ESSENTIALS OF GLYCOBIOLOGY LECTURE 21 MAY 4, 2004 Richard D. Cummings, Ph.D.

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. 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

3. 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 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 Dr. Cummings

4. Definition of the Galectins and Sequence Motifs Conserved Carbohydrate-Recognition Domain (CRD) of Galectins F M L C V I P L V A H I C F R M N L G E K S T E Q — H— —N— —R—X —V— —N— —X —W— —X— —X 4 5-10 N Q E G S K V L I V M F C D E N K H S L I V M F P C T F R K E G H — —X — — — —X— —X — — — 3 3-6 Human Galectin-1 69 -WGTEQREAV--FPFQPGSVAEVCITFDQANLT---VKLPDGYEFKFPNRL- -WGTEQRETV--FPFQKGAPIEITFSINPSDLT---VHLP-GHQFSFPNRL- 70 Chicken 14K Galectin Dr. Cummings

5. Different Subfamilies of Galectins Dr. Cummings

6. Different Subfamilies of Galectins Dr. Cummings

7. Structures of Galectins Crystal Structure (1.7 Å) of Dimeric Human Galectin-1 With Bound Lactose COO- NH2 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

8. 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

9. Structures of Galectins Amino Acids in Human Galectin-1 That Interact with Lactose N62 N62 W69 W69 E72 E72 H2O H45 H2O H45 R74 R74 H2O H53 H53 N47 D55 N47 D55 R49 R49 Without Lactose With Lactose Dr. Cummings

10. 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

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

12. Biosynthesis and Secretion of Galectins 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 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 1 2 2 2 >20 >20 Dr. Cummings

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

15. 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

16. 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

17. 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

18. 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 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 srbc’s.(From Sano et al (2003) J Clin Invest. 2003 Aug;112(3):389-97) Dr. Cummings

19. 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

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

21. Functions of Galectins Uniform Exposure of Phosphatidylserine (PS) in Apoptotic Neutrophils Hoechst Staining For DNA 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 ) Example of a Merged Image Single Cell From: Dias-Baruffi et al (2003) J. Biol. Chem.278(42):41282-93 Dr. Cummings

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

23. Functions of Galectins Praeparesis of Human Leukocytes Induced with Dimeric, but not Monomeric, Human Galectin-1 Enhances Their Phagocytosis by Activated Macrophages Background A HL-60 Cells Untreated Untreated dsHL -60 dGal-1 = Dimeric Gal-1 + dGal-1 dsHL -60 + dGal -1 + dGal-1 + Lactose dsHL -60 + dGal -1 + Lactose mGal-1 = Monomeric Gal-1 + mGal-1 dsHL -60 + mGal -1 + Camptothecin HL-60 + Camptothecin Aged Neutrophils Aged Neutrophils 0 10 20 30 40 Human Neutrophils B Resting Cells Resting Neutrophils Activated Cells Activated Neutrophils Activated Cells + dGal-1 Activated Neutrophils + dGal-1 Activated Cells + dGal-1 + Lactose Activated Neutrophils + dGal-1 + Lactose Activated Cells + mGal-1 Activated Neutrophils + mGal-1 Aged Neutrophils Aged Neutrophils From Dias-Baruffi et al (2003) J. Biol. Chem.278(42):41282-93 0 10 20 30 40 % Phagocytosis % Phagocytosis

24. Functions of Galectins

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