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J Biol Chem, Vol. 273, Issue 21, 13047-13052, May 22, 1998
From the Galectins are a family of lectins which share
similar carbohydrate recognition domains (CRDs) and affinity for small
Galectin-3 is a member of the galectin family of lectins defined
by a conserved ~14-kDa carbohydrate recognition domain
(CRD)1 showing affinity for
Galectin-3 is unique among the known galectins in that, in addition to
the canonical CRD (located at the C terminus), it contains an
unrelated, non-carbohydrate-binding N-terminal domain of between 120 (in human) and 166 (in dog) amino acids (3, 19). In contrast, galectins-1 and -2 are homodimers composed of the CRD alone, while galectins-4, -6, -8, and -9 possess an N- and C-terminal CRD linked in
tandem by a short polypeptide segment (18). The galectin-3 CRD shows
sequence identity ranging from 30-40% with galectins-4 through -10, to 20-25% with galectins-1 and -2. It has an affinity for lactose
(Lac, Kd = 1 mM), and
N-acetyllactosamine (LacNAc, Kd = 0.2 mM) similar to that of other galectins, but has a distinct
profile for larger oligosaccharides (20, 21), including
polyNAc-lactosaminoglycan, a polymer of Intact galectin-3, but not its CRD alone, shows avidity for multivalent
glycoconjugates (10, 11), modulates cell adhesion (14), and induces
intracellular signals (15). Thus it is thought that the N-terminal
domain of galectin-3 promotes the formation of dimers or higher order
oligomers, thereby permitting multivalent interactions essential for
its biological activities.
We report here the x-ray crystal structure of the CRD of human
galectin-3 in complex with Lac and LacNAc at 2.1-Å resolution. Previously we and others showed that galectin-1 (22, 23) and galectin-2
(24) are 2-fold symmetric homodimers of the canonical 14-kDa CRD. We
now show that, although the galectin-3 CRD is similar to that found in
galectin-1 and galectin-2, it displays structural features which
provide an explanation for the known differences in galectin-3
carbohydrate-binding specificity and mode of self-association.
Protein Purification and Crystallization--
Intact human
galectin-3 was expressed in Escherichia coli strain
BL21(DE3), followed by purification on a lactosyl-Sepharose column
essentially as described previously (11). A C-terminal fragment of
human galectin-3 (galectin-3-C; residues 107-250) containing the CRD
was then produced by collagenase (type VII, Sigma) digestion of the
intact molecule followed by repurification on lactosyl-Sepharose (11).
Galectin-3-C was dialyzed against crystallization buffer containing 10 mM Tris-HCl, pH 7.5, 10 mM 2-mercaptoethanol,
and 0.02% NaN3 to remove lactose, and concentrated to
10-15 mg/ml using a Centricon-10 (Amicon, Beverly, MA) ultrafiltration unit. Crystals were grown using the hanging drop vapor diffusion method
from drops containing equal volumes of the protein (10-15 mg/ml) in
crystallization buffer containing 1 mM lactose or 30 mM N-acetyllactosamine, and solution from the
well composed of 27-35% PEG 4000-6000, 100 mM Tris-HCl,
pH 8.5, 100 mM MgCl2, and 8 mM
2-mercaptoethanol. The crystals attain dimensions of 0.5 × 0.4 × 0.3 mm3 within 1 to 2 weeks and diffract to
approximately 1.9-Å resolution. They grow in space group
P212121 with unit cell dimensions
of a = 37.6 Å, b = 58.4 Å, and
c = 64.0 Å. There is one molecule in the asymmetric
unit resulting in a solvent content of 48%.
