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Purification of galectin-3 from ovine placenta: developmentally regulated expression and immunological relevance
Introduction
Results
Discussion
Materials and methods
Acknowledgments
Abbreviations
References
Purification of galectin-3 from ovine placenta: developmentally regulated expression and immunological relevance
Galectins, [beta]-galactoside-binding lectins, are extensively distributed in the animal kingdom and share some basic molecular properties. Galectin-3, a member of this family, is generally associated with differentiation, morphogenesis, and metastasis. In this study, galectin-3 was isolated from ovine placental cotyledons round the middle of the gestation period by lactose extraction followed by affinity chromatography on lactosyl-agarose, and separated from galectin-1 by size exclusion chromatography on a Superose 12 column. Under native conditions this lectin behaved as a monomer with an apparent molecular weight of ~29,000 and an isoelectric point of 9.0. The partial amino acid sequence of the peptides obtained by tryptic digestion of this protein followed by HPLC separation showed striking homology with other members of the galectin-3 subfamily. Furthermore, ovine placental galectin-3 exhibited specific mitogenic activity toward rat spleen mononuclear cells. Besides, this protein strongly reacted with a rabbit antiserum raised against a chicken galectin. Results obtained by Western blot analysis showed that its expression was greatly decreased in term placenta with respect to the middle of the gestation period, suggesting a regulated expression throughout development. Key words: animal lectin/[beta]-galactoside-binding lectin/galectin-3/mitogenic activity/ovine placenta
Introduction
Galectin-3 is a member of a growing family of [beta]-galactoside-binding lectins (Barondes et al., 1994), with an approximate molecular weight of 30,000. It has previously been known under different names, including CBP-35 (Jia and Wang, 1988), IgE-binding protein (Albrandt et al., 1987), Mac-2 (Cherayil et al., 1989), L-29 (Leffler et al., 1989), L-34 (Raz et al., 1989), and CBP-30 (Sato and Hughes, 1992). It was originally identified in mouse 3T3 fibroblasts (Roff and Wang, 1983), and later found in breast carcinoma (Oda et al., 1991), basophilic leukemia cells (Cherayil et al., 1989), human and murine tumor cells (Raz et al., 1989, 1991), human and rat lung (Sparrow et al., 1987; Leffler et al., 1989), human HeLa cells (Robertson et al., 1990), etc.
A variety of biological functions have been proposed for this protein, such as an involvement in the cell growth regulation (Moutsatsos et al., 1987), pre-mRNA splicing (Dagher et al., 1995), cell adhesion to basement membranes (Woo et al., 1990) and mast cells, neutrophils and monocytes activation (Frigeri et al., 1993; Liu et al., 1995; Yamaoka et al., 1995). Moreover, galectin-3 is generally associated with tissues in processes of differentiation and upregulated in neoplastic transformation (Raz et al., 1987).
Recently it has been shown that transformed T-cell lines infected with human T-cell leukemia virus type I express high levels of galectin-3, in significant contrast to uninfected cells, which do not express detectable amounts of this protein (Hsu et al., 1996). Furthermore, when these cells were transfected with galectin-3 cDNA, they displayed higher growth rates in comparison to control transfectans (Yang et al., 1996) and showed resistance to apoptosis induced by anti-Fas antibody and staurosporine. Hence, galectin-3 functions as a regulator of cell growth and apoptosis through its identity to the NWGR sequence motif of Bcl-2, a well-characterized proto-oncogen, suppressor of programmed cell death (Yang et al., 1996). On the other hand, Perillo et al. (1995) reported the induction of apoptosis in immune cells by human galectin-1. Thus, the potential coexistence of galectin-1 and -3, such filogenetically conserved proteins, in thesame tissue could represent an alternative pathway in the control of life and death.
However, Hirabayashi and Kasai (1984) identified galectin-1 in human term placenta, but found no evidence of the presence of galectin-3. Recently, we reported the isolation of galectin-1 from ovine placenta (Ivanovic et al., 1996), and in the present study we describe the purification and characterization of galectin-3 from ovine placental cotyledons. Furthermore, we compare its differential expression in cotyledons at mid-gestation and at term placenta and show evidence supporting its role as a cell growth regulator.
Results
Purification and molecular weight determination of galectin-3
After affinity chromatography on lactosyl-agarose the purified material was submitted to SDS-PAGE (Figure 1, lane c) showing two main bands, one with a molecular weight of about 16,000, corresponding to galectin-1 (Ivanovic et al., 1996) and the other of 29,000, similar to galectin-3.
