©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Galectin-1, a -Galactoside-binding Lectin in Chinese Hamster Ovary Cells
I. PHYSICAL AND CHEMICAL CHARACTERIZATION (*)

(Received for publication, October 27, 1994; and in revised form, January 9, 1995)

Moonjae Cho Richard D. Cummings

From the University of Oklahoma Health Sciences Center, Department of Biochemistry and Molecular Biology, Oklahoma Center for Molecular Medicine, Oklahoma City, Oklahoma 73190

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We report our studies on the characterization of an 14-kDa lectin, termed galectin-1, that we have found to be expressed by Chinese hamster ovary (CHO) cells. cDNA for galectin-1 from CHO cells was prepared and sequenced, and a recombinant form (rGal-1) was expressed in Escherichia coli. A mutated form of the protein that fully retained activity was also constructed (termed C2SrGal-1) in which Cys-2 was changed to Ser-2. rGal-1 was stable in the presence of reducing agent, but it quickly lost all activity in the absence of reducing agent. In contrast, glycoprotein ligands, such as basement membrane laminin, stabilized the activity of rGal-1 in the absence of reducing agent (t = 2 weeks). C2SrGal-1 was stable in the presence or absence of either ligand or reducing agent. Unexpectedly, galectin-1 was found to exist in a reversible and active monomer-dimer equilibrium with a K approx 7 µM and an equilibration time of t approx 10 h. Addition of haptenic sugars did not affect this equilibrium. Galectin-1 isolated from the cytosol of CHO cells was found to exist as monomers and dimers. These studies demonstrate that galectin-1 binding to a biological ligand stabilizes its activity and that the monomer/dimer state of the protein is regulated by lectin concentration.


INTRODUCTION

A variety of vertebrate cells and tissues synthesize a class of beta-galactoside binding proteins termed galectins, which occur in low (galectin-1 and -2) and high molecular weight forms (galectin-3 and -4) (Barondes, 1984; Barondes et al., 1994). Recently, galectins have also been found in lower invertebrates, such as sponge and nematode (Hirabayashi et al., 1992a, 1992b; Pfeifer et al., 1993). Galectins form a family of proteins (Gitt and Barondes, 1986) and are distinct from other animal lectins such as the Ca-dependent lectin family (C-type lectins) (Drickamer, 1988).

There have been several peculiar observations made about galectin-1 from diverse sources. (i) Galectin-1 is found mainly in the cytosol of most cells in which it occurs (Briles et al., 1979), (ii) the lectin has free sulfhydryls and is inactive in the absence of reducing agents (Hirabayashi and Kasai, 1991; Barondes, 1984), (iii) the lectin lacks an identifiable signal sequence and does not appear to be secreted through the normal secretory pathway in differentiated myoblasts (Cooper and Barondes, 1990), (iv) the lectin binds to beta-galactosyl-containing glycoconjugates on the cell surface and in the extracellular matrix (Cerra et al., 1984; Merkle and Cummings, 1988; Do et al., 1990; Cooper et al., 1991; Zhou and Cummings, 1993), and (v) galectin-1 is a dimer but disulfides are not involved (Liao et al., 1994).

These facts about galectin-1 raise many questions about its structure and function. If the lectin requires reducing agents to maintain its activity, how can the lectin function outside the cells and in the extracellular matrix? How is the activity of the extracellular form regulated? Does the lectin occur in some cases as a monomer or even higher oligomers? What structural forms of the lectin occur in the cytosol? Does the extracellular form of the lectin arise through secretion or through cellular injury?

These questions about the structure and function of galectin-1 have led to the present studies. We have discovered that Chinese hamster ovary (CHO) (^1)cells synthesize relatively large amounts of galectin-1 and have cloned and sequenced the cDNA for the lectin. A recombinant form of the protein was prepared to aid in understanding its structure. Contrary to expectations, we found that the lectin is highly stable in the absence of reducing agents when associated with a biological ligand, such as laminin. Furthermore, galectin-1 exists in a reversible monomer-dimer equilibrium. CHO cells are a convenient cell type in which to study the structure and function of galectin-1. The wild type CHO cells produce poly-N-acetyllactosamine-containing glycoconjugates bound by galectin-1, whereas some mutant CHO cell lines resistant to the cytotoxic effects of plant lectins lack glycoconjugates bound by galectin-1 and quantitatively secrete the protein. In the accompanying manuscript (Cho and Cummings, 1995), we take advantage of this system to study the biosynthesis and localization of galectin-1 and the role of glycoconjugates in these processes.


