Mechanism of multivalent glycoconjugate-lectin interaction: An update.

Lectins are predominantly oligomeric proteins with several binding sites per molecule. Glycoconjugates are their natural ligands, which often possess multiple binding epitopes. Thus, lectin-glycoconjugate interactions are mostly multivalent in nature. The mechanism of multivalent binding is fundamentally different from those described for monovalent interactions in textbooks and research papers. Over the years, binding studies that make use of different lectins and a variety of multivalent glycoconjugate ligands were conducted in order to understand the underlying principles of multivalency. Starting with seemingly simple synthetic multivalent analogs, systematic studies were carried out using natural glycoconjugate ligands with increasing valency and complexity. Those ligands included multivalent glycoproteins, polyvalent polysaccharides, including glycosaminoglycans, as well as supra-valent mucins and proteoglycans. Models and mechanisms of multivalent binding derived from quantitative data are summarized in the present updated review.

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 cross-linking 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 cross-linking 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.

Mechanism of Mucin Recognition by Lectins: A Thermodynamic Study.

Isothermal titration microcalorimetry (ITC) can directly determine the thermodynamic binding parameters of biological molecules including affinity constant, binding stoichiometry, heat of binding (enthalpy) and indirectly the entropy, and free energy of binding. ITC has been extensively used to study the binding of lectins to mono- and oligosaccharides, but limitedly in applications to lectin-glycoprotein interactions. Inherent experimental challenges to ITC include sample precipitation during the experiment and relative high amount of sample required, but careful design of experiments can minimize these problems and allow valuable information to be obtained. For example, the thermodynamics of binding of lectins to multivalent globular and 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 leading to increased affinity. Importantly, the mechanism of binding of lectins to mucins appears similar to that for a variety of protein ligands binding to DNA. Recent results also show that high-affinity lectin-mucin cross-linking 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 that facilitate binding interactions.

Tn and STn are members of a family of carbohydrate tumor antigens that possess carbohydrate-carbohydrate interactions.

The mucin-type O-glycome in cancer aberrantly expresses the truncated glycans Tn (GalNAcα1-Ser/Thr) and STn (Neu5Acα2,6GalNAcα1-Ser/Thr). However, the role of Tn and STn in cancer and other diseases is not well understood. Our recent discovery of the self-binding properties (carbohydrate-carbohydrate interactions, CCIs) of Tn (Tn-Tn) and STn (STn-STn) provides a model for their possible roles in cellular transformation. We also review evidence that Tn and STn are members of a larger family of glycan tumor antigens that possess CCIs, which may participate in oncogenesis.

Interactions of mucins with the Tn or Sialyl Tn cancer antigens including MUC1 are due to GalNAc-GalNAc interactions.

The molecular mechanism(s) underlying the enhanced self-interactions of mucins possessing the Tn (GalNAcα1-Ser/Thr) or STn (NeuNAcα2-6GalNAcα1-Ser/Thr) cancer markers were investigated using optical tweezers (OT). The mucins examined included modified porcine submaxillary mucin containing the Tn epitope (Tn-PSM), ovine submaxillary mucin with the STn epitope (STn-OSM), and recombinant MUC1 analogs with either the Tn and STn epitope. OT experiments in which the mucins were immobilized onto polystyrene beads revealed identical self-interaction characteristics for all mucins. Identical binding strength and energy landscape characteristics were also observed for synthetic polymers displaying multiple GalNAc decorations. Polystyrene beads without immobilized mucins showed no self-interactions and also no interactions with mucin-decorated polystyrene beads. Taken together, the experimental data suggest that in these molecules, the GalNAc residue mediates interactions independent of the anchoring polymer backbone. Furthermore, GalNAc-GalNAc interactions appear to be responsible for self-interactions of mucins decorated with the STn epitope. Hence, Tn-MUC1 and STn-MUC1 undergo self-interactions mediated by the GalNAc residue in both epitopes, suggesting a possible molecular role in cancer. MUC1 possessing the T (Galß1-3GalNAcα1-Ser/Thr) or ST antigen (NeuNAcα2-3Galß1-3GalNAcα1-Ser/Thr) failed to show self-interactions. However, in the case of ST-MUC1, self-interactions were observed after subsequent treatment with neuraminidase and ß-galactosidase. This enzymatic treatment is expected to introduce Tn-epitopes and these observations thus further strengthen the conclusion that the observed interactions are mediated by the GalNAc groups.

