Interaction of human erythrocyte Band 3 with Ricinus communis agglutinin and other lectins.

Agglutination and competition studies suggest that human erythrocyte Band 3 can interact with both mannose/glucose- and galactose-specific lectins. Purified Band 3 reconstituted into lipid vesicles binds concanavalin A, but the nonspecific binding component, measured in the presence of alpha-methylmannoside, is very high. This glycoprotein also carries binding sites for the galactose-specific lectin Ricinus communis agglutinin. Binding was inhibited poorly by lactose, but much more effectively by desialylated fetuin glycopeptides, suggesting that the lectin recognizes a complex oligosaccharide sequence on Band 3. The glycoprotein bears two separate classes of binding sites for R. communis agglutinin. High-affinity binding sites exist which show strong positive cooperativity and correspond in number to the outward-facing Band 3 molecules. A low-affinity binding mode is abolished by 40% ethyleneglycol, suggesting the involvement of hydrophobic lectin-glycoprotein interactions. Studies on binding of R. communis agglutinin to human erythrocytes indicate positively cooperative binding to 7 X 10(5) very-high-affinity sites per cell, and lectin binding is completely inhibitable by lactose. Based on its binding characteristics in vesicles, it seems likely that Band 3 forms the major receptor for this lectin in human erythrocytes. Properties such as positive cooperativity thus appear to be a common feature of the interaction of Band 3 with a variety of lectins of different specificity, both in erythrocytes and lipid bilayers.

Effects of bovine serum albumin on the interaction of concanavalin A and succinyl-concanavalin A with phospholipid bilayers.

Under physiological conditions, concanavalin A interacts with the surface of phospholipid liposomes through two distinct classes of binding sites, a relatively small number of high affinity sites and a much larger number of lower affinity sites. Addition of bovine serum albumin induces extensive additional binding of concanavalin A to liposomal membranes and this binding is saturable and "specific" (alpha-methyl mannoside inhibitable). Fraction V and high purity albumin both induce almost identical levels of concanavalin A binding to liposomes. Scatchard plots of the binding data demonstrated the induction of a large number of new, relatively high affinity lectin-binding sites on addition of albumin. Albumin-induced binding of concanavalin A to the bilayer surface shows a broad pH optimum and is not inhibited by 40% (w/v) ethylene glycol, suggesting that hydrophobic forces are relatively unimportant. In contrast, divalent succinyl-concanavalin A shows very little tendency to bind to liposomes, either in the absence or presence of albumin. Passage of high purity albumin down a concanavalin A affinity column or treatment with periodate completely eliminates the additional lectin binding. It thus seems likely that albumin-induced concanavalin A binding to liposomes is related to the presence of a concanavalin-A-binding component. This phenomenon may have important implications for lectin-binding studies carried out on membranes which have been exposed to serum proteins.

Lipid-protein interactions of the human erythrocyte concanavalin A receptor in phospholipid bilayers.

The interaction of the human erythrocyte concanavalin A receptor (a subpopulation of Band 3) with phospholipids has been investigated using differential scanning microcalorimetry of reconstituted vesicles prepared by detergent dialysis. The mean diameter of dialyzed phospholipid vesicles jumps dramatically on inclusion of the concanavalin A receptor and then increases linearly with the fraction of protein in the bilayer. The glycoprotein has a dramatic effect on the phospholipid gel to liquid-crystalline phase transition, and delta H decreases linearly with increasing mole fraction of protein up to a protein/lipid mole ratio of around 1:1160. Extrapolation of this data indicates that each concanavalin A receptor is able to perturb about 685 molecules of dimyristoylphosphatidylcholine, withdrawing them from the main phase transition. The cooperativity of phospholipid melting is profoundly disrupted by small amounts of glycoprotein, with the cooperative unit dropping to less than half its initial values at a protein/lipid mole ratio of 1:3800. A break occurs in the delta H curve as the protein/lipid mole ratio is increased above 1:1160, and delta H then increases linearly with increasing amounts of concanavalin A receptor in the bilayer. This phenomenon may be interpreted in terms of protein-protein aggregation which occurs in the phospholipid bilayer above a certain critical mole fraction of concanavalin A receptor, resulting in perturbed phospholipids being returned to the phase transition. In addition, the hydrophilic domains of the glycoprotein may exist in two different conformations depending on the protein concentration in the bilayer, and these may differ in their ability to interact with phospholipid headgroups at the membrane surface.

The concanavalin A receptor from human erythrocytes in lipid bilayer membranes. Interaction with concanavalin A and succinyl-concanavalin A.

The concanavalin A receptor from human erythrocyte membranes has been isolated by affinity chromatography using the mild, readily-dialyzable detergent dodecyltrimethylammonium bromide. The purified protein has been reincorporated into large unilamellar phospholipid vesicles using a detergent dialysis technique. The mean diameter of these vesicles increases as the lipid: protein ratio decreases. Binding of succinyl-concanavalin A to these vesicles was quantitated using 125I-labelled lectin in a filtration assay. The concanavalin A receptor in lipid bilayer vesicles provides specific high affinity binding sites for succinyl-concanavalin A with an association constant of 2.13 . 10(6) M-1. Scatchard plots indicate positive cooperativity of binding at very low lectin concentrations, a characteristic also seen in concanavalin A binding to intact human erythrocytes. The presence of bovine serum albumin has little effect on lectin binding and is not required for expression of cooperativity. Concanavalin A effectively competes with succinyl-concanavalin A for binding to the vesicles with an association constant of 4.83 . 10(6) M-1. Receptor-bearing vesicles are readily agglutinated by concanavalin A but not by its succinylated derivative. The kinetics of vesicle agglutination are biphasic, with an initial rapid phase followed by a pseudo-first order process. We suggest that studies on reassembled receptor proteins in lipid bilayers can provide valuable insight into receptor involvement in transmembrane signalling events and the factors involved in cell membrane behaviour and cell agglutination.