Identification of insulin-like growth factor 2 mRNA-binding protein 3 as a radioresistance factor in squamous esophageal cancer cells.

Identification of reliable markers of radiosensitivity and the key molecules that donate susceptibility to anticancer treatments to esophageal cancer cells would be highly desirable. We found that the mRNA expression of insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) was higher in radioresistant TE-5 and TE-9 cells than in radiosensitive TE-12 cloneA1 cells. Conversely, knocking down expression of IGF2BP3 mRNA in TE-5 and TE-9 cells using small interfering RNA significantly enhanced their radiosensitivity. Furthermore, patients with squamous cell esophageal cancers strongly expressing IGF2BP3 tended to respond poorly to chemoradiation. These data suggest that IGF2BP3 may be a key marker of radiosensitivity that diminishes the susceptibility of squamous cell esophageal cancer cells to radiotherapy. IGF2BP3 may, thus, be a useful target for improving radiotherapy for patients with esophageal squamous cell carcinoma.

Remodeling of the major pig xenoantigen by N-acetylglucosaminyltransferase III in transgenic pig.

We have been successful in generating several lines of transgenic mice and pigs that contain the human beta-d-mannoside beta-1,4-N-acetylglucosaminyltransferase III (GnT-III) gene. The overexpression of the GnT-III gene in mice and pigs reduced their antigenicity to human natural antibodies, especially the Galalpha1-3Galbeta1-4GlcNAc-R, as evidenced by immunohistochemical analysis. Endothelial cell studies from the GnT-III transgenic pigs also revealed a significant down-regulation in antigenicity, including Hanganutziu-Deicher antigen, and dramatic reductions in both the complement- and natural killer cell-mediated pig cell lyses. Changes in the enzymatic activities of other glycosyltransferases, such as alpha1,3-galactosyltransferase, GnT-IV, and GnT-V, did not support cross-talk between GnT-III and these enzymes in the transgenic animals. In addition, we demonstrated the effect of GnT-III in down-regulating the xenoantigen of pig heart grafts, using a pig to cynomolgus monkey transplantation model, suggesting that this approach may be useful in clinical xenotransplantation in the future.

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.

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.

Kinetic basis for the donor nucleotide-sugar specificity of beta1, 4-N-acetylglucosaminyltransferase III.

The kinetic basis of the donor substrate specificity of beta1, 4-N-acetylglucosaminyltransferase III (GnT-III) was investigated using a purified recombinant enzyme. The enzyme also transfers GalNAc and Glc moieties from their respective UDP-sugars to an acceptor at rates of 0.1-0.2% of that for GlcNAc, but Gal is not transferred at a detectable rate. Kinetic analyses revealed that these inefficient transfers, which are associated with the specificity of the enzyme, are due to the much lower V(max) values, whereas the K(m) values for UDP-GalNAc and UDP-Glc differ only slightly from that for UDP-GlcNAc. It was also found that various other nucleotide-Glc derivatives bind to the enzyme with comparable affinities to those of UDP-GlcNAc and UDP-Glc, although the derivatives do not serve as glycosyl donors. Thus, GnT-III does not appear to distinguish UDP-GlcNAc from other structurally similar nucleotide-sugars by specific binding in the ground state. These findings suggest that the specificity of GnT-III toward the nucleotide-sugar is determined during the catalytic process. This type of specificity may be efficient in preventing a possible mistransfer when other nucleotide-sugars are present in excess over the true donor.

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.

Masking and reduction of the Galactose-alpha1,3-Galactose (alpha-Gal) epitope, the major xenoantigen in swine, by the glycosyltransferase gene transfection.

The alpha-Gal epitope (Gal-alpha1-3Gal-beta1-4-GlcNAc-R), which is biosynthesized by the UDP-Gal:alpha1-3-galactosyltransferase (alpha1, 3GT), is highly associated with hyperacute rejection in swine to human xenotransplantation. A variety of strategies have been pursued to reduce or eliminate this epitope from swine tissues. Since swine ES cells are not available at present, the targeted knock out of the alpha1,3GT is restricted. Other strategies, such as enzyme competition of the alpha1,3GT with other glycosyltransferases and/or control of sugar processing by the glycosyltransferases, provide a new insight into the downregulation of the alpha-Gal epitope. This review will focus on this type of strategy, which involves a gene transfection of variety of glycosyltransferases as competitors against alpha1,3GT.

