Adult-onset depletion of sulfatide leads to axonal degeneration with relative myelin sparing.

3-O-sulfogalactosylceramide (sulfatide) constitutes a class of sphingolipids that comprise about 4% of myelin lipids in the central nervous system. Previously, our group characterized a mouse with sulfatide's synthesizing enzyme, cerebroside sulfotransferase (CST), constitutively disrupted. Using these mice, we demonstrated that sulfatide is required for establishment and maintenance of myelin, axoglial junctions, and axonal domains and that sulfatide depletion results in structural pathologies commonly observed in Multiple Sclerosis (MS). Interestingly, sulfatide is reduced in regions of normal appearing white matter (NAWM) of MS patients. Sulfatide reduction in NAWM suggests depletion occurs early in disease development and consistent with functioning as a driving force of disease progression. To closely model MS, an adult-onset disease, our lab generated a "floxed" CST mouse and mated it against the PLP-creERT mouse, resulting in a double transgenic mouse that provides temporal and cell-type specific ablation of the Cst gene (Gal3st1). Using this mouse, we demonstrate adult-onset sulfatide depletion has limited effects on myelin structure but results in the loss of axonal integrity including deterioration of domain organization accompanied by axonal degeneration. Moreover, structurally preserved myelinated axons progressively lose the ability to function as myelinated axons, indicated by the loss of the N1 peak. Together, our findings indicate that sulfatide depletion, which occurs in the early stages of MS progression, is sufficient to drive the loss of axonal function independent of demyelination and that axonal pathology, which is responsible for the irreversible loss of neuronal function that is prevalent in MS, may occur earlier than previously recognized.

Overexpression of GalNAc-transferase GalNAc-T3 promotes pancreatic cancer cell growth.

O-linked glycans of secreted and membrane-bound proteins have an important role in the pathogenesis of pancreatic cancer by modulating immune responses, inflammation and tumorigenesis. A critical aspect of O-glycosylation, the position at which proteins are glycosylated with N-acetyl-galactosamine on serine and threonine residues, is regulated by the substrate specificity of UDP-GalNAc:polypeptide N-acetylgalactosaminyl-transferases (GalNAc-Ts). Thus, GalNAc-Ts regulate the first committed step in O-glycosylated protein biosynthesis, determine sites of O-glycosylation on proteins and are important for understanding normal and carcinoma-associated O-glycosylation. We have found that one of these enzymes, GalNAc-T3, is overexpressed in human pancreatic cancer tissues and suppression of GalNAc-T3 significantly attenuates the growth of pancreatic cancer cells in vitro and in vivo. In addition, suppression of GalNAc-T3 induces apoptosis of pancreatic cancer cells. Our results indicate that GalNAc-T3 is likely involved in pancreatic carcinogenesis. Modification of cellular glycosylation occurs in nearly all types of cancer as a result of alterations in the expression levels of glycosyltransferases. We report guanine the nucleotide-binding protein, α-transducing activity polypeptide-1 (GNAT1) as a possible substrate protein of GalNAc-T3. GalNAc-T3 is associated with O-glycosylation of GNAT1 and affects the subcellular distribution of GNAT1. Knocking down endogenous GNAT1 significantly suppresses the growth/survival of PDAC cells. Our results imply that GalNAc-T3 contributes to the function of O-glycosylated proteins and thereby affects the growth and survival of pancreatic cancer cells. Thus, substrate proteins of GalNAc-T3 should serve as important therapeutic targets for pancreatic cancers.

Sulfatide is essential for the maintenance of CNS myelin and axon structure.