Figure
The separation of these galectins was achieved by size exclusion chromatography on a Superose 12 HR column and its elution profile is shown in Figure 3. The results of the SDS-PAGE show that each of the isolated peaks (Figure 1, lanes d and e) corresponds to one of the protein bands. This method not only separates both proteins, but also allowed us, on the basis of its elution volume, to establish that galectin-3 behaves as a monomer of a molecular weight of ~29,000 under physiological conditions. On the other hand, galectin-1 behaves as a dimer with a molecular weight of ~32,000. The first peak (galectin-1) showed specific hemagglutinating activity, as described previously (Ivanovic et al., 1996), while the second peak (galectin-3) did not, in agreement with its monomeric condition. The relative proportion of the two peaks round the middle of the gestational period was approximately 4:1 (galectin-1 to galectin-3).
Western blot analysis
In order to assess the immunological relationship between purified galectins, the material eluted from the affinity column was submitted to SDS-PAGE and blotted onto nitrocellulose membranes. Both protein bands strongly reacted with a rabbit anti-galectin serum, as determined by Western blot analysis (Figure 2A, lane c). Consequently, both galectins-1 and -3 separated by FPLC (Figure 3) also cross-reacted with this antiserum, indicating that they belong to the same family, as expected from their common affinity for the lactose-agarose column.
Figure
Figure
Partial amino acid sequence and isoelectric point of galectin-3
In order to confirm that the purified protein was indeed galectin-3, the band from SDS-PAGE was blotted onto a polyvinylidene difluoride membrane and applied to the automatic sequencer. No PTH-amino acids were detected in significant amount, indicating that the N-terminus was blocked. Therefore, the protein was submitted to enzymatic digestion in order to obtain internal sequence. A tryptic digest of this protein was fractionated by reverse phase HPLC on a C18 column, and the elution profile is shown in Figure 4. Selected peptides were sequenced, and the comparison of their amino acid sequences with the corresponding regions of galectin-3 from human HeLa cells (Robertson et al., 1990) and rat basophylic leukemia cells (Albrandt et al., 1987) is shown in Figure 5. Identities of 89% and 84% could be deduced after sequence comparison of the purified ovine placenta protein with human and rat galectins-3, respectively. All the peptides obtained belong to the C-terminal region of the molecule, since the N-terminal region, known as hnRNP-like domain, contains a repetitive sequence, rich in proline and glycine, with no trypsin-sensitive bonds (Barondes et al., 1994).
Figure
Figure
The isoelectric point of galectin-3, determined by isoelectric focusing of the purified galectin, was 9.0, in contrast to the acidic isoelectric point of galectin-1, which was approximately 5.2, in agreement with those reported for human galectins (Lutomski et al., 1996).
Cell growth regulatory activity
To examine whether galectin-3 was able to regulate cell growth in vitro proliferation, Concanavalin A (Con A) stimulated (5 µg/ml) and nonstimulated rat spleen mononuclear cells (SpMs) were cultured for 72 h in the absence or in the presence of increasing concentrations (1.5 and 3 µg/ml) of affinity purified galectin-3 and monitored by [3H]TdR incorporation.
As shown in Figure 6, when purified galectin-3 was added to nonstimulated resting SpMs cultures, we detected strong mitogenic activity at a threshold of 3 µg/ml with an approximately 8-fold increase in the proliferative response compared to SpMs cultured in medium alone. Then, when SpMs were simultaneously incubated with Con A (5 µg/ml) and galectin-3 (3 µg/ml), an additive effect on the proliferative response could be detected (Figure 6). Moreover, lactose (100 mM) was able to partially prevent galectin-3-induced proliferation incontrast to nonspecific sugars such as fucose and glucose (data not shown).
Figure
Morphological changes induced after 72 h culture by galectin-3 and the generation of cell clusters are documented in Figure 7. Galectin-3 treated cells (Figure 7B) showed distinct cell clusters characterized by their particular size and refringence, as compared with typical blasts induced by Con A (Figure 7C). In contrast, untreated cells incubated with medium alone did not show any particular clusters (Figure 7A). Strikingly, cells simultaneously incubated with Con A and galectin-3 exhibited the two different patterns of cell agglutination described above, as indicated in Figure 7D.
Regulated expression of galectin-3 at two different placental developmental stages
In order to elucidate whether galectin-3 expression was differentially regulated throughout development, Western blot analysis was performed from mid-gestation and term placenta. When the initial lactose extraction supernatant was obtained from the latter, it was necessary to apply 5-fold more protein to the SDS-PAGE in order to detect potential immunoreactivity (Figure 2B). As judged by the quantity of sample resolved and the densitometric analysis of the blot, there was a 35-fold decrease in the content of galectin-3 at term placenta with respect to that found round the middle of the gestation period. In contrast, no significant decrease in galectin-1 expression was observed. Similar results were obtained from three separate experiments using different placentas under the same conditions.
