ESSENTIALS OF GLYCOBIOLOGY

1
ESSENTIALS OF GLYCOBIOLOGY LECTURE 21 MAY 7,
2002 Richard D. Cummings, Ph.D. University of
Oklahoma Health Sciences Center College of
Medicine Oklahoma Center for Medical
Glycobiology THE S-TYPE LECTINS (GALECTINS)
2
The Large and Growing Galectin Family of Lectins
Human Galectin-1
LNHUGB
14.9 kDa Subunit
Human Galectin-2
XP_009968
14.4
Human Galectin-3 (Mac-2 antigen)
XP_007333
27.5
Human Galectin-4
NP_006140
35.5
Rat Galectin-5
NP_037108
16.0
Human Galectin-6
XP_008181
48.8
Human Galectin-7
I55469
15.0
Human Galectin-8
XP_002045
34.9
3
The Large and Growing Galectin Family of Lectins
Human Galectin-9
XP_006105
35.5
(short form)
XP_006104
39.1
(long form)
Human Galectin-10
XP_009208
15.6
Rat Galectin-11 (GRIFIN-(Galectin-Related
Inter-fiber protein)
AAC71765
15.8
Human Galectin-12
AAG40863
34.6
(splice variant)
AAG40864 AF314686
34.5
(splice variant)
Human Galectin-13
NP_037400
15.3
Sheep Galectin-14
17.0
Human Urate Transporter/Channel
36.3
U67958
4
Distribution of Galectins
Type Source Distribution Remarks Galectin-1 h,r,
m, most tissues Most common and hab,p
abundant galectin Galectin-2 h,m small
intestine Minor rel. to Gal-1 Galectin-3 h,r,m,
macrophage, N-terminal domain d,ha colon,
most tiss. is collagen-like Galectin-4
h,r,p,m alimentary tract Linkage of two CRDs is
protease-sensitive Galectin-5 r erythrocytes
85 identical to C- term. CRD of
Gal-9 Galectin-6 m gastrointestinal 85 ident. to
Gal-4 Galectin-7 h,r skin marker of stratified
epithelia Galectin-8 h,r liver,
kidney,lung Galectin-9 h,r,m thymus,
kidney C-terminal domain 85 Hodgkins lymp.
identical to Gal-5 Galectin-10 h eosinophil,
Charcot-Leyden basophil crystal
protein Galectin-11 r lens (Grifin) may
represent a new lens crystallin,galectin-
related inter-fiber protein
H-humanm-mousem-monkeyr-rat b-bovinep-porcine
d-dogha-hamster
5
Conserved Carbohydrate-Recognition Domain 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 RX 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---VKLPDGYEFKFPNR
L- -WGTEQRETV--FPFQKGAPIEITFSINPSDLT---VHLP-GHQFS
FPNRL-
70
Chick-14K Galectin
6
phylogenetic tree of galectin family members
percentages of protein identities among CRDs of
human galectin family members
7
Crystal Structure (1.7 Å) of Dimeric Human
Galectin-1
8
Crystal Structure (1.7 Å) of Dimeric Human
Galectin-1 With Bound Lactose
Sideview
Turned 90
9
Amino Acids in Human Galectin-1 That Interact
with Lactose
N62
N62
W69
W69
E72
E72
H45
H45
R74
R74
H2O
H53
H53
N47
D55
N47
D55
R49
R49
Without Lactose
With Lactose
10
Biosynthesis of Galectins
Kd 7mM
?
Monomer
Dimer
Inactive Forms
Metastable Intermediate
Kd 1mM

Glycoprotein Ligand
Secretion Mechanism?
