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Laboratory of Immunology, Department of Clinical Biochemistry, Faculty of Chemical Sciences, National University of Cordoba, Cordoba, Argentina;
Department of Biological Chemistry, Faculty of Pharmacy and Biochemistry, National University of Buenos Aires, Buenos Aires, Argentina; and
Centro de Excelencia en Productos y Procesos de Córdoba (CEPROCOR), Cordoba, Argentina.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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15,000 Da by SDS-PAGE that immunoreacted strongly
with the anti-chicken galectin serum. Gel filtration studies
revealed that the protein behaved like a dimer under native conditions,
and saccharides bearing a ß-D-galactoside
configuration were able to inhibit the hemagglutinating activity
displayed by the purified galectin. In agreement with its isoelectric
point of 4.8, the amino acid analysis showed a definitive acidic
pattern. Internal amino acid sequencing of selected peptides obtained
by proteolytic cleavage revealed that this carbohydrate-binding protein
shares all the absolutely preserved and critical residues found in
other members of the mammalian galectin-1 subfamily. Finally,
biochemical and ultrastructural evidence, obtained by genomic DNA
fragmentation and transmission electron microscopy, are also provided
to show its potential implications in the apoptotic program of T cells.
This effect was quantified by using the terminal deoxynucleotidyl
transferase-mediated dUTP biotin nick end-labeling assay and was found
to be associated to the specific carbohydrate-binding properties of
galectin. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Presently, 10 mammalian galectins have been well characterized (12).
The best studied among them, galectin-1, is a homodimer composed by
subunits of 134 amino acids, which belongs to the prototype
subfamily and is not subjected to posttranslational modifications (3).
Complete primary structures have been determined by direct peptide
analysis or deduced from cDNA nucleotide sequence from human lung (13, 14), human placenta (15), human HL60 promyelocytic leukemia (16), human
brain (17), a bovine fibroblast cell line (18), bovine heart (19), rat
lung and uterus (20), mouse 3T3 fibroblasts (21), and mouse
uv-fibrosarcoma (22). They are all structurally related (50–60%) to
the nonmammalian chicken isolectins
CLL-I4 or C-16 (23) and
CLL-II or C-14 (24).
Galectin-1 had shown therapeutic activity against autoimmune disease in two experimental models, i.e., experimental autoimmune myasthenia gravis (10) and experimental autoimmune encephalomyelitis (11). However, the molecular mechanisms involved in these immunomodulatory properties still remain to be elucidated. In this context, hypotheses have been raised concerning the ability of galectin-1 to affect processes in T cell suppressor commitment (11) and in sensitization or deletion of Ag-specific T cells (10). On the basis of recent investigations, there is evidence for specific cell growth-inhibitory activity of galectin-1 (7, 8) and its implication in apoptosis of activated T cells (25) and a particular subset of immature thymocytes (26).
However, despite considerable progress, several questions remain to be
addressed. Because galectin-1 had evidenced such
immunomodulatory properties, could it be possible to identify
its presence in key immunoregulatory cells such as macrophages (M
s)?
And if so, could it be possible to directly prove its functional
properties toward target cells? An investigation was conducted to
explore this field.
We have recently identified the presence of a soluble protein with key
features of mammalian galectins in rat peritoneal Ms, so-called
RMGal (27), by using a polyclonal Ab raised against the
extensively studied CLL-I (23, 28). It is particularly attractive
that RMGal total and surface expression were increased fivefold
in phorbol ester (PMA)- and chemotactic peptide
(FMLP)-"activated" Ms and twofold in peptone-elicited
"inflammatory" Ms (27).
