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ESSENTIALS OF GLYCOBIOLOGY LECTURE 21 MAY 4, 2004 Richard D. Cummings, Ph.D. University of Oklahoma Health Sciences Center College of Medicine Oklahoma Center for Medical Glycobiology “GALECTINS” Dr. Cummings Outline Historical Background Definition of the Galectins and Sequence Motifs Different Subfamilies of Galectins Prototype Galectins (Monomers) Chimeric Galectins Tandem Repeat Galectins Tertiary/Quaternary Structures of Galectins Galectins in Diverse Species: From Early Metazoans to Plants to Humans Biosynthesis and Secretion of Galectins Carbohydrate Ligands for Galectins Functions of Galectins Intracellular Functions Extracellular Functions Dr. Cummings Historical Background Prototypical Galectins: The Galactose-binding Lectins (Galectin-1 and -3) First discovered in electric organ of electric eel by Teichberg et al in 1975 as an agarose-binding protein capable of agglutinating trypsintreated rabbit erythrocytes Electrolectin required reducing conditions during storage for retention of activity Protein with similar properties subsequently isolated from bovine and chicken organs (termed L14) now called galectin-1 A larger protein (termed CBP35 and L-29) now called galectin-3 isolated from murine fibroblasts Other related proteins identified in many organisms, including C. elegans, zebrafish (Danio rerio), Drosophila, and sponges All members share sequence termed S-type (thiol) carbohydraterecognition domain (CRD) recognized by Drickamer in 1988 Group subsequently renamed galectins, since all members appear to recognize terminal, non-reducing galactose residues and have the ability to agglutinate cells Dr. Cummings Definition of the Galectins and Sequence Motifs Conserved Carbohydrate-Recognition F P C Domain (CRD) of Galectins M L F G L V R S — H— C —N— A —R—X —V— M—N— —X —W— E —X— E —X T 4 Q 5-10 K V H N I I L N D L L Q P E I I E R C N V G — K—X — — V —G —X— —X — — — H 3 K M 3-6 T M S E F H F F K S C V Human Galectin-1 69-WGTEQREAV--FPFQPGSVAEVCITFDQANLT---VKLPDGYEFKFPNRL-WGTEQRETV--FPFQKGAPIEITFSINPSDLT---VHLP-GHQFSFPNRL70 Chicken 14K Galectin Dr. Cummings Different Subfamilies of Galectins Dr. Cummings Different Subfamilies of Galectins Dr. Cummings Structures of Galectins Crystal Structure (1.7 Å) of Dimeric Human Galectin-1 With Bound Lactose COONH2 NH2 COO- Sideview Turned 90˚ Galectins have a highly conserved secondary structure with internally oriented hydrophobic residues in ß strands in the ß-sandwich of the galectin fold (Lobsanov et al., 1993; Liao et al., 1994; Leonidas et al., 1998). Dr. Cummings Structures of Galectins Comparative Structures of a Leguminous Plant Lectin Con A (an L-type Lectin) and Human Galectin-1 Con A Dimer Bovine Galectin-1 Dimer Both b-barrel proteins with no a-helix Dr. Cummings Structures of Galectins Amino Acids in Human Galectin-1 That Interact with Lactose N62 N62 W69 E72 W69 E72 H45 R74 R74 H2O H2O H45 H2O H53 D55 H53 N47 R49 With Lactose D55 N47 R49 Without Lactose Dr. Cummings Galectins in Diverse Species: From Early Metazoans to Plants to Humans From: The Website of Dr. Kurt Drickamer at http://ctld.glycob.ox.ac.uk/ctld/classes/Galectin2.html Dr. Cummings Biosynthesis and Secretion of Galectins Kd ~7mM Dimer Kd ~1mM Glycoprotein Ligand ? Monomer “Metastable Intermediate” Inactive Forms * Secretion Mechanism? Extracellular Cytosol N Dimer Monomer 3’ 5’ mRNA (Galectins lack a signal sequence) Dr. Cummings Biosynthesis and Secretion of Galectins Old Galectin Dogma New Info about Galectins Require reducing conditions for activity Can retain activity without reducing conditions in presence of ligands Occur only as soluble proteins Spliced forms may generate membraneanchored proteins Bind terminal Gal residues Bind GalNAc, GlcNAc, and Gal at internal and terminal positions, and sialylated Gal(NAc) Not post-translationally modification, other than Nterminal acetylation Some galectins are phosphorylated, glutathionylated, or cross-linked by transglutaminase Dr. Cummings Carbohydrate Ligands for Galectins Examples of Candidate Macromolecular Ligands Laminin Fibronectin Lysosome-associated membrane glycoproteins (LAMPs) CD7 CD43 CD45 Glycans Relative Binding Affinity To Galectin-1 1 2 2 2 >20 >20 Dr. Cummings Functions of Galectins Extracellular Galectin Intracellular Galectin CELL CELL Extracellular Matrix Dr. Cummings Functions of Galectins Cell-cell adhesion (galectins can agglutinate cells) Cell-matrix interaction (galectins can link cells to extracellular matrix (ECM) in an integrin- and Ca2+-independent fashion) RNA transport and splicing Cytoskeletal organization Many galectins are upregulated in tumor cells Dr. Cummings Functions of Galectins Cell signaling Growth arrest (galectins can arrest growth of embryonic fibroblasts) Mitogenesis (galectins can stimulate growth of some cells, e.g. lymphocytes) Apoptosis Galectins can induce death of some cells (in some cases this appears to be by apoptotic processes) Galectins can also