Galactocerebroside (GalC) and sulfatide are abundant myelin lipids. In mice incapable of synthesizing these lipids, myelin is thin and regionally unstable and exhibits several subtle structural abnormalities. Although galactolipid-null mice have been beneficial in the analysis of galactolipid function, it has not been possible to differentiate between the functions of GalC and sulfatide with these mice alone. In the present work, we have analyzed a murine model that forms normal levels of GalC but is incapable of synthesizing sulfatide. By comparing a plethora of morphological features between the galactolipid-null and the sulfatide-null mice, we have begun to differentiate between the specific functions of these closely related lipids. The most striking difference between these two mutants is the reduction of myelin developmental abnormalities (e.g., redundant and uncompacted myelin sheaths) in young adult sulfatide-null mice as compared with the galactolipid-null animals. Although sulfatide appears to play a limited role in myelin development, this lipid is essential for myelin maintenance, as the prevalence of redundant, uncompacted, and degenerating myelin sheaths as well as deteriorating nodal/paranodal structure is increased significantly in aged sulfatide-null mice as compared with littermate wildtype mice. Finally, we show that the role played by sulfatide in CNS maintenance is not limited to the myelin sheath, as axonal caliber and circularity are normal in young adult mutant mice but are significantly altered in aged sulfatide-null animals.

The enzymatic basis for the conversion of nonfucosylated to fucosylated alpha-fetoprotein by acyclic retinoid treatment in human hepatoma cells: activation of alpha1-6 fucosyltransferase.

The purpose of the present study was to investigate the mechanism by which nonfucosylated alpha-fetoprotein (AFP) is converted to fucosylated AFP in human hepatoma cell lines exposed to acyclic retinoid (AR), an effective drug for the secondary prevention of hepatocellular carcinoma. AR treatment (100 microM) of HepG2 and Hep3B cells significantly increased the activity and mRNA levels of alpha1-6 fucosyltransferase (alpha1-6 FucT), the enzyme responsible for the fucosylation of AFP, leading to an increase in fucosylated glycoproteins as evidenced by lectin binding measurements. Lectin immunoelectrophoresis of AFP obtained from culture media indicated that the relative percentage of nonfucosylated AFP (L1 fraction) was decreased and alpha1-6 fucosylated AFP (L3 fraction) was increased in these hepatoma cell lines after treatment with AR. The total AFP levels were, however, markedly suppressed by AR treatment, and therefore the absolute L3 fraction on the basis of the total AFP present was extremely low. These results demonstrate that AR enhances the conversion of the L1 to the L3 fraction due to the activation of alpha1-6 FucT in human hepatoma cell lines despite clinical outcome with AR treatment and the L3 fraction of AFP. Even though the dramatic decrease in AFP is the limiting factor in the synthesis of the L3 fraction and, therefore, the absolute value of fucosylated AFP is extremely low, the conversion from L1 to L3 as judged by lectin immunoelectrophoresis represents a good marker for the progress of AR treatment.

Homozygosity and linkage disequilibrium mapping of autosomal recessive distal myopathy (Nonaka distal myopathy).

Autosomal recessive distal myopathy or Nonaka distal myopathy (NM) is characterized by its unique distribution of muscular weakness and wasting. The patients present with spared quadriceps muscles even in a late stage of the disease. The hamstring and tibialis anterior muscles are affected severely in early adulthood. We have localized the NM gene to the region between markers D9S319 and D9S276 on chromosome 9 by linkage analysis. To further refine the localization of the NM gene, we conducted homozygosity and linkage disequilibrium analysis for 14 patients from 11 NM families using 18 polymorphic markers. All of the patients from consanguineous NM families were found to be homozygous for six markers located within the region between markers D9S2178 and D9S1859. We also provided evidence for significant allelic associations between the NM region and five marker loci. Examination of the haplotype analysis identified a predominant ancestral haplotype comprising the associated alleles 199-160-154-109 (marker order: D9S2179-D9S2180-D9S2181-D9S1804), present in 60% of NM chromosomes and in 0% of parent chromosomes. On the basis of the data obtained in this study, the majority of NM chromosomes were derived from a single ancestral founder, and the NM gene is probably located within the 1.5-Mb region between markers D9S2178 and D9S1791.

A glycomic approach to the identification and characterization of glycoprotein function in cells transfected with glycosyltransferase genes.