Data Collection, Structure Determination, and
Refinement--
Five heavy atom derivatives were used to calculate the
initial phases (see Table I). With the exception of dimethylmercury, the heavy atom compounds were dissolved in artificial mother liquor containing 32-35% PEG 6000, 100 mM Tris-HCl, pH 8.5, and
100-150 mM MgCl2, at concentrations of 1-3
mM, and soaked into crystals for 2-3 days. The
dimethylmercury derivative was obtained through vapor diffusion. After
mounting a crystal in a standard glass x-ray capillary, a small volume
(~2 µl) of dimethylmercury was pipetted into the end of the
capillary before sealing it with wax and epoxy. The crystal was then
allowed to equilibrate for 48 h before data collection. Native and
derivative data sets were collected at room temperature on a Siemens
multiwire area detector with a conventional rotating anode x-ray
source, and reduced using XDS (25). Heavy atom parameter refinement,
phase calculations, and solvent flattening were performed using PHASES
(26). An initial phase set was calculated using the isomorphous and
anomalous components of the derivative data sets (Table I) in
conjunction with solvent flattening. The resulting electron density map
at 2.5-Å resolution was of very high quality permitting an unambiguous chain tracing. The initial model was built using O (27). Model refinement was performed with X-PLOR (28) using the simulated annealing
and conventional energy minimization protocols with Fobs > 1 Structure Description--
The structure of galectin-3-C has been
determined in the presence of both Lac and LacNAc, and in both cases
refined at 2.1-Å resolution with good geometry (Table
I). Analysis using PROCHECK (32) shows
that all non-proline and non-glycine residues are found in the most
favored or additionally allowed regions of the Ramachandran plot. In
both complexes, Leu-114 is the first residue for which electron density
is observed, and hence, the first 6 residues are presumed to be
disordered. Well defined electron density is observed for all other
residues, including the C-terminal residue Ile-250. Both complexes are
very similar to each other, and all further reference to the structure
will pertain to the LacNAc complex unless otherwise indicated.
X-ray Crystal Structure of the Human Galectin-3 Carbohydrate
Recognition Domain at 2.1-Å Resolution*
,,,
,**
Departments of Molecular and Medical
Genetics and Biochemistry, University of Toronto, Toronto, Ontario, M5S
1A8, Canada and the § Center for Neurobiology and
Psychiatry,
ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References
-galactosides, but which show significant differences in binding
specificity for more complex glycoconjugates. We report here the x-ray
crystal structure of the human galectin-3 CRD, in complex with lactose and N-acetyllactosamine, at 2.1-Å resolution. This
structure represents the first example of a CRD determined from a
galectin which does not show the canonical 2-fold symmetric dimer
organization. Comparison with the published structures of
galectins-1 and -2 provides an explanation for the differences in
carbohydrate-binding specificity shown by galectin-3, and for the fact
that it fails to form dimers by analogous CRD-CRD interactions.
INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References
-galactosides (1, 2). Abundantly expressed in a few cell types, such
as macrophages and polarized epithelial cells in adults (2, 3) and
others during embryogenesis (4), it tends to be localized in the
cytoplasm and the nucleus. Although functions for galectin-3 have been
proposed in each of these subcellular locations (5-7), it is also
secreted by a nonclassical pathway (8, 9) and is found on the cell
surface and in the extracellular matrix. There it binds and cross-links
selected carbohydrate-containing ligands (10, 11) and is thought to modulate cell adhesion (12-14) and cell signaling (15, 16). Many
groups are currently studying the roles and uses of galectin-3 in
cancer, inflammation, host-pathogen interaction, and nerve injury,
among others (17, 18).
(1,3)-linked LacNAc units
found on many extracellular matrix and cell surface molecules.
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References
(Fobs)
between 8.0- and 2.1-Å resolution. Restrained atomic temperature
factors were refined using Fobs > 2(Fobs) between 5.0- and 2.1-Å resolution.
Water molecules were selected based on difference electron density and
hydrogen bond geometry and assigned an occupancy of 0.6 (29). All
figures were made with SETOR (30) with the exception of Figs. 4 and 7, which were created with GRASP (31).
RESULTS AND DISCUSSION
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Abstract
Introduction
Procedures
Results & Discussion
References
Structure determination and refinement
-sheets which
associate in a -sandwich arrangement (Fig.
1). The C
atoms of 43 homologous
residues in the -sheet core of galectin-3-C align with root mean
square deviations of 0.52 and 0.61 Å with those of human galectin-2
(24) and bovine galectin-1 (22), respectively (Fig. 1). In the
canonical dimeric galectins-1 and -2, -strands F1 and S1 from each
monomer extend the antiparallel -strand interactions across the
2-fold symmetric dimer interface (S1-S1' and
F1-F1' in Fig. 1), whereas the S1 and F1 -strands of
galectin-3-C form a solvent-exposed surface as discussed below in
detail.