In the present study, we confirm that this rat M protein is indeed a
"galectin-1-like" and report its purification, characterization,
and internal amino acid sequence. We also provide evidence showing its
implication in T cell apoptosis, an intrinsic cell suicide program that
has been as carefully regulated and preserved as galectins throughout
animal evolution (29, 30).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HBSS, RPMI 1640, PMA, FMLP, protease inhibitors, iodoacetamide, sugars, N-tosyl-L-phenylalanine chloromethyl ketone-treated trypsin, horseradish peroxidase-conjugated protein A, 4-chloro-1-naphthol, DNase-free RNase A, proteinase K, and Con A were purchased from Sigma (St. Louis, MO). Electrophoretic reagents were from Bio-Rad (Richmond, CA). FCS and L-glutamine were from Life Technologies (Paisley, U.K.). Trifluoroacetic acid (TFA) was from Baker (Phillipsburg, NJ), acetonitrile was HPLC grade, and all other chemical reagents were commercially available analytical grade.
Animals
Female Wistar rats (8 to 12 wk old; average weight, 250 g) were used in this study. Animals were housed and cared for at the Animal Resource Facilities, Department of Clinical Biochemistry, Faculty of Chemical Sciences, National University of Cordoba, in accordance with institutional guidelines.
Purification and activation of rat peritoneal macrophages
Peritoneal cells (PCs) were harvested from rats as previously
described (27). RBC were removed by the addition of lysis buffer (0.15
M NH4Cl, 1 mM KHCO3), and the M
population was purified from PCs by adherent culture in 100-mm-diameter
tissue culture dishes in RPMI 1640 supplemented with 20 µg/ml
gentamicin, 2 mM L-glutamine, and 10% heat-inactivated
FCS. Nonadherent cells were removed after 2 h at 37°C, and
monolayers of adherent cells were incubated overnight in the same
medium. The resultant M monolayer was 98% pure according to
morphologic and phagocytic criteria. Activated Ms were obtained by
in vitro treatment of cultured cells with 1) 1 µg/ml PMA for 120 min
at 37°C and 5% CO2 or 2) 200 nM FMLP for 120 min at
37°C and 5% CO2. Cells incubated with medium alone were
used as a control of in vitro activation.
Purification of the galectin-1-like protein from rat activated
Ms
Activated Ms (3 x 107 cells) were
washed twice with PBS (125 mM NaCl, 25 mM
Na2HPO4/NaH2PO4, pH
7.2) and sequentially homogenized with PBS containing 4 mM 2-ME, 2 mM
EDTA, and protease inhibitors (MEPBS) supplemented with 100 mM lactose.
Cells were disrupted by five cycles of sonication at 12 kHz for 5
s at 4°C, and the total cell lysate was centrifuged at 600 x
g for 10 min to remove cell debris. To obtain the soluble
fraction, cell lysates were then centrifuged at 100,000 x
g for 60 min. The lactosyl-Sepharose matrix was prepared by
lactose immobilization on divinyl sulfone-activated Sepharose 6B
(Sigma), and the affinity chromatography was performed on the
lactosyl-Sepharose matrix as previously described (31). Briefly,
cytosolic fractions from activated Ms were dialyzed against MEPBS to
remove lactose and applied at a flow rate of 5 ml/h to the
lactosyl-Sepharose column (0.9 x 10 cm) previously equilibrated
with MEPBS. The column was thoroughly washed with MEPBS until no
absorbance at 280 nm was detected in the effluent. Finally, the
adsorbed material was specifically eluted with MEPBS containing 100 mM
lactose and collected in 1-ml fractions. To stabilize the
carbohydrate-binding activity sensitive to oxidation of sulfhydryl
groups, the purified lectin was treated for 1 h with 0.1 M
iodoacetamide. Then, after extensive dialysis against MEPBS, fractions
monitored for protein content at 280 nm that displayed hemagglutinating
activity were pooled and concentrated using a Centripep 10 M. All
procedures were conducted at 4°C unless stated otherwise.
Hemagglutination assay
Lectin activity was determined as hemagglutinating activity
following the procedure described by Nowak et al. (32), using twofold
serial dilutions of samples in microtiter U plates and trypsin-treated
glutaraldehyde-fixed rabbit erythrocytes. To analyze the
inhibitory effect of sugars on hemagglutinating activity of the
purified M lectin, saline was replaced by different sugar solutions.