Discussion
In multicellular organisms, homeostasis is maintained through a balance between cell proliferation and cell death (Evan et al., 1995). In the present study, we describe the coexistence in ovine placenta of galectin-1 and -3, two highly conserved carbohydrate binding proteins that represent alternative signals in the decision between life and death (Perillo et al., 1995; Yang et al., 1996).
Herein, we describe the first purification of galectin-3 from ovine placental tissue, its identification based on its affinity for lactose, its molecular weight of [cong]29,000, its basic isoelectric point, its immunological cross-reactivity with other members of this lectin family, and the striking homology of its partial amino acid sequence with those found in rat and human galectin-3. In addition, size exclusion chromatography showed that it behaves as a monomer at physiological conditions. The presence of a single carbohydrate-recognition domain per molecule (Barondes et al., 1994), is in broad agreement with its lack of hemagglutinating activity. The galectin-3 molecule can be divided into two domains: an N-terminal half, which is rich in proline and glycine, and a C-terminal half, which is similar to the carbohydrate-recognition domain (CRD) of other members of the galectin family. Since the CRD is highly conserved among different species (Hirabayashi and Kasai, 1996), it is not surprising that anti-galectin serum immunoreacted with both ovine galectin-1 and -3, as evidenced by Western blot analysis.
It has recently been observed that galectin-3 is downregulated in breast cancer (Castronovo et al., 1996) and it was suggested that its decreased expression is associated with the acquisition of the invasive and metastatic phenotype. Its presence has also been described in embryos (Colnot et al., 1996) as well as in trophoblast cells (Van Den Brule et al., 1994). Consequently, the identification of galectin-3 in a proliferating tissue such as placenta makes this system attractive to study processes such as cell growth regulation and immunomodulation. In our study we detected a significant increase in lymphocyte proliferative response upon incubation with galectin-3 purified from mid-gestation placenta and an additive effect on the mitogenic activity of Con A. Consequently, we suggest that galectin-1 and -3 could positively or negatively affect the engagement of the apoptotic program of the cell, according to physiological needs.
Due to the particular features of placental tissue, it was interesting to study the developmentally regulated expression of galectin-3 throughout different stages of pregnancy. The results obtained by Western blot analysis show that its expression is greatly decreased in term placenta with respect to the middle of the gestation period. This observation could also explain the lack of evidence of its presence in human term placenta (Hirabayashi and Kasai, 1984).
Finally, our study is the first evidence of the presence of galectin-3 in ovine placental tissue; its developmentally regulated expression and cell growth regulatory activity suggest that it could play an important role in fundamental biological processes.
Figure
Materials and methods
Materials
Phenylmethylsulfonylfluoride, lactosyl-agarose, dithiothreitol, iodoacetamide, ethylenediaminetetraacetic acid, 2-mercaptoethanol, molecular weight markers, TPCK-treated trypsin, anti-rabbit IgG (whole molecule) alkaline phosphatase conjugate, 5-bromo-4-chloro-3-indolyl phosphate, nitroblue tetrazolium, nitrocellulose membranes, RPMI 1640 medium, and concanavalin A (Con A) were purchased from Sigma Chemical Co. St. Louis, MO. Centricon 10 M tubes were from Amicon (Danvers, MA). Falcon microtiter U plates were form Becton Dickinson (Oxnard, CA), and 96-well microtiter plates used for cell culture were from Corning (Corning, NY). Fetal calf serum (FCS) and l-glutamine were from Gibco Lab (Scotland, UK). Trifluoroacetic acid (TFA) was from Baker, (Phillipsburg, NJ); acetonitrile was HPLC grade, and all other chemicals were AR grade.
Ovine placental cotyledons were surgically removed from ewes around the middle of the gestation period and at full-term, immediately frozen and stored at -70°C prior to processing.
Purification of galectin-3 from ovine placental cotyledons
Galectins were obtained from ovine placental cotyledons round the middle of the gestation period by lactose extraction and affinity chromatography on a lactosyl-agarose column, as described previously (Ivanovic et al., 1996).
Galectins-1 and -3 were separated by size-exclusion chromatography on a Superose 12 HR 10/30 column.
Size exclusion chromatography
A Superose 12 HR 10/30 column and FPLC equipment (Pharmacia, Uppsala, Sweden) were used. A 100 µl sample was injected into the Superose 12 HR column preequilibrated with 20 mM Na phosphate buffer, pH 7.2, 150 mM NaCl, 2 mM dithiothreitol, 2 mM ethylenediaminetetraacetic acid. The flow rate was maintained at 0.5 ml/min and the eluent was monitored at 280 nm. It was calibrated using the following proteins as markers: [beta]-amylase, ovalbumin, bovine erythrocyte carbonic anhydrase, and bovine heart cytochrome c. Blue dextran and sodium azide were used to determine the void volume and the total solvent-accessible column volume, respectively.