The transfection of glycoprotein glycosyltransferase genes into cells leads to modification of both the structure and function of the glycoproteins and as a result, changes in glycome patterns. N-glycan branching enzymes hold some promise as a model system for the identification of glycome patterns. Both N-acetylglucosaminyltransferase III and alpha 1-6 fucosyltransferase are typical glycosyltransferases, which are involved in the branching of N-glycans. The resulting enzymatic products, bisecting N-GlcNAc and alpha 1-6 fucose residues, are no longer modified by other glycosyltransferases and it is a relatively simple task to identify their modification by means of lectins. In this review, the glycome patterns of glycosyltransferase gene transfectants and the non-transfectants were compared by two-dimensional gel electrophoresis and lectin staining, and the biological significance of the two genes are described. Analyses of glycome patterns by transfecting glycosyltransferase genes will lead to new fields of study in the area of postgenome research.

A remodeling system of the 3'-sulfo-Lewis a and 3'-sulfo-Lewis x epitopes.

It has been reported that the chemically synthesized 3'-sulfo-Le(a) and 3'-sulfo-Le(x) epitopes have a high potential as a ligand for selectins. To elucidate the physiological functions of 3'-sulfated Lewis epitopes, a remodeling system was developed using a combination of a betaGal-3-O-sulfotransferase GP3ST, hitherto known alpha1,3/1,4-fucosyltransferases (FucT-III, IV, V, VI, VII, and IX) and arylsulfatase A. The pyridylaminated (PA) lacto-N-tetraose (Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc) was first converted to 3'-sulfolacto-N-fucopentaose II (sulfo-3Galbeta1-3(Fucalpha1-4)GlcNAcbeta1-3Galbeta1-4Glc)-PA by sequential reactions with GP3ST and FucT-III. The 3'-sulfolacto-N-fucopentaose III (sulfo-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4Glc)-PA was then synthesized from lacto-N-neotetraose (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc)-PA by GP3ST and FucT-III, -IV, -V, -VI, -VII, or -IX in a similar manner. The substrate specificity for the 3'-sulfated acceptor of the alpha1,3-fucosyltransferases was considerably different from that for the non-substituted and 3'-sialylated varieties. When the GP3ST gene was introduced into A549 and Chinese hamster ovary cells expressing FucT-III, they began to express 3'-sulfo-Le(a) and 3'-sulfo-Le(x) epitopes, respectively, suggesting that GP3ST is responsible for their biosynthesis in vivo. The expression of the 3'-sialyl-Le(x) epitope on Chinese hamster ovary cells was attenuated by the introduction of GP3ST gene, indicating that GP3ST and alpha2,3-sialyltransferase compete for the common Galbeta1-4GlcNAc-R oligosaccharides. Last, arylsulfatase A, which is a lysosomal hydrolase that catalyzes the desulfation of 3-O-sulfogalactosyl residues in glycolipids, was found to hydrolyze the sulfate ester bond on the 3'-sulfo-Le(x) (type 2 chain) but not that on the 3'-sulfo-Le(a) (type 1 chain). The present remodeling system might be of potential use as a tool for the study of the physiological roles of 3'-sulfated Lewis epitopes, including interaction with selectins.

Down-regulation of the alpha-Gal epitope expression in N-glycans of swine endothelial cells by transfection with the N-acetylglucosaminyltransferase III gene. Modulation of the biosynthesis of terminal structures by a bisecting GlcNAc.

