J. Biochem, 2003, Vol. 134, No. 3 395-402
© 2003 Japanese Biochemical Society
BIOCHEMISTRY |
Characterization of Functional Domains of the Hemolytic Lectin CEL-III from the Marine Invertebrate Cucumaria echinata
1 Laboratory of Biochemistry, Faculty of Agriculture, Graduate School, Kyushu University, Hakozaki 6-10-1 Higashi-ku, Fukuoka 812-8581; and 2 Department of Applied Chemistry, Faculty of Engineering, Nagasaki University, Nagasaki 852-8521
CEL-III is a Ca2+-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.
* To whom correspondence should be addressed at the present address: College of Agriculture, Ibaraki University, 3-21-1 Chuo, Ami-machi, Inashiki-gun, Ibaraki, 300-0393. Fax: +81-29-888-8682, E-mail: kouzuma{at}mx.ibaraki.ac.jp
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