The reciprocal of the highest dilution of the lectin showing visible
agglutination was recorded as the titer, and the concentration of
sugars that inhibited hemagglutinating activity by 50% was also
determined.
Chicken galectin Ab preparation
A rabbit antiserum was raised against a 16,000 Da chicken galectin purified from adult liver (CLL-I or C-16), and used for immunochemical and immunocytochemical studies as previously described (27, 31, 33, 34).
Protein determination and amino acid analysis
Protein concentration was estimated by the method of Bradford (35), using BSA as standard, and by amino acid composition. Amino acid analysis was performed in triplicate by hydrolysis of the samples with 6 N HCl vapor at 110°C for 24 h under reduced pressure. The hydrolysates were analyzed on a model 420A amino acid analyzer (Applied Biosystems, Foster City, CA).
SDS-PAGE and Western blot
SDS-PAGE was performed in a Miniprotean II electrophoresis
apparatus (Bio-Rad) as described (36). Briefly, M lysates, purified
RMGal, and CLL-I were diluted in sample buffer and resolved on a 12.5%
separating polyacrylamide slab gel. Protein bands were detected using
Coomassie Brilliant Blue R250. After electrophoresis, the separated
proteins were transferred onto nitrocellulose membranes and probed with
a 1:250 dilution of the anti-galectin serum as previously described
(27). Blots were then incubated with 1 µg/ml horseradish
peroxidase-conjugated protein A and developed with 4-chloro-1-naphthol.
Control of specific immunoreaction was performed by incubation of the
blots with a rabbit preimmune serum.
Gel filtration analysis
Purified RMGal was submitted to fast protein liquid chromatography on a Superose 12 HR 10/30 column (Pharmacia LKB, Uppsala, Sweden) to determine its m.w. under nondenaturing conditions. The column was equilibrated and eluted with PBS containing 2 mM DTT and 2 mM EDTA at a flow rate of 0.5 ml/min. Standards used for calibration of the column were: human IgG (150,000 Da); BSA (66,000 Da); carbonic anhydrase (29,000 Da); cytochrome c (12,400 Da).
Isoelectric focusing (IEF)
Isoelectric point determinations were conducted in a Phast System (Pharmacia LKB) using a PhastGel IEF of 3.75 to 9.30 and pI calibration kit for PhastGel IEF (pH range covered, 3.50–8.65). Protein bands were detected by Coomassie Brilliant Blue R250 staining.
Enzymatic digestion and peptide purification
To determine its internal sequence, the protein was reduced, carbamidomethylated, and digested with trypsin at a 1:20 enzyme-substrate ratio in 2 M urea, 0.1 M ammonium bicarbonate at 37°C for 20 h (37). Tryptic peptides were separated by reverse phase HPLC (Applied Biosystems model 140A) on a Brownlee C18 column (2.1 x 220 mm) equilibrated with 95% solvent A and 5% solvent B (solvent A, 0.1% (v/v) TFA in water; solvent B, 80% (v/v) acetonitrile, 0.08% (v/v) TFA in water). Elution was performed at a flow rate of 0.8 ml/min with a 10 to 60% solvent B gradient for 70 min, and the eluent was monitored at 220 nm.
Amino acid sequencing and computerized sequence comparisons
Selected peptides were applied to a Polybrene-coated glass filter and sequenced in an Applied Biosystems model 477A automatic sequencer. Searches for homologies to the determined sequence were performed with the aid of the SWISS PROT and DNASIS protein sequence databases.
