Lectins. Lectins and beyond. Lectins. Lectins are present in all organisms

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1 Lectins Lectins and beyond Lectins of plants Lectins of animals Lectins in signaling, immune response, inflammation 3/10/11 Lectins Glycan binding proteins Translate the Glycome Carbohydrate binding proteins that are not antibodies or enzymes Latin: lectus, meaning to gather or select Relatively high dissociation constants (ca 100 µm) Carbohydrate recognition domains are small Most lectins are multivalent Soluble Glycan-Binding Proteins After Varki Lectins are present in all organisms Lectin Families Viruses--Influenza hemaglutinin/neuraminidase Bacteria--binding of microbe or toxin to host cells Plant Many have been purified and characterized Physiological functions are generally unknown Animal Growing families of proteins with a wide variety of functions 1

2 Lectin Families (cont d) Lectins? GAG-binding proteins Plant Lectins Structure of Plant lectins Leguminosae ConA (Concanavalin A from Jack bean) Phaseolus Vulgaris (PHA-L and PHE) Soy bean agglutinin Graminae Wheat germ agglutinin Solanaceae Tomato lectin Potato lectin Compact β-barrel, no alpha helices Anti-parallel beta-sheets Many require metals (leguminosae) Ca ++ and Mn ++ Metals do not participate directly in the binding but are required for tertiary structure Structure of concanavalin A (ConA), a legume seed lectin N-glycans recognized by concanavalin A (ConA) and Galanthus nivalis agglutinin (GNA) Fig.29.1 After Varki After Varki 2

3 N-glycans recognized by L-PHA, E-PHA, and DSA Functions of Plant lectins Little is known In legume seeds lectins can comprise up to 30% of the total protein They are expressed in other parts of the plant Nodulation factor in roots May function in pathogen defense Some lectins possess other activities besides carbohydrate binding RCAII (Ricin) RNA-N-glycosidase DBA has an adenine binding site in addition to CRD After Varki Uses of Plant lectins Agglutination of cells and blood typing Cell separation and analysis Bacterial typing Identification and selection of mutated cells with altered glycosylation Toxic conjugates for tumor cell killing Cytochemical characterization/staining of cells and tissues Mitogenesis of cells Mapping neuronal pathways Purification of glycoconjugates Assays of glycosyltransferases and glycosidases Defining glycosylation status of target glycoconjugates Animal lectins are everywhere in the organism Essentials of Glycobiology Second Edition 3

4 Type 2 C-type Lectin Binding Domain distributed homology that forms a unique recognition fold Mannose Binding Protein--a collectin Expressed on macrophages and antigen presenting cells Mannose binding protein is trimeric, each subunit binds to Man (also GlcNAc, ManNAc, Glc, Fuc) with low affinity Collagen like tails drive trimerization and then facilitate formation of trimers of trimers, generating bouquet-like structures that bind poly-man glycans extremely well--spacing of low affinity interactions yields high specificity After Esko MBP Collectin Bouquets achieve high specificity through multiple low-affinity interactions C-type Lectin Domains are found in association with a variety of other protein motifs Many fungal pathogens express mannans (Man polymers) Generally don t encounter poly-mannac, poly-glc, poly-fuc, or poly- GlcNAc (chitin?) so MBP specificity results from the existence of multivalent Mannose ligands Cytokine and chemokine expression recruits leukocytes to sites of inflammation--a multistep process initiated by carbohydrate recognition between leukocytes and endothelial cells The real thing garlandscience 4

5 Capture of lymphocytes at sites of peripheral inflammation requires endothelial P-selectin interaction with PSGL-1 on T-cells Lymphocyte extravasation in lymphatic tissues occurs at specialized endothelial domains called high endothelial venules (HEVs), post capillary venules characterized by cuboidal cells Lymphocytes express their own selectin, known as L-selectin. L-selectin binds to a group of ligands known as PNAds which are mucin-type proteins expressed on high endothelial cells. The shared characteristic of the PNAd proteins is a common carbohydrate structure. Zollner, T. (2003) JCI 112, 980 mmg.uci.edu/immunology/architecture/ Reciprocal ligand-selectin pairs encode different homing specificities L-selectin on lymphocytes recognizes specific glycans on PNAd proteins expressed on endothelial cells at the HEV of lymph nodes. P-selectin on endothelial cells at sites of inflammation recognizes specific glycans on the PSGL-1 protein expressed on lymphocytes. Note: Cytokines produced by macrophages and by activated granulocytes induce expression of P-selectin on endothelial cells and of the enzymes needed to build the recognized carbohydrate structures in lymphocytes. McEver, R. (2005) Nature Immunology 6, 1067 Essentials of Glycobiology Second Edition Galectin Binding Domain distributed homology that forms a shallow β-sheet recognition fold Human Galectins 5

