cDNA library was divided into eight parts, and each part was amplified separately in Escherichia coli DH10B (Life Technologies, Inc.). After purification with Qiagen Tip (Qiagen), 100 μg of the plasmid DNA was introduced into 5 × 106 3LL-HK46 cells by electroporation (180 V, 600 μF). Thirty-six to 48 h after transfection, the cells were harvested, washed with PBS(−), and reacted with anti-GM3 mAb M2590 for 30 min on ice and stained with FITC-conjugated rabbit anti-mouse IgG/A/M (Zymed Laboratories Inc.) for 30 min on ice. The immunologically stained cells were isolated using an EPICS Elite ESP cell sorter (Coulter), and the plasmid DNA was rescued from the 5% of the cells sorted into the strongest fluorescent fields. After two rounds of the transfection and the sorting, 5 × 1063LL-HK46 cells were co-transfected with both 100 μg of the plasmid DNA rescued from the strongly fluorescent cells and 20 μg of pBKCMVh2,8ST, harboring human GD3 synthase cDNA (19Nara K. Watanabe Y. Maruyama K. Kasahara K. Nagai Y. Sanai Y. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7952-7956Crossref PubMed Scopus (123) Google Scholar), and GD3 expression on the host cell surface was detected using anti-GD3 mAb R24 as a specific marker for enrichment of the targeted cDNA. The 0.6% of the total cells applied that were distributed into the strongest fluorescent fields were sorted using a FACS Vantage cell sorter (Becton Dickinson). E. coliDH10B cells were electroporated with the plasmid DNA recovered after this sorting and seeded into nine 96-well microplates to yield 100 colonies/well, and the subpool showing the strongest positive signals in the flow cytometric profile was chosen using sibling selection. Finally, using 3LL-ST28 as well as 3LL-HK46 cell lines as host cells, a single candidate cDNA, pCEV4C7, was cloned by seeding 24 96-well microplates with one colony per well. 3LL-HK46 and 3LL-ST28 cells were transfected with 100 μg of either pCEV18 or pCEV4C7 by electroporation. After 36 h, the cells were harvested and used as an enzyme source. The enzymatic activity of GM3 synthase was measured as described previously (19Nara K. Watanabe Y. Maruyama K. Kasahara K. Nagai Y. Sanai Y. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7952-7956Crossref PubMed Scopus (123) Google Scholar). Protein concentrations were estimated by the Pierce protein assay procedure with bovine serum albumin as a standard. MTN blots (CLONTECH) were used to determine the tissue distribution of GM3 synthase mRNA. A 2,066-bpEcoRI-EcoRI fragment of the pCEV4C7 cDNA was gel-purified, labeled with [α-32P]dCTP, and used as a probe. The blots were also hybridized with a radiolabeled human glyceraldehyde-3-phosphate dehydrogenase gene probe to control for the amount and quality of RNA in each sample. We first found that newly synthesized ganglioside GM3 was barely detectable on the surface of recipient 3LL cells using anti-GM3 monoclonal antibody M2590. We therefore devised a strategy for cloning the GM3 synthase gene on the basis of the facts that anti-GD3 monoclonal antibody R24 is an IgG protein, whereas M2590 mAb is an IgM protein, and that the GD3 synthetic sialyltransferase (sialyltransferase-II, SAT-II) cDNA had been successfully cloned and was available for our use (21Nakamura M. Tsunoda A. Sakoe K. Gu J. Nishikawa A. Taniguchi N. Saito M. J. Biol. Chem. 1992; 267: 23507-23514Abstract Full Text PDF PubMed Google Scholar); the polyoma virus large T antigen was first introduced into the mouse lung carcinoma cell line 3LL-J5 (22Inokuchi J. Jimbo M. Kumamoto Y. Shimeno H. Nagamatsu A. Clin. Exp. Metastasis. 1993; 11: 27-36Crossref PubMed Scopus (13) Google Scholar) to produce the 3LL-HK46 cell line, both of which were completely deficient in gangliosides but rich in lactosylceramide (the precursor of ganglioside GM3). Thereafter, transfecting human GD3 synthase cDNA into 3LL-HK46 cells, we successfully established the 3LL-ST28 cell line, which stably expressed ganglioside GD3 synthase activity. 