DNA extraction and electrophoretic analysis of DNA fragmentation
Spleen mononuclear cells (SpMs) and T cell-enriched populations were obtained from normal rats as previously described (27). Cell viability assessed by means of the trypan blue exclusion test was consistently >95%. To analyze DNA fragmentation, nonstimulated and mitogenically stimulated (Con A, 2.5 µg/ml) total SpMs or T cell-enriched populations were cultured in 24-well plates at a density of 2 x 107 cells per well in 1 ml of complete medium (RPMI 1640 plus 10 mM HEPES, 2 mM L-glutamine, 50 µM 2-ME, and 100 µg/ml gentamicin, supplemented with 10% heat-inactivated FCS), in the absence or in the presence of increasing concentrations of purified RMGal (4 or 6 µg/ml) for 3, 6, and 18 h, at 37°C in a humidified atmosphere of 5% CO2 in air. Optimal experimental conditions were found to be as follows. Galectin was incubated for 6 h at a concentration of 4 µg/ml (25 µM). Cells were then harvested, washed with TNE buffer (10 mM Tris-HCl pH 7.5, 100 mM NaCl, 2 mM EDTA, pH 8), and lysed by addition of 0.5% SDS. Then, cell lysates were incubated at 56°C for 3 h in the presence of 100 µg/ml proteinase K. After digestion, DNA was purified by successive phenol-chloroform extractions, and the resultant aqueous phase was mixed with 3 M sodium acetate (pH 5.2) and absolute ethanol. The mixture was incubated at -20°C overnight, and the ethanol-precipitated DNA was washed with 70% (v/v) ethanol. The purified DNA was resuspended in TE buffer (10 mM Tris-HCl and 1 mM EDTA, pH 7.5), and treated with 5 µl of 1 mg/ml DNase-free RNase A for 1 h. Samples were finally resuspended in loading buffer and resolved on a 1.8% agarose gel containing 0.5 µg/ml ethidium bromide. Electrophoresis was conducted in TBE buffer (89 mM Tris-HCl, 89 mM boric acid, and 2 mM EDTA, pH 8.4), and DNA visualization was accomplished under UV light.
TUNEL assay
Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP biotin nick end-labeling (TUNEL) assay was performed by a modification of the method of Gorczyca et al. (38). Samples consisting of nonstimulated or Con A-stimulated (2.5 µg/ml) T cell-enriched population (2 x 107 cells/well) were incubated for 6 h at 37°C in a humidified atmosphere of 5% CO2 in air, in the presence or absence of RMGal (4 µg/ml). To test the specificity of the apoptotic effect, cells were also incubated either with the galectin Ab (dilutions ranging from 1:25 to 1:250) or with galectin-specific (lactose) or nonspecific (glucose, galactose, and fucose) sugars at concentrations ranging from 10 to 100 mM. Cells were then harvested, washed with PBS containing 0.2% BSA, and fixed for 30 min at 4°C by using 2% buffered paraformaldehyde (pH 7.4). After permeabilization with PBS containing 0.2% BSA and 0.1% Triton X-100 for 15 min, cells were processed for TUNEL assay by using the MEBSTAIN Apoptosis Kit (Immunotech, Marseille, France, Cat. No. 1946), according to the manufacturer’s recommended protocol for flow cytometry. Incorporation of biotinylated dUTP by exogenous TdT was detected using FITC-conjugated avidin, and the green fluorescence was then analyzed in a Cytoron Absolute cytometer (Ortho Diagnostic System, Raritan, NJ). Cells treated with DNase I (1 µg/ml) for 1 h at 37°C after permeabilization were used as positive controls, whereas negative controls involved the omission of the TdT enzyme during TUNEL reaction.
Transmission electron microscopy
Ultrastructural features of apoptosis were studied on T cells cultured in the absence or in the presence of RMGal (4 µg/ml). After 6 h of incubation, medium was removed and cells were fixed by immersion in 1% glutaraldehyde diluted in 0.1 M cacodylate buffer (pH 7.3). Samples were postfixed in 1% OsO4, dehydrated, and embedded in Araldite (Ciba-Geigy, Summit, NJ). Thin sections were cut in a Porter-Blum MT2 ultramicrotome and examined in a Zeiss 109 electron microscope (Zeiss, Overkochen, Germany). Photographs were taken on a Kodak electron imaging film (Eastman Kodak, Rochester, NY).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To further explore its properties and functions, RMGal was
purified by a single-step affinity chromatography from activated Ms.
