Download The galectin family of mammalian carbohydrate

Biochemical Society Transactions
The galectin family of mammalian carbohydrate-binding molecules
R. C. Hughes
National Institute for Medical Research, Mill Hill, London NW7 IAA, U.K.
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T h e galectins are a family of P-galactoside-binding proteins (see [ l ] for recent review). T h e
family contains nine or ten members that have
distinct and dynamic patterns of expression
during development, which suggests diverse and
important roles in embryogenesis and tissue formation. Table 1 shows a partial list of galectin
expression in adult tissues. We have focused on
galectin-3, which is present on various inflammatory cells and is also widely distributed on
branching epithelia, including kidney tubules, as
discussed later.
Structure of the galectins
T h e general structure of the galectins is shown
in Figure 1. T h e carbohydrate-recognition
domains (CRDs) are derived from two exons and
show extensive sequence homology within the
family. Recent crystallographic studies show that
the CRDs of galectins 1, 2, 3 and 4 are homologous 12 P-stranded structures [2]. T h e family
divides into three subtypes. Galectins 1, 2 and 5
have a single CRD preceded by relatively short
N-terminal domains. Under normal conditions
these are dimeric molecules and hence are funcAbbreviations used: CRD, carbohydrate-recognition
domain; TGB, thioglycollate broth; MDCK, MadinDerby canine kidney; HGF, human growth factor.
tionally bivalent. Galectins 4, 6, 8 and 9 are constitutively bivalent: they have two CRDs joined
by a linker sequence and preceded by short
N-terminal domains. Galectin-3 is, so far, unique
in having an extra-long N-terminal domain that
is encoded within a single exon 111 and contains
multiple repeats of a nine-residue sequence rich
in glycine, proline and tyrosine. Structural
studies, including NMR, show this domain to be
largely unstructured and flexible ([3]; B. Birdsell,
J. Feeney, S. Bawumia and R. C. Hughes, unpublished work). T h e function of this domain is not
understood but it appears to be important for the
self-assembly of galectin-3 monomers and hence
a switch to multi-valency that occurs at high concentrations. Galectin-3 dimers and trimers are
stabilized in solution by cross-linking of glutamine residues in the repetitive sequence to
unidentified lysine residues in the globular CRD
by tissue-type transglutaminase [4].
Carbohydrate binding
Galectin binding to carbohydrates is calcium
independent, in contrast with C-type lectins such
as the selectin family. Galactose is a necessary
but insufficient requirement for high-affinity
binding. T h e amino acid residues in the carbohydrate-binding pocket of the CRDs responsible
for galactose recognition are completely conserved for all the galectins so far sequenced [5].
Table I
Partial list of galectin expression in adult tissues
Galectin
Tissue distribution
I
Skeletal/smooth muscle, motor/sensory neurons, kidney, placenta,
thymus
Hepatoma, GI tract (especially ileum)
Activated macrophages, eosinophils, neutrophils, mast cells, epithelium
of GI and respiratory tracts, some sensory neurons
Intestinal and oral epithelium
Erythrocytes, reticulocytes
Intestinal epithelium
Keratinocytes
Lung, liver, kidney, heart, brain
Liver, small intestine, kidney, lymphoid tissue, lung, cardiac/skeletal
muscle
2
3
4
5
6
7
8
9
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The Role of Glycosylation in Biomolecular Interactions
Figure I
Overall structure of the galectins
The CRD encompasses the light- and dark-stippled boxes. N-terminal domains are shown as cross-hatched or unshaded boxes. The extra
N-terminal domain of galedin-3 and the linker domain of other galectins are shown in black. AA, approximate numbers of amino acid
residues in each domain.
Exon
11
III
Iv
-30
-60
-50
N
V
VI
AA
Galectins 1, 2 , 7
Galectin 5
Exon II
III
Galectin 3
-1I5AA
Repeats
Y P G * * * P G A
Galectins 4, 6, 8, 9
However, there is a higher level of complexity
involved for carbohydrate recognition than just
the central galactose. Typically the galectins bind
to Type I Galb1,3 GlcNAc or Type I1 GalP1,4
GlcNAc chains, and with higher-affinity polylactosamine chains. However, extension at the nonreducing end of the disaccharide units with
NeuNAca2,3 or with GalNAccr1,3 and Fuccr1,Z
substituents enhances affinity to galectin-3 but
has a smaller effect or reduces galectin-1 binding
[6,7]. Hence, human blood group type A epitopes
are good ligands for galectin-3 but not galectin-1.
