|Budget Amount *help
¥3,300,000 (Direct Cost : ¥3,300,000)
Fiscal Year 1998 : ¥1,400,000 (Direct Cost : ¥1,400,000)
Fiscal Year 1997 : ¥1,900,000 (Direct Cost : ¥1,900,000)
This work was done to reveal the structure-function relationship of the hemolytic lectin CEL- III from the marine invertebrate Cucumaria echinata CEL-III is a Ca^<++> -dependent and Gal/GalNAc specific lectin with a molecular weight of 47, 500. This lectin has a hemolytic activity, especially toward rabbit and human erythrocytes. In the term of the present project, following results were obtained.
First of all, the partial amino acid sequence of CEL-III was analyzed. Although the structure of the N-terminal 40 residue remains undetermined. the amino acid sequence corresponding to 90% of the wholesequence of CEL-III was determined. The undetermined N-terminal residue of CL-ill suggests the occurrence of the modified N-terminus which may be produced by post translational modification of protein, From the determined partial structure, it was suggested that CEL-III has several domain structures.
Analytical data of the interaction of CEL-III with carbohydrates suggest that CEL-III has two car
bohydrate recognition sites per molecule. Spectroscopic and chemical modification studies clearly indicated the significance of the tryptophan residue in the carbohydrate binding. With an active fragment found in the papain digest of GEL-Ill, it was suggested that one of the two carbohydrate- binding sites exists in the N-terminal region of GEL-III.
By analyzing the action profile of CEL-III for liposome as models of erythrocyte membrane, GEL-ill was found to be a "pore-forming protein" which forms ion-permeable pores for liposomes containing glycolipid, leading to the rupture of lipid membranes. Immunoblotting analyses of CEL-III after interaction with liposomes indicate that CEL-III was oligomerized in liposomes. It was also found that oligomerization accompanied the great conformational change of the CEL-III molecule. Significance of amino groups in such a oligomerization process of CEL-III was also suggested.
Finally, CEL-III was found to be a cytotoxic protein, and showed the cytotoxicity for several cultured cells such as Hela cells and Vero cells. Less