CHEMICAL MODIFICATION OF THE HEMOLYTIC LECTIN CEL-III BY SUCCINIC ANHYDRIDE - INVOLVEMENT OF AMINO-GROUPS IN THE OLIGOMERIZATION PROCESS

CEL-III is a Ca2+-dependent lectin from a marine invertebrate, Cucumar ia echinata, which shows strong hemolytic activity toward human and ra bbit erythrocytes. After binding to carbohydrate receptors, CEL-III ol igomerizes in the erythrocyte membrane to form ion-permeable pores, le ading to the colloid osmotic rupture of the cells. Since hemolysis was greatly increased in the alkaline pH, especially above pH 9, involvem ent of amino groups of CEL-III in its hemolytic activity was evaluated using chemical modification by succinic anhydride. After modification of 7 amino groups per protein molecule, the hemolytic activity of CEL -III was reduced to 23% of the native protein, but hemagglutinating an d carbohydrate-binding activities were only slightly affected even aft er modification of 14 amino groups. A circular dichroism spectrum of m odified CEL-III showed almost no change in the secondary structure fro m that of the native protein, indicating that the decrease of hemolyti c activity was not caused by partial unfolding of the protein. Immunob lotting analysis of the erythrocyte membrane treated with modified CEL -III showed a decrease in the formation of CEL-III oligomer in the mem brane in parallel with the decrease in hemolytic activity. These resul ts suggest that amino groups of GEL-III are involved in its oligomeriz ation in the cell membrane, and their modification leads to inactivati on of the protein without much influence on the carbohydrate-binding a ctivity.