EXPERIMENTAL PROCEDURES

Materials

Mouse Engelbreth-Holm-Swarm laminin, molecular mass markers (14-200 kDa) for SDS-polyacrylamide gel electrophoresis, and tissue culture reagents were purchased from Life Technologies, Inc. Bovine serum albumin (fraction V), asialofetuin (from fetal calf serum, type III), Sephadex G-25, 2-mercaptoethanol, trifluoroacetic acid, phenylmethylsulfonyl fluoride, pepstatin, aprotinin, leupeptin, rabbit anti-mouse laminin, and ethanolamine were obtained from Sigma. In addition, Affi-Gel 15 and the Western blot kit containing 5-bromo-4-chloro-3`-indolyl phosphate p-toluidine salt and nitro blue tetrazolium chloride was obtained from Bio-Rad Laboratories. Lactose, acetone, EDTA-disodium salt, and sodium bicarbonate were obtained from Baker Chemical Co., and the BCA reagent for protein determination was purchased from Pierce. CNBr-Sepharose was obtained from Pharmacia Biotech Inc. S-Protein Labeling Mix ([S]Met/Cys, 1, 200 Ci/mmol) was purchased from DuPont NEN.

Cell Culture and Cell Radiolabeling

Chinese hamster ovary cells (CHO-K1 cells) and CHO mutant Lec8 cells were cultured in alpha-minimal essential media containing 10% fetal calf serum. CHO and Lec8 CHO cells were grown for 16 h in low methionine-Dulbecco's modified Eagle's media containing S-Protein Labeling Mix (10-20 µCi/ml) and 10% fetal calf serum.

Coupling of Asialofetuin and Laminin to Sepharose

Asialofetuin (10 mg/ml) and laminin (2 mg/ml) were dissolved in 0.1 M NaHCO(3) buffer (pH 8.0) and immediately coupled to CNBr-Sepharose (1 ml) according to the manufacturer's instructions. The coupling efficiency was greater than 90% as estimated by determining the amount of uncoupled protein by the BCA protein assay. The final densities of asialofetuin-Sepharose and laminin-Sepharose were approx10 mg/ml and approx2 mg/ml, respectively.

Cloning, Mutation, Expression, and Purification of Recombinant Galectin-1 (rGal-1) and C2S Recombinant Galectin-1 (C2SrGal-1)

The sequence of rat galectin-1 (Clerch et al., 1988) was used to design redundant oligonucleotide primers to hybridize to DNA encoded by each terminal sequence of galectin-1 in CHO cells. A BamHI site was placed in the 5` primer and a HindIII site was placed in the 3` primer (5` primer for rGal-1, 5`-GGGGGATCCGCCTGTGGTCTGGTCGCA; 5` primer for C2SrGal-1, 5`-GGGGGATCCGCCTCTGGTCTGGTCGCA; 3` primer for both rGal-1 and C2SrGal-1, 5`-GGGAAGCTTTCACTCAAAGGCCACGCA). Primers were synthesized by Operon Technologies, Inc. (Alameda, CA). Total RNA from CHO cells was isolated by the technique of Chirgwin et al.(1979), and cDNA was prepared using murine leukemia virus reverse transcriptase (Life Technologoes, Inc.) as described by Moremen(1989). The polymerase chain reaction (PCR) was used to amplify the galectin-1 gene region from this cDNA. CHO galectin-1 cDNA was modified using PCR primer-directed mutagenesis to place a serine codon at the second cysteine position to produce the C2S galectin-1. Separate constructs were prepared containing the CHO galectin-1 and C2S galectin-1 cDNA ligated into the BamHI and HindIII sites of pQAE9 (Qiagen).

E. coli strain M15 (Qiagen) were transformed with these plasmids to express recombinant galectin-1 (rGal-1) and C2S galectin-1 (C2SrGal-1) at high levels. rGal-1 and C2SrGal-1 were purified from sonicated E. coli cell extracts on a column of asialofetuin-Sepharose (1.5 times 30 cm). The column was washed with 5 column volumes of SPB-azide (6.7 mM KH(2)PO(4), 150 mM NaCl, pH 7.4, containing 14 mM 2-mercaptoethanol and 0.02% NaN(3)) and 3 volumes of SPB-azide containing 1 M NaCl. The bound rGal-1 or C2SrGal-1 were eluted with SPB-azide containing 0.1 M lactose and dialyzed against SPB-azide. Approximately 30 ml of rGal-1 (1.13 mg/ml) and 30 ml of C2SrGal-1 (1.3 mg/ml) were obtained from each 1-liter culture of transfected E. coli. To stabilize the rGal-1 against oxidative inactivation, all buffers used during purification contained beta-mercaptoethanol. S-Labeled rGal-1 and C2SrGal-1 were produced by growing E. coli in the minimal media (1 liter) containing NaSO(4) (10 mCi) until the A reached 0.5. An equal volume of methionine-deficient minimal essential media (1 liter) containing 1 mCi of S-Protein Labeling Mix and isopropyl-1-thio-beta-D-galactopyranoside (2 mM) was added, and the cells were incubated for another 5 h with shaking. The E. coli were harvested and lysed by sonication, and the extracts were applied to a column of asialofetuin-Sepharose (1.5 times 30 cm containing 10 mg/ml conjugated protein and a total volume of 100 ml) to purify radiolabeled rGal-1 or C2SrGal-1. Specific activity of purified C2SrGal-1 and rGal-1 were 10,922 cpm/µg and 9,745 cpm/µg, respectively. Protein concentration was determined by the BCA assay.

