Tarun K. Dam

Lectins as pattern recognition molecules: The effects of epitope density in innate immunity

The innate immune response of multicellular organisms is initiated by the binding of soluble and membrane-bound host molecules including lectins to the surface of pathogenic organisms. Until recently, it was believed that the epitopes recognized by host molecules were uniquely associated with the pathogenic organisms. Hence, the term pattern recognition receptors (PRRs) was used to describe their binding specificities. However, with an expanding number of lectin classes including C-type lectins, siglecs, and galectins recognized as PRRs, it is apparent that many of the glycan epitopes recognized on foreign pathogens are present in the host and involved in cellular functions. Hence, the molecular basis for pattern recognition by lectins of carbohydrate epitopes on pathogens is in question. A number of studies indicate that the density and number of glycan epitopes in multivalent carbohydrates and glycoprotein receptors determine the affinity of lectins and their effector functions. This paper reviews lectins that are involved in innate immunity, mechanisms of enhanced affinity and cross-linking of lectins with density-dependent glycan epitopes, density-dependent recognition of glycan receptors by lectins in host systems and lectin-glycan interactions in foreign pathogens. Evidence indicates that lectin pattern recognition in innate immunity is part of a general mechanism of density-dependent glycan recognition. This leads to a new definition of lectin receptor in biological systems, which considers the density and number of glycan epitopes on the surface of cells and not just the affinity of single epitopes.

Binding of multivalent carbohydrates to concanavalin A and Dioclea grandiflora lectin. Thermodynamic analysis of the "multivalency effect".

Binding of a series of synthetic multivalent carbohydrate analogs to the Man/Glc-specific lectins concanavalin A andDioclea grandiflora lectin was investigated by isothermal titration microcalorimetry. Dimeric analogs possessing terminal α-D-mannopyranoside residues, and di-, tri-, and tetrameric analogs possessing terminal 3,6-di-O-(α-d-mannopyranosyl)-α-d-mannopyranoside residues, which is the core trimannoside of asparagine-linked carbohydrates, were selected in order to compare the effects of low and high affinity analogs, respectively. Experimental conditions were found that prevented precipitation of the carbohydrate-lectin cross-linked complexes during the isothermal titration microcalorimetry experiments. The results show that the value of n, the number of binding sites on each monomer of the lectins, is inversely proportional to the number of binding epitopes (valency) of each carbohydrate. Hence,n values close to 1.0, 0.50, and 0.25 were observed for the binding of mono-, di-, and tetravalent sugars, respectively, to the two lectins. Importantly, differences in the functional valency of a triantennary analog for concanavalin A and D. grandifloralectin are observed. The enthalpy of binding, ΔH, is observed to be directly proportional to the number of binding epitopes in the higher affinity analogs. For example, ΔH of a tetravalent trimannoside analog is nearly four times greater than that of the corresponding monovalent analog. Increases inK a values of the multivalent carbohydrates relative to monovalent analogs, known as the “multivalency effect,” are shown to be due to more positive entropy (TΔS) contributions to binding of the former sugars. A general thermodynamic model for distinguishing binding of multivalent ligands to a single receptor with multiple, equal subsitesversus binding to separate receptor molecules is given.

Thermodynamics of Multivalent Carbohydrate−Lectin Cross-Linking Interactions: Importance of Entropy in the Bind and Jump Mechanism

The high affinity (K(d) = 0.2 nM) of the soybean agglutinin (SBA), a tetrameric GalNAc specific lectin, for a modified form of porcine submaxillary mucin, a linear glycoprotein, with a molecular mass of approximately 10(6) Da and approximately 2300 GalNAcalpha1-O-Ser/Thr residues (Tn-PSM) has been ascribed to an internal diffusion mechanism that involves binding and jumping of the lectin from GalNAc to GalNAc residue of the mucin [Dam, T. K., et al. (2007) J. Biol. Chem. 282, 28256-28263]. Hill plot analysis of the raw ITC data shows increasing negative cooperativity, which correlates with an increasing number of lectin-mucin cross-linking interactions and decreasing favorable binding entropies. However, the affinity of bound SBA for other Tn-PSM molecules during cross-linking is much higher than that of free SBA for GalNAcalpha1-O-Ser, a monovalent analogue. The high affinity of bound SBA for GalNAc residues on other Tn-PSM molecules appears to be due to the favorable entropy of binding associated with the internal diffusion mechanism. Furthermore, the increasing negative cooperativity of SBA binding to Tn-PSM correlates with a decreasing level of internal diffusion of the lectin on the mucin as cross-linking occurs. These findings indicate the importance of the internal diffusion mechanism in generating large, favorable entropies of binding that drive lectin-mucin cross-linking interactions. The results are important for understanding the energetics of lectin-mucin cross-linking interactions that are associated with biological signaling on the surface of cells and the role of the internal diffusion mechanism in ligand-biopolymer interactions in general.

