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

Toshiaki K Shibata,Fumiko Matsumura,Ping Wang,Shinyi Yu,Chi-Chi Chou,Kay-Hooi Khoo,Kazuko Kitayama,Tomoya O Akama,Kazuhiro Sugihara,Naohiro Kanayama,Kyoko Kojima-Aikawa,Peter H Seeberger,Minoru Fukuda,Atsushi Suzuki,Daisuke Aoki,Michiko N Fukuda

doi:10.1074/jbc.m111.305334

Abstract

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 SO(3) → 3Galβ1 → 4GlcNAcβ1 → 3(±SO(3) → 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.

Highlights

We produced a humanized monoclonal antibody, designated HMOCC-1, against cell surface carbohydrates presented by malignant ovarian cancer
B3GNT7 (Fig. 1B, panel h) or GAL3ST3 (Fig. 1B, panel l), suggesting that these two enzymes are necessary for HMOCC-1 antigen biosynthesis
When HEK293T cells were transfected with a mixture lacking CHST1, immunostaining intensity was weakened, but the proportion of HMOCC-1-positive cells increased (Fig. 1B, panel i), suggesting that CHST1 plays a regulatory role in antigen formation

Summary

Introduction

We produced a humanized monoclonal antibody, designated HMOCC-1, against cell surface carbohydrates presented by malignant ovarian cancer. Results: Co-transfection experiments predicted HMOCC-1 antigenic oligosaccharides structures, which were chemically synthesized for testing antibody binding. 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 suggested that the sulfotransferase CHST1 regulates the abundance and intensity of HMOCC-1 antigen. When cells were first co-transfected with GAL3ST3 and B3GNT7 and with CHST1, the resulting cells strongly expressed HMOCC-1 antigen. When cells were transfected with a mixture of GAL3ST3 and CHST1 before or after transfection with B3GNT7, the number of anti-

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Conclusion