Preparation of Antibodies to Galectin-1

An anti-galectin-1 antiserum was raised in a rabbit by an initial subcutaneous injection of 250 µg of purified protein in complete Freund's adjuvant, followed by two more boosts over a 5-week period with 250 µg of purified protein in incomplete Freund's adjuvant administered in the same route. Serum was collected 10 days after the final boost. Monospecific anti-galectin-1 antibody was purified by affinity chromatography of serum over a column (1 times 5 cm) containing galectin-1 conjugated to Affi-Gel 15. Galectin-1 was conjugated by the manufacturer's instructions in the presence of 0.1 M lactose to a final density of 5 mg/ml. Antibody bound to immobilized galectin-1 was eluted with 0.1 M glycine buffer (pH 2.5) and dialyzed against PBS-azide.

Separation of Monomer and Dimer of Galectin-1 on Size Exclusion HPLC

Separation of monomer and dimer forms of rGal-1 and C2SrGal-1 was performed by size exclusion HPLC using a TSK-GEL SW 2000 size exclusion column (7.5 mm times 30 cm, Beckman). The column was equilibrated with PBS-azide, and separation was carried out isocratically for 20 min with Beckman System Gold HPLC. The column flow rate was 1 ml/min. The fractions were monitored by absorbance at 280 nm (or at 214 nm when the amount of protein was low).

ELISA Assay for Binding of Galectin-1 to Laminin

Laminin was diluted in 0.1 M NaHCO(3) buffer (pH 9.6) to 1 µg/ml, and 100-µl aliquots were used to coat a 96-well microtiter plate (Immulon 2) overnight at 4 °C. The wells were washed twice and blocked with 1% bovine serum albumin in PBS-azide for 1 h at room temperature. After blocking, different amounts of C2SrGal-1 were added and incubated for 2-20 h. The wells were washed three times with washing buffer (PBS-azide containing 0.05% Tween 20) to remove unbound galectin-1. Rabbit anti-galectin-1 antibody (1:16,000 dilution in a blocking buffer) was added and incubated for 1 h, and the wells were washed three times with the same buffer. Anti-rabbit IgG antibody conjugated with alkaline phosphatase (1:5,000 dilution) in PBS-azide containing 1% bovine serum albumin was added and incubated at room temperature for 1 h. After washing three times with washing buffer and once with distilled water, the wells were incubated with 100 µl of 1 mg/ml p-nitrophenyl phosphate in 0.1 M NaHCO(3) buffer (pH 9.6). The absorbance of each well was read at 405 nm using an automated microtiter plate reader (Bio-Tek, Model EL 309).

Evaluation of Galectin Stability under Nonreducing Conditions

Stability of lectins was examined by both (i) hemagglutinating activity in the absence of reducing agent (2-mercaptoethanol) and (ii) asialofetuin binding activity. For assaying lectin, we used a version of the previously described hemagglutination method (Merkle et al., 1989). Briefly, 10 µl of a 10% suspension of washed rabbit erythrocytes was placed on a glass plate and mixed with 10 µl of PBS-azide and 10 µl of a solution of lectin in PBS-azide or SPB-azide. Hemagglutination activity was defined as 4+ when the hemagglutination of rabbit erythrocytes was detected as soon as lectin was added, 3+ when hemagglutination required 30-60 s, 2+ when hemagglutination required 60-90 s, 1+ when hemagglutination required more than 90-180 s, and 0+ when no hemagglutination was detected within 180 s. For the asialofetuin binding, lectin solutions (1.1 mg/ml) in SPB-azide were applied to a column of asialofetuin-Sepharose (1 ml of 10 mg/ml coupled asialofetuin), then the column was washed with either PBS-azide or SPB-azide. The run-through and PBS-azide or SPB-azide wash solutions were collected, then 2 ml of either PBS-azide or SPB-azide were added to the column and the column was incubated for 24 h at 4 °C. After 24 h, either PBS-azide or SPB-azide was collected and the column was washed with 2 ml of either buffer three times. The incubation and washing of the column with buffer was repeated each day up to 10 days, and the galectin-1 remaining on the column after 10 days was eluted with 0.1 M lactose in PBS-azide. Each fraction was analyzed by SDS-PAGE and Coomassie Blue staining (50% MeOH, 20% acetic acid, 0.1% Coomassie Blue) and destained with destaining solution (30% MeOH, 7% acetic acid). A similar type of experiment was done with metabolically radiolabeled [S]Met/Cys-labeled galectin, and fractions were monitored by determining radioactivity.

