Identification of Mono- and Disulfated N-Acetyl-lactosaminyl Oligosaccharide Structures as Epitopes Specifically Recognized by Humanized Monoclonal Antibody HMOCC-1 Raised against Ovarian Cancer

A humanized monoclonal antibody raised against human ovarian cancer RMG-I cells and designated as HMOCC-1 (Suzuki, N., Aoki, D., Tamada, Y., Susumu, N., Orikawa, K., Tsukazaki, K., Sakayori, M., Suzuki, A., Fukuchi, T., Mukai, M., Kojima-Aikawa, K., Ishida, I., and Nozawa, S. (2004) Gynecol. Oncol....

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Bibliographic Details
Published in:The Journal of biological chemistry 2012-02, Vol.287 (9), p.6592-6602
Main Authors: Shibata, Toshiaki K., Matsumura, Fumiko, Wang, Ping, Yu, ShinYi, Chou, Chi-Chi, Khoo, Kay-Hooi, Kitayama, Kazuko, Akama, Tomoya O., Sugihara, Kazuhiro, Kanayama, Naohiro, Kojima-Aikawa, Kyoko, Seeberger, Peter H., Fukuda, Minoru, Suzuki, Atsushi, Aoki, Daisuke, Fukuda, Michiko N.
Format: Article
Language:English
Subjects:
Amino Sugars - chemical synthesis
Amino Sugars - immunology
Animals
Antibodies, Monoclonal - immunology
Antibody Specificity - immunology
Carbohydrate
Carbohydrate Chemistry
Carbohydrate Glycoconjugate
Carbohydrate Glycoprotein
Carbohydrate Sequence
Carbohydrate Sulfotransferases
CHO Cells
Cricetinae
Disulfides - chemical synthesis
Disulfides - immunology
Epitope Mapping
Epitopes - immunology
Female
Glycobiology and Extracellular Matrices
Glycosyltransferase
HEK293 Cells
Humans
Molecular Sequence Data
N-Acetylglucosaminyltransferases - genetics
N-Acetylglucosaminyltransferases - metabolism
N-Acetylneuraminic Acid - metabolism
Oligosaccharides - chemical synthesis
Oligosaccharides - immunology
Ovarian Cancer
Ovarian Neoplasms - immunology
Ovarian Neoplasms - pathology
Polylactosamine
RNA, Small Interfering - pharmacology
Sulfotransferase
Sulfotransferases - genetics
Sulfotransferases - metabolism
Tumor Cells, Cultured
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Description
Summary:A humanized monoclonal antibody raised against human ovarian cancer RMG-I cells and designated as HMOCC-1 (Suzuki, N., Aoki, D., Tamada, Y., Susumu, N., Orikawa, K., Tsukazaki, K., Sakayori, M., Suzuki, A., Fukuchi, T., Mukai, M., Kojima-Aikawa, K., Ishida, I., and Nozawa, S. (2004) Gynecol. Oncol. 95, 290–298) was characterized for its carbohydrate epitope structure. Specifically, a series of co-transfections was performed using mammalian expression vectors encoding specific glycosyltransferases and sulfotransferases. These experiments identified one sulfotransferase, GAL3ST3, and one glycosyltransferase, B3GNT7, as required for HMOCC-1 antigen formation. They also suggested that the sulfotransferase CHST1 regulates the abundance and intensity of HMOCC-1 antigen. When HEK293T cells were co-transfected with GAL3ST3 and B3GNT7 expression vectors, transfected cells weakly expressed HMOCC-1 antigen. When cells were first co-transfected with GAL3ST3 and B3GNT7 and then with CHST1, the resulting cells strongly expressed HMOCC-1 antigen. However, when cells were transfected with a mixture of GAL3ST3 and CHST1 before or after transfection with B3GNT7, the number of antigen-positive cells decreased relative to the number seen with only GAL3ST3 and B3GNT7, suggesting that CHST1 plays a regulatory role in HMOCC-1 antigen formation. Because these results predicted that HMOCC-1 antigens are SO3→3Galβ1→4GlcNAcβ1→3(±SO3→6)Galβ1→4GlcNAc, we chemically synthesized mono- and disulfated and unsulfated oligosaccharides. Immunoassays using these oligosaccharides as inhibitors showed the strongest activity by disulfated tetrasaccharide, weak but positive activity by monosulfated tetrasaccharide at the terminal galactose, and no activity by nonsulfated tetrasaccharides. These results establish the HMOCC-1 epitope, which should serve as a useful reagent to further characterize ovarian cancer. We produced a humanized monoclonal antibody, designated HMOCC-1, against cell surface carbohydrates presented by malignant ovarian cancer. Co-transfection experiments predicted HMOCC-1 antigenic oligosaccharides structures, which were then chemically synthesized for testing antibody binding. HMOCC-1 antigen is the glycan structure composed of SO3→3Galβ1→4GlcNAcβ1→3(±SO3→6)Galβ1→4GlcNAcβ1→. The approach employed in this study will be useful in determining specificity of an undefined monoclonal anti-carbohydrate antibody.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M111.305334