Multivalent lectin-carbohydrate interactions energetics and mechanisms of binding.

The biological signaling properties of lectins, which are carbohydrate-binding proteins, are due to their ability to bind and cross-link multivalent glycoprotein receptors on the surface of normal and transformed cells. While the crosslinking properties of lectins with multivalent carbohydrates and glycoproteins are relatively well understood, the mechanisms of binding of lectins to multivalent glycoconjugates are less well understood. Recently, the thermodynamics of binding of lectins to synthetic clustered glycosides, a multivalent globular glycoprotein, and to linear glycoproteins (mucins) have been described. The results are consistent with a dynamic binding mechanism in which lectins bind and jump from carbohydrate to carbohydrate epitope in these molecules. Importantly, the mechanism of binding of lectins to mucins is similar to that for a variety of protein ligands binding to DNA. Recent analysis also shows that high-affinity lectin-mucin crosslinking interactions are driven by favorable entropy of binding that is associated with the bind and jump mechanism. The results suggest that the binding of ligands to biopolymers, in general, may involve a common mechanism that involves enhanced entropic effects which facilitate binding and subsequent complex formation including enzymology.

Multivalent Lectin—Carbohydrate Interactions

The biological signaling properties of lectins, which are carbohydrate-binding proteins, are due to their ability to bind and cross-link multivalent glycoprotein receptors on the surface of normal and transformed cells. While the crosslinking properties of lectins with multivalent carbohydrates and glycoproteins are relatively well understood, the mechanisms of binding of lectins to multivalent glycoconjugates are less well understood. Recently, the thermodynamics of binding of lectins to synthetic clustered glycosides, a multivalent globular glycoprotein, and to linear glycoproteins (mucins) have been described. The results are consistent with a dynamic binding mechanism in which lectins bind and jump from carbohydrate to carbohydrate epitope in these molecules. Importantly, the mechanism of binding of lectins to mucins is similar to that for a variety of protein ligands binding to DNA. Recent analysis also shows that high-affinity lectin-mucin crosslinking interactions are driven by favorable entropy of binding that is associated with the bind and jump mechanism. The results suggest that the binding of ligands to biopolymers, in general, may involve a common mechanism that involves enhanced entropic effects which facilitate binding and subsequent complex formation including enzymology.

Binding Studies of α-GalNAc-specific Lectins to the α-GalNAc (Tn-antigen) Form of Porcine Submaxillary Mucin and Its Smaller Fragments