Reversibility of Monomer-Dimer Equilibrium and Asialofetuin Binding Activity of the Monomer

Reversibility of monomer-dimer equilibrium was studied as follows. [S]Met/Cys-labeled C2SrGal-1 (100 µg/ml in PBS-azide) was diluted 100-fold in PBS-azide and incubated at 4 °C for 20 h to reach equilibrium. This sample was applied to the size exclusion HPLC column, and fractions (0.166 ml) were collected. The fractions were monitored at A, and the radioactivity of each fraction was determined by scintillation counting. To the same 100-fold diluted [S]Met/Cys-labeled C2SrGal-1 (200 µl), 100 µl of unlabeled C2SrGal-1 stock solution (1.1 mg/ml) was added and the mixture was incubated for 20 h at 4 °C to reach equilibrium. From this mixture, 100 µl was analyzed as described above. To evaluate the binding activity of monomer for asialofetuin, the [S]Met/Cys-labeled C2SrGal-1 was diluted to 100-fold and incubated for 20 h at 4 °C to allow all the galectin-1 to shift to monomer. Into this monomer solution, 100 µl of asialofetuin-Sepharose was added, incubated for 15 min, and centrifuged, and the supernatant was collected. The beads were washed once and the wash solution was added to the supernatant. The total radioactivity in the supernatant was compared to the initial radioactivity added and the percent of unbound lectin calculated.

Detection of Cytosolic Galectin-1 by ELISA

The Lec8 CHO cells were grown in 100-mm dishes to 100% confluence, and the cells were solubilized by adding 500 µl of lysate buffer (PBS-azide containing 1% Triton X-100, 2 mM phenylmethylsulfonyl fluoride). Within 15 min, the extract was analyzed by size exclusion HPLC and fractions (320 µl) were collected. One hundred microliters of each fraction was transferred to a 96-well microtiter plate to coat the wells for 1 h at room temperature. Each fraction was monitored by ELISA, as described above, using monospecific antibodies to galectin-1. One hundred microliters of each fraction was used for Western blot analysis.

Western Blot Analysis of Galectin-1

Cell extracts or fractions from the size exclusion HPLC columns were precipitated with 10% trichloroacetic acid and analyzed by SDS-PAGE followed by transfer to nitrocellulose paper overnight at 20 V. The paper was incubated with washing buffer containing 5% nonfat dry milk for 2 h, washed 5 times with washing buffer, and overlaid with anti-galectin-1 antibody (8 µg/ml) in washing buffer containing 0.5% nonfat dry milk for 1 h. After 3 washes with washing buffer, goat anti-rabbit antibody conjugated with alkaline phosphatase was added and incubated for 1 h. The paper was subsequently washed three times in washing buffer. Galectin-1 was detected by color development using the Western blot kit (5-bromo-4-chloro-3`-indolyl phosphate p-toluidine salt and nitro blue tetrazolium) and following instructions from the manufacturer.


RESULTS

Cloning and Sequencing of cDNA for Galectin-1 in CHO Cells and Expression of Recombinant Galectin-1

We initially discovered that CHO cells synthesized galectin-1 by examining protein in extracts of the cells after affinity chromatography over a column of immobilized asialofetuin. A 14-kDa protein was bound by the column and eluted with lactose (data not shown), using methods identical with those used to identify galectin-1 in pig heart tissue (Merkle et al., 1989). However, we found that it was difficult to isolate from the cultured cells sufficient quantities of the protein for detailed biochemical analyses and to obtain enough material for sequencing. In addition, galectins typically have blocked N termini (Hirabayashi and Kasai, 1993). We chose, therefore, to isolate cDNA for galectin-1 from CHO cells both for the purposes of obtaining the predicted sequence of the protein and for preparing a recombinant form of the lectin expressed in bacteria. Other than blockage of the N terminus, galectin-1 is not known to have any post-translational modifications, such as glycosylation or acylation, which would preclude production of the lectin in a prokaryotic host (Tracey et al., 1992). cDNA for galectin-1 was amplified by PCR using primers prepared from the rat galectin-1 sequence and cDNA derived from mRNA of CHO cells. Three independent PCR amplification products were cloned and sequenced and found to be identical. The nucleotide sequence and deduced amino acid sequence for galectin-1 from CHO cells is shown in Fig. 1and compared to the rat sequence (Clerch et al., 1988). The CHO galectin-1 contains 134 amino acids (minus the initiating methionine) and 6 relatively conserved Cys residues at positions 2, 16, 42, 60, 88, and 130. The CHO and rat cDNAs are 89% homologous in nucleotide sequence, and the encoded proteins have 94% identity.


Figure 1: The cDNA sequence and encoded amino acid sequence of galectin-1 from CHO cells compared to rat galectin-1.



To allow more definitive biochemical characterization and generate a more stable form of galectin-1, we also constructed a mutated form of the protein in which the conserved Cys-2 residue was converted to Ser-2, as described under ``Experimental Procedures.'' This form of the lectin was termed C2SrGal-1. It has been shown that Cys residues are not critical for lectin activity but they contribute to instability of the protein in the absence of reducing agents (Hirabayashi and Kasai, 1991; Abbott and Feizi, 1991). We constructed two expression plasmids using pQAE 50 and cDNA for galectin-1 (rGal-1) and cDNA for mutated galectin-1 (C2SrGal-1). Both transformed E. coli strains produced the recombinant lectins in the presence of 2 mM isopropyl-1-thio-beta-D-galactopyranoside. These recombinant lectins had specific activities similar to the native galectin-1, in both hemagglutinating assays and binding assays to immobilized asialofetuin and laminin.

