Dr. Saito photo
The forefront of carbohydrate science

Cloning of a novel sialyltransferase gene

---An interview with Professor Masaki Saito

Four years of research activity on "Sugar chain genes and their biological functions," funded by a Grant in Aid for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture, performed since 1993 and involving most Japanese glycoscientists in carbohydrate science, achieved remarkable results. In particular, the cloning of various glycosyltransferase genes, related to the biosynthesis of glycoconjugates and advances in research on functions of glycoconjugates functions utilizing the expression system of sugar transferases, has given rise to great expectations for future developments. Against such a background, the Gordon Research Conference for "Structure and Biological Function of Glycolipids and Sphingolipids" (Chairman: Dr. Yoshitaka Nagai) was held for the first time in Japan (Gifu), gathering together over 150 distinguished scientists from all of the world. In addition, an international symposium entitled "Glycosyltransferases and cellular communications" was held in March 1997 in Osaka, and was indeed an epoch making event in the investigation of glycogenes. The elucidation of glycogenes is related to clarifying the functions or control mechanisms of complex carbohydrates, and thus will play an important role in advancing our knowledge of glycobiology.

We interviewed Professor Masaki Saito of Hokkaido University, who recently succeeded in cloning a sialyltransferase among various sugar transferases, regarding the significance of cloning this enzyme and the background of his research.
Q 
Professor Saito, we heard that you succeeded in cloning a novel sialyltransferase in your laboratory. What kind of properties does this enzyme have? Could you tell us about the process leading to the successful cloning of this enzyme?
This enzyme transfers sialic acid to the galactose molecule in lactosylceramide to synthesize of a ganglioside called GM3, which is a microconstituent of cell surface membranes. Almost all major sialyltransferases belonging to ganglioside biosynthesis have been cloned. However, the cloning of the GM3 synthetase gene was difficult since this enzyme functions first in ganglioside synthesis. We have been involved in research on the physiological functions of glycosphingolipids, especially GM3, for a long time, and have been pursuing the sialyltransferase gene, which is related to the biosynthesis of sugar chains. We have continued this research for about six and a half years together with a research group at Jichi Medical School, where I previously worked. However, we had technical difficulties in studying this enzyme. During the course of our research, we were able win the cooperation of each researcher who had investigated essential issues related to our study such as anti-GM3, or anti-GD3 antibodies, GM3 deficient cell lines and the GD3 synthesizing gene. Moreover, we devised a method of expression cloning. These matters seem to have worked out successfully in our research. Namely, the expression cloning of GM3 synthese gene is difficult to detect using an anti-GM3 antibody. Therefore, we used another antibody that detected GD3, which is a sugar chain consisting of two sialic acid molecules synthesized by introducing the second sialyltransferase gene. Using this method, we were able to select cells bearing the sialyltransferase genes among cells in which the c-DNA library of HL-60 cells was induced, and concentrated on such cells. Finally, we were able to locate genes suitable for our purpose after sib selection using E. coli.
QAre there any differences in this enzyme in comparison to previously identified sialyltransferases?
It was found that this enzyme has two sialyl motifs, called L and S, and type II membrane enzyme having a transmembrane domain clearly at the N-terminal end observed in previously identified enzymes, and its C-termini were anchored to Goldi membranes. The greatest difference was the structural substitution of histidine with asparagine in sialyl motif L. This enzyme can only recognize the whole structure of lactosylceramide with high specificity.
QWhat kind of background did you have when you began the study of GM3? Please talk a bit more about the motives for starting this research.
Previously, I found an enzyme bound to mitochondrial membranes during the course of purification. This finding caused me to become interested in biomembranes. In those days, Dr. Yoshitaka Nagai, who was an associate professor at the Institute of Medical Science of the University of Tokyo, taught me the importance of glycosphingolipids, and the bases of the experiments. Thereafter, I made advances on his findings in the field of glycolipids in cerebral tissues, hemocytes and tumor cells. At that time, Molt 4 of human T cell line or HL-60 cells of promyelocytic leukemia cell line had been developed, and circumstances that allowed research on glycolipids using leukemia cell membranes were gradually improving. When HL-60 cells were differentiated into the monocyte series by a chemical substance, the GM3 contents of the cells increased. In those days, we obtained a large amount of canine red blood cells, then purified approximately 1 g of GM3 from those RBC. Using those purified GM3, we studied what would happen when GM3 was added to the hemocyte culture system. When we added GM3 to the pure culture system of HL-60 cells, we found morphological changes in HL-60 cells. We continued our experiments because we initially suspected that the admixture of some impurities might have caused those morphological changes. However, it was found that other gangliosides did not cause such changes, and chemically synthesized GM3 could induce similar changes. Therefore, we concluded that the morphological changes observed in HL-60 cells were due to the effects of GM3 itself. Thereafter, differentiation of HL-60 cells into neutrophils was reported in a culture system after gangliosides of the neolacto series were added, demonstrating that these glycolipids serve not only as lymphocytic cell surface markers, but also as glycoconjugates which can induce cell differentiation.