The down-regulation of the alpha-Gal epitope (Galalpha1,3Galbeta-R) in swine tissues would be highly desirable, in terms of preventing hyperacute rejection in pig-to-human xenotransplantation. In an earlier study, we reported that the introduction of the beta1,4-N-acetylglucosaminyltransferase (GnT) III gene into swine endothelial cells resulted in a substantial reduction in the expression of the alpha-Gal epitope. In this study, we report on the mechanism for this down-regulation of the alpha-Gal epitope by means of structural and kinetic analyses. The structural analyses revealed that the amount of N-linked oligosaccharides bearing the alpha-Gal epitopes in the GnT-III-transfected cells was less than 10% that in parental cells, due to the alteration of the terminal structures as well as a decrease in branch formation. In addition, it appeared that the addition of a bisecting GlcNAc, which is catalyzed by GnT-III, leads to a more efficient sialylation rather than alpha-galactosylation. In vitro kinetic analyses showed that the bisecting GlcNAc has an inhibitory effect on alpha-galactosylation, but does not significantly affect the sialylation. These results suggest that the bisecting GlcNAc in the core is capable of modifying the biosynthesis of the terminal structures via its differential effects on the capping glycosyltransferase reactions. The findings may contribute to the development of a novel strategy to eliminate carbohydrate xenoantigens.

Suppression of integrin expression and tumorigenicity by sulfation of lactosylceramide in 3LL Lewis lung carcinoma cells.

To investigate the cellular functions of sulfated glycosphingolipids, we introduced the cerebroside sulfotransferase (CST) gene into J5 cells, a subclone of 3LL Lewis lung carcinoma cells. The J5 cells lack acidic glycosphingolipids but accumulate their common biosynthetic precursor, lactosylceramide. We established the stable CST transfectants, J5/CST-1 and J5/CST-2 clones, highly expressing sulfated lactosylceramide (SM3). Both clones exhibited more spherical morphology in comparison to mock transfectant, and their adhesiveness to fibronectin and laminin was significantly lower. The loss of cell-substratum interactions in these SM3-expressing cells could be attributed to decreased expression of integrins (alpha(5), alpha(6), and beta(1)) on the cell surface and their whole cellular levels. However, the levels of H-2K(b) and H-2D(b) antigens remained unchanged. Reverse transcriptase-polymerase chain reaction and Northern blot analyses for these integrins exhibited significant decrease of beta(1) gene expression in J5/CST-1 and 2, but there was no change in the levels of alpha(5) and alpha(6) transcripts. Deglycosylation by endoglycosidase H treatment clearly demonstrated that the precursor form of beta(1) integrin, possessing high mannose oligosaccharide chains, was preferentially decreased in the CST transfectants. These results demonstrate that endogenous SM3 negatively regulates beta(1) integrin expression at the transcriptional level, and the decrease of alpha integrin proteins in the CST transfectants was due to the post-transcriptional modification. We suggest the putative importance of the intracellular pre-beta(1) integrin pool for normal integrin maturation and subsequent function. Although the rates of cell proliferation in vitro for mock and CST transfectants were similar, tumorigenicity of J5/CST-1 and -2 cells inoculated into syngeneic C57/BL6 mice was greatly decreased or even absent. This was probably due to global loss of the efficient cell-matrix interactions, which are essential for the development of malignant tumors in vivo. Thus, we showed the evidence that cellular SM3 negatively regulates the cell-substratum interaction, resulting in the loss of tumorigenicity.

Ectopic expression of alpha1,6 fucosyltransferase in mice causes steatosis in the liver and kidney accompanied by a modification of lysosomal acid lipase.

The alpha1,6 fucosyltransferase (alpha1,6 FucT) catalyzes the transfer of a fucose from GDP-fucose to the innermost GlcNAc residue of N-linked glycans via an alpha1,6 linkage. alpha1,6 FucT was overexpressed in transgenic mice under the control of a combined cytomegalovirus and chicken beta-actin promoter. Histologically numerous small vacuoles, in which lipid droplets had accumulated, were observed in hepatocytes and proximal renal tubular cells. Electron microscopic studies showed that the lipid droplets were membrane-bound and apparently localized within the lysosomes. Cholesterol esters and triglycerides were significantly increased in liver and kidney of the transgenic mice. Liver lysosomal acid lipase (LAL) activity was significantly lower in the transgenic mice compared to the wild mice, whereas LAL protein level, which was detected immunochemically, was increased, indicating that the specific activity of LAL was much lower in the transgenic mice. In all of the transgenic and nontransgenic mice examined, the activity of liver LAL was negatively correlated with the level of alpha1,6 FucT activity. As evidenced by lectin and immunoblot analysis, LAL was found to be more fucosylated in the transgenic mice, suggesting that the aberrant fucosylation of LAL causes an accumulation of inactive LAL in the lysosomes. Such an accumulation of inactive LAL could be a likely cause for a steatosis in the lysosomes of the liver and kidney in the case of the alpha1,6 FucT transgenic mice.