Antibody Production

Antiserum was raised in rabbits against rGal-1, and monospecific antibodies to the protein were purified by affinity chromatography of on a column of rGal-1-Affi-Gel 15 (1 mg/ml). These purified antibodies were specific for the galectin-1 in CHO cells by two criteria. In Western blot analysis against total cellular material from CHO cells, the purified antibodies reacted only with galectin-1 and did not cross-react with another soluble 30-kDa lectin (galectin-3), which is also expressed by these cells (Fig. 2). In addition, purified antibody immunoprecipitated only galectin-1 from [S]Met/Cys-labeled CHO cells (see Cho and Cummings(1995)).


Figure 2: Demonstration of antibody specificity by Western blotting. Monospecific antibodies (80 µg/ml) toward galectin-1 were obtained from rabbit sera after purification on a column of rGal-1-Affi-Gel 15 (1 mg/ml), as described under ``Experimental Procedures.'' CHO cells extracts (50 µg) were analyzed by SDS-PAGE and transferred to nitrocellulose. Strips from a single blot were cut and stained with a monospecific antibody (1:10 dilution) (A) and corresponding preimmune rabbit IgG (B). (Migration positions are indicated for protein molecular mass standards of 200, 116, 94, 68, 43, 29, and 14 kDa.)



Glycoprotein Ligand Stabilizes Galectin-1 in the Absence of Reducing Agents

We initially sought to characterize the stability of galectin-1 in a variety of conditions and explore the possibility that reducing conditions might not be required, as thought previously, for retention of lectin activity in vitro. In solution without 2-mercaptoethanol, rGal-1 lost all hemagglutinating activity within 12 h (Fig. 3A). We estimated that the t of rGal-1 in the absence of reducing buffer was 6-10 h. rGal-1 was also unable to bind to a column of asialofetuin-Sepharose within 12 h of treatment in buffer lacking 2-mercaptoethanol (data not shown).


Figure 3: Stability of the rGal-1 and C2SrGal-1 under various conditions. A, the hemagglutination activity of rGal-1, stored in the absence or presence of reducing solutions, was assayed over a period of 48 h, as described under ``Experimental Procedures.'' The hemagglutinating activity of the lectin is scored as 0-4+, with 4+ indicating maximal activity and 0+ indicating no activity. B, [S]Met/Cys-labeled rGal-1 and C2SrGal-1 were applied to columns of either asialofetuin-Sepharose or laminin-Sepharose and eluted daily with reducing or nonreducing buffer over a period of 10 days, as described under ``Experimental Procedures'' and as discussed in the text. The percent of radiolabeled lectin remaining bound at each time point is indicated. bullet, rGal-1 bound to asialofetuin-Sepharose in reducing buffer; up triangle, rGal-1 bound to asialofetuin-Sepharose in nonreducing buffer; circle, rGal-1 bound to laminin-Sepharose in nonreducing buffer; box, C2SrGal-1 bound to asialofetuin-Sepharose in nonreducing buffer.



We then explored whether binding of the lectin to a glycoconjugate ligand could stabilize activity of the lectin. To test this possibility, 50 µg of rGal-1 was bound to a 1-ml column of asialofetuin-Sepharose containing 5 mg/ml ligand in either the presence or absence of 2-mercaptoethanol. The column was washed with buffer either with or without 2-mercaptoethanol. This column was washed every 24 h to remove lectin that had become inactive. The rGal-1 washed from the column without hapten was collected and analyzed by SDS-PAGE. At the end of the experiment, the column was eluted with buffer containing 100 mM lactose, to estimate the amount of residual lectin left on the support. When the rGal-1 was bound to a column of asialofetuin-Sepharose, approximately one-half of the lectin appeared to retain its activity for 150 h in the absence of 2-mercaptoethanol (data not shown). In the presence of 2-mercaptoethanol, the vast majority of rGal-1 appeared to retain its activity during the 150 h (data not shown).

To directly quantify the stability of the lectin when bound to columns of immobilized ligands, [S]Met-labeled rGal-1 and C2SrGal-1 were prepared by metabolic radiolabeling of E. coli expressing the recombinant proteins. The radiolabeled lectins were bound to a column of asialofetuin-Sepharose with or without 2-mercaptoethanol, and, every 24 h, the column was washed with either reducing or nonreducing buffer to remove lectin that had become inactive. Fifty percent of the [S]rGal-1 remained bound at 5 days to the asialofetuin-Sepharose in the absence of reducing agent (Fig. 3B). In the presence of reducing agent, more than 80% of the lectin was still bound to the asialofetuin-Sepharose column (Fig. 3B). We then tested whether a more active ligand, the basement membrane glycoprotein laminin, could stabilize the lectin even further. Virtually all the rGal-1 remained bound to the column of laminin-Sepharose in the absence of reducing agent, and the t was estimated to be >2 weeks (Fig. 3B). This may reflect the higher affinity exhibited by galectin-1 for laminin in comparison to asialofetuin. Laminin contains poly-N-acetyllactosamine, a high affinity determinant for galectin-1 binding, whereas asialofetuin lacks this structural feature and binds relatively poorly to galectin-1 (Zhou and Cummings, 1990).