3LL-HK46 cells were used as host cells for the first stage of our expression cloning, in which co-transfection was performed with GD3 synthase cDNA to detect the GD3expression on the cell surface. Then, in the second stage, 3LL-ST28 cells served as recipient cells to efficiently isolate the GM3 synthase gene. The cDNA library was prepared in the vector pCEV18 from poly(A)+ RNA of HL-60 cells that had been differentiated along the monocytic lineage with phorbol ester and was introduced into the host cells by electroporation. For the first two cycles of cell sorting, the transfected cells were stained with M2590 followed by a FITC-conjugated second antibody and subjected to cell sorting. The 5% of the cells sorted into the strongest fluorescent fields was recovered, and the plasmid DNA was rescued by the method of Hirt (25Hirt B. J. Mol. Biol. 1967; 26: 365-369Crossref PubMed Scopus (3352) Google Scholar). Thus enriched plasmid DNA was co-transfected into the host cells with the plasmid pBKCMVh2,8ST (19Nara K. Watanabe Y. Maruyama K. Kasahara K. Nagai Y. Sanai Y. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7952-7956Crossref PubMed Scopus (123) Google Scholar), which harbors human GD3 synthase cDNA. After sorting the cells that were stained with anti-GD3 monoclonal antibody R24 and FITC-conjugated anti-mouse IgG antibody, we obtained a single candidate clone (termed 4C7) via two sequential sibling selections using 3LL-ST28 cells as the hosts. Fig. 1 shows the FACS profile of 3LL cells transiently transfected with clone 4C7. Strongly positive cells were found only when ST28 cells, and not HK46 cells, were used as the hosts. This result of the difficulty in detecting GM3 molecule by FACS suggests different localization or organization of the GM3 on the cell surface in different cell lines (23Ichikawa S. Sakiyama H. Suzuki G. Hidari K.I.P. Hirabayashi Y. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4638-4643Crossref PubMed Scopus (221) Google Scholar). The insert DNA of clone 4C7 spans 2,359 bp with a single open reading frame of 1,089 bp starting at nucleotide 278, which is compatible with Kozak's rule (26Kozak M. J. Cell Biol. 1989; 108: 229-241Crossref PubMed Scopus (2810) Google Scholar) (Fig. 2 A). The predicted primary sequence contains 362 amino acids with a molecular mass of 41.7 kDa, which is very close to that of the deglycosylated form (43 kDa) of the purified GM3 synthase from rat liver microsomes (27Melkerson-Watson L.J. Sweeley C.C. J. Biol. Chem. 1991; 266: 4448-4457Abstract Full Text PDF PubMed Google Scholar). As shown by the hydropathy analysis (Fig. 2 B), there exists a single anchor helix led by cationic amino acid residues in the N-terminal portion, suggesting that the 4C7 product might be a type II transmembrane protein (28Klein P. Kanehisa M. Delisi C. Biochim. Biophys. Acta. 1985; 815: 468-471Crossref PubMed Scopus (629) Google Scholar). The characteristic sequences found in all the sialyltransferases so far cloned, sialylmotifs L and S (29Drickamer K. Glycobiology. 1993; 3: 2-3Crossref PubMed Scopus (91) Google Scholar), were also found in the center and the C-terminal region of the 4C7 product, respectively, and it should be noted that there was one drastic and nonconservative substitution, of His for Asp (at position 177 in the peptide, in sialylmotif L), which is invariant in all the other known sialyltransferases (11Kim Y.-J. Kim K.-S. Do S.-I. Kim C.-H. Kim S.-K. Lee Y.-C. Biochem. Biophys. Res. Commun. 1997; 235: 327-330Crossref PubMed Scopus (50) Google Scholar, 12Scheidegger E.P. Sternberg L.R. Roth J. Lowe J.B. J. Biol. Chem. 1995; 270: 22685-22688Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 13Nakayama J. Fukuda M.N. Fredette B. Ranscht B. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7031-7035Crossref PubMed Scopus (218) Google Scholar, 14Sasaki K. Watanabe E. Kawashima K. Sekine S. Dohi T. Oshima M. Hanai N. Nishi T. Hasegawa M. J. Biol. Chem. 1993; 268: 22782-22787Abstract Full Text PDF PubMed Google Scholar, 15Kitagawa H. Paulson J.C. Biochem. Biophys. Res. Commun. 1993; 194: 375-382Crossref PubMed Scopus (124) Google Scholar, 16Kitagawa H. Paulson J.C. J. Biol. Chem. 1994; 269: 17872-17878Abstract Full Text PDF PubMed Google Scholar, 17Giordanengo V. Bannwarth S. Laffont C. Van Miegem V. HarduinLepers A. Delannoy P. Lefebvre J.-C. Eur. J. Biochem. 1997; 247: 558-566Crossref PubMed Scopus (44) Google Scholar, 18Stamenkovic I. Asheim H.C. Deggerdal A. Blomhoff H.K. Smeland E.B. Funderud S. J. Exp. Med. 1990; 172: 641-643Crossref PubMed Scopus (49) Google Scholar, 19Nara K. Watanabe Y. Maruyama K. Kasahara K. Nagai Y. Sanai Y. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7952-7956Crossref PubMed Scopus (123) Google Scholar, 20Haraguchi M. Yamashiro S. Yamamoto A. Furukawa K. Takamiya K. Lloyd K.O. Shiku H. Furukawa K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10455-10459Crossref PubMed Scopus (179) Google Scholar) (Fig. 2, A and C). Data base searches revealed that the 4C7 protein shared 27.1–41.2% sequence similarity with other sialyltransferases that catalyze formation of the α-2,3 and α-2,6 linkages of sialic acid to the acceptor galactose moiety, but not with enzymes that catalyze formation of the α-2,8 linkage to the nonreducing terminal sialic acid residue of glycosphingolipids and glycoproteins. These results strongly indicate that the 4C7 protein should be classified into the sialyltransferase family.Figure 2The nucleotide and its deduced amino acid sequences of clone 4C7. The nucleotide sequence is numbered on the left, with sequences upstream of the putative initiating ATG assigned negative numbers. The amino acid sequence is numbered on the right. The predicted transmembrane domain isunderlined and the potential N-glycosylation sites are marked with triple dots below the Asn residues. Sialylmotifs L and S are double-underlined, and the characteristic His residue in the sialylmotif L is boxed.A, the sequence of clone 4C7. B, hydropathy plot of the 4C7 product analyzed by the method of Hopp and Woods (30Hopp T.P. Woods K.R. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 3824-3828Crossref PubMed Scopus (2921) Google Scholar). Thesolid bars below the plot indicate the locations of sialylmotifs L and S. C, comparison of amino acid sequence of sialylmotif L in human sialyltransferases so far cloned.SAT-I, clone 4C7 product; ST3N-1, Galβ1–3(4)GlcNAc α2,3-ST (15Kitagawa H. Paulson J.C. Biochem. Biophys. Res. Commun. 1993; 194: 375-382Crossref PubMed Scopus (124) Google Scholar); ST3N-2, Galβ(1–3/1–4)GlcNAc α2,3-ST (14Sasaki K. Watanabe E. Kawashima K. Sekine S. Dohi T. Oshima M. Hanai N. Nishi T. Hasegawa M. J. Biol. Chem. 1993; 268: 22782-22787Abstract Full Text PDF PubMed Google Scholar); ST3O-1, Galβ1–3GalNAc α2,3-ST-1 (16Kitagawa H. Paulson J.C. J. Biol. Chem. 1994; 269: 17872-17878Abstract Full Text PDF PubMed Google Scholar); ST3O-2, Galβ1–3 GalNAc α2,3-ST-2 (17Giordanengo V. Bannwarth S. Laffont C. Van Miegem V. HarduinLepers A. Delannoy P. Lefebvre J.-C. Eur. J. Biochem. 1997; 247: 558-566Crossref PubMed Scopus (44) Google Scholar); SThM, sthm gene (GenBankTM data base, accession number U14550);ST6N, Galβ1–4GlcNAc α2,6-ST (18Stamenkovic I. Asheim H.C. Deggerdal A. Blomhoff H.K. Smeland E.B. Funderud S. J. Exp. Med. 1990; 172: 641-643Crossref PubMed Scopus (49) Google Scholar); SAT-II, GD3 synthase (19Nara K. Watanabe Y. Maruyama K. Kasahara K. Nagai Y. Sanai Y. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7952-7956Crossref PubMed Scopus (123) Google Scholar, 20Haraguchi M. Yamashiro S. Yamamoto A. Furukawa K. Takamiya K. Lloyd K.O. Shiku H. Furukawa K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10455-10459Crossref PubMed Scopus (179) Google Scholar); STX, stxgene (12Scheidegger E.P. Sternberg L.R. Roth J. Lowe J.B. J. Biol. Chem. 1995; 270: 22685-22688Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar); ST8SiaIII, Siaα2–3Galβ1–4GlcNAc α2,8-ST (GenBankTM data base, accession number AF004668); PST-1, polysialyltransferase (13Nakayama J. Fukuda M.N. Fredette B. Ranscht B. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7031-7035Crossref PubMed Scopus (218) Google Scholar); ST8SiaV, GM1b/GD1a/GT1b/GD3α2,8-ST (11Kim Y.-J. Kim K.-S. Do S.-I. Kim C.-H. Kim S.-K. Lee Y.-C. Biochem. Biophys. Res. Commun. 1997; 235: 327-330Crossref PubMed Scopus (50) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To confirm that the 4C7 product catalyzes the synthesis of ganglioside GM3, we assayed the enzymatic activity in 3LL cells transiently transfected with pCEV4C7. As shown in Fig. 3, both 3LL-HK46 and 3LL-ST28 cells exhibited GM3 synthase activity only when the cells were transfected with pCEV4C7. An additional band with the same Rf value as ganglioside GT3 was observed when transfected 3LL-ST28 cells were used as the enzyme source (Fig. 3, lane 4), which suggests that GD3 synthase might catalyze the synthesis of ganglioside GT3. It is controversial whether or not GD3 synthase is identical with GT3 synthase (9Nara K. Watanabe Y. Kawashima I. Tai T. Nagai Y. Sanai Y. Eur. J. Biochem. 