ESSENTIALS OF GLYCOBIOLOGY LECTURE 21 MAY 4, 2004 Richard D. Cummings, Ph.D. University of Oklahoma Health Sciences Center College of Medicine Oklahoma.

ESSENTIALS OF GLYCOBIOLOGY

LECTURE 21

MAY 4, 2004

Richard D. Cummings, Ph.D.University of Oklahoma Health Sciences Center

College of MedicineOklahoma Center for Medical Glycobiology

“GALECTINS”

Dr. Cummings

Historical Background Definition of the Galectins and Sequence Motifs Different Subfamilies of Galectins

Prototype Galectins (Monomers)Chimeric GalectinsTandem 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 FunctionsExtracellular Functions

Outline

Dr. Cummings

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

-WGTEQREAV--FPFQPGSVAEVCITFDQANLT---VKLPDGYEFKFPNRL-

-WGTEQRETV--FPFQKGAPIEITFSINPSDLT---VHLP-GHQFSFPNRL-

Human Galectin-1

Chicken 14K Galectin

69

— H— —N— —R—X —V— —N— —X —W— —X— —X

FMLCVI

PLVAHI

5-10 4

CFRMNL

ST

GEK

EQ

70

— —X — — — —X— —X — — —3-6

RKE

PCTF

LIVMF

NQEGSKV

GH 3

DENKHS

LIVMFC

Conserved Carbohydrate-Recognition Domain (CRD) of Galectins

Definition of the Galectins and Sequence Motifs

Dr. Cummings

Different Subfamilies of Galectins

Dr. Cummings

Different Subfamilies of Galectins

Dr. Cummings

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

NH2

NH2

COO-

COO-

Bovine Galectin-1 DimerCon 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


Dr. Cummings

W69

H45

H2O

N47

R49

H53D55

R74

E72

N62

Amino Acids in Human Galectin-1 That Interact with Lactose

With Lactose Without Lactose

W69

H45

N47

R49

H53D55

R74

E72

N62


Dr. Cummings

H2O H2O

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

Cytosol

Extracellular

mRNA

Kd ~1M

SecretionMechanism?

Dimer Inactive Forms

Dimer

Kd ~7M

3’

?

Monomer

GlycoproteinLigand

Monomer 5’

“Metastable Intermediate”

N

Biosynthesis and Secretion of Galectins

*

(Galectins lack a signal sequence)Dr. Cummings

Require reducing conditions for activity

Occur only as soluble proteins

Bind terminal Gal residues

Not post-translationallymodification, 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 GalectinsOld Galectin Dogma

Biosynthesis and Secretion of Galectins

Dr. Cummings

Carbohydrate Ligands for Galectins

Examples of Candidate Macromolecular Ligands

LamininFibronectinLysosome-associated membrane glycoproteins (LAMPs)CD7CD43CD45

Glycans

RelativeBinding

Affinity To Galectin-1

2 >20>2021 2

Dr. Cummings

Extracellular Galectin

CELL

CELL

Intracellular Galectin

Functions of Galectins

Extracellular Matrix

Dr. Cummings

Cell-cell adhesion(galectins can agglutinate cells)

Cell-matrix interaction(galectins can link cells to extracellular matrix (ECM) in anintegrin- and Ca2+-independent fashion)

RNA transport and splicing

Cytoskeletal organization

Many galectins are upregulated in tumor cells


Dr. Cummings

Cell signalingGrowth 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

Potential Involvement of Galectins in Immune Regulation and Cancer

Galectin-1 may be a negative regulator of immune responseinduces death of T- cellsinhibits cell growthInhibits 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 deathmay 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 interactionsis chemoattractantpromotes leukocyte activation


Dr. Cummings


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


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


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

Galectin Cross-Linking and Signal Transduction

Monomeric Mutants of Galectin Lack Signaling Functions!


Dr. Cummings

Annexin V-FITCStaining

Hoechst StainingFor DNA

Uniform Exposure of Phosphatidylserine (PS) in Apoptotic Neutrophils

Example of a Merged Image Single Cell


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 (Kd estimated at 5 x 10-10 M )

Polarized Exposure of PS in Galectin-1-Treated Activated Neutrophils

Annexin V-FITCStaining

Hoechst Staining

Example of a Merged Image Single Cell


Dr. Cummings

From Dias-Baruffi et al (2003) J. Biol. Chem. 278(42):41282-93

0 10 20 30 40

Resting Neutrophils

Activated Neutrophils

Activated Neutrophils + dGal-1

Activated Neutrophils + dGal-1 + Lactose

Activated Neutrophils + mGal-1

Aged Neutrophils

% Phagocytosis

0 10 20 30 40

Untreated dsHL-60

dsHL-60 + dGal-1

dsHL-60 + dGal-1 + Lactose

dsHL-60 + mGal-1

HL-60 + Camptothecin

Aged Neutrophils

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 CellsActivated Cells + dGal-1

Activated Cells + dGal-1 + Lactose

Activated Cells + mGal-1Aged 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



Galectins and Their Effects on Cellular Homeostasis

From: Hsu and Liu (2004) Glycoconjugate J. 19, 507–515

Dr. Cummings


ESSENTIALS OF GLYCOBIOLOGY LECTURE 21 MAY 4, 2004 Richard D. Cummings, Ph.D. University of Oklahoma Health Sciences Center College of Medicine Oklahoma.

Documents

cells galectins

renamed galectins

cummings slide

cells prototypical galectins

helix structures of

growth arrest galectins

medical glycobiology

apoptotic processes