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Sialyl-glycoconjugates in cholesterol-rich microdomains of P388 cells are the triggers for apoptosis induced by Rana catesbeiana oocyte ribonuclease

Glycoconjugate Journal volume 31, pages 171–184 (2014)Cite this article

Abstract

SBL/RC-RNase was originally isolated from frog (Rana catesbeiana) oocytes and purified as a novel sialic acid-binding lectin (SBL) that displayed strong anti-cancer activity. SBL was later shown to be identical to a ribonuclease (RC-RNase) from oocytes of the same species. The administration of SBL/RC-RNase induced apoptosis (with nuclear condensation and DNA fragmentation) in mouse leukemia P388 cells but did not kill umbilical vein endothelial or fibroblast cells derived from normal tissues. The cytotoxic activity of SBL/RC-RNase was inhibited by desialylation of P388 cells and/or the co-presence of free bovine submaxillary mucin. FACS analysis showed that SBL/RC-RNase was incorporated into cells after attachment to cholesterol-rich microdomains. Addition of the cholesterol remover methyl-β-cyclodextrin reduced SBL/RC-RNase-induced apoptosis. Apoptosis occurred through the caspase-3 pathway following activation of caspase-8 by SBL/RC-RNase. A heat shock cognate protein (Hsc70) and a heat shock protein (Hsp70) (each 70 kDa) on the cell membrane were shown to bind to SBL/RC-RNase by mass spectrometric and flow cytometric analyses. Quercetin, an inhibitor of Hsc70 and Hsp70, significantly reduced SBL/RC-RNase-induced apoptosis. Taken together, our findings suggest that sialyl-glycoconjugates present in cholesterol-rich microdomains form complexes with Hsc70 or Hsp70 that act as triggers for SBL/RC-RNase to induce apoptosis through a pathway involving the activation of caspase-3 and caspase-8.

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Abbreviations

FACS:

Fluorescence-activated cell sorting

GSL:

Glycosphingolipid

HSC70:

70-kDa heat shock cognate protein

HSP70:

70-kDa heat shock protein

MβCD:

Methyl β-D-cyclodextrin

NHDF:

Normal human epidermal fibroblast

NHEM:

Normal human epidermal melanocyte

NHEK:

Normal human keratinocyte cell

RC-RNase:

Rana catesbeiana ribonuclease

SBL:

Sialic acid-binding lectin

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Acknowledgments

This study was supported in part by Grants-In-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) Japan, the Japan Society for the Promotion of Science (JSPS) and Nagasaki International University. The authors are grateful to Dr. Stephen Anderson for English editing of the manuscript.

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Authors and Affiliations

  1. Divisions of Microbiology and Functional Morphology, Department of Pharmacy, Faculty of Pharmaceutical Science, Nagasaki International University, 2825-7 Huis Ten Bosch, Sasebo, Nagasaki, 859-3298, Japan

    Y. Ogawa, Y. Fujii & H. Kobayashi

  2. Division of Cell Recognition Study, Institute of Molecular Biomembrane and Glycobiology, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai, 981-8558, Japan

    S. Sugawara, T. Tatsuta, M. Hosono & K. Nitta

  3. Division of Proteomics and BioMolecular Science, Faculty of Medicine, Juntendo University, Hongo, Bunkyo-ku, 113-8421, Japan

    T. Fujimura & H. Taka

  4. Laboratory of Glycobiology and Marine Biochemistry, Department of Life and Environmental System Science, Graduate School of NanoBio Sciences, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama, 236-0027, Japan

    Y. Koide, I. Hasan, R. Matsumoto, H. Yasumitsu, S. M. A. Kawsar & Y. Ozeki

  5. Department of Biochemistry and Molecular Biology, Faculty of Science, Rajshahi University, Rajshahi, 6205, Bangladesh

    I. Hasan

  6. Laboratory of Microbiology and Environmental Molecular Toxicology, Department of Life and Environmental System Science, Graduate School of NanoBio Sciences, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama, 236-0027, Japan

    R. A. Kanaly

  7. Laboratory of Carbohydrate and Protein Chemistry, Department of Chemistry, Faculty of Science, University of Chittagong, Chittagong, 4331, Bangladesh

    S. M. A. Kawsar

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  1. Y. Ogawa
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  2. S. Sugawara
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  3. T. Tatsuta
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  4. M. Hosono
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  5. K. Nitta
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  6. Y. Fujii
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Correspondence to Y. Ogawa.

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Y. Ogawa and S. Sugawara made equal contributions to the study and are both considered as first authors.

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Ogawa, Y., Sugawara, S., Tatsuta, T. et al. Sialyl-glycoconjugates in cholesterol-rich microdomains of P388 cells are the triggers for apoptosis induced by Rana catesbeiana oocyte ribonuclease. Glycoconj J 31, 171–184 (2014). https://doi.org/10.1007/s10719-013-9513-7

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  • DOI: https://doi.org/10.1007/s10719-013-9513-7

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