From the Genome Research Era to the Glycome
In Japan, many studies on complex carbohydrates have long been accomplished by members of the research groups supported by the Ministry of Education, Science, Sports and Culture (MESSC), and excellent results have been produced. Currently, a project "Sugar remodeling and cellular communications", has been formed and tackling the subject. We interviewed Professor Naoyuki Taniguchi, a prominent researcher in this field, asked some questions about professor's research history, current matters of concern, and future study subjects in the glycoscience field.
How did you become involved in the study of sugar?
-glutamyl
transpeptidase (-GTP), an enzyme that degrades glutathione,
was the subject of my thesis. I then continued to study this subject under
the guidance of Professor Meister Cornell University Medical College
and Professor Makita of the Cancer Institute at Hokkaido University, and
focused on the examination of cancer-associated changes of -GTP
and its enzymatic mechanism. 
-GTP
happened to be a glycoprotein, didn't it?-GTP isolated from fetal and cancer
cells, we were, at that time, unable to analyze the sugar chain. Fortunately,
Professor Kobata (Kobe University), who was interested in carbohydrate component
of the -GTP, offered a cooperative study. During our
collaboration, we discovered a bisecting GlcNAc structure, which was, I
think, how our current study came about.After moving to Osaka University, we developed a method for measuring the activity of GnT-III, an N-acetylglucosamine transferase (GnT) that produces bisecting GlcNAc, and from there our work progressed to enzyme purification, cDNA cloning, and gene cloning.
-GTP?-GTP (which has complex
type sugar chains), exists in a large quantity only in that isolated from
hepatic cancer. With cancer, there is approximately a 100-fold increase
in GnT-III activity in the liver. This enzyme does not exist in the normal
liver, but exists in the greatest amount in the kidney. Therefore, purification
of this enzyme was initiated using rat kidneys. We spent about 3 years on
isolation and purification of this enzyme.We have also succeeded in the gene cloning of glycosyltransferase. After GnT-III, we succeeded in cloning GnT-V. Unfortunately, this was accomplished by another group just 1 month earlier. However, we independently have succeeded in gene cloning after isolating the same enzyme from human tissue.
After this, we succeeded in the purification of
-1,6-fucosyltransferase
(FucT), an enzyme that transfers a fucose to the linkage chitobiose of -fetoprotein
(AFP). AFP increases when hepatic cancer appears and is also detected in
the blood when acute hepatitis or liver cirrhosis occurs. Therefore, AFP
is, of course, useful as a tumor maker, but the accuracy of diagnosing cancer
may only be about 65-70%.On the other hand, AFP with
-1,6 fucose is fairly specific
to hepatic cancer. Since we thought it interesting to examine the mechanism
of -1,6 fucosylation, we conducted gene cloning after
the purification of 1,6 FucT from human gastric cancer cells and the porcine brain.In cancer, abnormalities often occur at the branching site of the sugar chain. For this reason, we are interested in enzymes involved in the branching of the sugar chain. That is to say, branching is involved in all events of our interest.
For all studies hitherto mentioned, we did not conduct so-called "homology cloning" using EST. Rather, our strategy was to conduct gene cloning after isolating protein and examining the properties of it.
Glyco-genes are expressed in a manner specific to organs, tissues, or cells. Therefore, their activities may completely differ depending on the site of their expression, such as in cancer, in the kidney, or in the brain.
Since the
-1,6 Gn sugar chain is involved in cancer
metastasis, GnT-V that synthesizes this sugar chain is considered to be
a bad guy. However, GnT-III is regarded as a good guy since it inhibits
the production of -1,6 Gn by forming -1,4
bisecting GlcNAc Here, it should be noted that in transgenic mice, in whom GnT-III
is overexpressed, new diseases may appear such as fatty liver resulting
from lipid accumulation. In this case, GnT-III is not a good guy. It is
not yet known what happens when GnT-III is expressed in other organs, although
there may be other findings that suggest that GnT-III inhibits the sensitivity
to spleen NK cells, and that the overexpression of -1,6 FucT leads to Wolman
disease, a disease characterized by fat accumulation.Therefore, I think there are considerable differences in the functions of glyco-genes depending on the organs or cells. The significance of their presence may also differ.
-1,6
FucT are located on different chromosomes. It is possible that they may
be related to some diseases, but at present, there is no evidence to support
this. Their functions are completely different, probably because they are
differentiated at a relatively early evolutionary stage.Concerning glyco-genes in general, the mechanism of gene regulation is only partially known for GalT, at present. Unfortunately in GnT-III gene, also, the mechanism of regulation has not yet been clarified.
-1,3 GalT and -1,4 GalT. And the
x-ray crystallographic analysis of GnT-I has just been completed. For GnT-III
and V, conducting the x-ray crystallographic analysis is difficult since
they have sugars. For -1,6 FucT, analysis may be possible because -1,6 FucT
has no sugars. Since the domain structure of the genes for glycosyltransferases
is similar, domains such as acceptor recognition sites and donor recognition sites
may be found one right after the other. Thus, there may be a great advance
in the understanding of enzyme reaction. However, questions addressing the
manner in which these enzymes are located in the cell or in the Golgi, what
kind of cluster they form, and how they actually synthesize the sugar chain,
may take some time to be answered.At present, only a little is known about where lectins, which recognize sugar chains are present and their properties. For example, although the
-1,6 branched structure abundantly appears in metastatic
cancer, how the cancer cells recognize it has not been clarified. We are
thinking of carrying out studies in this area in the future. The target of our current group is to examine the individuals, cells, phenotypes after introduction of a gene into the cells, and the results of knockout animals. Also, we initially proposed to study specific intercellular ligands, sialyl Lewis x, midkine, growth factors, and interactions and signaling through proteoglycans.