6 Possible biosynthetic routes for galectins in animal cells Functional interactions of galectins with cell-surface glycoconjugates and extracellular glycoconjugates Galectins facilitate cell adhesion and signaling. Interactions with surface ligands regulate signaling thresholds through cross-linking and receptor recycling. Known and putative functions of galectins in the immune system Essentials of Glycobiology Second Edition Domain structures of the known Siglecs in humans and mice Biological Interactions Involving Siglecs (CD22ish) From: Crocker P, Paulson J. & Varki, A. Nature Reviews Immunol. 7: ,

7 Biological functions mediated by sialoadhesin (Siglec-1): Interactions of macrophages with cells and pathogens Biological functions mediated by myelin-associated glycoprotein (MAG, Siglec-4) Siglec-4 ligand loss leads to myelin degradation Lectin binding characteistics and characterization Sheikh, KA, et al. (1999) PNAS 96, 7532 Lectin binding characteristics Lectin-Glycan interactions displace and co-opt water After Schnaar Essentials of Glycobiology Second Edition 7

8 Lectin binding characteristics Cholera Toxin Shallow binding/recognition sites Distributed protein sequence motifs Binding mediated by salt bridges, metal coordination, Van der Waals interactions, aromatic stacking, hydrogen bonding Generally low affinity Strong binding results from multivalency, BUT Cholera toxin exists as a AB 5 complex, each B subunit binds to a single molecule of ganglioside GM1 The glycan binds to shallow pockets near the surface Kd of monomer for GM1 ~40 nm, very unusual (usually µm to mm) After Esko Cholera Toxin Multivalency raises affinity of interaction to ~40 pm Only 3 sugars are bound Selectivity is high, since binding occurs with great precision GalNAc After Esko Electrostatics H-bonding van der waals interactions Tyr, Phe, or alkyl side chains Water and divalent cations can act as bridging groups that increase selectivity Gal Gal Neu5Ac Glc After Esko Affinity/Avidity/Multivalency Affinity determines experimental approach 8

9 Washing can kill you Characterizing lectin binding Equilibrium dialysis against labeled hapten Equilibrium binding, stop by PEG with centrifugation (solubilized receptor) Equilibrium binding, stop by filtration (membranes) Multivalent ligands Multivalent receptor probes Biacore realtime kinetics Cell adhesion, flow under shear to immobilized glycan or receptor Cell adhesion, static adhesion to immobilized glycan X-ray co-crystallography, NMR, and MS mapping of relevant contacts and protein dynamics Screening for lectins, high throughput, no assumptions Lectins and Lectin Characterization: General Principles Lectins generally bind with low affinity but achieve high avidity through multi-valency Lectin motifs comprise distributed sequence similarities and structural homologies; extended primary amino acid sequence conservation is not generally associated with lectin-like activities Methods for lectin characterization must consider affinity, valency, and avidity The development of multivalent probes, as well as methods for determining static and dynamic adhesion have been essential for defining lectin binding specificity and lectin function To define the paradigm by which glycan binding proteins modulate cell function, especially of immune cells and immune responses The Prime Directive--Consortium for Functional Glycomics Lectins and immune regulation: General Principles Cell-surface glycans participate in all arms of the immune system Glycan recognition is an integral component of innate (antibody-independent) immunity Specific glycan structures modify humeral immune (antibody-dependent) responses Differentiation of immune cell types requires appropriate glycan expression Cell-cell interactions that lead to immune cell activation are modulated by carbohydrate 9