In contrast to rGal-1, the hemagglutinating activity of C2SrGal-1 was stable when the lectin was stored in solution in the absence of reducing agent for 48 h (data not shown). In addition, more than 80% of the [S]Met-labeled C2SrGal-1 in nonreducing solutions remained firmly bound to a column of asialofetuin-Sepharose for up to 10 days (Fig. 3B). These results demonstrate that cysteine at position 2 in the hamster galectin-1, as also observed for the human and bovine galectin-1, is critical in causing instability of the lectin in solutions lacking reducing agents. It has been observed by Liao et al.(1994), in a crystal structure of bovine galectin-1, that this Cys-2 is disordered, whereas the other 5 cysteine residues are either buried in the molecule or oxidized and solvated. Taken together, the results demonstrate that galectin-1 is inactive in the absence of reducing agents, but, more importantly, they show that reducing conditions are not required to maintain activity of the lectin in vitro when it is associated with high affinity glycoconjugate ligands.

Kinetics of Galectin-1 Binding to Immobilized Glycoconjugates

To estimate the functionality of galectin-1 and its kinetics of binding to glycoconjugates, we used microtiter plates containing immobilized laminin. Wells coated with differing amounts of Engelbreth-Holm-Swarm laminin were tested for binding rGal-1. The binding of C2SrGal-1 (5 µg/well) to immobilized laminin was rapid, and a plateau was reached within 1 h (Fig. 4A). Lactose (100 mM) could compete off most of the bound lectin from laminin within 30 min (Fig. 4A). We then measured the binding of differing concentrations of rGal-1 to laminin in a 2-h assay (Fig. 4B) and found that saturated binding occurred with approx20 µg per well. Unexpectedly, saturation was achieved at lower amounts of lectin (5-10 µg/well) when the incubation time was increased from 2 hr to 20 h (Fig. 4B). In either case, however, the amount of lectin bound at saturation was identical. This anomalous binding behavior led us to consider the possibility that the time-dependent change in binding of galectin-1 to laminin might be due to changes in the dimeric structure of the lectin, in which the dimer might dissociate into monomers with time and the apparent binding would be enhanced. We therefore investigated further the physical state of galectin-1 in solution and its physiological significance.


Figure 4: Kinetics of binding of C2SrGal-1 to immobilized laminin. A, 5 µg of C2SrGal-1 was applied per microtiter plate well precoated with laminin (500 ng/well) and incubated for different times, as indicated. The amount of bound lectin was determined using antibody to the lectin in an ELISA-type format, as described under ``Experimental Procedures.'' Lactose (25 mM final concentration) was added after 360 min to dissociate bound lectin. In B, microtiter plate wells were coated with laminin (500 ng/well), and different amounts of C2SrGal-1 were added and incubated at 4 °C for either 2 or 20 h, as indicated. Each point represents an average of triplicate wells.



Galectin-1 Is a Monomer at Low Lectin Concentration

Early studies on galectin-1 suggested that the protein is primarily a homodimer in solution, and homodimer formation does not depend on the formation of disulfide bonds (Teichberg and Levi, 1981; Briles et al., 1977). Crystal structures of bovine galectin-1 and human galectin-2 are consistent with this prediction (Liao et al., 1994; Lobsanov et al., 1993). We analyzed C2SrGal-1 on either nonreducing or reducing SDS-PAGE for the status of the protein under different protein concentrations. The lectin had similar electrophoretic mobility under either nonreducing or reducing conditions with the major band migrating as a 14-kDa protein, as expected (data not shown). Similar results were found for rGal-1. We did observe a faint band corresponding to protein with slower electrophoretic mobility of approximately 30 kDa, which might represent a dimer of the protein that was not fully dissociated under the conditions of the experiment.

To directly investigate the possibility of monomer and dimer equilibrium of galectin-1, we used high performance size exclusion chromatography to analyze rGal-1 or C2SrGal-1 serially diluted in buffer with or without reducing agent. The diluted samples were kept at 4 °C for 24 h to promote equilibrium, and each sample was analyzed by size exclusion HPLC in a 15-min run. C2SrGal-1 was recovered as two peaks in this column with the first peak eluting at 7 min 30-45 s and the second peak at 8 min 35-50 s (Fig. 5). A number of standard proteins ranging in size from 13.7 to 150 kDa were analyzed to standardize the size exclusion properties of the TSK column. Based on the elution positions of these molecular mass standards, we estimated that the first eluted peak of C2SrGal-1 represented a 30.5-kDa protein and the later peak a 14.9-kDa protein (Fig. 5, inset). These correspond to the predicted sizes of the dimer and monomer forms of C2SrGal-1, respectively.


