Characterization of functional domains of the hemolytic lectin CEL-III from the marine invertebrate Cucumaria echinata.
CEL-III is a Ca(2+)-dependent, galactose/N-acetylgalactosamine (GalNAc)-specific lectin isolated from the marine invertebrate Cucumaria echinata. This lectin exhibits strong hemolytic activity and cytotoxicity through pore formation in target cell membranes. The amino acid sequence of CEL-III revealed the N-terminal two-thirds to have homology to the B-chains of ricin and abrin, which are galactose-specific plant toxic lectins; the C-terminal one-third shows no homology to any known proteins. To examine the carbohydrate-binding ability of the N-terminal region of CEL-III, the protein comprising Pyr1-Phe283 was expressed in Escherichia coli cells. The expressed protein showed both the ability to bind to a GalNAc-immobilized column as well as hemagglutinating activity for rabbit erythrocytes, confirming that the N-terminal region has binding activity for specific carbohydrates. Since the C-terminal region could not be expressed in E. coli cells, a fragment containing this region was produced by limited proteolysis of the native protein by trypsin. The resulting C-terminal 15 kDa fragment of CEL-III exhibited a tendency to self-associate, forming an oligomer. When mixed with erythrocytes, the oligomer of the C-terminal fragment caused hemagglutination, probably due to hydrophobic interaction with cell membranes, while the monomeric fragment did not. Chymotryptic digestion of the preformed CEL-III oligomer induced upon lactose binding also yielded an oligomer of the C-terminal fragment comprising six molecules of the 16 kDa fragment. These results suggest that after binding to cell surface carbohydrate chains, CEL-III oligomerizes through C-terminal domains, leading to the formation of ion-permeable pores by hydrophobic interaction with the cell membrane.
Kouzuma
Yoshiaki
Y
Laboratory of Biochemistry, Faculty of Agriculture, Graduate School, Kyushu University, Hakozaki 6-10-1 Higashi-ku, Fukuoka 812-8581. kouzuma@mx.ibaraki.ac.jp
Suzuki
Yota
Y
Nakano
Masahiro
M
Matsuyama
Kayo
K
Tojo
Sumiki
S
Kimura
Makoto
M
Yamasaki
Takayuki
T
Aoyagi
Haruhiko
H
Hatakeyama
Tomomitsu
T
D000036:Abrin / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D050260:Carbohydrate Metabolism; D002942:Circular Dichroism; D004910:Erythrocyte Membrane / Q000378:metabolism; D006461:Hemolysis; D006863:Hydrogen-Ion Concentration; D037102:Lectins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D011817:Rabbits; D012276:Ricin / Q000737:chemistry; D012616:Sea Cucumbers / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
C402644:CEL-III lectin, Cucumaria echinata; D037102:Lectins; D000036:Abrin; D012276:Ricin