Extracellular
Cytosol
N
3
5
Monomer
Dimer
mRNA
11
SOME PROPOSED FUNCTIONS OF GALECTINS
Extracellular Galectin
Intracellular Galectin
CELL
CELL

• Cell-cell adhesion
• Cell-matrix interaction
• Cell signaling
• Growth arrest
• Mitogenesis
• Apoptosis
• RNA transport and splicing
• Cytoskeletal organization

12
Require reducing conditions for
activity Occur only as soluble proteins Bind
terminal Gal Not post-translationally modified,
other than N-terminal acetylation
Can retain activity without reducing conditions
in presence of ligands Spliced forms may
generate membrane-anchored proteins Bind
GalNAc, Gal, at internal and terminal positions,
and sialylated Gal(NAc) Some galectins may be
phosphorylated, glutathionylated, cross-linked by
transglutaminase
New Info about Galectins
Old Galectin Dogma
13
Are galectins always dispersed in the cytosol
and localized in the nucleus? Galectin-1 is
localized to myofibrils of porcine cardiac cells
Background Staining 2? Ab only (H/E Stained)
Acetone Fixed Frozen Section
1? mAb to Galectin-1 2? Ab (H/E Stained)
14
Background on Galectin-1
Galectin-1 originally isolated from electric
organ of electric eel (called electrolectin)
(Teichberg et al, 1975). Subsequently identified
in mammalian heart and lung (de Waard and
Kornfeld, 1976) and embryonic chick muscle (Nowak
and Barondes, 1976). Galectin-1 binds laminin, a
basement glycoprotein(Zhou and Cummings, 1990)
lamp glycoproteins (Do, Smith Cummings, 1990)
fibronectin (Ozeki, et al, 1995) and a7b1
integrin (Gu et al, 1994) ganglioside (Kopitz et
al, 1998) and CD45 (Perillo et al,
1995). Galectin-1 (and others) secreted by
non-classical pathway (Hughes, 1994 Cho and
Cummings, 1995).
15
Background on Galectin-1
Galectin-1 is mitogenic for lymphocytes (Pitts
and Yang, 1981), but has a growth-inhibitory
activity for some mammalian cells (Wells
Mallucci, 1991) which is apparently independent
of their beta-galactoside binding site (Scott
Zhang, 2002). Galectin-1 is able to induce
apoptosis in some types of cells (certain T-cell
subsets) (Rappl et al, 2002)
16
Background on Galectin-1
Galectin-1 activates the neutrophil respiratory
burst and may have proinflammatory functions
(Almkvist et al, 2002) activates Ca2
intracellular rise (Walzel et al,
1996). Galectin-1 in drosophila is expressed in
a developmentally-regulated tissue- and cell
specific manner (Pace et al, 2002)
17
Background on Galectin-3
Galectin-3 originally identified as macrophage
antigen Mac-2and occurs both on the cell surface
and intracellularly. Upon apoptotic stimulation,
galectin 3 becomes enriched in the mitochondria
and prevents mitochondrial damage and cytochrome
c release (Yu et al, 2002) Galectin-3 contains
the NWGR motif shared by bcl-2 family members
gal3 binds bcl-2 in a lactose-inhibitable manner
(Yang et al, 1996 Akahani et al, 1997)
18
Background on Galectin-3
Galectin-3 suppresses apoptosis (anti-apoptotic)
and anoikis such suppression may contribute to
cell survival during metastatic cascades this
suppression requires phosphorylation of Ser6
(Yoshii et al, 2002) Galectin-3 proposed to play
a role in b2-integrin-independent neutrophil
extravasation (Sato et al, 2002) During
involution of the mammary gland galectin-3
expression is up-regulated and occurs in
non-apoptotic cells (Mengwasser et al, 2002)
19
Background on Galectin-3
An alternatively spliced form of chicken
galectin-3 contains a predicted transmembrane
spanning domain and leucine zipper motif (Gorski
et al, 2002) Galectin-3 ligands include Lamp
glycoproteins, IgE, laminin, intestinal mucins,
and Mac2 binding protein and cytokeratin (Goletz
et al, 1997).