10 Selected topics Thymic selection of self vs. non-self Generation and function of the immune synapse Regulation of antibody production and B-cell responsiveness Thymic selection Thymic selection Thymic involution is a normal component of aging The thymus has a lobular architecture with trabeculae providing entry points to each lobe

11 Distinct regions can be discerned within each lobe Extensive network of thymic epithelial cells provides large surface area for interaction with thymocytes Self vs. non-self selection T-cell maturation and selection begins in the cortex Immature T-cells arrive from the bone marrow as double negative cells, lacking expression of both CD4 and CD8, as well as the CD3 component of the TCR. What does restricted mean? MHC I, MHC II, Cd1d? TCR rearrangement occurs in the cortex, producing receptors for MHC displayed on cortical thymic epithelial cells. Thymocytes that generate TCRs capable of recognizing MHC-I begin to express CD8 and thymocytes that generate TCRs capable of recognizing MHC-II begin to express CD4. CD4+ and CD8+ thymocytes are attracted to the cortico-medullary junction by chemokines produced by macrophages and dendritic cells located in the medulla. Thymic selection for self vs. nonself requires two steps Core-2 synthesis generates ligands for Galectins Step 1: Positive selection for thymocytes expressing rearranged TCRs that bind self-mhc presenting self-peptide on cortical thymic epithelial cells. Cells that can interact with MHC survive, those that can t apoptose; a positive survival signal. Cells that interact very strongly with self MHC and self-peptide are retained in the cortex. Step 2: Negative selection for thymocytes that express rearranged TCRs that bind self-peptides presented by self-mhc on medullary epithelial cells, macrophages, or marrow-derived dendritic cells. Cells that interact strongly are killed, cells that do not interact or interact weakly are matured into CD4+ or CD8+ T-cells. Core2 GlcNAcT is upregulated in cortical thymocytes 11

12 C2GlcNAcT activity in relation to thymic selection C2GlcNAcT expression by naïve T-cells in the thymic cortex increases with residence time and decreases as the differentiating T-cell migrates to the medulla Strong TCR activation by self-bound MHC results in retention of the naïve T-cell in the cortex and maintenance of C2GlcNAcT activity (negative selection) Weak TCR activation results in migration of the T-cell to the thymic medulla and down-regulation of C2GlcNAcT activity (positive selection) C2GlcNAcT activity generates substrates for Galectin binding, enhancing signaling through receptors that lead to apoptosis of cortical T-cells with self-specificity Antigen presentation and the adaptive immune response How is antigen presented? After Varki, A. Glycans are essential for peptide loading onto MHC-I Formation of the immunological synapse Rudd, P.,

13 The immunological synapse is the communication point for activation of specific T-cells Red/brown = cell adhesion molecules, integrins, Ig-CAM Green/yellow = TCR, MHC I/II White = outlines of T-cell on an APC Dustin, M., 2000 The synapse must accommodate glycans Dustin, M., 2000 Rudd, P., 2001 Galectins may organize components of the synapse, providing a regulatory matrix that modulates signaling levels--glcnactv KO has overactive signaling T-cell activation modulates B-cell responses Lowe, J.,

14 Three Possible Models for Functions of CD22-Sialic (Siglec-2) Acid Interactions Th1--IFNγ, IL12, microbial responses, auto-immunity Th2--IL4, 5, 6, 9, 10, 13, parasite responses, asthma, allergy Collins, B., 2002 Proposed Biological functions mediated by CD22: CD22 glycan-dependent homotypic interactions in equilibrium with CD22 BCR interactions General Principles related to glycans and immune function Cell-surface glycans participate in all arms of the immune system, where they: Mediate or regulate cell adhesion Modulate cell signaling events By regulating signaling and adhesion, progenitor cells are driven toward specific differentiation/activation by glycan expression Method Overview--Flow cytometry and FACS 14

15 Forward Scattering=cell size Side Scattering=cellular granularity Cell Sorting is a preparative method that yields specific cell populations BD Biosciences Stain cells with two (or more) fluorescent markers and analyze (or sort) based on fluorescence intensity Gating on one population eliminates irrelevant background and allows dissection of specific cell populations Flow cytometry (and cell-sorting) can be quantitative Ormerod, M.G., Flow Cytometry; 15