Briefly, peritoneal Ms were cultured, activated with PMA or FMLP,
and disrupted by sonication in the presence of MEPBS containing 100 mM
lactose as previously described (27). To avoid the presence of the
recently cloned galectin-5 (39), erythrocytes were removed in all the
cases by the addition of lysis buffer. After extensive dialysis against
MEPBS to remove lactose, cytosolic fractions that showed
hemagglutinating activity were applied to a lactosyl-Sepharose matrix.
The column was extensively washed with MEPBS, and finally the
hemagglutinating activity was quantitatively eluted in a single peak
with MEPBS containing 100 mM lactose. A typical purification profile is
depicted in Figure 1
. The purified M
galectin accounted for 0.05% of total soluble proteins present in cell
lysates, yielding an average recovery of 10 µg from 3 x
107 activated cells. The concentration of sugars that
inhibited 50% of hemagglutinating activity of the purified RMGal was
determined after dialysis against MEPBS (Table I). As expected, the most potent
inhibitors were sugars bearing a ß-D-galactoside
configuration such as thiodigalactoside and lactose, resembling the
carbohydrate-binding properties of other members of this protein
family. Other sugars such as galactose, melibiose, and
N-acetylgalactosamine were much less effective inhibitors.
On the other hand, early fractions representing the run-through
material neither exhibited hemagglutinating activity nor immunoreacted
with the anti-galectin serum (results not shown).
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To characterize the purified galectin, the following properties of RMGal were determined.
Molecular weight determination.
The single-step affinity chromatography procedure resulted in the
isolation of a homogeneous purified galectin resolved as a sharp single
protein band corresponding to an apparent subunit molecular weight of
15,000 ± 500 by SDS-PAGE (Fig. 2A, lanes 1 and
2). The starting material, consisting of total
soluble proteins of activated Ms, was also resolved by SDS-PAGE at
two concentrations (Fig. 2A, lanes 3 and
4). As a strategy to discriminate between the range
14,000 to 16,000 Da, a mixture (5:1) of affinity-purified CLL-I
(Mr 16,000) and RMGal was resolved
under identical conditions (Fig. 2B).
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). The elution profile showed a peak of
absorbance at 280 nm with Mr 32,000,
suggesting that RMGal behaves like a dimer under physiologic
conditions. Besides, this dimeric form showed the specific
hemagglutinating activity exhibited by other members of the galectin-1
family. Moreover, migration of RMGal as a monomer by SDS-PAGE under
both reducing (Fig. 2
) and nonreducing (data not shown) conditions
suggests that the apparent dimeric structure in the native state is due
to noncovalent associations.
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. The M galectin
focused as a single band corresponding to an isoelectric point of
4.8, thus confirming the homogeneity of the preparation. In broad
agreement, amino acid analysis of the purified protein evidenced a
definitive acidic pattern. The amino acid composition was characterized
by a high content of glycine, serine, and glutamic and aspartic acids
(data not shown).
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galectin-like protein in total
cells lysates by a strong cross-reactivity with a polyclonal Ab raised
against CLL-I, a nonmammalian galectin (27). We herein determined that
the isolated RMGal also reacted with the same anti-chicken galectin
Ab. Immunoreactive profiles of affinity-purified CLL-I and RMGal are
shown in Figure 5 (lanes 1and 2, respectively). As a control of specific
reaction, purified RMGal was probed with a preimmune serum at the same
dilution (lane 3).