Recent homology modelling and oligosaccharide
docking studies together with site-directed mutagenesis have identified likely contact residues in
the extended binding pocket of galectin-3 CRD
responsible for recognition of these more complex carbohydrates (K. Henrick, S. Bawumia, E.
A. M. Barboni, B. Mehul and R. C. Hughes,
unpublished work). These residues include, in
the hamster sequence: Arg- 13 and Glu- 160 for
Fuccc1,Z; Arg-139 and Ile-141 for GalNAca1,3;
and Arg-139, Glu-230 and Ser-232 for
NeuNAca2,3 substituents on the primary galactose.
Secretion
The galectins are found in the cell cytoplasm.
They are made on free ribosomes [8], lack trans-
membrane sequences or signal sequences for
co-translational transfer into the endoplasmic
reticulum and are not glycosylated although
several have consensus sites for N-glycosylation.
Despite these features galectins are secreted
from cells by a novel, incompletely understood
mechanism independent of the classical secretory pathway through the endoplasmic reticulumGolgi complex [9- 111. Immunocytochemical
studies have indicated that galectin-1 and galectin-3, before secretion, accumulate at sites at the
cytoplasmic side of plasma membranes [9,10,12].
This step is rate limiting in galectin-3 secretion
from macrophages as well as from Cos cells
transfected with galectin-3 constructs, and is
strongly up-regulated by heat shock and calcium
ionophores [10,13]. The next step in galectin
secretion appears to be evagination of plasma
membranes
specifically
at
those
sites
[9,10,12,13], a process that, at least for galectin3, critically requires N-terminal domains. Thus,
Cos cells transfected with galectin-3 CRD cDNA
can be induced to deliver the expressed protein
to plasma membrane sites, but evagination and
subsequent secretion are blocked [ 131. Furthermore, fusion proteins of a cytoplasmic indicator
protein such as chloramphenicol acetyltransferase with the N-terminal half of galectin-3 are
very efficiently exported from transfected Cos
cells [ 131. Pulse-chase and cytochemical data
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indicate that the final steps in galectin secretion
are the pinching off of evaginating plasma membrane domains and the release, either spontaneous or assisted, of galectin from the externalized
vesicles. Thus a light vesicular fraction isolated
on sucrose gradients from conditioned medium
of transfected Cos cells secreting hamster galectin-3, or mouse macrophages secreting endogenous
galectin-3,
contains
lectin
in
a
protease-resistant form unless detergent is added
to the incubation mixture [13]. Under physiological conditions galectin-3 is rather quickly
released (half-life 60-90 min) from the vesicles
into a soluble form that is fully sensitive to protease. T h e results suggest that vesicular release
plays a significant role in secretion of galectin-3,
although the possibility that direct transfer
through the plasma membrane at the evaginating
sites or elsewhere also takes place cannot be
excluded. In yeast, a novel transmembrane protein was recently identified by expression cloning
that appears to be critical for galectin-1 secretion
from transfected cells [ 141.
Extracellular functions
Once exported from the cell, galectins are free to
combine with appropriately glycosylated proteins
at cell surfaces or in the extracellular matrix.