Reduction of the major swine xenoantigen, the alpha-galactosyl epitope by transfection of the alpha2,3-sialyltransferase gene.

alpha2,3-Sialyltransferase represents a putative enzyme that reduces the Galalpha1-3Gal beta1-4GlcNAc-R (the alpha-galactosyl epitope) by intracellular competition with alpha1,3-galactosyltransferase for a common acceptor substrate. This study demonstrates that the overexpression of the alpha2,3-sialyltransferase gene suppresses the antigenicity of swine endothelial cells to human natural antibodies by 77% relative to control cells and by 30% relative to cells transfected with alpha1,2-fucosyltransferase, and in addition, it reduces the complement-mediated cell lysis by 75% compared with control cells and by 22% compared with cells transfected with alpha1, 2-fucosyltransferase. The mechanism by which the alpha-galactosyl epitope was reduced was also studied. Suppression of alpha1, 3-galactosyltransferase activity by 30-63% was observed in the transfectants with alpha2,3-sialyltransferase, and mRNA expression of the alpha1,3-galactosyltransferase gene was reduced as well. The data suggest that the alpha2,3-sialyltransferase effectively reduced the alpha-galactosyl epitope as well as or better than the alpha1, 2-fucosyltransferase did and that the reduction of the alpha-galactosyl epitope is due not only to substrate competition but also to an overall reduction of endogenous alpha1, 3-galactosyltransferase enzyme activity.

Structure of the main saccharide chain in the acrosome reaction-inducing substance of the starfish, Asterias amurensis.

The structure of the main saccharide chain of the acrosome reaction-inducing substance in the egg jelly coat of the starfish, Asterias amurensis, is composed of the following pentasaccharide repeating units (Structure I). A polymer consisting of 10-11 repeating units has been observed to induce the acrosome reaction in starfish sperm at high calcium concentrations. [STRUCTURE I:see text] The identities and linkage positions of constituent sugars were established using sugar, methylation, and sulfate analyses together with one- and two-dimensional nmr spectroscopy. The structure was supported by the data obtained for desulfation products and the Smith degradation of the polysaccharide.

Age-related changes in ganglioside composition in human lens.

We previously reported that human lens accumulates gangliosides in association with aging and senile cataract progression. Structural analysis revealed that gangliosides in human cataractous lenses were composed of ganglio-series gangliosides, such as GM3, GM2, GM1 and GD1a, and sialyl-Lewisx-containing neolacto-series gangliosides. Although Lewisx-containing neolacto-series glycolipid was found to accumulate in association with aging and cataract progression, the sialyl-Lewisx gangliosides did not show much accumulation in individual lenses from subjects between 16 and 80 years old. The content of sialyl-Lewisx gangliosides was about two to four times higher than that of Lewisx glycolipids, suggesting the possibility that the increase in Le(x) glycolipid is partly due to the desialylation of sialyl-Le(x) gangliosides. On the other hand, the expression of ganglio-series gangliosides increased in an age-related manner. Thus, age-related changes in lens glycolipids may modify the cell-to-cell interaction induced by cell surface sugar chains, leading to the initiation and progression of cataract.

Identification of new mutagenic heterocyclic amines and quantification of known heterocyclic amines.