Figure 5: Monomer and dimer forms of galectin-1 in solution. A 100-µl sample of C2SrGal-1 containing 10 µg of lectin was analyzed by size exclusion HPLC through a SW 2000 column, and elution was monitored by A (OD214). The column was calibrated with molecular mass markers as indicated in the inset (bovine -globulin, 158 kDa; bovine serum albumin, 67 kDa; chicken ovalbumin, 44 kDa; equine myoglobin, 25 kDa; chymotrypsinogen, 17 kDa; ribonuclease A, 13.7 kDa).



The formation of monomers and dimers was totally dependent on the concentration of C2SrGal-1. Most of the C2SrGal-1 existed as a dimer in the undiluted stock solution (protein concentration of 80 µM) (Fig. 6A). In contrast, at low concentration (80 nM) the monomeric form was dominant. The amount of the monomeric form of the protein at different concentrations of C2SrGal-1 was determined, the chromatograms are shown in Fig. 6A, and a compilation of the results are shown in Fig. 6B. It can be estimated form these data that the apparent K(d) of dissociation of the dimer to monomer is approx7 µM.


Figure 6: Size exclusion HPLC and concentration-dependent dimerization of the C2SrGal-1. A, C2SrGal-1 (80 µM) was diluted in PBS-azide to various concentrations and allowed to sit at 4 °C for 20 h to equilibrate. From each sample, 100 µl was analyzed by size exclusion HPLC, as in Fig. 5. At high concentrations, A was used for protein determination, and, at low protein concentrations, A was used. B, the peak area was integrated, and the concentration dependence of the monomer formation was plotted.



Slow Interconversion of Monomer and Dimer

We measured the rate of formation of monomer from dimer by diluting the dimer form of the protein to a low concentration (0.8 µM) and measuring the conversion to monomer with time. Immediately after the sample was diluted (0 h), most of the C2SrGal-1 was still present as a dimer (Fig. 7). The equilibrium slowly shifted with time to the monomer. Within 3 h, a significant amount of monomer was observed, and, by 36 h, the protein was mostly monomeric (Fig. 7). These experiments were performed with C2SrGal-1 under nonreducing conditions because rGal-1 is unstable under nonreducing conditions and loses activity before it can reach equilibrium (Fig. 3). To demonstrate, however, that the monomer-dimer equilibrium was not unique to the C2S mutation, we performed the size exclusion HPLC in reducing conditions using rGal-1 at two different lectin concentrations (8 µM and 160 nM) and at 0 h and 40 h (Fig. 8). These studies demonstrate that rGal-1, like C2SrGal-1, also undergoes a monomer-dimer equilibrium that is concentration-dependent. In a variety of such experiments comparing rGal-1 with C2SrGal-1, we could find no differences in the monomer-dimer equilibrium kinetics.


Figure 7: Time-dependent monomer formation of the C2SrGal-1 dimers. C2SrGal-1 was diluted to 0.8 µM in PBS-azide, size exclusion HPLC was performed following different times of incubation, as indicated, and A was monitored.




Figure 8: Time- and concentration-dependent monomer formation of rGal-1 dimers in reducing buffers. The monomer formation of rGal-1 in reducing buffer was analyzed by size exclusion HPLC by varying the concentration of lectin and incubation times, as indicated, and A was monitored.



Monomer-Dimer Equilibrium Is Reversible

To investigate the reversibility of this equilibrium and demonstrate formation of dimer from monomer, [S]Met-labeled C2SrGal-1 was prepared and diluted to 0.8 µM. The sample was incubated for 20 h at 4 °C to allow equilibrium. One-half of this sample was then analyzed by size exclusion HPLC. Each fraction was monitored by both A and radioactivity. A majority of the [S]Met-labeled protein co-eluted with the A peaks, and the major peak of the monomeric protein, as expected (Fig. 9A). To the other half of the sample, unlabeled C2SrGal-1 was added (final concentration 80 µM) to increase the concentration of lectin, and the mixture was incubated at 4 °C for 20 h to reach equilibrium. The mixture was analyzed by HPLC and monitored by the same methods. As shown in Fig. 9B, the [S]Met-labeled material was recovered in the dimer, demonstrating that the monomeric [S]Met-labeled C2SrGal-1 was able to reform dimers at higher lectin concentrations. These results demonstrate that the monomer-dimer equilibrium of the galectin-1 is concentration-dependent and reversible.


