During my graduate and post-doctoral years, I was trained as a protein chemist. I purified a lectin called SHA (Streptomyces hemagglutinin) which I screened from 333 strains, and an enzyme called beta-galactosyl-transferase to homogeneity by manually-prepared affinity gels. Those trainings had led to my first major achievement as an independent researcher, which was the purification of biologically active insulin receptors from human placental membranes. The purified receptor not only had a theoretically reasonable number of insulin binding sites and contained an intact β subunit which exhibited tyrosine kinase activity. We next purified human IGF-I receptor which led to successful cloning of its cDNA in collaboration with Dr. Ullrich at Genentech, and to production of monoclonal antibodies (mAbs) that have been widely marketed. Those early studies were supported by two NIH R01 grants.
Based on a working hypothesis that increased IGF-I receptor signaling must be contributing to cancer cell growth and cancer progression, we constructed IGF-II ribozymes and a recombinant anti-IGF-I receptor antibody from 1H7 mAb and showed that they inhibited prostate and breast cancer growth, respectively. These projects were funded by grants from DOD U.S. Army Prostate Cancer Research Program and California Breast Cancer Research Program.
As for insulin-action-related programs, we purified cGMP-inhibited phosphodiesterase 3A (PDE3A) from human placentas and revealed human PDE3A genomic DNA structure. This work was supported by ADA and JDRI.
My major focus since 1990s has been cancer-targeted antibody-engineering. When I left City of Hope in 2000, I continued the anti-IGF-I receptor program, and added a new project to produce anti-carbohydrate antibodies by phage-display technology. The production of human single-chain variable fragments (scFv) against carbohydrate antigens by phage-display technology was a logical strategy towards the development of antibody therapeutics since carbohydrates are self-antigens. Panning and screening of phage-displayed human scFvs using a variety of probes presenting structurally-defined carbohydrates resulted in identifying a number of candidate phage clones as judged by DNA sequences and specific binding to carbohydrate moieties of interest. Expression of candidate scFv genes was not straightforward. The best expression system and fine adjustments for the conditions to prepare active forms had to be determined for each scFv protein. The successful production of active scFv proteins seems to be dependent on their DNA and/or amino acid sequences.
After carrying out a decade of laborious and continuous hard work and identifying several human anti-carbohydrate scFv genes that may contribute to future developments in basic and clinical research, I must admit that our original idea of producing a variety of carbohydrate-specific antibodies by phage-display technology was rather naïve. During those studies, however, we isolated cDNA from hybridoma producing MLS128 which is an IgG3 specific for oncofetal carbohydrate antigen called Tn. MLS128 mAb suppressed colon cancer cell growth by binding to 110 KDa surface glycoprotein. Interaction between MLS128 scFv and Tn-antigens is now being investigated by NMR. Characterization and identification of 110 KDa glycoprotein is also under intensive investigation. This was one of the post-genome projects supported by CREST from the Japanese Science and Technology Agency.
Antibody therapeutics and diagnostics
Since we have recombinant scFv genes for anti IGF-I receptor 1H7 and 3B7 as well as anti-Tn antigen MLS128 which were derived from mouse mAbs, and other human type anti-carbohydrate scFv genes, future antibody-based diagnostics and therapeutics are currently under consideration.
MLS128 anti-Tn antigen mAb and its colon cancer-specific receptor
Anti-Tn antigen MLS128 mAb was produced two decades ago by immunizing mice with “cancerous antigens” derived from LS180 colon cancer cells. Our studies demonstrated that MLS128 bound to 110 kDa glycoprotein (GP) in colon cancer cells, thereby inhibiting cell growth. Extensive attempts have been made towards understanding the inhibitory action of MLS128 on colon cancer cell growth and solving the primary structure of 110 kDa GP. Since limited proteolysis of 110 kDa GP was observed in microdomain fractions that had been kept frozen for several years, the recent work was carried out to determine susceptibility of 110 kDa GP to proteases as well as N-glycosidase F. These studies revealed : 110 kDa GP contains N-glycans. It does not contain inter-disulfide bonds but appears to have intra-disulfides. It seems to contain cleavage sites for cathepsin D which could cause limited digestion. LS180 cells derived from the laboratory of Prof. Akiyama, Univ. of Tokyo, produced a limited proteolysis product-like 75 kDa GP.
To determine the amino acid sequence of 110 kDa GP, a new strategy has been initiated which takes advantage of a smaller fragment, 75 kDa GP, derived from LS180 cells cultured in Akiyama laboratory. The isolation of 110 KDa GP by two-dimensional electrophoresis has been carried out by Prof. Yamamoto, Univ. of Tokyo.