Domain-specific mutations of a transforming growth factor (TGF)-beta 1 latency-associated peptide cause Camurati-Engelmann disease because of the formation of a constitutively active form of TGF-beta 1.

Transforming growth factor (TGF)-beta1 is secreted as a latent form, which consists of its mature form and a latency-associated peptide (beta1-LAP) in either the presence or the absence of additional latent TGF-beta1-binding protein. We recently reported that three different missense mutations (R218H, R218C, and C225R) of beta1-LAP cause the Camurati-Engelmann disease (CED), an autosomal dominant disorder characterized by hyperosteosis and sclerosis of the diaphysis of the long bones. Pulse-chase experiments using fibroblasts from CED patients and expression experiments of the mutant genes in an insect cell system suggest that these mutations disrupt the association of beta1-LAP and TGF-beta1 and the subsequent release of the mature TGF-beta1. Furthermore, the cell growth of fibroblasts from a CED patient and mutant gene-transfected fibroblasts was suppressed via TGF-beta1. The growth suppression observed was attenuated by neutralizing antibody to TGF-beta1 or by treatment of dexamethasone. On the other hand, the proliferation of human osteoblastic MG-63 cells was accelerated by coculture with CED fibroblasts. These data suggest that the domain-specific mutations of beta1-LAP result in a more facile activation of TGF-beta1, thus causing CED.

Overexpression of N-acetylglucosaminyltransferase III enhances the epidermal growth factor-induced phosphorylation of ERK in HeLaS3 cells by up-regulation of the internalization rate of the receptors.

N-Acetylglucosaminyltransferase III (GnT-III) is a key enzyme that inhibits the extension of N-glycans by introducing a bisecting N-acetylglucosamine residue. In this study we investigated the effect of GnT-III on epidermal growth factor (EGF) signaling in HeLaS3 cells. Although the binding of EGF to the epidermal growth factor receptor (EGFR) was decreased in GnT-III transfectants to a level of about 60% of control cells, the EGF-induced activation of extracellular signal-regulated kinase (ERK) in GnT-III transfectants was enhanced to approximately 1.4-fold that of the control cells. A binding analysis revealed that only low affinity binding of EGF was decreased in the GnT-III transfectants, whereas high affinity binding, which is considered to be responsible for the downstream signaling, was not altered. EGF-induced autophosphorylation and dimerization of the EGFR in the GnT-III transfectants were the same levels as found in the controls. The internalization rate of EGFR was, however, enhanced in the GnT-III transfectants as judged by the uptake of (125)I-EGF and Oregon Green-labeled EGF. When the EGFR internalization was delayed by dansylcadaverine, the up-regulation of ERK phosphorylation in GnT-III transfectants was completely suppressed to the same level as control cells. These results suggest that GnT-III overexpression in HeLaS3 cells resulted in an enhancement of EGF-induced ERK phosphorylation at least in part by the up-regulation of the endocytosis of EGFR.

Molecular cloning and characterization of a human beta-Gal-3'-sulfotransferase that acts on both type 1 and type 2 (Gal beta 1-3/1-4GlcNAc-R) oligosaccharides.