Probing lectin-mucin interactions by isothermal titration microcalorimetry.

Isothermal titration microcalorimetry (ITC) can directly determine the thermodynamic binding parameters of biological molecules including affinity constant, binding stoichiometry, and heat of binding (enthalpy) and indirectly the entropy and free energy of binding. ITC has been extensively used to study the binding of lectins to mono- and oligosaccharides, but limited applications to lectin-glycoprotein interactions. Inherent experimental challenges to ITC include sample precipitation during the experiment and relative high amount of sample required, but careful design of experiments can minimize these problems and allow valuable information to be obtained. For example, the thermodynamics of binding of lectins to multivalent globular and 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 leading to increased affinity. Importantly, the mechanism of binding of lectins to mucins appears similar to that for a variety of protein ligands binding to DNA. Recent results also show that high affinity lectin-mucin cross-linking 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 that facilitate binding interactions.

Single molecule study of heterotypic interactions between mucins possessing the Tn cancer antigen.

Mucins are linear, heavily O-glycosylated proteins with physiological roles that include cell signaling, cell adhesion, inflammation, immune response and tumorgenesis. Cancer-associated mucins often differ from normal mucins by presenting truncated carbohydrate chains. Characterization of the binding properties of mucins with truncated carbohydrate side chains could thus prove relevant for understanding their role in cancer mechanisms such as metastasis and recognition by the immune system. In this work, heterotypic interactions of model mucins that possess the Tn (GalNAcαThr/Ser) and T (Galß1-3GalNAcαThr/Ser) cancer antigens derived from porcine submaxillary mucin (PSM) were studied using atomic force microscopy. PSM possessing only the Tn antigen (Tn-PSM) was found to bind to PSM analogs possessing a combination of T, Tn and STn antigens as well as biosynthetic analogs of the core 1 blood group A tetrasaccharide (GalNAcα1-3[Fucα1-2] Galß1-3GalNAcαSer/Thr). The rupture forces for the heterotypic interactions ranged from 18- to 31 pN at a force-loading rate of ∼0.5 nN/s. The thermally averaged distance from the bound complex to the transition state (xß) was estimated to be in the range 0.37-0.87 nm for the first barrier of the Bell Evans analysis and within 0.34-0.64 nm based on a lifetime analysis. These findings reveal that the binding strength and energy landscape for heterotypic interactions of Tn-PSM with the above mucins, resemble homotypic interactions of Tn-PSM. This suggests common carbohydrate epitope interactions for the Tn cancer antigen with the above mucin analogs, a finding that may be important to the role of the Tn antigen in cancer cells.

Density-dependent lectin-glycan interactions as a paradigm for conditional regulation by posttranslational modifications.

Mice with null mutations in specific Golgi glycosyltransferases show evidence of glycan compensation where missing carbohydrate epitopes are found on biosynthetically related structures. Repetitive saccharide sequences within the larger glycan structures are functional epitopes recognized by animal lectins. These studies provide the first in vivo support for the existence of a feedback system that maintains and regulates glycan epitope density in cells. Receptor regulation by lectin-glycan interactions and the Golgi provides a mechanism for the adaptation of cell surface receptors and solute transporters in response to environmental cues and intracellular signaling. We suggest that other posttranslational modification systems might have similar conditional features regulated by density-dependent ligand-epitope interactions.

Enhanced self-association of mucins possessing the T and Tn carbohydrate cancer antigens at the single-molecule level.

