Identification of mono- and disulfated N-acetyl-lactosaminyl Oligosaccharide structures as epitopes specifically recognized by humanized monoclonal antibody HMOCC-1 raised against ovarian cancer

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.

FIGURE 1.

Immunocytochemistry for HMOCC-1 of transfected HEK293T cells. Transfected cells were treated with the HMOCC-1 antibody (human IgM) and stained using the immunoperoxidase method. Hematoxylin was employed as a counterstain. A, cells were transfected by the following expression vectors: mock (panel a); a mixture of eight GTs plus six STs (panel b); a mixture of 8 GTs (panel c), and a mixture of 6 STs (panel d). Arrow in panel b shows a positively stained cell. B, cells were transfected by a mixture of eight GTs and six STs lacking the following: none (panel a); FUT1 (panel b); FUT2 (panel c); GCNT1 (panel d); GCNT3 (panel e); B3GNT4 (panel f); B3GNT6 (panel g); B3GNT7 (panel h); CHST1 (panel i); CHST2 (panel j); CHST4 (panel k); GAL3ST3 (panel l); Chst5 (panel m); or CH6ST6 (panel n). C, GAL3ST3 only (panel a; CHST1 only (panel b); B3GNT7 only (panel c); CHST1 + GAL3ST3 (panel d); CHST1 + B3GNT7 (panel e), and B3GNT7 + GAL3ST3 (panel f). D, GAL3ST3 + B3GNT7 (panel a); CHST1 + B3GNT7 and then GAL3ST3 (panel b); CHST1 and then B3GNT7 + GAL3ST3 (panel c); GAL3ST3 + B3GNT7 and then CHST1 (panel d); B3GNT7 and then CHST1 + GAL3ST3 (panel e), and CHST1 + GAL3ST3 and then B3GNT7 (panel f).

FIGURE 2.

Immunocytochemistry for HMOCC-1 of RMG-I cells and CHO Lec2 cells. Cells were treated with the HMOCC-1 antibody (human IgM) and stained using the immunoperoxidase method. Hematoxylin was employed as a counterstain. A, immunocytochemistry of RMG-I cells before (panels a and b) and after (panels c and d) mild acid hydrolysis with (panels b and d) or without (panels a and c) HMOCC-1 antibody. B, CHO Lec2 cells transfected with mock vector (panel a) or co-transfected with GAL3ST3 plus B3GNT7 expression vectors (panel b).

FIGURE 3.

Chemical synthesis of sulfated N-acetyl-lactosaminyl tetrasaccharides. See supplemental material for more details.

FIGURE 4.

ELISA inhibition assay for HMOCC-1 using chemically synthesized oligosaccharides. RMG-I cells cultured in 96-well tissue culture plates were fixed and reacted with HMOCC-1 antibody followed by peroxidase-conjugated anti-human IgM antibody. ELISA inhibition assays were performed in the presence of synthetic oligosaccharides at indicated concentrations. A, comparison between nonsulfated (blue) and monosulfated (green) oligosaccharides. B, comparison between monosulfated (green) and disulfated (purple) oligosaccharides. C, comparison between two disulfated oligosaccharides (purple and red).

FIGURE 5.

LC-MS/MS analysis of permethylated disulfated glycans from ovarian cancer tissues. The LC-MS profile shown was summed over a period of time where the most abundant disulfated glycans were eluted and detected as doubly charged [M − 2H]²⁻ molecular ions in negative ion mode. Parent ions that afforded the doubly charged fragment ion at m/z 521 and were determined by MS/MS as carrying disulfated dilacNAc are circled in red. Each symbol represents: yellow circle, Hex; blue square, HexNAc; pink diamond, NeuNAc; circled S, SO₃.

FIGURE 6.

Agarose gel electrophoresis of RT-PCR products amplified from RMG-I cells in the presence and absence of RT. A, RT-PCR for GAL3STs. Theoretical lengths for each product are as follows: GALST1, 180 bp; GAL3ST2, 228 bp; GAL3ST3, 182 bp; GAL3ST4, 205 bp; and GAPDH (internal control), 359 bp. B, RT-PCR for B3GNT2 and B3GNT7. Theoretical lengths for each product are as follows: B3GNT2, 225 bp; B3GNT7, 178 bp, and GAPDH (internal control), 359 bp.

FIGURE 7.

HMOCC-1 antigen expressed by siRNA transfected RMG-I cells. A, immunohistochemistry of RMG-I cells transfected by none (panel a), control siRNA (panel b), and siRNAs each specific to GAL3ST3 (panel c), GAL3ST4 (panel d), B3GNT2 (panel e), and B3GNT7 (panel f). B, quantitative analysis of immunocytochemistry data obtained by five transfection experiments. Each image was analyzed by ImageJ, and numbers obtained were summarized by Prism program. Two-tailed Student's t test was applied for statistical analysis. Asterisks show statistically significant differences (p < 0.05). N.S., not significant or p > 0.05.

FIGURE 8.

Proposed structures and biosynthetic pathways for HMOCC-1 antigens. The absence of CHST1 and GAL3ST3 adds sulfate onto the Gal3 position of the LacNAc terminal. In the presence of CHST1, two types of sulfation occur, one at the terminal galactose (left) and the other at an internal galactose (right). Addition of sulfate to the Gal6 terminal (left) prohibits further modification by GAL3ST3. When sulfate is added to an internal Gal6, GAL3ST3 modifies the product by adding sulfate onto the Gal3 position of sulfated LacNAc, resulting in production of a strong HMOCC-1 antigen.