GM3 is a ganglioside that is distributed ubiquitously in all animal cells. However, once the bioactivities of GM3 had been clarified, the basic enzyme synthesizing this molecule became important. Namely, we thought that switching GM3 synthtase gene on and off was important.
QCould you give us your opinions regarding the significance of the research on such sugar chain genes and comment on the future prospects of this research ?
Due to the progress in molecular biology, vital phenomena including genesis, differentiation, growth, proliferation, maintenance of homeostasis, aging and malignancy development, can be elucidated at the gene level. Sugar chains contained in glycoconjugates play an important role in these vital phenomena.

However, the elucidation of these sugar chains was delayed due to their markedly varied molecular structures or expression mechanisms, which are the secondary products of genetic information and controlled by unknown factors. To elucidate the biological functions of the sugar chains, systemic decoding of genetic information contained in the sugar chain genes and molecular biological analyses based on the above systemic decoding are necessary. Recently, dozens of sugar transferases have been cloned, and more than half of them were cloned by Japanese researchers. This will facilitate research elucidating the roles of glycoconjugates by controlling the expression of specific genes in cells, i.e. it will become possible to perform experiments elucidating the role of sugar chains during the genesis of animals using transgenic or knockout mice. The role of sugar chains in glycoconjugates is expected to develop significantly in medicine and pharmacology.

Previous results of gene research have been further developed by the new Grant in Aid for Scientific Research on Priority Areas entitled "Sugar chain remodeling", which is promoting the development of new subjects. I hope that younger investigators who will be responsible for next generation of research will become involved in the study of carbohydrates and pursue unique research of their own in the future.



Brief personal history of Professor Masaki Saito
Professor Saito graduated from the Faculty of Medicine of the University of Tokyo in 1967, and completed the doctoral course of medicine at the University of Tokyo in 1972. He was then appointed an assistant at the Institute of Medical Science of the University of Tokyo in 1972 and studied at Harvard University between 1972 and 1974. Prof. Saito became a chief researcher at Tokyo Metropolitan Institute of Gerontology in 1974. He was later appointed section chief at the Tokyo Metropolitan Institute of Gerontology in 1977. In 1980, he became an instructor of hematology at Jichi Medical School and in 1983, he was appointed as a professor of hematology at Jichi Medical School. Since 1995, Prof. Saito has served as professor at the School of Medicine of Hokkaido University. He received a Grant in Aid for Scientific Research on Priority Areas entitled "Sugar chain genes" from the Ministry of Education, Science and Culture between 1993 and 1996.
References
1) Bioactive Gangliosides: Differentiation inducers for hematopoietic cells and their mechanism(s) of actions. Saito Masaki. Advances in Lipid Research 25, 303-327
2) Interleukin-3-associated expression of gangliosides in mouse myelogenous leukemia NFS60 cells introduced with interleukin-3 gene: expression of ganglioside GD1a and key involvement of CMP-NeuAc: lactosylceramide alpha 2-->3-sialyltransferase in GD1a expression. Tsunoda A., Nakamura M, Kirito K, Hara K, Saito M. Biochemistry 34, 9356-9367, 1995

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