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To assure identity of the isolated protein, partial amino acid
sequence was determined at the protein level and compared with galectin
sequences already established. The 15,000-Da galectin band from
SDS-PAGE was first blotted onto a polyvinylidene difluoride membrane
and then applied to the automatic sequencer. No
phenylthiohydantoin-amino acid peaks were detected in significant
amounts, indicating that the N terminus was blocked. Hence, the
internal amino acid sequence of RMGal was determined by tryptic
digestion of the protein. The elution profile of the resultant
peptides, fractionated by reverse phase HPLC, is shown in Figure 6. Selected peptides were then sequenced
by automated Edman degradation.
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galectin and galectin-1 from different sources showed relevant
overlapping sequences (Fig. 7). A high
degree of identity could be found between RMGal and galectin-1 from rat
lung and uterus (81.3%), mouse 3T3 fibroblasts (M3T3, 78.0%), bovine
fibroblasts (92.3%), human placenta and brain (84.6%), chicken skin
(CLL-II or C-14, 61.0%), and chicken liver (CLL-I or C-16, 56.0%),
the antigen for which the Ab was raised. For comparison purposes, two
consensus sequences were included: the first is restricted to mammalian
galectin-1; and the second shows all invariant amino acids preserved in
most 14,000- to 16,000-Da galectins analyzed thus far.
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The next issue we attempted to elucidate was related to the
functional significance of galectin overexpression in activated Ms.
The fact that galectins have recently been proposed to be involved in
the regulation of apoptosis (25, 26, 34, 40) and the idea that Ms
are key immunoregulatory cells able to trigger "activation" or
"death" (41) prompted us to investigate the ability of the purified
RMGal to induce T cell apoptosis.
For this purpose, mitogenically stimulated and nonstimulated SpMs were
cultured in the presence of optimal concentrations of RMGal and then
processed for DNA fragmentation, TUNEL assay, and transmission electron
microscopy. The electrophoretic pattern of genomic DNA extracted after
6 h of cell culture is shown in Figure 8. The highly characteristic DNA cleavage
into oligonucleosomal-size fragments of 180 to 200 bp was
intensified in SpMs stimulated with Con A and exposed to RMGal (Fig. 8, lane 3), and in SpMs incubated with RMGal alone at
two concentrations (lanes 4 and 5,
respectively). In contrast, ladder-type DNA fragmentation was almost
not observed in DNA extracted from SpMs cultured in medium alone (Fig. 8, lane 1), whereas cells stimulated with Con A but
not treated with RMGal showed the typical low intensity pattern of
fragmentation (42), characteristic of cell death following activation
with mitogenic stimuli (lane 2). The same
pattern was clearly evidenced when the T cell population was purified
from total SpMs. Genomic DNA fragmentation was found to be particularly
increased when RMGal was added to stimulated (lane
8) and nonstimulated (lane 9) T
cells, in comparison with controls of T cells in medium alone
(lane 6) and T cells stimulated with Con A
(lane 7).
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, samples exposed to RMGal showed 33.4%
of apoptotic cells, evidenced by specific incorporation of biotinylated
dUTP into DNA strand breaks (Fig. 9B), whereas
control samples cultured in medium alone showed 5.0% of spontaneous
apoptosis (Fig. 9A). Moreover, the proportion of
TUNEL-positive cells increased to 38.0% when cells were simultaneously
incubated with Con A and RMGal (Fig. 9D), in
comparison with cells cultured in the presence of Con A but unexposed
to RMGal (Fig. 9C, 9.5%). To explore the specificity of the
apoptotic effect and to investigate whether the carbohydrate
recognition domain of RMGal is involved in this galectin function,
lactose was added to cell cultures at increasing concentrations. The
specific disaccharide was able to inhibit galectin-induced apoptosis
when added at a concentration of 100 mM, exerting a decrease from
33.4% to 11.8% of TUNEL-positive cells (Fig. 9, B vs
E). In contrast, nonspecific sugars could not prevent
this effect at any of the concentrations tested (data not shown). Thus,
induction of apoptosis by RMGal was highly specific and related to its
carbohydrate-binding properties. On the other hand, the effect was not
significantly blocked when cells were exposed to RMGal in the presence
of the galectin Ab (Fig. 9, B vs F, 33.4% vs
31.7%). Apoptosis was further confirmed by ultrastructural studies of
morphologic changes induced by RMGal. Representative electron
micrographs of T cells exposed to RMGal (4 µg/ml) for 6 h are
shown in Figure 10 (Fig. 10A), in
comparison with untreated cells (Fig. 10B).
