Purification and characterisation of a lectin isolated from the Manila clam Ruditapes philippinarum in Korea.
The characteristics of a lectin from the marine bivalve Ruditapes philippinarum (Manila clam) were investigated in this study. A method was developed for the isolation of the Manila clam lectin (MCL). Affinity chromatography using mucin-Sepharose, ion-exchange chromatography with DEAE-Toyoperl, and gel filtration with Superose 6 were used for MCL isolation. SDS-PAGE showed that the MCL protein had a molecular mass of 138 kDa, and consisted of 74-, 34-, and 30-kDa subunits. The native lectin in solution behaved as a 274-kDa protein in gel filtration chromatography. The lectin activity of MCL was Ca2+ -dependent, and the optimal Ca2+ concentration for MCL activity was 20 mM. MCL activity was stable between pH 6 and pH 9, and was temperature-dependent; incubation of MCL at 90 degrees C led to irreversible denaturation. The activity of MCL was not inhibited by the presence of monosaccharides, such as Man, Fuc, Gal, Glc, GlcNAc, and NeuNAc. In contrast, the lectin activity of MCL was strongly inhibited by the presence of porcine mucins. MCL activity was also inhibited by N-acetyl-d-galactosamine, human embryonic alpha-1-acid glycoprotein, and highly branched mannans from marine halophilic bacteria. It appears that MCLs have unusual carbohydrate specificities for N-acetyl-d-galactosamine, which contains both mucin-type carbohydrate chains and highly branched mannans. Immunofluorescence staining revealed that MCL was bound to the surfaces of purified hypnospores from Perkinsus sp., which is a protozoan parasite of Manila clams.
Bulgakov
Alexander A
AA
School of Applied Marine Science, College of Ocean Science, Cheju National University, Jeju, Republic of Korea.
Park
Kyung-Il
KI
Choi
Kwang-Sik
KS
Lim
Hee-Kyoung
HK
Cho
Moonjae
M
D000117:Acetylglucosamine / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D049872:Bivalvia / Q000737:chemistry* / Q000469:parasitology; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D056890:Eukaryota / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006386:Hemagglutination Tests; D006863:Hydrogen-Ion Concentration; D007723:Korea; D037102:Lectins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification*; D008024:Ligands; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020539:Sequence Analysis, Protein
D037102:Lectins; D008024:Ligands; D000117:Acetylglucosamine