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Fig. 1.
Stereo view of the galectin-1 dimer
superimposed on the galectin-3-C/LacNAc complex. The C traces
of human galectin-3-C and bovine galectin-1 (22) are shown in
bold and thin lines, respectively.
F1/S1 and F1'/S1' label the
-strands forming the dimer interface in galectin-1. These
-strands extend the antiparallel -sheet interactions across the
2-fold symmetric dimer interface. Gal and Nag
label the galactose and N-acetylglucosamine moieties of the
bound LacNAc in the galectin-3-C structure.
Primary Carbohydrate Binding Site--
As shown in Fig. 1 the
Lac/LacNAc binding site is formed by -strands S4-S6a/S6b. With S3
these -strands define a carbohydrate-binding cassette, encoded for
by a single DNA exon (2, 24), which is evolutionarily conserved among
members of the galectin family. The amino acids making direct
interaction with the bound carbohydrate are highly conserved among all
galectins sequenced to date and are contained on these -strands. The
galactose moiety of Lac/LacNAc is most deeply buried in the binding
site (Fig. 2); 166 Å2 of its
total 230-Å2 surface area is buried by the protein. Its
C-4 hydroxyl group plays a central role in binding, likely accepting
hydrogen bonds from the highly conserved residues His-158 and Arg-162,
while donating hydrogen bonds to Asn-160 and W1 (Table
II). The galactose C-6 hydroxyl group
also displays this cooperative hydrogen bonding pattern (33),
interacting with Glu-184, Asn-174, and W3. The planar C-3, C-4, C-5,
and C-6 carbon atoms of the galactose moiety are in van der Waals
contact with the aromatic side chain of Trp-181 in a fashion similar to
that seen in a number of other galactose and lactose binding lectins
(34).
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values for the
(1,4)-glycosidic linkage of Lac and LacNAc, respectively, are
70°/103° and 68°/103°, close to the calculated minima
for the saccharides in solution (36).
Extended Ligand Binding Site--
Examination of the
solvent-exposed surfaces of galectin-3-C, shows that the Lac/LacNAc
binding site, formed by -strands S4-S6, is a cleft, open at both
ends (see Fig. 3). At the nonreducing end
(galactose) of the bound carbohydrate the cleft is extended by residues
on -strands S1-S3, whereas at the reducing (GlcNAc) end it is open
to the surrounding solution. The carbohydrate binding site is similar
in galectin-1 and galectin-2, consistent with the demonstrated ability
of galectins-1 and -3 to bind longer oligosaccharides such as
polylactosaminoglycans (21, 37-39). These lectins, however, do show
differences in affinity for longer oligosaccharides, particularly those
substituted on the O-3 of the nonreducing galactose moiety (21, 38).
Galectin-3, for example, binds GalNAc1-3(Fuc1-2)Gal1-4Glc
with almost 100-fold higher affinity than does galectin-1 (20, 21). The
-linked GalNAc moiety would be expected to interact with residues in
the extended cleft formed by -strands S1-S3. As shown in Fig.
4, although the identity and conformation
of residues involved in binding the Lac/LacNAc moiety in the primary
binding site of galectin-1 and galectin-3 are very similar, they differ
in the vicinity of the galactose O-3. Galectin-3 has an arginine
residue at position 144 which is well positioned to interact with the
GalNAc moiety or other saccharide residues linked to the galactose O-3
(Fig. 4). The serine found in galectin-1 is presumably unable to make similar interactions. In addition, the bulky leucine residue at position 31 in galectin-1 is reduced to an alanine (Ala-146) in galectin-3, creating more space for O-3 substituents (Fig. 4).