Galectin-treated cells revealed the typical ultrastructural features of
apoptosis, including reduction of the cytoplasmic volume, loss of
surface microvilli, chromatin condensation, and margination along the
inner surface of the nuclear envelope.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study involved the purification, characterization, and
protein sequence analysis of a galectin-1-like protein from activated
rat Ms and strong evidence of its implications in T cell
apoptosis.
Until recently, it was relatively easy to recognize the same galectin
among different mammalian species because of great amino acid sequence
conservation. However, the immunologic relationship of particular
mammalian galectins to those found in lower vertebrates and
invertebrates is still a matter of controversy (2, 3). In our approach,
we succeeded in identifying a rat galectin-1-like protein in peritoneal
Ms by means of a strong cross-reactivity with a polyclonal Ab raised
against the "nonmammalian" CLL-I or C-16. Despite the fact that
RMGal shares all the absolutely critical residues of galectin-1, we do
not rule out the possibility that it will be an alternative isoform of
galectin-1, as described for chicken isolectins (28). Strikingly,
functional activation of Ms by protein kinase C agonists and
inflammatory agents was accompanied by substantial remodeling of RMGal
total and surface expression. This type of regulated expression,
extensively documented in our previous work (27) and deeply studied for
other galactoside-binding proteins (44, 45), made this system
attractive for purification of this protein at conditions in which
cells were activated; hence, it reached its maximum levels.
Therefore, RMGal was purified from chemically activated Ms by a
single-step affinity chromatography on a lactosyl-Sepharose matrix,
resulting in the isolation of a sharp protein band of a subunit
molecular weight of 15,000 immunoreactive with an anti-galectin
serum, an isoelectric point of 4.8 consistent with an acidic amino
acid composition, and a high hemagglutinating activity specifically
inhibited by saccharides bearing a ß-D-galactoside
configuration. Furthermore, gel filtration studies showed that the
galectin behaves as a dimer under nondenaturing conditions. Taken
together, all the described properties of RMGal resemble those
exhibited by mammalian galectin-1. Definitive data on its nature and
identity were obtained by microsequencing tryptic peptides derived from
the affinity-purified protein. Computer-assisted sequence library
searches revealed extensive alignments with mammalian galectin-1,
indicating that galectin-encoding genes may have derived from a single
ancestor gene that has evolved by several duplications and being an
explanation for the shared antigenicity of these molecules (2, 17).