2-amino-1-methyl-6-(4-hydroxyphenyl)imidazo[4,5-b]pyridine (4'-OH-PhIP) was mutagenic, inducing 180 revertants of Salmonella typhimurium TA98 per 100 micrograms with S9 mix and was formed by heating a mixture of creatine, tyrosine and glucose. It was detected in broiled beef at a level of 21.0 ng per g of broiled beef, which is comparable to the level of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Two new mutagens were isolated from bacteriological-grade beef extract using a new Salmonella tester strain, YG1024, which has a much higher O-acetyltransferase level than TA98. These mutagens were identified as 2-amino-4-hydroxymethyl-3,8-dimethylimidazo[4,5-g]quinoxaline (4-CH2OH-8-MeIQx) and 2-amino-1,7,9-trimethylimidazo[4,5-g]quinoxaline(7,9-DiMeIgQx++ +). The amounts of these mutagenic heterocyclic amines (HCAs) in beef extract were 6.0 ng and 53 ng per g of beef extract, respectively. 4-CH2OH-8-MeIQx induced 326,000 revertants of YG1024 and 99,000 revertants of TA98 per microgram with S9 mix, while 7,9-DiMeIgQx induced 13,800 and 670 revertants of YG1024 and TA98, respectively, per microgram in the presence of S9 mix. The levels of nine previously reported HCAs in cooked meats and fish and in beef extract were determined quantitatively. The level of PhIP was highest (0.56 approximately 69.2 ng/g), followed by that of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) (0.64 approximately 6.44 ng/g), and those of other HCAs were 0.03 approximately 2.50 ng/g. Mainstream smoke condensates of five Japanese brands of cigarettes contained four HCAs, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-9H-pyrido[2,3-b]indole (A alpha C) and 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA alpha C), at levels of 0.02 approximately 13.5 ng per cigarette and sidestream smoke condensates of two brands of cigarettes contained these HCAs at levels of 0.14 approximately 2.72 ng per cigarette. PhIP was not detected in any sample of mainstream or sidestream smoke condensate.

Structural determination of a new mutagenic heterocyclic amine, 2-amino-1,7,9-trimethylimidazo[4,5-g]quinoxaline (7,9-DiMeIgQx), present in beef extract.

We previously found two new mutagens, compounds I and II, in bacteriological-grade beef extract by monitoring the mutagenicity to a new Salmonella strain, YG1024; compound I was identified as 2-amino-4-hydroxymethyl-3,8-dimethylimidazo[4,5-f]quinoxaline (4-CH2OH-8-MeIQx). In the present study, we isolated compound II from the beef extract, which accounted for 2% of the total mutagenicity of materials adsorbed on blue cotton. Further, we found that a large quantity of compound II was produced by heating a mixture of creatine, threonine and glucose (1:1:0.5) at 200 degrees C for 5 h, the level being 860-fold of that in the beef extract. The structure of this compound was determined to be 2-amino-1,7,9-trimethylimidazo[4,5-g]quinoxaline (7,9-DiMeIgQx) by X-ray crystallography. The amount of 7,9-DiMeIgQx in bacteriological-grade beef extract was estimated to be 53 ng/g. This compound induced 13 800 and 670 revertants of S. typhimurium YG1024 and TA98 respectively, per micrograms in the presence of S9 mix.

Isolation and identification of a new mutagen, 2-amino-4-hydroxy-methyl-3,8-dimethylimidazo[4,5-f]quinoxaline (4-CH2OH-8-MeIQx), from beef extract.

By monitoring the mutagenicity to a new Salmonella tester strain, YG1024, which has a much higher level of O-acetyltransferase activity than S.typhimurium TA98, we found two new mutagenic compounds in bacteriological-grade beef extract. One of them (compound I), which had a similar UV spectrum to that of 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), was isolated and shown to account for approximately 2% of the total mutagenicity of the materials adsorbed to blue cotton, and its concentration was estimated to be 6.0 ng/g beef extract. This amount of compound in beef extract was insufficient to allow measurements of various spectra, but its level was increased approximately 9-fold by heating beef extract with creatine and threonine at 200 degrees C for 5 h. From UV and mass spectra of the compound obtained from beef extract heated with creatine plus threonine, it was deduced to be a hydroxymethyl derivative of aminodimethylimidazo-quinoxaline. Compound I was isolated from the urine of rats given 4,8-DiMeIQx and identified as 2-amino-4-hydroxymethyl-3,8-dimethylimidazo[4,5-f]quinoxaline (4-CH2OH-8-MeIQx) by 1H-NMR analysis. 4-CH2OH-8-MeIQx induced 326,000 revertants of YG1024 and 99,000 revertants of TA98 per micrograms in the presence of S9 mix.