A comparison of the cell-binding characteristics of the mitogenic and nonmitogenic lectins from lima beans.

Using the two lectins from lima bean, we have tested the model for mitogenic stimulation of lymphocytes proposed by Prujansky et al. (Prujansky, A., Ravid, A., and Sharon, N. (1978) Biochim. Biophys. Acta 508, 137-146). The lectins used, a tetramer with two saccharide-binding sites and an octamer with four binding sites, are specific for N-acetyl-D-galactosamine. Our results show that cooperative binding may not be a prerequisite for mitogenicity of all lectins. We found that neither the weakly mitogenic tetramer nor the potently mitogenic octamer bound cooperatively to bovine lymphocytes. The strong mitogen bound with a higher affinity than the weak mitogen and fewer mitogen molecules bound to the lymphocyte surface at saturation. Competitive binding experiments indicated that both lectins bound to the same receptors. Our results suggest that the mitogenic lectin is able to bind and cross-link more membrane receptors. We have also studied the binding of the lima bean lectins to human red blood cells of types A, AB, B, and O. Both lectins bound cooperatively to type A and type AB cells and our data indicate that the lima bean lectins bind predominantly to the type A determinant.

A comparison of the interactions of the mitogenic and nonmitogenic lima bean lectins with human lymphocytes.

Binding and mitogenic characteristics of the 2 Phaseolus lunatus (lima bean) lectins have been examined with human peripheral blood lymphocytes. Chemical cross-linking of the nonmitogenic lima bean lectin produced a species that stimulated human lymphocytes as well as or better than the mitogenic lima bean lectin, which is a T lymphocyte mitogen with requirement for monocyte participation. The maximal stimulation and the dose response to the cross-linked lima bean lectin did not significantly differ from that observed with the mitogenic lectin. We have used fluorescein-labeled lima bean lectins to show that both lectins share mutually exclusive binding sites on lymphocytes. Our results strongly support the concept that multiple valence of lectins is important in inducing mitogenesis. Both the mitogenic and nonmitogenic lectins demonstrated selective binding by labeling only 70% od human peripheral blood lymphocytes. The fraction not binding lectin is a population of T lymphocytes. Competitive binding studies with the lima bean lectins and other N-acetyl-D-galactosamine-specific lectins suggest that the cell surface receptors for these various lectins may be quite different. We have also studied the response of human lymphocytes to the lima bean lectins and soybean agglutinin after neuraminidase treatment. As previously demonstrated (22), neuraminidase treatment of the cells drastically altered the binding and mitogenic response to soybean agglutinin. Lima bean lectin binding to the treated cells was also markedly increased, but the mitogenic response was essentially unaffected.

Activation of concanavalin A by Cd2+.

Binding of Cd2+ to concanavalin A and the subsequent induction of saccharide-binding activity has been studied at pH 6.5. We found that Cd2+ bound to both metal sites, S1 and S2, and that Cd2+ alone would induce sugar binding in concanavalin A. Using the fluorescent sugar 4-methylumbelliferyl alpha-D-mannopyranoside we determined that full saccharide-binding activity was obtained only when the total bound Cd2+ stoichiometry reached 2 ions/concanavalin A subunit. We also report evidence suggesting that the binding of Cd2+ to S2 is the crucial step in activation and that Cd2+ binding to S1 induces a form of concanavalin A similar to that induced by Zn2+, Ni2+, or Co2+ and different from that induced by Mn2+.

Mn2+ and Ca2+ binding to the lima bean lectins.

We have purified the two lectins from lima beans (Phaseolus lunatus) and studied the binding of Ca2+ and Mn2+ ions to each. The 120,000-dalton tetrameric lima bean lectin (LBL4) and the 240,000-dalton octamer (LBL8) were purified by chromatography on Ultrogel and sulfopropyl Sephadex. Using equilibrium dialysis, we have found that demetallized LBL4 (apo-lbl4) will bind either 2 Mn2+ or 4 Ca2+ ions. The Ca2+ binding is strongly cooperative while the Mn2+ binding is not. If apo-LBL4 is presaturated with Mn2+ (Mn2+-LBL4), it will bind only 2 Ca2+ ions in a noncooperative manner. Apo-LBL4 presaturated with Ca2+ (Ca2+-LBL4) does not bind Mn2+. The metal stoichiometry for LBL8 is double that for LBL4 under all of the above conditions.

Calcium-induced cooperativity of manganese binding to concanavalin A.

Titrations employing electron spin resonance spectroscopy and equilibrium dialysis studies have revealed that Mn2+ binding to concanavalin A is cooperative in the presence and noncooperative in the absence of Ca2+. The degree of cooperativity increases with increasing pH. Hill coefficients range from 1.4 at pH 5.0 to 1.8 at pH 6.85. In addition to inducing cooperativity in Mn2+ binding, Ca2+ influences the pH dependence and increases the affinity of Mn2+ binding. In contrast to previous suggestions based mostly on work conducted near pH 5, demetallized concanavalin A does bind Ca2+ with an appreciable binding constant. These observations indicate that at physiological pH the role of metal ions in determining functional properties of concanavalin A is different from that suggested by metal binding studies conducted at lower pH values.