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Generation and characterization of a series of monoclonal antibodies that specifically recognize [HexA(±2S)-GlcNAc]n epitopes in heparan sulfate

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Abstract

Five monoclonal antibodies AS17, 22, 25, 38 and 48, a single monoclonal antibody ACH55, and three monoclonal antibodies NAH33, 43, 46, that recognize acharan sulfate (IdoA2S-GlcNAc)n, acharan (IdoA-GlcNAc)n and N-acetyl-heparosan (GlcA-GlcNAc)n, respectively, were generated by immunization of mice with keyhole limpet hemocyanin-conjugated polysaccharides. Specificity tests were performed using a panel of biotinylated GAGs that included chemically modified heparins. Each antibody bound avidly to the immunized polysaccharide, but did not bind to chondroitin sulfates, keratan sulfate, chondroitin nor hyaluronic acid. AS antibodies did not bind to heparan sulfate or heparin, but bound to 6-O-desulfated, N-desulfated and re-N-acetylated heparin to varying degrees. ACH55 bound to tri-desulfated and re-N-acetylated heparin but hardly bound to other modified heparins. NAH antibodies did not bind to heparin and modified heparins but bound to heparan sulfate to varying degrees. NAH43 and NAH46 also bound to partially N-de-acetylated N-acetyl-heparosan. Immunohistochemical analysis in rat cerebella was performed with the antibodies. While NAH46 stained endothelia, where heparan sulfate is typically present, neither ACH55 nor AS25 stained endothelia. On the contrary ACH55 and AS25 stained the molecular layer of the rat cerebella. Furthermore, ACH55 specifically stained Purkinje cells. These results suggest that there is unordinary expression of IdoA2S-GlcNAc and IdoA-GlcNAc in specific parts of the nervous system.

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Abbreviations

NAc-HSs:

acharan sulfate, acharan and N-acetyl-heparosan

HS:

heparan sulfate

GlcA:

D-glucuronic acid

IdoA:

L-iduronic acid

HexA:

unspecified hexuronic acid

ΔHexA:

4,5-unsaturated hexuronic acid

GlcNAc:

N-acetyl-D-glucosamine

GlcNH2 :

N-unsubstituted D-glucosamine

2S:

2-O-sulfated

6S-:

6-O-sulfated

NS:

N-sulfated

GAGs:

glycosaminoglycans

NDS/NAc-Hep:

N-desulfated/N-acetylated heparin

6DS-Hep:

6-O-desulfated heparin

6DS/NAc-Hep:

6-O-desulfated/N-acetylated heparin

6DS/NDS/NAc-Hep:

6-O-desulfated/N-desulfated/N-acetylated heparin

2DS/NDS/NAc-Hep:

2-O-desulfated/N-desulfated/N-acetylated heparin

6DS/2DS-Hep:

6-O-desulfated/2-O-desulfated heparin

6DS/2DS/NAc-Hep:

6-O-desulfated/2-O-desulfated/N-acetylated heparin

TriDS/NAc-Hep:

6-O-desulfated/2-O-desulfated/N-desulfated/N-acetylated heparin

PDeNAcNAH:

partially N-de-acetylated N-acetyl-heparosan

HPLC:

high-performance liquid chromatography

PDP:

2-pyridyldisulfide-propionated

KLH:

keyhole limpet hemocyanin

ELISA:

enzyme-linked immunosorbent assay

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Authors and Affiliations

  1. Central Research Laboratories, Seikagaku Corporation, 3-1253 Tateno, Higashiyamato, Tokyo, 207-0021, Japan

    Kiyoshi Suzuki, Koji Yamamoto, Yutaka Kariya, Hiroshi Maeda & Mamoru Kyogashima

  2. Research Laboratories Seikagaku Biobusiness Corporation, 3-1253 Tateno, Higashiyamato, Tokyo, 207-0021, Japan

    Takeshi Ishimaru & Shuichi Miyaura

  3. Division of Molecular Pathology, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya, Aichi, 464-8681, Japan

    Masahiro Fujii, Akiko Yusa, Reiji Kannagi & Mamoru Kyogashima

  4. Division of Surgical Oncology, Department of Surgery, Nagoya University Graduate School of Medicine, 65, Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan

    Masahiro Fujii

  5. Natural Products Research Institute, College of Pharmacy, Seoul National University, 20-313, 599 Gwanangno, Gwanak-gu, Seoul, 151-742, Korea

    Eun Ji Joo & Yeong Shik Kim

  6. Institute for Molecular Science of Medicine, Aichi Medical University, Nagakute, Aichi, 480-1195, Japan

    Koji Kimata

  7. Department of Oncology, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, 467-8603, Japan

    Mamoru Kyogashima

Authors
  1. Kiyoshi Suzuki
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  2. Koji Yamamoto
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  3. Yutaka Kariya
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  4. Hiroshi Maeda
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  5. Takeshi Ishimaru
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  6. Shuichi Miyaura
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  7. Masahiro Fujii
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  8. Akiko Yusa
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  9. Eun Ji Joo
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  10. Koji Kimata
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  11. Reiji Kannagi
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  12. Yeong Shik Kim
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  13. Mamoru Kyogashima
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Corresponding author

Correspondence to Mamoru Kyogashima.

Additional information

Suzuki and Yamamoto contributed equally to this study.

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Suzuki, K., Yamamoto, K., Kariya, Y. et al. Generation and characterization of a series of monoclonal antibodies that specifically recognize [HexA(±2S)-GlcNAc]n epitopes in heparan sulfate. Glycoconj J 25, 703–712 (2008). https://doi.org/10.1007/s10719-008-9130-z

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  • DOI: https://doi.org/10.1007/s10719-008-9130-z

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