Recent evidence indicates that galectins play
diverse and potentially important roles subsequent to their ligation of biologically active
receptors. In particular, galectin-3 appears to be
a multi-purpose signalling molecule. It modulates growth and apoptosis of human leukaemia
cells [15]. When added to human monocytes exogenously at low, probably monomeric concentrations, galectin-3 triggers superoxide production
and potentiates the lipopolysaccharide-induced
release of interleukin-1 up to 2-fold [16,17]. At
higher concentrations it binds to and cross-links
IgE receptors on basophilic cells and induces
cytotoxicity towards intracellular parasites, degranulation and serotonin release [ 18,191. Galectin3 binds to the NCA 160 surface antigen of
human neutrophils, a member of the carcinoembryonic antigen family of cell-adhesion molecules, and triggers an oxidative burst [ZO]. On
human T-cells (Jurkat cells) the lectin binds to
the heavy chain of the CD98 T-cell activation
antigen and induces an uptake of extracellular
calcium [21]. Of course, a direct link between
galectin-3 ligation of these and other surface
antigens and the biological effect is not implied,
except the effect on mast cells which is most
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likely mediated by IgE cross-linking as are other
degranulation-effectors. However, the results
amply illustrate the diversity of surface receptors
recognized by galectin-3 and the range of downstream effects reported.
Galectins also bind to certain glycoforms of
extracellular matrix components, such as laminin
and fibronectin [7] or tenascin [22] as well as
cell-surface adhesion molecules. A major receptor on mouse macrophages is the a-subunit of
the C D l l b / l 8 integrin, the Mac 1 antigen [23].
Interestingly, surface expression of galectin-3 is
a developmental marker on primary peritoneal
mouse macrophages [ 11,24,25]. Although it is
made and secreted by cells at various stages of
activation elicited with different inflammatory
agents, surface expression is rather tightly
restricted to peritoneal macrophages elicited by
thioglycollate broth (TGB). This implies that
glycosylation of galectin-3 receptors, including
CD1 lb, is modulated developmentally and that
provision of high-affinity lectin-binding glycans
on these receptors may be specifically associated
with elicitation by TGB. A correlation of these
findings with the reported up-regulation of a
specific a 1,3-gaIactosyl transferase gene in TGBelicited mouse macrophages [26] is rather striking, although so far no direct linkage between the
two observations has been described. However,
the transferase would be capable of extending
P-galactose terminals on N-glycans of surface
receptors such as C D l l b with a1,3 galactose
substituents, thereby increasing their affinity for
galectin-3 and boosting surface retention; this
applies especially under high-stringency conditions such as those which are likely to occur in
tissues. In humans, where the a 1,3 galactosyl
transferase is absent, other high-affinity glycans
may, by analogy, appear transiently on activated
macrophages. In pulmonary fibrosis a subset of
alveolar macrophages transiently expressing
glycan structures recognized by the plant lectin
DBA and possibly related to blood-group A epitopes, have been identified [27].
TGB-elicited macrophages may represent a
population of cells that have been recently
recruited into the tissue space by extravasation
through an endothelial monolayer and penetration of the underlying basement membrane. T h e
C D l l b / l 8 integrin is a key player in these events
and modulation of its activity by ligation with
galectin-3 is an intriguing possibility. Several
studies have shown that galectins, when added
exogenously to various cell-adhesion or motility
The Role of Glycosylation in Biomolecular Interactions
assays, can either antagonize or promote adhesions in a concentration-dependent manner
[ 11,281. For example, galectin-1 and galectin-3
can weaken cell interactions with laminin substrata in simple in vitro assays. In the former
case a direct inhibitory effect on the ct7/fil integrin, the major laminin receptor in the myoblasts
used for this study, was shown. Similarly the
migration of human metastatic breast tumour
cell lines through a Matrigel barrier in a Transwell assay is increased by nanomolar concentrations of galectin-3 consistent with a weakening of
cell-substratum adhesions, but is inhibited at
higher concentrations [29]. T h e simplest interpretation of these findings is that galectins may
sterically block integrin-matrix interactions upon
binding to one or more of the interacting partners. At higher concentrations bivalent galectins
may stimulate adhesions by cross-linking
specifically glycosylated cell surface and matrix
proteins.
Galectin-3 and epithelial polarity
Galectin-3 is expressed widely in epithelia,
usually on apical plasma membranes domains of
polarized tissues [30]. In the kidney its expression is regulated developmentally. Using reverse
transcription-PCR the first transcripts in mouse
embryonic kidney are detected at Ell-12, expression is maximal at E15-16 and decreases in newborns (P. Winyard, A. s. Wolf, Q. Bao and R. C.