suppress apoptosis in some cells Galectins can also induce exposure of phosphatidylserine (PS)on cell surfaces - often used as a marker of apoptosis) Phagocytosis and Clearance (galectin induction of PS can lead to recognition and phagocytosis of cells by professional phagocytes Dr. Cummings Functions of Galectins Potential Involvement of Galectins in Immune Regulation and Cancer Galectin-1 may be a negative regulator of immune response induces death of T- cells inhibits cell growth Inhibits cell-matrix interactions In experimental animal models of inflammation galectin-1 reduces tissue injury Galectin-1, -7, -8, -9, and -12 all are reported have apoptosis-inducing activity to certain types of cells By contrast, Galectin-3 has proinflammatory properties. inhibits T cell death may interact with bcl-2 to promote anti-apoptotic activity (Gal-3 contains the antideath Asp-Trp-Gly- Arg (NWGR) motif that is conserved in the Bcl-2 homology domain (BH1) of the Bcl-2 family) promotes T cell-matrix interactions is chemoattractant promotes leukocyte activation Dr. Cummings Functions of Galectins Potential Involvement of Galectins in Immune Regulation and Cancer Galectin-3 null mice have reduced leukocyte infiltration in experimental models of inflammation (peritoneal inflammation) Macrophages from Galectin-3 null mice have reduced phagocytic activity, associated with reduced intracellular Galectin-3 levels (LEFT FIGURE) Reduced phagocytosis of apoptotic thymocytes by gal3–/– macrophages. (a) In vitro phagocytosis of apoptotic thymocytes by macrophages was performed and the phagocytic index was calculated following 20-minute incubation of these cells. Data shown represent the mean ± SD from six experiments (P < 0.001). (b) Scatter plots of the percentage of macrophages containing phagocytosed apoptotic thymocytes and/or apoptotic bodies 30 minutes after injection of the cells into the peritoneum of wild-type and gal3–/– mice with sterile peritonitis, shown as mean (heavy horizontal lines) ± SD. Each data point represents the result from one mouse; seven mice of each genotype were used (P < 0.01). (RICHT FIGURE) Distribution of galectin-3 (upper panels; green) and F-actin (middle panels; red) inside wildtype macrophages. An overlay of these images demonstrates colocalization of F-actin and galectin-3 (lower panels; yellow). Panels on the left show wild-type macrophages before stimulation and those on the right show 1 minute after stimulation with opsonized srbc’s.(From Sano et al (2003) J Clin Invest. 2003 Aug;112(3):389-97) Dr. Cummings Functions of Galectins Potential Involvement of Galectins in Immune Regulation and Cancer Galectin-3 null mice have abnormalities in chondrocyte proliferation and extracellular matrix in the the hypertrophic zone of long bones, associated with decreased chondrocyte survival/turnover Galectin-3 overexpression correlated with enhanced metastatic potential implicated in many aspects of tumor biology While Galectin-4 is normally expressed primarily in the alimentary tract, it shows strong expression of galectin-4 in cancers from other tissues including breast and liver Galectin-9 (also called ecalectin) was identified as a T cell-derived potent eosinophil chemoattractant Dr. Cummings Functions of Galectins Galectin Cross-Linking and Signal Transduction Monomeric Mutants of Galectin Lack Signaling Functions! Dr. Cummings Functions of Galectins Uniform Exposure of Phosphatidylserine (PS) in Apoptotic Neutrophils Annexin V-FITC Staining Annexin V is a 35 kDa protein with a high affinity for PS (Kd estimated at 5 x 10-10 M ) From: Dias-Baruffi et al (2003) J. Biol. Chem. 278(42):41282-93 Hoechst Staining For DNA Example of a Merged Image Single Cell Dr. Cummings Functions of Galectins Polarized Exposure of PS in Galectin-1-Treated Activated Neutrophils Annexin V-FITC Staining From Dias-Baruffi et al (2003) J. Biol. Chem. 278(42):41282-93 Hoechst Staining Example of a Merged Image Single Cell Dr. Cummings Functions of Galectins Praeparesis of Human Leukocytes Induced with Dimeric, but not Monomeric, Human Galectin-1 Enhances Their Phagocytosis by Activated Macrophages Background HL-60 Cells A Untreated dsHL-60 Untreated + dGal-1 dGal-1 = Dimeric Gal-1 dsHL-60 + dGal-1 dsHL-60 + + dGal-1 + Lactose + dGal-1 Lactose + mGal-1 +dsHL-60 mGal-1 HL-60 + Camptothecin + Camptothecin mGal-1 = Monomeric Gal-1 Aged Neutrophils Aged Neutrophils Human Neutrophils 0 10 0 10 20 30 40 20 30 % Phagocytosis 40 B Resting Neutrophils Resting Cells Activated Neutrophils Activated Cells Activated Neutrophils + dGal-1 Activated Cells + dGal-1 Activated Neutrophils + dGal-1 Activated Cells + dGal-1 + Lactose + Lactose + mGal-1 ActivatedActivated CellsNeutrophils + mGal-1 Aged Neutrophils Aged Neutrophils From Dias-Baruffi et al (2003) J. Biol. Chem. 278(42):41282-93 % Phagocytosis Functions of Galectins Functions of Galectins Galectins and Their Effects on Cellular Homeostasis From: Hsu and Liu (2004) Glycoconjugate J. 19, 507–515 Dr. Cummings