Mucins are linear O-glycosylated glycoproteins involved in inflammation, cell adhesion, and tumorigenesis. Cancer-associated mucins often possess increased expression of the T (Galß1,3GalNAcαThr/Ser) and Tn (GalNAcαThr/Ser) cancer antigens, which are diagnostic markers for several cancers, including colon cancer. We have used AFM based single-molecule forced unbinding under near physiological conditions to investigate the self-interactions between porcine submaxillary mucin (PSM) as well as between PSM analogs possessing various carbohydrates including the T- and Tn-antigen. Distributions of unbinding forces and corresponding force loading rates were determined for force loading rates from 0.18 nN/s to 39 nN/s, and processed to yield most probable unbinding forces f* and lifetimes of the interactions. Parameter f* varied in the range 27 to 50 pN at force loading rates of about 2 nN/s among the various mucins. All mucin samples investigated showed self-interaction, but the tendency was greatest for PSM displaying only the Tn-antigen (Tn-PSM) or a mixture of Tn-, T-antigen, and the trisaccharide Fucα1,2Galß1,3GalNAc (Tri-PSM). Weaker self-interactions were observed for native PSM (Fd-PSM), which consists of a nearly equal mixture of the longer core 1 blood group A tetrasaccharide (GalNAcα1,3(Fucα1,2)Galß1,3GalNAcαSer/Thr) and Tn-antigen. The data are consistent with the truncated Tn and T glycans enhancing self-interaction of the mucins. These carbohydrate cancer antigens may, thus, play an active role in the disease by constitutively activating mucin and mucin-type receptors by self-association on cells.

Fine specificities of two lectins from Cymbosema roseum seeds: a lectin specific for high-mannose oligosaccharides and a lectin specific for blood group H type II trisaccharide.

The legume species of Cymbosema roseum of Diocleinae subtribe produce at least two different seed lectins. The present study demonstrates that C. roseum lectin I (CRL I) binds with high affinity to the "core" trimannoside of N-linked oligosaccharides. Cymbosema roseum lectin II (CRL II), on the other hand, binds with high affinity to the blood group H trisaccharide (Fucα1,2Galα1-4GlcNAc-). Thermodynamic and hemagglutination inhibition studies reveal the fine binding specificities of the two lectins. Data obtained with a complete set of monodeoxy analogs of the core trimannoside indicate that CRL I recognizes the 3-, 4- and 6-hydroxyl groups of the α(1,6) Man residue, the 3- and 4-hydroxyl group of the α(1,3) Man residue and the 2- and 4-hydroxyl groups of the central Man residue of the trimannoside. CRL I possesses enhanced affinities for the Man5 oligomannose glycan and a biantennary complex glycan as well as glycoproteins containing high-mannose glycans. On the other hand, CRL II distinguishes the blood group H type II epitope from the Lewis(x), Lewis(y), Lewis(a) and Lewis(b) epitopes. CRL II also distinguishes between blood group H type II and type I trisaccharides. CRL I and CRL II, respectively, possess differences in fine specificities when compared with other reported mannose and fucose recognizing lectins. This is the first report of a mannose-specific lectin (CRL I) and a blood group H type II-specific lectin (CRL II) from seeds of a member of the Diocleinae subtribe.

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.

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.

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.

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.

Effects of clustered epitopes in multivalent ligand-receptor interactions.

Many biological ligands are composed of clustered binding epitopes. However, the effects of clustered epitopes on the affinity of ligand-receptor interactions in many cases are not well understood. Clustered carbohydrate epitopes are present in naturally occurring multivalent carbohydrates and glycoproteins, which are receptors on the surface of cells. Recent studies have provided evidence that the enhanced affinities of lectins, which are carbohydrate binding proteins, for multivalent carbohydrates and glycoproteins are due to internal diffusion of lectin molecules from epitope to epitope in these multivalent ligands before dissociation. Indeed, binding of lectins to mucins, which are large linear glycoproteins, appears to be similar to the internal diffusion mechanism(s) of protein ligands binding to DNA, which have been termed the "bind and slide" or "bind and hop" mechanisms. The observed increasing negative cooperativity and gradient of decreasing microaffinity constants of a lectin binding to multivalent carbohydrates and glycoproteins result in an initial fraction of lectin molecules that bind with very high affinity and dynamic motion. These findings have important implications for the mechanisms of binding of lectins to mucins, and for other ligand-biopolymer interactions and clustered ligand-receptor systems in general.

Phosphorylated human galectin-3: facile large-scale preparation of active lectin and detection of structural changes by CD spectroscopy.

Galectin-3 has a unique modular design. Its short N-terminal stretch can be phosphorylated, relevant for nuclear export and anti-anoikis/apoptosis activity. Enzymatic modification by casein kinase 1 at constant ATP concentration yielded mg quantities of mono- and diphosphorylated derivatives at Ser5/Ser11 in a 2:1 ratio. Their carbohydrate-inhibitable binding to asialofetuin, cell surfaces of three tumor lines, rabbit erythrocytes leading to haemagglutination and cytoplasmic sites in fixed tissue sections was not markedly altered relative to phosphate-free galectin-3. Spectroscopically, phosphorylation induced alterations in the far UV CD, indicative of an increase in ordered structure. This is accompanied by changes in the environment of aromatic amino acids signified by shifts in the near UV CD.