Hughes, unpublished work). Immunocytochemistry of sections of newborn kidney shows that
galectin-3 expression is highest in ureteric bud
derivatives i.e., collecting ducts and connecting
segments of distal tubules. These results suggest
that galectin-3 may play a role in establishing or
maintaining the epithelial architecture of collecting ducts during kidney differentiation.
Recent work, using the collecting ductderived Madin-Darby canine kidney (MDCK)
cell line as a model, is consistent with this idea
[31]. When cells are seeded at low density within
three-dimensional collagen gels, the cells grow
clonally and form small aggregates that, in time,
become organized into polarized cysts with a
central lumen and a basal surface in contact with
the surrounding matrix, as well as a laminin-rich
basement membrane deposited by the cells.
Treatment of the cyst cultures with human
growth factor (HGF) scatter factor induces the
cysts to form sprouts that by further cell division
and migration elongate into tubule-like structures that eventually fuse and form a large syncy-
tium.
Immunocytochemistry
shows
that
galectin-3 is excluded from the lumenal/apical
surface of the cysts whereas the lateral and basal
surfaces are heavily stained. In the HGF/scatter
factor-treated cultures the baso-lateral staining
in the cyst bodies is retained but there is a
marked down-regulation of galectin-3 expression
at the sprout sites and in the forming tubules.
Thus high galectin-3 expression is associated
with sites of tight adhesions in the developing
epithelium, leading to the possibility that the
lectin may serve to synergize with or activate
other adhesive interactions. These interactions
include cell-cell adhesions at lateral surfaces
mediated by cadherin family members and cellmatrix adhesions at basal surfaces involving
integrins and matrix components. At sites of cell
movement and re-organization occurring during
sprouting and tubule formation, where strength
of adhesion would need to be lessened, lectin
expression is reduced at the interactive plasma
membrane domains. In support of this idea treatment of cyst cultures in MDCK cells with galectin-3-blocking antibodies does indeed speed up
the growth of the cysts as compared with the
control culture. Conversely addition of high concentrations of recombinant galectin-3 retards
cyst growth. Similarly, a ricin-resistant MDCK
cell line that produces and secretes wild-type
levels of galectin-3, but fails to galactosylate
glycoproteins, forms cysts in collagen cultures
that grow very quickly and adopt very irregular
shapes (Q. Bao and R. C. Hughes, unpublished
work). These results show that loss of galectin-3
binding receptors at the cell surface or within
the extracellular matrix is correlated with
abnormal cyst formation in this experimental
system.
Some recent findings relate these results to
the potential roles of galectin-3 in formation of
epithelia in the embryonic kidney (P. Winyard, A.
S. Wolf, Q. Bao and R. C. Hughes, unpublished
work). During human kidney ontogeny galectin-3
is first expressed in the epithelia of the mesonephric duct and is absent in the intermediate
mesoderm. In human metanephric kidney galectin-3 is found predominantly at lumenal/apical
domains of ureteric bud branches in the nephrogenic cortex. Interestingly, this subcellular localization shifts to a more baso-lateral pattern as the
medullary collecting duct lineage matures. However, in human multicystic dysplastic kidney
disease the malformed dysplastic tubules and
cysts retain an immature apical expression of
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galectin-3. It is tempting to see parallels in these
expression patterns occurring during normal and
diseased kidney differentiation and the MDCK
model. Galectin-3 on basal domains of maturing
collecting duct epithelia would be in a position to
influence cell interactions with basement membranes, particularly laminin, that is firmly implicated in epithelial maturation based on in vitro
studies [32]. Conversely, apically expressed
galectin-3 would be functionless as a negative
growth regulator of cyst expansion. Further
studies are directed at defining the signals
involved in targeted secretion of galectin-3 from
apical or baso-lateral domains of polarizing epithelia and the effects of recombinant lectin and
blocking antibodies on ureteric b u d development
in explants of kidneys of normal and dysplastic
mice as well as mice having null mutations in the
galectin-3 gene [331.
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_ _ _ _ _ _ _ ~
Received 26 June 1997