Cytotoxicity and glycan-binding profile of a d-galactose-binding lectin from the eggs of a Japanese sea hare (Aplysia kurodai)

Sarkar M.A. Kawsar, Ryo Matsumoto, Yuki Fujii, Haruki Matsuoka, Naoko Masuda, Iwahara Chihiro, Hidetaro Yasumitsu, Robert A. Kanaly, Shigeki Sugawara, Masahiro Hosono, Kazuo Nitta, Naoto Ishizaki, Chikaku Dogasaki, Jiharu Hamako, Taei Matsui, Yasuhiro Ozeki

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16 Citations (Scopus)

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 × 10 3 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.

Original languageEnglish
Pages (from-to)509-519
Number of pages11
JournalProtein Journal
Volume30
Issue number7
DOIs
Publication statusPublished - 10-2011

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Bioengineering
  • Biochemistry
  • Organic Chemistry

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