Purification and Biochemical Characterization of a D-Galactose Binding Lectin from Japanese Sea Hare (Aplysia kurodai) Eggs

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S. M. A. Kawsar¹, R. Matsumoto¹, Y. Fujii¹, H. Yasumitsu¹, C. Dogasaki², M. Hosono³, K. Nitta³, J. Hamako⁴, T. Matsui⁵, N. Kojima⁶, and Y. Ozeki^1*

¹Laboratory of Marine Biochemistry, Department of Environmental Biosciences, International Graduate School of Arts and Sciences, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan; fax: +81-45-787-2413; E-mail: ozeki@yokohama-cu.ac.jp

²Department of Food and Hygiene, Faculty of Environmental Health Science, Azabu University, 1-17-71, Fuchinobe, Sagamihara City, Kanagawa 229-8501, Japan; E-mail: dogasaki@azabu-u.ac.jp

³Division of Cell Recognition Study, Institute of Molecular Biomembrane and Glycobiology, Tohoku Pharmaceutical University, 4-4-1 Komatsusima, Aoba-ku, Sendai 981-8558, Japan; E-mail: mhosono@tohoku-pharm.ac.jp

⁴Faculty of Medical Management and Information Science, School of Health Sciences, Fujita Health University, Toyoake, Aichi 470-1192, Japan; E-mail: jhamako@fujita-hu.ac.jp

⁵Department of Biology, School of Health Sciences, Fujita Health University, Toyoake, Aichi 470-1192, Japan; E-mail: tmatsui@fujita-hu.ac.jp

⁶Laboratory of Environmental Biosciences, Y. S. F. H., 6 Ono, Tsurumi-ku, Yokohama 230-0046, Japan; E-mail: no02-kojima@city.yokohama.jp

^* To whom correspondence should be addressed.

Received September 19, 2008; Revision received February 18, 2009

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A lectin was purified from Japanese sea hare Aplysia kurodai by lactosyl-agarose affinity chromatography. The molecular mass of the lectin was determined to be 56 and 32 kDa by SDS-PAGE under non-reducing and reducing conditions, respectively. It was found to agglutinate trypsinized and glutaraldehyde-fixed rabbit and human erythrocytes in the absence of divalent cations. The lectin exhibited stable thermo-tolerance as it retained hemagglutinating activity for 1 h even at 80°C and showed stability at pH 10. By contrast, it was very sensitive at pH less than 5 and in the presence of the sulfhydryl-group preserving reagent, β-mercaptoethanol. The hemagglutinating activity by the lectin was specifically inhibited by D-galactose, galacturonic acid, methyl-α- and methyl-β-D-galactopyranoside, lactose, melibiose, and asialofetuin. The association rate constant (k_ass) and dissociation rate constant (k_diss) were determined for the lectin to be 4.3·10⁵ M^–1·sec^–1 and 2.2·10^–3 sec^–1, respectively, using a surface plasmon resonance biosensor. The lectin moderately inhibited cell proliferation in the P388 cell line dose dependently. Interestingly, lectin-treated cells did not show a fragmented DNA ladder as is caused by apoptosis, suggesting that the cell proliferation inhibition was caused by another unknown mechanism.

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KEY WORDS: Aplysia kurodai, cell proliferating inhibition, D-galactose-binding lectin, galacturonic acid, sea hare, surface plasmon resonance

DOI: 10.1134/S0006297909070025

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