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

S. M. A. Kawsar1, R. Matsumoto1, Y. Fujii1, H. Yasumitsu1, C. Dogasaki2, M. Hosono3, K. Nitta3, J. Hamako4, T. Matsui5, N. Kojima6, and Y. Ozeki1*

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
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 (kass) and dissociation rate constant (kdiss) 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.
KEY WORDS: Aplysia kurodai, cell proliferating inhibition, D-galactose-binding lectin, galacturonic acid, sea hare, surface plasmon resonance

DOI: 10.1134/S0006297909070025