Hemagglutination assays
These were performed as described previously (Iglesias et al., 1996), using rabbit erythrocytes treated with trypsin and glutaraldehyde.
Protein determination
Protein content was estimated by the method of Bradford (1976) with bovine serum albumin as a standard, and by acid hydrolysis followed by determination of amino acid composition. This was performed on a model 420A Amino Acid Analyzer (Applied Biosystems).
Antiserum preparation
A rabbit antiserum against a chicken galectin was raised as described previously (Castagna and Landa, 1994a), and its immunochemical and immunocytochemical properties have been reported (Castagna and Landa, 1994b; Rabinovich et al., 1996).
SDS-PAGE and Western blot analysis
Dialyzed and freeze-dried samples were subjected to SDS-PAGE at room temperature in slab gels as described by Schagger and von Jagow (1987). The gels were stained with Coomassie brilliant blue R-250.
Separated proteins were electroblotted onto a nitrocellulose membrane as previously described (Iglesias et al., 1996) and the electrotransference time was adjusted according to the proteins to be transferred. The membrane was blocked with 3% milk in 50 mM Tris-HCl buffer, pH 7.6, 150 mM NaCl, and probed with a 1:200 dilution of rabbit anti-galectin serum (Castagna and Landa, 1994a). The blots were developed with alkaline phosphatase-conjugated anti-rabbit immunoglobulin (1:10,000 dilution) using 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium as substrate. Chicken galectin was used as a control of positive immunoreaction, and negative controls were performed by incubation of blots with rabbit preimmune serum.
Quantification of the level of galectin-3 was performed by using a Hewlett Packard ScanJet IIP and analysis by a SigmaScan program.
Isoelectric focusing was carried out in a Phast System (Pharmacia LKB) on a PhastGel IEF 3.75-9.30.
Cell proliferation assay
Spleen mononuclear cells (SpMs) were obtained in sterile conditions from normal rats by Ficoll-Hypaque gradient centrifugation, washed, and resuspended in RPMI 1640 medium. Cell viability assessed by means of the trypan blue exclusion test was consistently greater than 95%.
To examine cell growth regulatory activity, cells were cultured in 96-well microtiter plates, at 5 × 106 cells/ml in 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 Con A (5 µg/ml). Simultaneously, galectin-3 was added to the cultures at two concentrations (1.5 and 3 µg/ml) (50 and 100 nM) and incubated at 37°C in a humidified atmosphere of 5% CO2 in air. Lactose and nonspecific sugars such as glucose and fucose were added (100 mM) to the culture medium in order to test whether growth regulatory effect of galectin-3 is related to lectin properties. After 72 h, the cultures were pulsed with 1 µCi/well [methyl-3H] thymidine ([3H]TdR sp. act.: 20.00 Ci/mmol) for an additional 18 h. Cells were then harvested and [3H]TdR incorporation was monitored by using a liquid scintillation counter. Results are expressed as mean c.p.m. ± SD of triplicate determinations from a representative out of four independent experiments. To rule out a possible toxic effect of galectin-3, cell viability was tested by means of trypan blue exclusion test, after incubation with galectin-3 at different concentrations for different culture periods.
The mean and SD were calculated, comparisons were made among groups, and significant differences were determined by one way analysis of variance (ANOVA). P values less than 0.05 were considered to be statistically significant.
nzymatic digestion and peptide purification
The purified protein was reduced, carbamidomethylated, and digested with trypsin at a 1:20 enzyme-to-substrate ratio in 2 M urea, 0.1 M ammonium bicarbonate, at 37°C for 20 h (Stone et al., 1989).
Tryptic peptides were separated by reverse phase HPLC (Applied Biosystems Model 140A) on a Brownlee C18 column (2.1 × 220 mm) equilibrated with 5% (v/v) acetonitrile, 0.1% TFA in water. Elution was performed at a flow rate of 0.2 ml/min with a 8-48% (v/v) acetonitrile, 0.1% (v/v) TFA linear gradient in 70 min.
Amino acid sequencing
Selected peptides were applied to a polybrene-coated glass filter and sequenced in an Applied Biosystems Model 477A Automatic Sequencer (Applied Biosystems, Foster City, CA) run according to the manufacturer's instructions.
Acknowledgments
We thank Lic. Alberto Valcarcel for the generous gift of ovine term placental cotyledons, and Miss Dora Beatti for excellent technical assistance. This work was supported by a grant from the Universidad de Buenos Aires. The amino acid sequencing was performed in the LANAIS-PRO (UBA-CONICET) (Facility for Protein Sequencing).
Abbreviations
AcN, acetonitrile; Con A, concanavalin A; CRD, carbohydrate recognition domain; FCS, fetal calf serum; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SpMs, spleen mononuclear cells; TFA, trifluoroacetic acid.
References
3To whom correspondence should be addressed
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