Sequence of SHA has been revealed after 40 years of storage
The Streptomyces lectin, SHA27S5, is a blood group B-specific hemagglutinin which from the culture supernatant of Streptomyces sp. 27S5. The hemagglutinating activity is inhibited by L-rhamnose, D-galactose, and L-arabinose, suggesting that the stereochemistry at C-2 and C-4 seems essential for the binding to this lectin. The unique feature is that this lectin is a very small monomeric protein with a molecular weight of 13,000 with two binding sites, which accounts for the hemmagglutinating activity. Although SHA27S5 was discovered and characterized more than 30 years ago, it is still unknown how such small-sized lectin can specifically bind to blood group B using two binding sites. Unfortunately, the Streptomyces sp. 27S5 strain is lost so that we cannot clone the gene encoding this lectin.
Although purification and characterization of SHA was well established which was my doctoral-theses, its primary structure had not been determined since due to the redundancy found in amino acid sequences of the peptides, I gave up the sequencing of SHA in 70s. After I returned to City of Hope in April, 2014, I started a fruitful collaboration with Dr. Markus Kalkum, the Director of Mass Spectrometry & Proteomics Core. I have kept over 50 mg of purified SHA in freezers while I had working as PI at City of Hope and Tokai University in Japan. The frozen SHA was turned out to be intact and still active. By sophisticated current mass spectrometric technologies, Dr. Kalkum’s group solved the primary structure of SHA as of May 2015
Abstract presented: Bagramyan, K.et al. Mass spectrometric resurrection of SHA, a L-rhamnose and β-D-galactose binding lectin from the lost strain Streptomyces 27S5. 63rd American Society for Mass Spectrometry Conference, ThP307, Saint Louis, 31 May - 4 June, 2015.
In collaboration with Drs Kalkum and Yoshiki Yamaguchi at Riken in Japan, we are trying to express recombinant SHA in E. coli or other cells. This has become an extremely simulative project in my research career, so we will plan not only basic research involving X-ray crystallography and NMR, but also translational research since recombinant lectins such as SHA can be valuable for glycobiology-based biomedical applications, including diagnostics and drug delivery approaches.
To achieve significant reduction in school children’s obesity by introducing innovative interventions
Childhood obesity has increased substantially. In 2012, more than one third of children and adolescents were overweight or obese. There is an urgent need for schools to take a leading role in the promotion of healthy living, which should lead to a life-long commitment to good health. This program will test the hypothesis that interventions at 1st graders are the key to successfully implement a healthy lifestyle for the rest of life such as waking up early and sleeping more than 8 hours, eating well-balanced meals, and a life-long continual physical activity as clearly illustrated in the leaflet.
Selected Publications related to Insulin and IGF-I receptors
Fujita-Yamaguchi, Y., Choi, S., Sakamoto, Y. and Itakura, K. (1983) Purification of insulin receptor with full binding activity. J.Biol.Chem., 258:5045-5049
Kasuga, M., Fujita-Yamaguchi, Y., Blithe, D.L., and Kahn, C.R. (1983) Tyrosine-specific protein kinase activity is associated with the purified insulin receptor. Proc.Natl. Acad.Sci., USA, 80:2137-2141.
Fujita-Yamaguchi, Y. (1984) Characterization of purified insulin receptor subunits. J.Biol.Chem. 259:1206-1211.
LeBon, T. R., Jacobs, S., Cuatrecasas, P., Kathuria, S. and Fujita-Yamaguchi, Y. (1986) Purification of insulin-like growth factor (IGF)-I receptor from human placental membranes. J.Biol.Chem., 261:7685-7689.
Fujita-Yamaguchi, Y., LeBon, T., Tsubokawa, M., Henzel, W., Kathuria, S., Koyal, D., and Ramachandran, J. (1986) Comparison of insulin-like growth factor (IGF)-I receptor and insulin receptor purified from human placental membranes. J.Biol.Chem., 261:16727-16731.
Ullrich, A., Gray, A., Tam, A.W., Yang-Feng, T., Tsubokawa, M., Collins, C., Henzel, W., LeBon, T., Kathuria, S., Chen, E., Jacobs, S., Francke, U., Ramachandran, J. and Fujita-Yamaguchi, Y. (1986) Insulin like growth factor I receptor primary structure: Comparison with insulin receptor suggests structural determinants define functional specificity. EMBO J. 5:2503-2512.
