John L. Wang

RELATIONSHIPS BETWEEN THE STRUCTURE AND ACTIVITIES OF CONCANAVALIN A

Concanavalin A (Con A ) , the lectin of the jack bean (Canavalia ensiformis), is one of the most widely used of the plant agglutinins in biological studies of eukaryotic cells. This protein has a number of activities that are useful for studies of cell surfaces and cell division. These activities, which may proceed by a variety of mechanisms, all appear to be dependent on the initial binding of Con A to specific saccharide-containing receptors on the cell surface, for they can be inhibited by appropriate specific monosaccharides such as D glucose and D-mannose, but no; by related compounds such as D-galactose.’ and germ line cells,3 presumably by virtue of its multiple valence for cell surface receptors, but frequently there are differences in the agglutinability of normal, trypsinized, or virus-transformed cells of the same cell line.4. These differences appear to arise from variations in the distribution, rather than in the number, of Con A receptors on the various types of cell.G-s Con A can also bind to adipocytes, with which it is as effective as insulin in enhancing the rate of glucose transport and inhibiting epinephrine-stimulated lipolysis.” Con A is a competitive inhibitor of insulin binding to these cells, and the lectin binding is itself inhibited by a-methylmannoside.” Perhaps the most extensively studied activity of Con A is its ability to induce mitogenesis in lymphocytes,10. l1 a phenomenon analogous to the stimulation of immune cells by specific antigens. It is striking that the binding of Con A to lymphocytes alters the mobility and distribution of various receptor molecules on the cell surface. Under appropriate conditions, Con A in low doses forms “caps” with its own glycoprotein receptors.12* l 3 On the other hand, higher concentrations of Con A inhibit the formation of “patches” and “caps” induced on lymphocytes by antibodies and other reagent^.^ All of these properties of Con A make it a powerful probe for studies of the cell surface and of the regulation of cell metabolism and cell division. In order to understand the molecular basis for the activities of Con A, we have undertaken extensive studies of its structure and function. We have recently reported the amino acid sequence and complete three-dimensional structure of Con A.I5 We have also prepared chemically modified forms of Con A, with altered subunit structure and altered biological properties.lX The results of these studies have suggested a number of useful hypotheses for further analysis of the biological activities of Con A. It has been found that Con A can agglutinate a variety of somatic *,

Kinetics of colchicine inhibition of mitogenesis in individual lymphocytes

Abstract Colchicine inhibits the mitogenic stimulation of lymphocytes from various species by lectins such as concanavalin A (ConA). Removal of ConA from the cell surface by competitive inhibition with α-methyl- d -mannoside revealed that longer exposures to the lectin result in an increase in the number of cells that are committed to blast transformation and thymidine incorporation. A similar kinetic analysis of the effects of colchicine addition without lectin removal showed that the drug blocks stimulation early in the sequence of events of lymphocyte activation and that the time of inhibition was correlated with the kinetics of cellular commitment to lectin stimulation. Moreover, simultaneous addition of both α-methyl- d -mannoside and colchicine did not alter the time course of commitment to mitogenesis, suggesting that both inhibitory effects occurred in similar or identical cell populations. The results are consistent with the hypothesis that the colchicine blockade occurs temporally near or at the event that commits the individual lymphocyte to undergo blast transformation and DNA synthesis. These observations support the hypothesis that cytoplasmic microtubular proteins play a role in the regulation of mitogenic stimulation.

Structure and Function of Concanavalin A

Lectins have been extensively used to analyze a variety of fundamental processes in cell biology. In conjuntion with our studies on the cell surface and mitosis, we have determined the amino acid sequence and three-dimensional struction of concanavalin A (Con A), the mitogenic lectin from the jack bean. Knowledge of the structure has been helpful in interpreting experiments on lymphocyte mitogenesis and the effects of Con A on cell surface receptor mobility. Con A subunits for molecular weight 25,500 are folded into dome-like structures of maximum dimensions 42 times 40 times 39 A. The domes are related by 222 symmetry to form roughly tetrahedral tetramers. Each subunit contains two large antiparallel pleated sheets, and subunits are joined to form dimers and tetramers by interactions involving one of these pleated sheets. We have examined the binding of a variety of carbohydrates to Con A and have obtained preliminary data which suggest that there are differences in the saccharide-binding behavior of Con A in solution and in the crystalline state. Dimeric chemical derivatives of Con A have been prepared and shown to have biological activities different from those of the native tetrameric protein. Under different conditions, native Con A exhibits two antagonistic activities on the lymphoid cell surface: the induction of cap formation by its own receptors and the inhibition of the mobility of a variety of receptors, including its own receptors. The dimeric derivative, succinyl-Con A, is just as effective a mitogen as the native lectin, but it lacks the ability to modulate cell surface receptor mobility. The data suggest that neither extensive immobilization of cell surface receptors nor cap formation is required for cell stimulation. Further studies on modulation of receptor translocation suggest that hypothesis that there exists a connecting network of colchicine-sensitive proteins that links receptors of different kinds and mediates their rearrangement. The degree of connectivity of this postulated network appears to be altered by changes in the state of attachment of various surface receptors to the network. Thus the network might provide the cell with a means of transmitting signals such as the stimulus for mitosis by lectins or antigens.

