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Immunization of A4galt-deficient mice with glycosphingolipids from renal cell cancers resulted in the generation of anti-sulfoglycolipid monoclonal antibodies

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Abstract

In this study, we immunized Gb3/CD77 synthase gene (A4galt) knockout (KO) mice with glycosphingolipids (GSLs) extracted from 3 renal cell cancer (RCC) cell lines to raise monoclonal antibodies (mAbs) reactive with globo-series GSLs specifically expressed in RCCs. Although a number of mAbs reactive with globo-series GSLs were generated, they reacted with both RCC cell lines and normal kidney cells. When we analyzed recognized antigens by mAbs that were specifically reactive with RCC, but not with normal kidney cells at least on the cell surface, many of them turned out to be reactive with sulfoglycolipids. Eight out of 11 RCC-specific mAbs were reactive with SM2 alone, and the other 3 mAbs were more broadly reactive with sulfated glycolipids, i.e. SM3 and SM4 as well as SM2. In the immunohistochemistry, these anti-sulfoglycolipids mAbs showed RCC-specific reaction, with no or minimal reaction with adjacent normal tissues. Thus, immunization of A4galt KO mice with RCC-derived GSLs resulted in the generation of anti sulfated GSL mAbs, and these mAbs may be applicable for the therapeutics for RCC patients.

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Acknowledgments

We thank T. Mizuno and Y. Nakayasu for technical assistance.

This study was supported by a Grant-in-Aid from the New Energy and Industrial Technology Development Organization of Japan (NEDO), and grants-in-Aid for Scientific Research B (15H04696) and Grant-in-aid for Scientific Research on Innovative Areas (23110008) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT).

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Authors and Affiliations

  1. Department of Biochemistry II, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya, 466-0065, Japan

    Reiko Ando, Noriyo Tokuda, Noboru Hashimoto, Xiaoen Fan, Keiko Furukawa & Koichi Furukawa

  2. Department of Urology, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya, 466-0065, Japan

    Tokunori Yamamoto & Momokazu Goto

  3. IMS, RIKEN Center for Integrative Medical Sciences, 1-7-22, Suehiro, Tsurumi-ku, Yokohama, 230-0045, Japan

    Kazutaka Ikeda

  4. Department of Biomedical Sciences, Chubu University College of Life and Health Sciences, 1200 Matsumoto, Kasugai, Aichi, 487-8501, Japan

    Ryo Taguchi, Keiko Furukawa & Koichi Furukawa

  5. Research Center of Biomedical Analysis and Radioisotope, Teikyo University School of Medicine, 2-11-1, Kuga, Itabashi-ku, Tokyo, 173-8605, Japan

    Yukio Niimura

  6. Institute of Glycoscience, Tokai University, 4-1-1 Kitakaname, Hiratsuka, Kanagawa, 259-1292, Japan

    Akemi Suzuki

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  1. Reiko Ando
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  2. Noriyo Tokuda
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Correspondence to Koichi Furukawa.

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Supplemental Table S1

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Supplemental Table S2

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Supplemental Table S3

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Supplemental Fig. S1

Immunization schedule of mice. A4galt KO mice were immunized intravenously or intraperitoneally with glycolipids derived from RCC cell lines as described in “Materials and methods”. Immunization times of mice used for the fusion were 11~14. Serum reactivity check by IF was performed at appropriate time points. (PDF 40 kb)

Supplemental Fig. S2

Specificity of mAb F722-3 analyzed by TLC-immunostaining. TLC-immunostaining was performed with mAb 722-3 (hybridoma supernatant at 1:2 dilution) as described in “Materials and Methods”. BBG, bovine brain gangliosides as standards. SM2 was prepared from African green monkey kidney cells, Verots S3 and its structure was defined as described in Ref. 19. (PDF 25 kb)

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Ando, R., Tokuda, N., Yamamoto, T. et al. Immunization of A4galt-deficient mice with glycosphingolipids from renal cell cancers resulted in the generation of anti-sulfoglycolipid monoclonal antibodies. Glycoconj J 33, 169–180 (2016). https://doi.org/10.1007/s10719-016-9654-6

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