Calcium ions stabilize a protein structure of hemolytic lectin CEL-III from marine invertebrate Cucumaria echinata.

CEL-III, a galactose/N-acetylgalactosamine (Gal/GalNAc)-specific lectin purified from a marine invertebrate, Cucumaria echinata, has a strong hemolytic activity, especially toward human and rabbit erythrocytes in the presence of Ca2+. We evaluated the role of Ca2+ in hemagglutinating and hemolytic activities of CEL-III. We found that Ca2+ is closely associated with both activities of CEL-III. The fluorescence spectra of CEL-III upon binding to Ca2+ were measured. The result showed a structural change of CEL-III in the presence of Ca2+. The structural change of CEL-III upon Ca2+ binding was further demonstrated by stabilization against urea denaturation and by insusceptibility to protease digestions. CEL-III was completely unfolded at a low concentration of 2 M urea, while CEL-III complexed with Ca2+ was stable in 6 M urea. As for protease digestions, CEL-III monomer and oligomer were readily digested by trypsin, chymotrypsin, and papain in the absence of Ca2+, while they were insusceptible to the three proteases in the presence of Ca2+. The papain digestion of the decalcified oligomer produced a large C-terminal peptide, suggestting that the C-terminal region of CEL-III may participate in oligomerization of CEL-III as a core domain.

Purification and identification of N-glycolylneuraminic acid (Neu5Gc) from the holothuroidea Gumi, Cucumaria echinata.

N-Glycolylneuraminic acid (Neu5Gc), precious sialic acid which could not be synthesized by a chemical method, occurrs in the body of holothuroidea, Gumi Cucumaria echinata. Gumi contains 85% of total sialic acid, as Neu5Gc, in the body. Neu5Gc was purified from dry powder of the body using Dowex 1-x8 (HCOO* form) anion exchange chromatography after mild acid hydrolysis with 0.1 N trifluoroacetic acid. Using GC-MS and 1H-NMR spectroscopy, the purified Neu5Gc was correctly identified to be Neu5Gc. The purity of Neu5Gc was more than 99%. This is the first report of purification and identification of Neu5Gc from holothuroidea by using anion exchange chromatography, GC-MS, and 1H-NMR.

Biological activities of the lectin, abrin-a, against human lymphocytes and cultured leukemic cell lines.

The cytoagglutination by abrin-a against human cultured cell lines derived from acute lymphoblastic leukemia (ALL) and human peripheral blood lymphocytes obtained from normal adults and from patients with adult T cell leukemia (ATL) was investigated. Among acute T lymphoblastic leukemia (T-ALL) cell lines, abrin-a showed strong cytoagglutination against relatively differentiated cell lines, such as Jurkat and CCRF-HSB-2. Among acute B lymphoblastic leukemia (B-ALL) cell lines, abrin-a strongly agglutinated an immature cell line, NALM6. In comparison with ALL cell lines, cytoagglutination by abrin-a against normal lymphocytes was weak. Abrin-a showed higher cytoagglutination against lymphocytes derived from ATL than lymphocytes derived from normal adults. In connection with the cytoagglutination, abrin-a-induced cytotoxicity against human cultured leukemic cell lines was evaluated. In proportion to the extent of cytoagglutination, abrin-a induced cytotoxicity in Jurkat, CCRF-HSB-2, MOLT-4, RPMI8402, and BALL-1 as well. Although CCRF-CEM and BALM-1 were both weakly agglutinated by abrin-a, these cell lines were very sensitive to the abrin-a-induced cytotoxicity. NALM6 was strongly agglutinated by abrin-a, but abrin-a exhibited less strong cytotoxicity against this cell line. These results suggest the feasible application of abrin-a as a tool to distinguish the human leukemic cells and its potential for clinical application.

Interaction of the hemolytic lectin, CEL-III, with cultured human leukemic cell lines.

We studied interaction of CEL-III with cultured human leukemic cell lines and lymphocytes from normal adults by evaluating the extent of cytotoxicity and cytoagglutination. Among acute T lymphoblastic leukemia (T-ALL) cell lines, CEL-III displayed increased toxicity against different acute lymphoblastic leukemia (ALL) cell lines as a function of increasing differentiation stage. In the case of acute B lymphoblastic leukemia (B-ALL) cell lines, CEL-III showed strong cytotoxicity against relatively immature cell lines. We found that CEL-III was more toxic for ALL cell lines than leukocytes obtained from peripheral blood of healthy adults. Strong influence of the additional amount of calcium ion on the extent of cytotoxicity was observed. In addition, we describe a new way to evaluate the extent of cytoagglutination in "% of agglutinated cells". These findings make CEL-III a promising candidate in research for lectins which bind to and destroy only the targeted leukemic cells.

Studies on the carbohydrate binding sites of the hemolytic lectin CEL-III isolated from the marine invertebrate Cucumaria echinata.

The binding of carbohydrates to the hemolytic lectin CEL-III isolated from the marine invertebrate Cucumaria echinata was studied. Equilibrium dialysis data suggest that CEL-III has two carbohydrate-binding sites with equal affinity. The binding of specific carbohydrates to CEL-III induces a decrease in the fluorescence intensity at 339 nm and the shift of the fluorescence emission maximum to a wavelength shorter by 3 nm, owing to the change in the environment of tryptophan. By analyzing the change in the fluorescence intensity at 339 nm as a function of the concentration of carbohydrates, the association constants for binding of individual carbohydrates to CEL-III were calculated. The results indicate that GalNAc, lactulose, and lactose are bound by CEL-III with fairly high affinity among the carbohydrates tested. The pH-dependence profile of the association constant of lactose suggests that CEL-III binds carbohydrates with highest affinity around pH 5.0. Modification of CEL-III with N-bromosuccinimide produces an oxidized derivative, in which four tryptophan residues/mol were oxidized and had no hemolytic activity. However, two out of these four tryptophans escaped from the modification in the presence of specific saccharides and the resulting derivative retained fairly high hemolytic activity.

Carbohydrate-binding properties of the hemolytic lectin CEL-III from the holothuroidea Cucumaria echinata as analyzed using carbohydrate-coated microplate.

The carbohydrate-binding properties of the hemolytic lectin CEL-III from the Holothuroidea Cucumaria echinata were studied using the microplate assay system which we have recently developed [Hatakeyama et al. (1996) Anal. Biochem. 237, 188-192]. When the binding of CEL-III to lactose covalently immobilized on a microplate was examined using colloidal gold solution, the binding was detected with as little as 1 microgram/ml protein. Affinity of several carbohydrates to CEL-III was assessed by means of an inhibition experiment using the lactose-coated plate and it was found that N-acetylgalactosamine has the highest affinity for CEL-III, followed by lactose and lactulose. Examination of the binding of CEL-III to the lactose-coated plate at various pH values and temperatures revealed that the affinity is higher in the acidic pH region and at lower temperatures. From the Ca(2+)-dependence profile for the binding of CEL-III to the lactose-coated plate, the apparent dissociation constant for Ca2+ was estimated to be 2.3 mM. These results suggested that the carbohydrate-binding properties of CEL-III are closely related to its hemolytic activity, although an additional interaction between the protein and the lipid bilayer, which is enhanced in the alkaline pH region, also seems to be necessary for its hemolytic action.