Chemical modification of the hemolytic lectin CEL-III by succinic anhydride: involvement of amino groups in the oligomerization process.
CEL-III is a Ca(2+)-dependent lectin from a marine invertebrate, Cucumaria echinata, which shows strong hemolytic activity toward human and rabbit erythrocytes. After binding to carbohydrate receptors, CEL-III oligomerizes in the erythrocyte membrane to form ion-permeable pores, leading to the colloid osmotic rupture of the cells. Since hemolysis was greatly increased in the alkaline pH, especially above pH 9, involvement 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 and carbohydrate-binding activities were only slightly affected even after modification of 14 amino groups. A circular dichroism spectrum of modified CEL-III showed almost no change in the secondary structure from that of the native protein, indicating that the decrease of hemolytic activity was not caused by partial unfolding of the protein. Immunoblotting analysis of the erythrocyte membrane treated with modified CEL-III showed a decrease in the formation of CEL-III oligomer in the membrane in parallel with the decrease in hemolytic activity. These results suggest that amino groups of CEL-III are involved in its oligomerization in the cell membrane, and their modification leads to inactivation of the protein without much influence on the carbohydrate-binding activity.
Hatakeyama
T
T
Department of Applied Chemistry, Faculty of Engineering, Nagasaki University, Japan. thata@net.nagasaki-u.ac.jp
Matsuyama
Y
Y
Funada
T
T
Fukuyama
S
S
Kuwahara
H
H
Aoyagi
H
H
Yamasaki
N
N
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002942:Circular Dichroism; D004912:Erythrocytes / Q000187:drug effects*; D006461:Hemolysis; D006801:Humans; D015151:Immunoblotting; D037102:Lectins / Q000737:chemistry*; D009842:Oligopeptides / Q000737:chemistry*; D011485:Protein Binding; D011817:Rabbits; D013387:Succinic Anhydrides
D000596:Amino Acids; D037102:Lectins; D009842:Oligopeptides; D013387:Succinic Anhydrides; C031801:succinic anhydride