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Possible Site of Interaction with RNA-- Mouse galectin-3 (formerly known as CBP35) has been shown to be part of the heterogeneous nuclear ribonucleoprotein complex (40, 41) and may be involved in pre-mRNA splicing in vitro (5). In addition, human galectin-3 has recently been shown to directly bind RNA fragments in a gel-shift assay (42). Electrostatic potential calculations (see Fig. 3.) show that the carbohydrate binding cleft of galectin-3 is flanked by a linear array of three positively charged arginine residues (Arg-186, Arg-162, and Arg-144). These residues are spaced approximately 5.3 and 7.9 Å apart, close to the phosphate repeat distance in RNA, leading to the possibility that the carbohydrate binding cleft may also be the RNA binding site. Consistent with this suggestion is the fact that the RNA splicing assay is inhibited by soluble oligosaccharides in a rank order reflecting their affinity for galectin-3 (5), even though the interaction of galectin-3 with heterogeneous nuclear ribonucleoprotein particles (41) and RNA fragments (42) appears not to be inhibited by lactose.
The Homologous Galectin-1/Galectin-2 Dimer Interface--
A
striking feature of galectin-3-C is that, in contrast with galectins-1
or -2, it is found to be monomeric in solution at protein
concentrations of up to approximately 0.1 mM (11). For this
reason it was of particular interest to examine the region in the
galectin-3 CRD corresponding to the canonical galectin-1 and -2 dimer
interface. As shown in Figs. 5 and
6 the galectin-1 dimer interface is a
very apolar surface composed of residues Leu-4, Ala-6, Leu-9, Phe-133,
Val-131, and Ile-128. In the galectin-3 CRD, the apolar nature of this
interface has largely been eliminated by a reduction of the F1-S1
-strand separation and the introduction of Tyr-118 and Tyr-247. In
both cases the ring hydroxyl group of the Tyr residues point into
solution creating a much more polar exposed surface (Fig. 6).
Furthermore, the canonical dimer interface is partially obstructed by
residues Leu-114, Ile-115, and Val-116, the end of the galectin-3
N-terminal domain (see Fig. 5). In fact, Val-116 is the start of a
conserved tripeptide sequence (V(L)PY) found in galectin-3, galectin-4,
and many other galectins, but not found in galectins-1 and -2. It makes
a cis-peptide linkage with the highly conserved Pro-117 which is in
turn followed typically by a tyrosine residue, in this case Tyr-118,
one of the two tyrosine residues responsible for the increased polarity
of the canonical dimer interface. Taken together it would appear that
the region corresponding to the dimer interface in galectin-1 and
galectin-2 does not serve a similar role in galectin-3.
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-sheet which may provide
a site for monomer-monomer interactions. Associations involving both
the N- and C-terminal domains could easily be achieved by a parallel
orientation of monomers, an arrangement seen in the collectins, soluble
C-type lectins with an N-terminal collagenous triple helical
oligomerization domain (44).
The CRDs of many lectin types have been found in different structural
arrangements among members of their respective families. In fact, the
precise structural arrangement of CRDs in multimeric lectins appears to
be an important means of conferring both specificity and affinity on
their interactions with multivalent carbohydrates (34). The fact that
galectin-3 does not possess the canonical 2-fold symmetric dimer
interface characteristic of galectins-1 and -2 suggests that the
galectins are no exception. Moreover, the fact that monomeric
galectin-3 is in equilibrium with higher order oligomers under
appropriate conditions, further suggests that in this case the valency
is dynamic.
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ACKNOWLEDGEMENT |
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We thank Robert Atchison for technical assistance in producing galectin-3.
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FOOTNOTES |
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* This work was supported by grants from the Medical Research Council of Canada and the National Cancer Institute of Canada (to J. M. R.) and a grant from the Cigarette and Tobacco Surtax Fund of the State of California through the Tobacco-related Disease Research Program of the University of California (to H. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates (code 1A3K) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
Current address: Dept. of Medical Microbiology, Section for
Clinical Immunology, Lund University, S-22362, Lund, Sweden.
** To whom correspondence should be addressed. Tel.: 416-978-0557; Fax: 416-978-6885; E-mail: james.rini{at}utoronto.ca.
1 The abbreviations used are: CRD, carbohydrate recognition domain; galectin-3-C, C-terminal fragment (amino acids 107-250) of galectin-3; Lac, lactose; LacNAc, N-acetyllactosamine; PEG, polyethylene glycol.
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REFERENCES |
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References
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