Isothermal titration microcalorimetry (ITC) and hemagglutination inhibition measurements demonstrate that a chemically and enzymatically prepared form of porcine submaxillary mucin that possesses a molecular mass of ∼106 daltons and ∼2300 α-GalNAc residues (Tn-PSM) binds to the soybean agglutinin (SBA) with a Kd of 0.2 nm, which is ∼106-fold enhanced affinity relative to GalNAcα1-O-Ser (Tn), the pancarcinoma carbohydrate antigen. The enzymatically derived 81 amino acid tandem repeat domain of Tn-PSM containing ∼23 α-GalNAc residues binds with ∼103-fold enhanced affinity, while the enzymatically derived 38/40 amino acid cleavage product(s) of Tn-PSM containing ∼11-12 α-GalNAc residues shows ∼102-fold enhanced affinity. A natural carbohydrate decorated form of PSM (Fd-PSM) containing 40% of the core 1 blood group type A tetrasaccharide, and 58% peptide-linked GalNAcα1-O-Ser/Thr residues, with 45% of the peptide-linked α-GalNAc residues linked α-(2,6) to N-glycolylneuraminic acid, shows ∼104 enhanced affinity for SBA. Vatairea macrocarpa lectin (VML), which is also a GalNAc binding lectin, displays a similar pattern of binding to the four forms of PSM, although there are quantitative differences in its affinities as compared with SBA. The higher affinities of SBA and VML for Tn-PSM relative to Fd-PSM indicate the importance of carbohydrate composition and epitope density of mucins on their affinities for lectins. The higher affinities of SBA and VML for Tn-PSM relative to its two shorter chain analogs demonstrate that the length of a mucin polypeptide and hence total carbohydrate valence determines the affinities of the three Tn-PSM analogs. The results suggest a binding model in which lectin molecules “bind and jump” from α-GalNAc residue to α-GalNAc residue along the polypeptide chain of Tn-PSM before dissociating. The complete thermodynamic binding parameters for these mucins including their binding stoichiometries are presented. The results have important implications for the biological activities of mucins including those expressing the Tn cancer antigen.

Diocleinae Lectins Are a Group of Proteins with Conserved Binding Sites for the Core Trimannoside of Asparagine-linked Oligosaccharides and Differential Specificities for Complex Carbohydrates*

The seed lectin from Dioclea grandiflora and jack bean lectin concanavalin A (ConA) are both members of the Diocleinae subtribe of Leguminosae lectins. Both lectins have recently been shown to possess enhanced affinities and extended binding sites for the trisaccharide, 3,6-di-O-(α-d-mannopyranosyl)-d-mannose, which is present in the core region of all asparagine-linked carbohydrates (Gupta, D., Oscarson, S., Raju, S., Stanley, P. Toone, E. J. and Brewer, C. F. (1996) Eur. J. Biochem.242, 320–326). In the present study, the binding specificities of seven other lectins from the Diocleinae subtribe have been investigated by hemagglutination inhibition and isothermal titration microcalorimetry (ITC). The lectins are from Canavalia brasiliensis, Canavalia bonariensis, Cratylia floribunda, Dioclea rostrata, Dioclea virgata, Dioclea violacea, and Dioclea guianensis. Hemagglutination inhibition and ITC experiments show that all seven lectins are Man/Glc-specific and have high affinities for the core trimannoside, like ConA and D. grandifloralectin. All seven lectins also exhibit the same pattern of binding to a series of monodeoxy analogs and a tetradeoxy analog of the trimannoside, similar to that of ConA and D. grandifloralectin. However, C. bonariensis, C. floribunda,D. rostrata, and D. violacea, like D. grandiflora, show substantially reduced affinities for a biantennary complex carbohydrate with terminal GlcNAc residues, whileC. brasiliensis, D. guianensis, and D. virgata, like ConA, exhibit affinities for the oligosaccharide comparable with that of the trimannoside. Thermodynamic data obtained by ITC indicate different energetic mechanisms of binding of the above two groups of lectins to the complex carbohydrate. The ability of the lectins to induce histamine release from rat peritoneal mast cells is shown to correlate with the relative affinities of the proteins for the biantennary carbohydrate.

Garlic (Allium sativum) lectins bind to high mannose oligosaccharide chains

Two mannose-binding lectins, Allium sativum agglutinin (ASA) I (25 kDa) and ASAIII (48 kDa), from garlic bulbs have been purified by affinity chromatography followed by gel filtration. The subunit structures of these lectins are different, but they display similar sugar specificities. Both ASAI and ASAIII are made up of 12.5- and 11.5-kDa subunits. In addition, a complex (136 kDa) comprising a polypeptide chain of 54 ± 4 kDa and the subunits of ASAI and ASAIII elutes earlier than these lectins on gel filtration. The 54-kDa subunit is proven to be alliinase, which is known to form a complex with garlic lectins. Constituent subunits of ASAI and ASAIII exhibit the same sequence at their amino termini. ASAI and ASAIII recognize monosaccharides in mannosyl configuration. The potencies of the ligands for ASAs increase in the following order: mannobiose (Manα1–3Man) < mannotriose (Manα1–6Manα1–3Man) ≈ mannopentaose ≪ Man9-oligosaccharide. The addition of two GlcNAc residues at the reducing end of mannotriose or mannopentaose enhances their potencies significantly, whereas substitution of both α1–3- and α1–6-mannosyl residues of mannotriose with GlcNAc at the nonreducing end increases their activity only marginally. The best manno-oligosaccharide ligand is Man9GlcNAc2Asn, which bears several α1–2-linked mannose residues. Interaction with glycoproteins suggests that these lectins recognize internal mannose as well as bind to the core pentasaccharide of N-linked glycans even when it is sialylated. The strongest inhibitors are the high mannose-containing glycoproteins, which carry larger glycan chains. Indeed, invertase, which contains 85% of its mannose residues in species larger than Man20GlcNAc, exhibited the highest binding affinity. No other mannose- or mannose/glucose-binding lectin has been shown to display such a specificity.

