1. 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

2. 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 Outline Dr. Cummings

3. First discovered in electric organ of electric eel by Teichberg et al in 1975 as an agarose-binding protein capable of agglutinating trypsin- treated 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) carbohydrate- recognition 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 Prototypical Galectins: The Galactose-binding Lectins (Galectin-1 and -3) Historical Background Dr. Cummings

4. -WGTEQREAV--FPFQPGSVAEVCITFDQANLT---VKLPDGYEFKFPNRL- -WGTEQRETV--FPFQKGAPIEITFSINPSDLT---VHLP-GHQFSFPNRL- Human Galectin-1 Chicken 14K Galectin 69 H N RX V N X W X X FMLCVIFMLCVI PLVAHIPLVAHI 5-10 4 CFRMNLCFRMNL STST GEKGEK EQEQ 70 X X X 3-6 RKERKE PCTFPCTF LIVMFLIVMF NQEGSKVNQEGSKV GHGH 3 DENKHSDENKHS LIVMFCLIVMFC Conserved Carbohydrate-Recognition Domain (CRD) of Galectins Definition of the Galectins and Sequence Motifs Dr. Cummings

5. Different Subfamilies of Galectins Dr. Cummings

6. Different Subfamilies of Galectins Dr. Cummings

7. Crystal Structure (1.7 Å) of Dimeric Human Galectin-1 With Bound Lactose 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). Structures of Galectins Dr. Cummings NH 2 COO -

8. Bovine Galectin-1 Dimer Con A Dimer Comparative Structures of a Leguminous Plant Lectin Con A (an L-type Lectin) and Human Galectin-1 Both -barrel proteins with no -helix Structures of Galectins Dr. Cummings

9. W69 H45 H2OH2O N47 R49 H53 D55 R74 E72 N62 Amino Acids in Human Galectin-1 That Interact with Lactose With Lactose Without Lactose W69 H45 N47 R49 H53 D55 R74 E72 N62 Structures of Galectins Dr. Cummings H2OH2O H2OH2O

10. Galectins in Diverse Species: From Early Metazoans to Plants to Humans Dr. Cummings From: The Website of Dr. Kurt Drickamer at http://ctld.glycob.ox.ac.uk/ctld/classes/Galectin2.html

11. Cytosol Extracellular mRNA K d ~1 M Secretion Mechanism? Dimer Inactive Forms Dimer K d ~7 M 3 ? Monomer Glycoprotein Ligand Monomer 5 Metastable Intermediate N Biosynthesis and Secretion of Galectins * (Galectins lack a signal sequence) Dr. Cummings

12. Require reducing conditions for activity Occur only as soluble proteins Bind terminal Gal residues Not post-translationally modification, other than N- terminal acetylation Can retain activity without reducing conditions in presence of ligands Spliced forms may generate membrane- anchored proteins Bind GalNAc, GlcNAc, and Gal at internal and terminal positions, and sialylated Gal(NAc) Some galectins are phosphorylated, glutathionylated, or cross-linked by transglutaminase New Info about Galectins Old Galectin Dogma Biosynthesis and Secretion of Galectins Dr. Cummings

13. 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 2>20 212 Dr. Cummings

14. Extracellular Galectin CELL Intracellular Galectin Functions of Galectins Extracellular Matrix Dr. Cummings

15. Cell-cell adhesion (galectins can agglutinate cells) Cell-matrix interaction (galectins can link cells to extracellular matrix (ECM) in an integrin- and Ca 2+ -independent fashion) RNA transport and splicing Cytoskeletal organization Many galectins are upregulated in tumor cells Functions of Galectins Dr. Cummings

16. 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 Functions of Galectins Dr. Cummings

17. 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 Functions of Galectins Dr. Cummings

18. 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 Functions of Galectins Dr. Cummings (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 wild- type 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 srbcs.(From Sano et al (2003) J Clin Invest. 2003 Aug;112(3):389-97)

19. 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 Functions of Galectins Dr. Cummings

20. Galectin Cross-Linking and Signal Transduction Monomeric Mutants of Galectin Lack Signaling Functions! Functions of Galectins Dr. Cummings

21. Annexin V-FITC Staining Hoechst Staining For DNA Uniform Exposure of Phosphatidylserine (PS) in Apoptotic Neutrophils Example of a Merged Image Single Cell Functions of Galectins Dr. Cummings From: Dias-Baruffi et al (2003) J. Biol. Chem. 278(42):41282-93 Annexin V is a 35 kDa protein with a high affinity for PS (K d estimated at 5 x 10 -10 M )

22. Polarized Exposure of PS in Galectin-1-Treated Activated Neutrophils Annexin V-FITC Staining Hoechst Staining Example of a Merged Image Single Cell Functions of Galectins Dr. Cummings From Dias-Baruffi et al (2003) J. Biol. Chem. 278(42):41282-93

23. 0102030 40 Resting Neutrophils Activated Neutrophils Activated Neutrophils + dGal-1 + Lactose Activated Neutrophils + mGal-1 Aged Neutrophils % Phagocytosis 0 10 20 3040 UntreateddsHL-60 dsHL-60 +dGal dsHL-60 +dGal-1 + Lactose dsHL-60 +mGal HL-60 +Camptothecin AgedNeutrophils A B Praeparesis of Human Leukocytes Induced with Dimeric, but not Monomeric, Human Galectin-1 Enhances Their Phagocytosis by Activated Macrophages Human Neutrophils HL-60 Cells % Phagocytosis Untreated + dGal-1 + dGal-1 + Lactose + mGal-1 + Camptothecin Aged Neutrophils Resting Cells Activated Cells Activated Cells + dGal-1 Activated Cells + dGal-1 + Lactose Activated Cells + mGal-1 Aged Neutrophils dGal-1 = Dimeric Gal-1 mGal-1 = Monomeric Gal-1 Background From Dias-Baruffi et al (2003) J. Biol. Chem. 278(42):41282-93 Functions of Galectins

25. Galectins and Their Effects on Cellular Homeostasis From: Hsu and Liu (2004) Glycoconjugate J. 19, 507–515 Dr. Cummings Functions of Galectins