Isolation and Properties of a Mannan-Binding Lectin from the Coelomic Fluid of the Holothurian Cucumaria japonica

A. A. Bulgakov^1*, E. L. Nazarenko¹, I. Yu. Petrova², M. G. Eliseikina², N. M. Vakhrusheva¹, and V. A. Zubkov¹

¹Pacific Institute of Bioorganic Chemistry, Far East Branch of the Russian Academy of Sciences, pr. 100-letiya Vladivostoka 159, Vladivostok, 690022 Russia; fax: 7 (4232) 31-4050; E-mail: bulgak@piboc.marine.su

²Institute of Marine Biology, Far East Branch of the Russian Academy of Sciences, ul. Pal'chevskogo 17, Vladivostok, 690032 Russia; fax: 7 (4232) 31-0900; E-mail: aisa@vld.global-one.ru

^* To whom correspondence should be addressed.

Received September 6, 1999; Revision received January 14, 2000

---

A new 44-kD, C-type mannan-binding lectin (MBL-C) consisting of two identical subunits was isolated from the coelomic fluid of the holothurian Cucumaria japonica. In the direct hemagglutination assay, the lectin was effectively inhibited by highly branched mannans similar in structure to yeast mannans and composed of alpha-(1-->2)- and alpha-(1-->6)-bound D-mannopyranose residues. Hemagglutination was not inhibited by mannosaccharides, common constituents of the hydrocarbon chains of “normal” glycoproteins. The lectin reaction depends on Ca²⁺ concentration: maximum activity of MBL-C is observed at 10 mM Ca²⁺. The activity of MBL-C increases in the pH range from 5 to 7 and reaches maximum at pH 7.0. The lectin is sensitive to temperature. Heating of the lectin solution at temperatures above 40°C decreases activity, while incubation at 90°C for 1 h leads to complete irreversible inactivation. Carbohydrate specificity, Ca²⁺-dependence, and amino acid composition indicate that MBL-C belongs to the C-type mannan-binding lectins. Polyclonal antibodies against MBL-C revealed its immunochemical similarity to a mannan-binding lectin from another holothurian species, Stichopus japonicus; this provides evidence for structural homology between these proteins.

---

KEY WORDS: mannan-binding lectin, mannan-binding protein, holothurian, Cucumaria japonica, mannans

---