Thermodynamics of Lectin-Carbohydrate Interactions BINDING OF THE CORE TRIMANNOSIDE OF ASPARAGINE-LINKED CARBOHYDRATES AND DEOXY ANALOGS TO CONCANAVALIN A

The trisaccharide 3,6-di-O-(α-D-mannopyranosyl)-D-mannose, which is present in all asparagine-linked carbohydrates, was previously shown by titration microcalorimetry to bind to the lectin concanavalin A (ConA) with nearly −6 kcal mol−1 greater enthalpy change and 60-fold higher affinity than methyl-α-D-mannopyranoside (Mandal, D. K., Kishore, N., and Brewer, C. F. (1994) Biochemistry 33, 1149-1156). Similar studies of the binding of a series of monodeoxy derivatives of the α(1-3) residue of the trimannoside showed that this arm was required for high affinity binding (Mandal, D. K., Bhattacharyya, L., Koenig, S. H., Brown, R. D., III, Oscarson, S., and Brewer, C. F. (1994) Biochemistry 33, 1157-1162). In the present paper, a series of monodeoxy derivatives of the α(1-6) arm and “core” Man residue of the trimannoside as well as dideoxy and trideoxy analogs were synthesized. Isothermal titration microcalorimetry experiments establish that the 3-, 4-, and 6-hydroxyl groups of the α(1-6)Man residue of the trimannoside binds to the lectin, along with the 2- and 4-hydroxyl groups of the core Man residue and the 3- and 4-hydroxyl groups of the α(1-3)Man residue. Dideoxy analogs and trideoxy analogs showed losses of affinities and enthalpy values consistent with losses in binding of specific hydroxyl groups of the trimannoside. The free energy and enthalpy contributions to binding of individual hydroxyl groups of the trimannoside determined from the corresponding monodeoxy analogs are observed to be nonlinear, indicating differential contributions of the solvent and protein to the thermodynamics of binding of the analogs. The thermodynamic solution data agree well with the recent x-ray crystal structure of ConA complexed with the trimannoside (Naismith, J. H., and Field, R. A. (1996) J. Biol. Chem. 271, 972-976).

Isothermal titration calorimetry reveals differential binding thermodynamics of variable region-identical antibodies differing in constant region for a univalent ligand

The classical view of immunoglobulin molecules posits two functional domains defined by the variable (V) and constant (C) regions, which are responsible for antigen binding and antibody effector functions, respectively. These two domains are thought to function independently. However, several lines of evidence strongly suggest that C region domains can affect the specificity and affinity of an antibody for its antigen (Ag), independent of avidity-type effects. In this study, we used isothermal titration calorimetry to investigate the thermodynamic properties of the interactions of four V region-identical monoclonal antibodies with a univalent peptide antigen. Comparison of the binding of IgG1, IgG2a, IgG2b, and IgG3 with a 12-mer peptide mimetic of Cryptococcus neoformans polysaccharide revealed a stoichiometry of 1.9–2.0 with significant differences in thermodynamic binding parameters. Binding of this peptide to the antibodies was dominated by favorable entropy. The interaction of these antibodies with biotinylated peptides manifested greater enthalpy than for native peptides indicating that biotin labeling affected the types of Ag-Ab complexes formed. Our results provide unambiguous thermodynamic evidence for the notion that the C region can affect the interaction of the V region with an Ag.