A novel sulfotransferase gene (designated GP3ST) was identified on human chromosome 2q37.3 based on its similarity to the cerebroside 3'-sulfotransferase (CST) cDNA (Honke, K., Tsuda, M., Hirahara, Y., Ishii, A., Makita, A., and Wada, Y. (1997) J. Biol. Chem. 272, 4864-4868). A full-length cDNA was obtained by reverse transcription-polymerase chain reaction and 5'- and 3'-rapid amplification of cDNA ends analyses of human colon mRNA. The isolated cDNA clone predicts that the protein is a type II transmembrane protein composed of 398 amino acid residues. The amino acid sequence indicates 33% identity to the human CST sequence. A recombinant protein that is expressed in COS-1 cells showed no CST activity, but did show sulfotransferase activities toward oligosaccharides containing nonreducing beta-galactosides such as N-acetyllactosamine, lactose, lacto-N-tetraose (Lc4), lacto-N-neotetraose (nLc4), and Gal beta 1-3GalNAc alpha-benzyl (O-glycan core 1 oligosaccharide). To characterize the cloned sulfotransferase, a sulfotransferase assay method was developed that uses pyridylaminated (PA) Lc4 and nLc4 as enzyme substrates. The enzyme product using PA-Lc4 as an acceptor was identified as HSO(3)-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1- 4Glc-PA by two-dimensional (1)H NMR. Kinetics studies suggested that GP3ST is able to act on both type 1 (Gal beta 1-3GlcNAc-R) and type 2 (Gal beta 1-4GlcNAc-R) chains with a similar efficiency. In situ hybridization demonstrated that the GP3ST gene is expressed in epithelial cells lining the lower to middle layer of the crypts in colonic mucosa, hepatocytes surrounding the central vein of the liver, extravillous cytotrophoblasts in the basal plate and septum of the placenta, renal tubules of the kidney, and neuronal cells of the cerebral cortex. The results of this study indicate the existence of a novel beta-Gal-3'-sulfotransferase gene family.

A novel carbohydrate binding activity of annexin V toward a bisecting N-acetylglucosamine.

A bisecting GlcNAc-binding protein was purified from a Triton X-100 extract of a porcine spleen microsomal fraction using affinity chromatography, in conjunction with an agalacto bisected biantennary sugar chain-immobilized Sepharose. Since the erythroagglutinating phytohemagglutinin (E-PHA) lectin preferentially binds to sugar chains which contain the bisecting GlcNAc, during purification the binding activity of the protein was evaluated by monitoring the inhibition of lectin binding to the N-acetylglucosaminyltransferase III (GnT-III)-transfected K562 cells which express high levels of the bisecting GlcNAc. The molecular mass of the purified protein was found to be 33 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By sequencing analysis, the isolated protein was identified as annexin V. Flow cytometric analysis showed that fluorescein-labeled annexin V binds to the GnT-III-transfected cells but not to mock cells, and that the binding was not affected by the addition of phospholipids. Furthermore, surface plasmon resonance measurements indicated that annexin V binds to the agalacto bisected biantennary sugar chain with a K(d) of 200 microM while essentially no binding was observed in the case of the corresponding non-bisected sample. These results suggest that annexin V has a novel carbohydrate binding activity and may serve as an endogenous lectin for mediating possible signals of bisecting GlcNAc, which have been implicated in a variety of biological functions.

Molecular cloning and expression of cDNA encoding chicken UDP-N-acetyl-D-glucosamine (GlcNAc): GlcNAcbeta 1-6(GlcNAcbeta 1-2)- manalpha 1-R[GlcNAc to man]beta 1,4N-acetylglucosaminyltransferase VI.

A cDNA that encodes UDP-N-acetyl-d-glucosamine (GlcNAc):GlcNAcbeta1-6(GlcNAcbeta1-2)Manalpha1-R[GlcNA c to Man]beta1, 4N-acetylglucosaminyltransferase VI (GnT VI), which is responsible for the formation of pentaantennary asparagine-linked oligosaccharides (N-glycans), has been cloned from a hen oviduct cDNA library based on the partial amino acid sequences of the purified enzyme. The isolated cDNA clone contained an open reading frame encoding 464 amino acids, including all of the peptides that were sequenced. The deduced amino acid sequence predicts a type II transmembrane topology and contains two potential N-glycosylation sites. The primary structure was found to be significantly similar to human GnT IV-homologue, the gene for which was cloned from the deleted region in pancreatic cancer, and to human and bovine GnT IVs. Chicken GnT VI-transfected COS-1 cells showed a high GnT VI activity (26.8 pmol/h/mg protein), whereas nontransfected, mock-transfected, or human GnT IV-homologue-transfected COS-1 cells had no activity. Northern blot analysis using poly(A)(+) RNA from hen oviduct indicated that the size of GnT VI mRNA is 2.1 kilobases. Reverse transcription-polymerase chain reaction analysis showed that GnT VI mRNA was relatively highly expressed in oviduct, spleen, lung, and colon.

