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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)

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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