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Cytotoxicity and Glycan-Binding Profile of a d-Galactose-Binding Lectin from the Eggs of a Japanese Sea Hare (Aplysia kurodai)

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Abstract

A divalent cation-independent 16 kDa d-galactose binding lectin (AKL-2) was isolated from eggs of sea hare, Aplysia kurodai. The lectin recognized d-galactose and d-galacturonic acid and had a 32 kDa dimer consisting of two disulfide-bonded 16 kDa subunits. Eighteen N-terminus amino acids were identified by Edman degradation, having unique primary structure. Lectin blotting analysis with horseradish peroxidase-conjugated lectins has shown that AKL-2 was a glycoprotein with complex type oligosaccharides with N-acetyl d-glucosamine and mannose at non-reducing terminal. Two protein bands with 38 and 36 kDa in the crude extract of sea hare eggs after purification of the lectin was isolated by AKL-2-conjugated Sepharose column and elution with 0.1 M lactose containing buffer. It suggested that the lectin binds with an endogenous ligand in the eggs. AKL-2 kept extreme stability on haemagglutination activity if it was treated at pH 3 and 70 °C for 1 h. Glycan binding profile of AKL-2 by frontal affinity chromatography technology using 15 pyridylamine labeled oligosaccharides has been appeared that the lectin uniquely recognized globotriose (Galα1-4Galβ1-4Glc; Gb3) in addition to bi-antennary complex type N-linked oligosaccharides with N-acetyllactosamine. Surface plasmon resonance analysis of AKL-2 against a neo-glycoprotein, Gb3-human serum albumin showed the k ass and k diss values are 2.4 × 103 M−1 s−1 and 3.8 × 10−3 s−1, respectively. AKL-2 appeared cytotoxicity against both Burkitt’s lymphoma Raji cell and erythroleukemia K562. The activity to Raji by the lectin was preferably cancelled by the co-presence of melibiose mimicing Gb3. On the other hand, K562 was cancelled effectively by lactose than melibiose. It elucidated that AKL-2 had cytotoxic ability mediated glycans structure to cultured cells.

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Abbreviations

AKL:

Sea hare (Aplysia kurodai) eggs lectin

ASF:

Asialofetuin

BSM:

Bovine submaxillary mucin

ConA:

Concanavalin A

FACT:

Frontal affinity chromatography technology

Gb3:

Globotriose (Galα1-4Galβ1-4Glc)

GPC:

Gel permeation chromatography

PA:

Pyridylamine

PSM:

Porcine stomach mucin

PVDF:

Polyvinyllidene difluoride

SPR:

Surface plasmon resonance

WGA:

Wheat germ agglutinin

WST-8:

2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophhenyl)-2H-tetrazolium, monosodium salt

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Acknowledgments

This work was supported in part by a Grant-In-Aid for scientific research (No. 1021009100 and 22580226) from the Japan Society for the Promotion of Science (JSPS), Japanese Association for Marine Biology (JAMBIO, Project No.23-04) from Ministry of Education, Culture, Sports, Science and Technology (MEXT) Japan and Strategic Research Project (No. G2307) from Yokohama City University (YCU).

Author information

Authors and Affiliations

  1. Department of Chemistry, Graduate School of Science, The University of Chittagong, Chittagong, 4331, Bangladesh

    Sarkar M. A. Kawsar

  2. Laboratory of Glycobiology and Marine Biochemistry, Department of Genome System Sciences, Graduate School of NanoBiosciences, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama, 236-0027, Japan

    Sarkar M. A. Kawsar, Ryo Matsumoto, Yuki Fujii, Haruki Matsuoka, Naoko Masuda, Iwahara Chihiro, Hidetaro Yasumitsu & Yasuhiro Ozeki

  3. Laboratory of Environmental Microbiology and Molecular Toxicology, Department of Genome System Sciences, Graduate School of NanoBiosciences, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama, 236-0027, Japan

    Robert A. Kanaly

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

    Shigeki Sugawara, Masahiro Hosono & Kazuo Nitta

  5. Department of Food and Hygiene, Faculty of Environmental Health Science, Azabu University, 1-17-71 Fuchinobe, Sagamihara City, Kanagawa, 229-8501, Japan

    Naoto Ishizaki & Chikaku Dogasaki

  6. Department of Biology, School of Health Sciences, Fujita Health University, Toyoake, Aichi, 470-1192, Japan

    Jiharu Hamako & Taei Matsui

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  1. Sarkar M. A. Kawsar
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  2. Ryo Matsumoto
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  9. Shigeki Sugawara
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  16. Yasuhiro Ozeki
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Correspondence to Yasuhiro Ozeki.

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Sarkar M. A. Kawsar and Ryo Matsumoto is equal contribution as the first author.

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Kawsar, S.M.A., Matsumoto, R., Fujii, Y. et al. Cytotoxicity and Glycan-Binding Profile of a d-Galactose-Binding Lectin from the Eggs of a Japanese Sea Hare (Aplysia kurodai). Protein J 30, 509–519 (2011). https://doi.org/10.1007/s10930-011-9356-7

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