Li, S.-L., Yan, P.-F., Paz, I.B., and Fujita-Yamaguchi, Y. (1992) Human insulin receptor β subunit transmembrane/cytoplasmic domain expressed in a baculovirus expression system: Purification, characterization, and polylysine effects on the protein tyrosine kinase activity. Biochemistry 31:12455-12462.
Yan, P.F., Li, S.-L., Liang, S.-J., Giannini, S., and Fujita-Yamaguchi, Y. (1993) The role of C-terminal and acidic domains in the activity and stability of human insulin receptor protein tyrosine kinase studied by purified deletion-mutants of the β subunit. J. Biol. Chem. 268:22444-22449.
Selected Publications related to antibody therapeutics and diagnostics
Fujita-Yamaguchi, Y. Production of Single-Chain Variable-Fragments against Carbohydrate Antigens. (2014) Antibodies 3, 155-168.
Fujita-Yamaguchi, Y. (2013) Renewed interest in basic and applied research involving monoclonal antibodies against an oncofetal Tn-antigen. J Biochem.152, 103-105.
Yuasa, N., Ogawa, H., Koizumi, T., Tsukamoto, K., Matsumoto-Takasaki, A., Asanuma, H., Nakada, H., and Fujita-Yamaguchi, Y. (2012) Construction and expression of anti-Tn-antigen-specific single chain antibody genes from hybridoma producing MLS128 monoclonal antibody. J Biochem. 151, 371-381.
Kusada, Y, Morizono, T., Matsumoto-Takasaki, A., Sakai, K., Sato, S., Asanuma, H.,Takayanagi, A., and Fujita-Yamaguchi, Y. (2008) Construction and characterization of single-chain antibodies against human insulin-like growth factor-1 Receptor from hybridomas producing 1H7 and 3B7 monoclonal antibody. J. Biochem. 143, 9-19.
Li, S.-L., Liang, S.-J., Guo, N., Wu, A. M., and Fujita-Yamaguchi, Y. (2000) Single chain antibodies against human insulin-like growth factor-I receptor: Expression, purification, and effect on tumor growth. Cancer Immunol Immunother., 49, 243-252.
Selected Publications related to MLS128 anti-Tn antigen mAb and its colon cancer-specific receptor
Morita, N., Yajima, Y., Asanuma, H., Nakada, H., and Fujita-Yamaguchi, Y. (2009) Inhibition of cancer cell growth by anti-Tn monoclonal antibody MLS128. Biosci. Trends. 3, 32-37
Zamri, N., Masuda, N., Oura, F., Yajima, Y., Nakada, H., and Fujita-Yamaguchi, Y.(2012) Effects of two monoclonal antibodies, MLS128 against Tn-antigen and 1H7 against insulin-like growth factor-I receptor, on the growth of colon cancer cells. Biosci Trends., 6, 303-312.
Zamri, N., Masuda, N., Oura, F., Kabayama, K., Yajima, Y., Nakada, H., Yamamoto, K., and Fujita-Yamaguchi, Y. (2013) Characterization of anti-Tn-antigen MLS128 binding proteins involved in inhibiting the growth of human colorectal cancer cells. Biosci Trend, 7, 221-229.
Oura, F., Yajima, Y., Nakata, M., Taniue, K., Akiyama, T., Nakada, H., Yamamoto, K., and Fujita-Yamaguchi, Y. (2015) Susceptibility to proteases of anti-Tn-antigen MLS128 binding glycoproteins expressed in human colon cancer cells. Biosci Trends. 9, 49-55.
Selected Publications related to sequence of SHA
Fujita, Y., Oishi, K., Suzuki, K., and Imahori, K. (1975) Purification and properties of anti-B hemagglutinin produced by Streptomyces sp. Biochemistry 14:4465-4470.
Fujita-Yamaguchi, Y., Oishi, K., Suzuki, K., and Imahori, K. (1982) Studies on carbohydrate-binding to a lectin purified from Streptomyces sp. Biochim.Biophys.Acta., 701:86-92.
Bagramyan, K., Hong, T.B.,Murad, J.P., Fujita-Yamaguchi, Y., and Kalkum, M. Mass spectrometric resurrection of SHA, a L-rhamnose and β-D-galactose binding lectin from the lost strain Streptomyces 27S5. 63rd American Society for Mass Spectrometry (ASMS) Conference, ThP307, Saint Louis, 31 May - 4 June, 2015.