20
Background on Galectins-7,-8,and -9
Galectin-7 (also called PIG1) intracellularly
expressed exhibits pro-apoptotic function through
JNK activation and mitochondrial cytochrome c
release (Kuwabara et al, 2002) Galectin-8
expression is inversely related to tumour growth
rate (Nagy et al, 2002) Galectin-9 (also known
as ecalectin) represents a novel class of
eosinophil chemoattractants (ECAs) produced by
activated T cells (Sato et al, 2002 Matsumoto et
al, 2002)
21
Further Background on Galectins
Intracellularly, galectins may exist in a
glutathionylated form (Fratelli et al,
2002) Galectin-14 expressed in eosinophils and
its release may contribute to eosinophil function
and allergic inflammation (Dunphy et al,
2002) Galectins-1, -3, and -14 contain a
Bcl-2-like motif through this motif these
galectins may regulate apoptosis (38-41). In
particular, it has been postulated that Bcl-2 and
galectin-3 may heterodimerize through this motif
to inhibit Fas-antibody-mediated apoptosis (Yang
et al, 1996 Akahani et al, 1997 Perillo et
al, 1997).
22
Further Background on Galectins
Many galectins have already been linked to
immunity (Vasta, et al, 1999). Galectins
regulate cytokine production (Cortegano et al,
19989 Vespa et al, 1999), stimulate thymocyte
apoptosis (Galectin-1 Chung et al,2000 Pace et
al 2000 Galectin-9 - Wada et al, 1997), activate
respiratory bursts of neutrophils and mast cells
(Yamaoka et al, 1995) and migration of
leukocytes (Matsushita et al, 2000 Sano, et al,
2000). Galectins 1, 3, 10, 11 and 14 appear to
localize simultaneously to the nucleus and
cytoplasm under various conditions (Dunphy, et
al, 2000).
23
Further Background on Galectins
The role galectins play in the nucleus remains
unclear, but it has been postulated that
galectins 1 and 3 regulate pre-mRNA splicing
(Dagher et al, 1995 Vyakarnam et al, 1998
Dunphy et al, 2000). The specific presence of
galectin-14 in eosinophils and its release during
inflammatory reactions into the lung fluid
indicate that the protein may play an important
role in allergic-type responses, in particular in
allergic asthma (Dunphy et al, 2002).
Unexpectedly, the C-terminus of the human urate
transporter/channel contains the galectin CRD
(related to galectin-5) in a transmembrane
protein (Leal-Pinto et al, 1997).
24
Information about Galectin Function from Mouse
Genetics
Galectin 3(-/-) mice develop fewer inflammatory
cell infiltrations in the peritoneal cavities
than the wild-type (gal3(/)) mice in response
to inflammatory stimuli, mainly due to lower
numbers of macrophages (m?). Also, when
compared to cells from gal3(/) mice,
thioglycollate-elicited inflammatory cells from
gal3(-/-) mice exhibited significantly lower
levels of NF-kappaB response.
25
Information about Galectin Function from Mouse
Genetics
Different cell-spreading phenotypes are observed
in cultured (m?) from the two genotypes (m?)
from gal3(/) mice exhibited well spread out
morphology, those from gal3(-/-) mice were often
spindle-shaped. Peritoneal (m?) from gal3(-/-)
mice are more prone to undergo apoptosis than
those from gal3(/) mice when treated with
apoptotic stimuli (Hsu et al, 2000).
26
Information about Galectin Function from Mouse
Genetics
Galectin 1(-/-) mice have generally normal
phenotypes and litter sizes (). Galectin
1(-/-) mice have disrupted axonal architecture
for a subset of olfactory neurons (Puche et al,
1996) this is supported by in vitro work which
showed that galectin-1 promotes binding between
olfactory neurons and a laminin glycoconjugate
that lines the axonal migration path in vivo
(Mahanthappa et al, 1994). In vitro galectin-1
can induce programmed cell death in T-cells in a
CD45-dependent manner (Perillo et al, 1995).