Binding studies of alpha-GalNAc-specific lectins to the alpha-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 approximately 10(6) daltons and approximately 2300 alpha-GalNAc residues (Tn-PSM) binds to the soybean agglutinin (SBA) with a K(d) of 0.2 nm, which is approximately 10(6)-fold enhanced affinity relative to GalNAcalpha1-O-Ser (Tn), the pancarcinoma carbohydrate antigen. The enzymatically derived 81 amino acid tandem repeat domain of Tn-PSM containing approximately 23 alpha-GalNAc residues binds with approximately 10(3)-fold enhanced affinity, while the enzymatically derived 38/40 amino acid cleavage product(s) of Tn-PSM containing approximately 11-12 alpha-GalNAc residues shows approximately 10(2)-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 GalNAcalpha1-O-Ser/Thr residues, with 45% of the peptide-linked alpha-GalNAc residues linked alpha-(2,6) to N-glycolylneuraminic acid, shows approximately 10(4) 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 alpha-GalNAc residue to alpha-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.

Galectin-3 and galectin-1 bind distinct cell surface glycoprotein receptors to induce T cell death.

Galectins are a family of mammalian beta-galactoside-binding proteins that positively and negatively regulate T cell death. Extracellular galectin-1 directly induces death of T cells and thymocytes, while intracellular galectin-3 blocks T cell death. In contrast to the antiapoptotic function of intracellular galectin-3, we demonstrate that extracellular galectin-3 directly induces death of human thymocytes and T cells. However, events in galectin-3- and galectin-1-induced cell death differ in a number of ways. Thymocyte subsets demonstrate different susceptibility to the two galectins: whereas galectin-1 kills double-negative and double-positive human thymocytes with equal efficiency, galectin-3 preferentially kills double-negative thymocytes. Galectin-3 binds to a complement of T cell surface glycoprotein receptors distinct from that recognized by galectin-1. Of these glycoprotein receptors, CD45 and CD71, but not CD29 and CD43, appear to be involved in galectin-3-induced T cell death. In addition, CD7 that is required for galectin-1-induced death is not required for death triggered by galectin-3. Following galectin-3 binding, CD45 remains uniformly distributed on the cell surface, in contrast to the CD45 clustering induced by galectin-1. Thus, extracellular galectin-3 and galectin-1 induce death of T cells through distinct cell surface events. However, as galectin-3 and galectin-1 cell death are neither additive nor synergistic, the two death pathways may converge inside the cell.

Galectins bind to the multivalent glycoprotein asialofetuin with enhanced affinities and a gradient of decreasing binding constants.

Our previous isothermal titration microcalorimetry (ITC) studies of the binding of synthetic multivalent carbohydrates to the Man/Glc-specific lectins concanavalin A (ConA) and Dioclea grandiflora lectin (DGL) showed negative binding cooperativity that was due to the carbohydrate ligands and not the proteins [Dam, T. K., et al. (2002) Biochemistry 41, 1351-1358]. The negative cooperativity was associated with the decreasing functional valence of the carbohydrates upon progressive binding of their epitopes. The present study also shows negative cooperativity in the ITC binding data of asialofetuin (ASF), a glycoprotein that possesses nine LacNAc epitopes, to galectin-1, -2, -3, -4, -5, and -7, and truncated, monomer versions of galectin-3 and -5, which are members of a family of animal lectins. Although the observed K(a) values for binding of ASF to the galectins and two truncated forms are only 50-80-fold greater than that of LacNAc, analysis of the data in terms of the relationship between the observed macroscopic free energy of binding and the decreasing microscopic free energies of binding of the epitopes shows that the first LacNAc epitope of ASF binds with approximately 6000-fold higher affinity than the last epitope. Thus, the microscopic binding constants of the galectins for the first epitope(s) of ASF are in the nanomolar range, with a gradient of decreasing binding constants of the remaining epitopes. The results indicate that the above galectins bind with fractional, high affinities to multivalent glycoproteins such as ASF, independent of the quaternary structures of the galectins. These findings have important implications for the binding of galectins to multivalent carbohydrate receptors.