1996; 238: 647-652Crossref PubMed Scopus (25) Google Scholar, 10Nakayama J. Fukuda M.N. Hirabayashi Y. Kanamori A. Sasaki K. Nishi T. Fukuda M. J. Biol. Chem. 1996; 271: 3684-3691Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 11Kim Y.-J. Kim K.-S. Do S.-I. Kim C.-H. Kim S.-K. Lee Y.-C. Biochem. Biophys. Res. Commun. 1997; 235: 327-330Crossref PubMed Scopus (50) Google Scholar). In contrast to the previous reports on purified GM3 synthase from rat liver (27Melkerson-Watson L.J. Sweeley C.C. J. Biol. Chem. 1991; 266: 4448-4457Abstract Full Text PDF PubMed Google Scholar) and rat brain (31Preuss U. Gu X. Gu T. Yu R.K. J. Biol. Chem. 1993; 268: 26273-26278Abstract Full Text PDF PubMed Google Scholar), the substrate specificity of the 4C7 product was found to be highly restricted to lactosylceramide as the acceptor. 2A. Ishii, K. Makino, C. Nakamura, and M. Saito, manuscript in preparation. It has not yet been determined whether the 4C7 product is active toward free lactose or N- and O-linked oligosaccharides of glycoproteins as substrates. However, this strict acceptor specificity for lactosylceramide, and not for the nonreducing terminal galactose moieties of other glycosphingolipids tested, together with previous reports on the substrate specificity of purified GM3synthase, suggest the existence of isozymes having broader substrate specificity than the 4C7 gene product. The glycosphingolipid composition in clone 4C7-transfected 3LL-HK46 cells was analyzed by TLC, and it was clearly demonstrated that the de novosynthesis of GM3 molecules occurred in the transfected cells, along with an observable decrease in neutral lactosylceramide. 2A. Ishii, K. Makino, C. Nakamura, and M. Saito, manuscript in preparation. The hydropathy plot (Fig. 2 B) showed three or four strongly hydrophobic segments in and around sialylmotif S near the C terminus of the enzyme. These results suggest that the enzyme might be anchored to the lumenal side of the Golgi membrane. The expression of GM3 synthase in human tissues was assessed by Northern blot analyses. A major transcript 2.4 kilobases in size was detected at various level in all human tissues tested; it was highly expressed in brain, placenta, skeletal muscle, and testis, whereas it was very weakly expressed in liver, kidney, pancreas, and colon. In some tissues, a minor band of 7 kilobases was detected (Fig. 4 A). To characterize in more detail the gene expression of GM3synthase in brain, Northern blotting analysis of various parts of the human brain was performed with the same probe. As shown in Fig. 4 B, GM3 synthase mRNA was widely distributed in human brain, but slightly elevated expression was observed in the cerebral cortex, temporal lobe, and putamen. This study describes the molecular cloning of a new human sialyltransferase involved in the biosynthesis of ganglioside GM3. In our expression cloning, it was found that newly synthesized ganglioside GM3 was barely detectable with anti-GM3 antibody M2590 on the surface of transfected 3LL-HK46 cells in FACS analysis (Fig. 1). This problem was overcome by co-transfecting or introducing in advance the GD3 synthase cDNA; the final product ganglioside GD3 was easily detected using its specific antibody R24 (Fig. 1). Such modifications of expression cloning are generally useful, and use of an analogous modification has also been reported (20Haraguchi M. Yamashiro S. Yamamoto A. Furukawa K. Takamiya K. Lloyd K.O. Shiku H. Furukawa K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10455-10459Crossref PubMed Scopus (179) Google Scholar). The human GM3 synthase cDNA cloned in the present study and its product showed some characteristics strikingly different from those of the other sialyltransferases so far cloned. An invariant aspartic acid residue was found in the C-terminal region of the sialylmotif L of all the other sialyltransferases (Fig. 2 C). This acidic amino acid residue was substituted by the basic amino acid (His) in GM3 synthetic sialyltransferase. More interestingly, this amino acid replacement is conserved in the GM3 synthases of mouse, rat, and green monkey origins. 