Which will hence be the physiologic relevance of such regulated
expression of a carbohydrate-binding protein in key immunoregulatory
cells? Resident, inflammatory, and activated Ms show different
profiles of enzymes and receptors that can be up- or down-regulated and
are closely related to functional competence (41). Because galectin-1
has recently been proposed to induce apoptosis (25, 26), we wondered
whether overexpression of RMGal in activated Ms and further binding
to target cells could be an alternative pathway in the generation of
death signals. Strong biochemical and ultrastructural evidence is
provided in this study to show that the purified M galectin is
clearly associated to a positive control in the apoptotic threshold of
T cells. The highly characteristic ladder pattern of DNA cleavage into
oligonucleosome-sized fragments of 180 to 200 bp was highly
intensified within 6 h of RMGal treatment in both stimulated and
nonstimulated SpMs and T cells. It suggests that in our experimental
conditions cell stimulation is not an esential step for RMGal-induced
apoptosis. Moreover, by using the TUNEL assay after correcting for
spontaneous apoptosis in control samples, we detected 28% of T
cells undergoing apoptosis on exposure to RMGal. This effect was highly
specific since lactose, a ß-galactoside-related sugar, was able to
prevent incorporation of biotinylated dUTP in the TUNEL assay,
suggesting that this galectin function is related to its
carbohydrate-binding properties. However, the galectin Ab was not able
to block apoptosis at any of the dilutions tested, raising the
possibility that it could not recognize an active domain involved in
this function, such as the carbohydrate recognition domain. In
addition, RMGal-treated cells displayed the typical ultrastructural
changes compatible with the development of an apoptotic cell death
program. Consistent with these findings, we recently provided evidence
in a biologically "hetero" system that the chicken galectin CLL-I
inhibits growth of rat T cells via apoptosis, which proved to be
controlled in time, lectin concentration, and in a saccharide-dependent
manner (34). In our study we succeeded in extrapolating those results
to an endogenous physiologic system (galectin and cells from the same
species). These achievements are worthwhile of discussion in terms of
the high conservation of this protein family.
It is now widely accepted that animal cells have the ability to self-destruct by activation of an intrinsic cell suicide program when they become seriously damaged, or are no longer needed, as in the case of potentially autoreactive lymphocytes and excess cells after the completion of an immune response (29, 46). Although diverse signals can induce apoptosis in a wide variety of cell types, a number of evolutionary conserved genes regulate a final common cell death pathway that is preserved from worms to humans (30). Suggestively, a novel 16,000-Da galectin has recently been identified in the nematode Caenorhabditis elegans (47), an advantageous system for the elucidation of complex biologic phenomena occurring in multicellular organisms, such as apoptosis. In support, an interesting report describes the overexpression of the human galectin-1 gene, encoding a ß-galactoside-binding lectin during the induction of apoptosis by glucocorticoids (48).
Finally and on the basis of our present knowledge, is it possible to envisage any physiologic role for galectins in vivo? Null-mutant mice as regards the galectin-1 gene have been generated (49), but these knockout mice were found to be completely vital and proliferative. Nevertheless, it seems likely that a novel paradigm is widening the horizons of galectin research. At present clear-cut evidence exists concerning the interrelationship of effector and repressor genes within each animal cell death pathway. In this context, the best defined genetic program exists in C. elegans, in which two effector genes, ced-3 and ced-4, and one protector gene, ced-9, are required for the regulation of programmed cell death (50). On the other hand, a model has been proposed in mammals in which the ratio of Bcl-2 to Bax, two members of the same family, determines survival or death following an apoptotic stimulus (51). As galectin-1 promotes T cell apoptosis (25, 26) and conversely, galectin-3, has been recently shown to confer resistance to programmed cell death through a cell death inhibition pathway involving Bcl-2 (40), it seems meaningful that the interplay between galectin-1 and galectin-3 could represent an alternative pathway in the normal control of cell growth that hinges on a delicate balance between cell proliferation and cell death.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Gabriel A. Rabinovich, Laboratorio de Inmunología, Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Ag. Postal 4, CC 61, (5000) Córdoba, Argentina. E-mail address:
3 N.M.M. and L.F.C. contributed equally to this work.
4 Abbreviations used in this paper: CLL-I, chicken lactose lectin-I; MEPBS, phosphate-buffered saline containing 4 mM 2-ME and 2 mM EDTA; Ms, macrophages; PCs, peritoneal cells; RMGal, rat macrophage galectin; SpMs, spleen mononuclear cells; TdT, terminal deoxynucleotidyl transferase; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end-labeling; TFA, trifluoroacetic acid; IEF, isoelectric focusing.
Received for publication July 30, 1997. Accepted for publication January 22, 1998.
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)) and protein determination (absorbance at 280 nm
(
)). Protein concentration was confirmed by the method of Bradford
(35) and by amino acid composition.