Reduction of the major xenoantigen on glycosphingolipids of swine endothelial cells by various glycosyltransferases.

The effect of the various glycosyltransferases on glycosphingolipids was examined, using transfected swine endothelial cell (SEC) lines. The reactivity of parental SEC to normal human serum (NHS) and Griffonia simplicifolia IB(4) (GSIB4) lectin, which binds to the Gal alpha1-3 Gal beta 1-4 GlcNAc-R (alpha-galactosyl epitope), was reduced by approximately 20% by the treatment with D-PDMP (D-threo-1-phenyl-2-decan- oylamino-3-morpholino-1-propanol), suggesting that glycosphingolipids contained by SEC have a considerable amount of the alpha-galactosyl epitope. The overexpression of two different types of glycosyltransferase, N-acetylglucosaminyl transferase III (GnT-III), as well as alpha2, 6-sialyltransferase (ST6Gal I), alpha2,3-sialyltransferase (ST3Gal III), and alpha1,2-fucosyltransferase (alpha1,2FT), suppresses the total antigenicity of SEC significantly. However, the reduction in reactivities toward NHS and GSIB4 lectin in the case of GnT-III transfectants was milder than those in other transfectants. Western blot analysis indicated that the glycoproteins in all transfectants had diminished reactivity to NHS and GSIB4 lectin to approximately the same extent. Therefore, the neutral glycosphingolipids of these transfectants were separated by thin layer chromatography, followed by immunostaining with NHS and GSIB4 lectin. The levels of the alpha-galactosyl epitope in glycosphingolipids were not decreased in the GnT-III transfectants but were in the ST6Gal I, ST3Gal III, and alpha1,2FT transfectants. These data indicate that ST6Gal I, ST3Gal III, and alpha1,2FT reduced the alpha-galactosyl epitope in both glycoproteins and glycosphingolipids, while GnT-III reduced them only in glycoproteins.

Cancer-associated alternative usage of multiple promoters of human GalCer sulfotransferase gene.

The galactosylceramide sulfotransferase (cerebroside sulfotransferase, CST) (EC 2.8.2.11) gene is highly expressed in human renal cancer cells. To elucidate the regulatory mechanism of its gene expression, we have determined the genomic organization of the human CST gene. The gene comprises at least four exons and spans about 20 kb. The coding region is located in exons 3 and 4. To determine the transcription initiation sites, 5'-rapid amplification of cDNA ends analysis was performed using mRNA obtained from four human renal cancer cell lines, SMKT-R1-R4, and normal human renal proximal tubular cells. We found four transcription initiation sites and alternative usage of six exons corresponding to the 5'-untranslated region in cancer cells. On the other hand, the only transcript beginning at exon 1a was observed in normal cells. Using reverse transcriptase-PCR analysis, we confirmed that all of the exons 1a-d, especially exons 1c and 1d, are used as a transcription initiation site in cancer cells, whereas only exons 1a and 1b, mostly 1a, are utilized in normal cells. Analyzing the protein production from the mRNA variants with different 5'-UTRs, we found that all the transcripts examined produced the identical proteins. These observations suggest that the aberrant usage of transcription initiation sites flanked with promoters/enhancers is involved in the cancer-associated expression of the CST gene. Furthermore, this gene was assigned to human chromosome 22q12 by means of fluorescence in situ hybridization.