3K. Matsuda, A. Ishii, and M. Saito, manuscript in preparation. Thus, this replacement must be critically relevant to the GM3 synthetic activity. It is, however, still unclear how this substitution effects the interaction between the enzyme and its substrate(s) and whether or not the substitution is limited to GM3 synthases of mammalian origin. Among all the glycosphingolipids tested, only lactosylceramide could serve as an acceptor for the sialylation catalyzed by the cloned human GM3 synthase. In contrast, it has been reported that the purified GM3 synthase from rat liver exhibited a broader substrate specificity (27Melkerson-Watson L.J. Sweeley C.C. J. Biol. Chem. 1991; 266: 4448-4457Abstract Full Text PDF PubMed Google Scholar) and that the enzyme from rat brain could utilize both galactosylceramide and asialoganglioside GM2(GA2) as acceptors as well as lactosylceramide (31Preuss U. Gu X. Gu T. Yu R.K. J. Biol. Chem. 1993; 268: 26273-26278Abstract Full Text PDF PubMed Google Scholar). Although we cannot rule out differences in the purity of the enzyme preparations, these discrepancies may imply the existence of isozymes with different substrate specificities for glycosphingolipids. Northern analysis indicated that the human GM3 synthase gene was expressed in a tissue-specific manner (Fig. 4 A). The high expression of GM3 synthase messenger RNA in brain (Fig. 4 B) may be responsible for the abundance in brain of GM3 itself and of other gangliosides with more complex oligosaccharide chains, which might be related to axonal (32Schwarz A. Rapaport E. Hirschberg K. Futerman A.H. J. Biol. Chem. 1995; 270: 10990-10998Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 33Harel R. Futerman A.H. J. Biol. Chem. 1993; 268: 14476-14481Abstract Full Text PDF PubMed Google Scholar) and dendritic outgrowth (34Furuya S. Ono K. Hirabayashi Y. J. Neurochem. 1995; 65: 1551-1561Crossref PubMed Scopus (67) Google Scholar). The expression of GM3 synthase was enhanced by the treatment of HL-60 and U937 cells with the differentiation inducer TPA (4Nojiri H. Takaku F. Terui Y. Miura Y. Saito M. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 782-786Crossref PubMed Scopus (218) Google Scholar). Although transcriptional regulation of GM3 synthase has not yet been demonstrated, elucidation of the mechanisms controlling expression of the GM3 synthase gene should yield insight into the mechanism(s) by which leukemic cells can be induced to differentiate into monocytoids. We thank Prof. Masaru Taniguchi (Chiba University) and Prof. Kazuo Maruyama (Tokyo Medical and Dental University) for providing the anti-GM3 mAb (M2590) and the plasmid pCEV18, respectively.
<div>Abstract<p>Abnormal activation of hypoxia-inducible factor-1 (HIF-1), one of the most important transcription factors for the adaptation of cells to hypoxia, is frequently observed in numerous types of solid tumors. Dysregulation of HIF-1 induces tumor angiogenesis and enhances the expression of anti-apoptotic proteins and glycolysis-associated enzymes in cancer cells, which in turn leads to the promotion of tumor growth. In the present study, we examined the pathophysiologic role of HIF-1 in multiple myeloma. Furthermore, we explored the possibility that HIF-1 may be a molecular target for myeloma therapy. We identified constitutive expression of the hypoxia-inducible factor-1 α (HIF-1α)-subunit in established myeloma cell lines and in primary myeloma cells. Treatment with insulin-like growth factor-1 (IGF-1) significantly increased HIF-1α expression through activation of the AKT and mitogen-activated protein kinase signaling pathways. Inhibition of HIF-1 function either by echinomycin, a specific HIF-1 inhibitor, or a siRNA against HIF-1α resulted in enhanced sensitivity to melphalan in myeloma cells. This inhibition of HIF-1 also reversed the protective effect of IGF-1 on melphalan-induced apoptosis. Inhibition of HIF-1 drastically reduced both basal and IGF-1–induced expression of survivin, one of the most important anti-apoptotic proteins in myeloma cells. We conclude that HIF-1 inhibition may be an attractive therapeutic strategy for multiple myeloma. [Mol Cancer Ther 2009;8(8):2329–38]</p></div>