There are already about 400 publications, and I think we have achieved good results concerning isolation, determination of functions, and identification of genes for carbohydrate synthesis. In a year-and-a-half, we will have more new discoveries.
By 2003, it is expected that the identification of all human genes will be completed. However, understanding of the cell biology solely through gene clarification may be almost impossible. Clarification of the cause of disease or the provision of a means for selecting therapeutic methods based only on the results may not be achieved.
The human body and its cells are full of diversification. Data for only one gene is probably not sufficient for the discussion of disease.
When a certain gene is knocked out, a specific disease ensues and a causal relation can be established. However, in the case of the sugar chain, even if a glyco-gene is knocked out, it does not bring about a direct result but rather reflects the final figure brought about by the indirect knockout of other genes. The sugar chain modifies glycoprotein, glycolipid, and proteoglycan, which all affect matters in various steps, particularly the final step. Therefore, to reiterate this idea in another way, the study of glyco-genes may be useful because of the higher probability that there will be elucidation of events which exist in the down stream, in the place closest to where diseases are genarated. This is a rather direct manifestation. Nowadays, I often think we should place a little more emphasis on this way of thinking. Although the cause of disease is very complicated, what really controls the cause is the final stop. Therefore, it is not true that one gene has one function, but rather that a various combination of genes controls the function. The sugar chain may possibly define a function. This is why nowadays I conclude that this way of thinking is rather important. The concept of glycomes may cover this idea.
Presently, I am involved in the study of pig-to-human xenotransplantation. The problem is that there is an
-1,3 Gal epitope sugar
chain on the cell surface in pigs and a natural antibody to this epitope
exists in humans, which leads to the occurrence of hyperacute rejection.
We do not have a technique to knockout pig genes that form this sugar chain
yet. However, it was found that the synthesis of -Gel epitope becomes negligible
when bisecting GlcNAc is introduced into sugar chain using GnT-III.
Therefore, I am currently studying the functional knockout of the epitope
using this technique. I would expect that the concept of glycomes will become very important as such studies or clarification of phenomena increase in number. In fact, glycome analysis using C. elegans has already been started overseas.
Brief personal history of Professor Naoyuki Taniguchi
The meeting president for Japanese Biochemical Society in 2001. Honorary member of the American Society for Biochemistry and Molecular Biology. The secretary general for International Union of Biochemistry and Molecular Biology, 2006. Editorial board members: J. Biol. Chem., Glycobiology, Glycoconjugate J.,etc.
| 1) | Yamashita, K., Hitoi, A., Taniguchi, N., Yokosawa, N., Tsukada. Y., and Kobata. A.: Comparative study of the sugar chains of gamma-glutamyltranspeptidases purified from rat liver and rat AH-66 hepatoma cells. Cancer Res. 1983, 43, 5059-5063 |
| 2) | Nishikawa, A., lhara, Y.. Hatakeyama, M, Kangawa. K.. and Taniguchi. N.: Purification, cDNA cloning, and expression of UDP-N-acetylglucosamine: beta-D-mannoside beta-l,4N-acetylglucosaminyltransferase III from rat kidney. J. Biol. Chem. 1992,267, 18199-18204 |
| 3) | Yoshimura, M.. Nishikawa, A., Ihara, Y., Taniguchi. S., and Taniguchi. N.: Suppression of lung metastasis of B 16 mouse melanoma by N-acetylglucosaminyltransferase III gene transfection. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 8754-8758 |
| 4) | Uozumi, N., Yanagidani, S.. Miyoshi, E., Ihara. Y.. Sakuma, T., Gao, CX., Teshima. T., Fujii. S., Shiba, T., and Taniguchi. N.: Purification and cDNA cloning of porcine brain GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha1-6-fucosyltransferase. J. Biol. Chem. 1996, 271, 27810-27817 |
| 5) | Tanemura. M., Miyagawa. S.. Koyota, S.. Koma. M.. Matsuda. H., Tsuji. S.. Shirakura. R., and Taniguchi, N.: Reduction of the major swine xenoantigen. the alpha-galactosyl epitope by transfection of the alpha2,3-sialyltransferase gene. J. Biol. Chem. 1998. 273. 16421-16425 |
| 6) | Taniguchi, N. and lkeda. Y.: gamma-Glutamyl transpeptidase: catalytic mechanism and gene expression. Adv. Enzymol. Relat. Areas Mol. Biol. 1998, 72, 239-728 |
| 7) | Taniguchi, N., Jain, S. K.. Takahashi, M., Ko. J.-H.. Sasai. K., Miyoshi, E.. and Ikeda. Y.: Glycosyltransferases: cell surface remodeling and regulation of receptor tyrosine kinase-induced signaling. Pure Appl. Chem. 1999.71. 719-728 |
| 8) | Taniguchi, N., Miyoshi, E., Ko, JH., Ikeda, Y., and Ihara, Y.: Implication of N-acetylglucosaminyltransferasesII and V in cancer: gene regulation and signaling mechanism. Blochim. Biophys. Acta 1999. 1455. 287-300 |
| 9) | Miyoshi, E., Noda, K., Yamaguchi, Y., Inoue, S., Ikeda. Y., Wang, W.. Ko. JH.. Uozumi, N., Li, W.. and Taniguchi, N.: The alphal-6-fucosyltransferase gene and its biological significance. Biochim. Biophys. Acta 1999. 1473, 9-20 |
| 10) | Miyagawa, S., Tanemura. M.. Koyota, S., Koma, M.. Ikeda, Y., Shirakura. R.. and Taniguchi, N.: Masking and reduction of the Galactose-alpha1,3-Galactose (alpha-Gal) epitope, the major xenoantigen in swine, by the glycosyltransferase gene transfection. Biochem. Biophys. Res. Commun. 1999, 264, 611-614 |
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