Favin, a crystalline lectin from Vicia faba

Abstract A lectin from the fava bean (Vicia faba) has been purified and crystallized in a form suitable for high-resolution crystallographic structure analysis. This protein binds glucose- and mannose-like saccharides, and it is mitogenic for lymphocytes. The fava lectin crystallizes in the orthorhombic space group. P212121 with unit cell dimensions a = 90.0, b = 89.3, and c = 67.4 A . The mass of protein in the asymmetric unit is 53,000 daltons, corresponding to the molecular weight of the protein in solution.

The effects of sodium metaperiodate on t and b lymphocytes.

Abstract The differential mitogenic response of T and B lymphocytes to sodium metaperiodate has been investigated. It was found that periodate treatment leads to lymphocyte stimulation in spleen cells from Balb/c mice but not in spleen cells from the congenitally athymic nu/nu mice. In addition, treatment of Balb/c spleen cells with anti-θ serum plus complement lowers the mitogenic response to periodate and to concanavalin A without affecting the response to lipopolysaccharide. These results suggest a requirement for the presence of T lymphocytes in the initiation of a response to periodate. Spleen cells from nude mice also react with periodate, and their ability to respond to B cell mitogens is impaired after treatment with the chemical reagent.

Evidence for Conformational Changes in Concanavalin A Upon Binding of Saccharides as Determined from Solvent Water Proton Magnetic Relaxation Rate Dispersion Measurements

In previous studies of the interaction of solvent water molecules with the Mn++ ion in Mn-Con A by observation of the dispersion of the spin-lattice relaxation rate (T) of the solvent water protons over a wide range of magnetic fields (Koenig et al., 1973), we have shown that this rate is dominated by the residence time of a single exchanging water ligand on the Mn++ ion. In limited measurements at low fields, we also observed that the binding of α- or β- methyl-D-glucopyranoside to Mn-Con A decreased the relaxation rate by approximately 15 percent. In the present study, we have measured the effects of binding of a series of mono- and oligosaccharides on the solvent water proton relaxation rate over a range of magnetic fields from 5 Oe to 12 KOe and show that the observed decrease in the relaxation rate is due to an increase in the residence time of the single exchanging water ligand. This effect is consistent with a conformational change in the protein upon binding of saccharides. We find that the binding of α- and β- methyl-D-glucopyranoside, α- methyl-D-mannopyranoside and β-(o-iodophenyl)-D-glucopyranoside produce the same increase in residence time and therefore the same conformational change in the protein, whereas galactose and β- (o-iodophenyl)-D-galactopyranoside show no effects. The same reduction in relaxation rate as that caused by the above monosaccharides was observed with the following oligosaccharides: D-maltose, D-maltotriose, D-maltotetraose, o-α-D-mannopyranosyl-(1→2)-D-mannose, o-α-D-mannopyranosyl-(1→2)-o-α-D-mannopyranosyl-(1→2)-D-mannose, o-α-D-mannopyran-osyl-(1→2)-o-α-D-mannopyranosyl-(1→2)-0-α-D-mannopyranosyl-(1→2)-D-mannose and melezitose. As observed by Goldstein and co-workers, the first three oligosaccharides have nearly the same affinity constant, whereas the α-(1,2) linked mannans show increasing affinity constants with increasing chain length. Melezitose also shows enhanced binding by a factor of four relative to α-methyl-D-glucopyranoside. The water relaxation data suggest that the above mono- and oligosaccharides bind to Con A by a similar mechanism involving only a single saccharide residue combined with the protein at one time. Determination of the enthalpy (∆H) and entropy (∆S) of binding of maltotriose and melezitose indicates that the factor of 8 difference in their affinity constants is due to different ∆S values. This suggests that the greater affinity of melezitose is due to the presence of two glucose residues in the molecule, either of which is capable of binding to the same site on the protein. The increased probability of binding for melezitose results in a larger forward rate constant relative to maltotriose which has only one glucose residue which can bind to the protein. Thus, the greater affinity of the α-(1→2)-mannose oligosaccharides appears to be due to a statistical increase in probability of binding because of the presence of more than one binding residue in the chain and not to an extended binding site on the protein.