Figure 9: Reversibility of the monomer-dimer equilibrium. A, 100 µl of a 1/100 dilution of [S]Met-labeled C2SrGal-1 (10 µg/ml) was applied to the size exclusion HPLC column, and fractions were collected. The fractions were monitored at A, and the radioactivity in each fraction was determined by liquid scintillation counting. B, to the same diluted sample of [S]Met-labeled C2SrGal-1 (200 µl), 100 µl of nonlabeled C2SrGal-1 (1.1 mg/ml) was added and incubated at 4 °C for 20 h to allow equilibration. One hundred µl of this mixture was analyzed as in A.



We tested the possibility that haptenic sugar might affect the monomer-dimer equilibrium for the lectin. A concentrated form of C2SrGal-1 in dimer form was diluted to a low concentration to promote monomer formation and incubated with or without 0.1 M lactose. The samples were analyzed by size exclusion HPLC using PBS-azide buffer either with or without 0.1 M lactose. The monomer formation of the lectin was not affected by lactose (Fig. 10).


Figure 10: Effect of lactose on monomer-dimer equilibrium. A dilute amount of C2SrGal-1 in a predominantly monomeric form was incubated with or without 0.1 M lactose overnight at 4 °C. The samples were then analyzed by size exclusion HPLC using PBS-azide buffer either with or without 0.1 M lactose.



Monomeric Galectin-1 Is Able to Bind Carbohydrate

We then determined whether the monomeric form of galectin-1 was able to bind carbohydrate ligands. The binding studies in Fig. 4suggest that at low lectin concentrations the lectin is able to bind laminin, but this type of analysis does not provide quantitative activity information about the total monomeric lectin pool. To more directly define the percentage of monomeric protein that is active, the [S]Met-labeled C2SrGal-1 was diluted to a concentration (100 nM) where it exists primarily as a monomer and equilibrated overnight at 4 °C. A portion was then analyzed by size exclusion HPLC, and, as expected, more than 80% of the protein was present in the monomeric form (data not shown). This monomeric lectin was then incubated for 15 min in solution with asialofetuin-Sepharose. The beads were separated from the solution by centrifugation and washed once with PBS-azide, and the radioactivity present on the beads and in the supernatants was determined. We found that 77 ± 3% of C2SrGal-1 was bound to asialofetuin-Sepharose. These results demonstrate that the monomeric form of galectin-1 is active in binding to carbohydrate ligands.

Galectin-1 Exists as Monomers and Dimers in the Cytosol of CHO Cells

As shown in our accompanying study (Cho and Cummings, 1995), galectin-1 in CHO cells is synthesized in the cytosol and secreted from the cells. We therefore sought to analyze the monomer-dimer status of galectin-1 in the cell cytosol. Normally, this type of analysis would be confounded because of the presence of carbohydrate ligands for the lectin in the cell extracts and the resulting association of the lectin with glycoproteins. We therefore chose to conduct our analyses with the Lec8 mutant of CHO cells. These cells are unable to galactosylate any glycolipids or glycoproteins (Briles et al., 1977; Stanley, 1984; Deutscher and Hirschberg, 1986) and do not contain glycoconjugate ligands for the lectin (Zhou and Cummings, 1993). The galectin-1 in Lec8 CHO cells is, however, functional as evidenced by the quantitative binding of lectin from solubilized cells to a column of asialofetuinSepharose (data not shown).

To analyze the monomer-dimer nature of the intracellular form of galectin-1 in Lec8 CHO cells, the cells were harvested, solubilized with 1% Triton X-100, and analyzed by size exclusion HPLC. Fractions were collected, and one-half of each fraction was transferred to a 96-well microtiter plate. Using anti-galectin-1 antibodies, each fraction was assayed for the lectin by ELISA. The results indicate that most of the detectable galectin-1 derived from Lec8 CHO cell extracts eluted from the HPLC column in the position of both monomers and dimers (Fig. 11). Each fraction was also analyzed by Western blot using antibodies to the lectin. The results showed that the major immunoreactive bands in Western blots corresponded exactly to the peaks of absorbance in the ELISA assay (data not shown). The monomer/dimer forms are unlikely to arise by interconversions occurring after cell lysis because of the slow rate observed for interconversion of monomer and dimer in the above experiments. Some of the lectin detected by the ELISA assay in Fig. 11may also be in a higher oligomeric form of unknown nature, but this possibility was not studied further at this time. Overall, the results indicate that galectin-1 in CHO cells exists as both monomer and dimer forms and that both forms are capable of binding glycoconjugates.


Figure 11: Monomer and dimer forms of galectin-1 in the cytosol of Lec8 CHO cells. Lec8 CHO cells were grown to confluence in a 100-mm dish, harvested, and solubilized with 1 ml of 1% Triton X-100/PBS-azide, and 100 µl was analyzed by size exclusion HPLC. Fractions (320 µl) were collected and 100 µl of each fraction was transferred to a 96-well microtiter plate. Galectin-1 was determined in each fraction using anti-galectin-1 antibodies in an ELISA-type format, as described under ``Experimental Procedures.''




DISCUSSION

We have discovered that galectin-1 from CHO cells is a monomeric protein that is able to form dimers in the micromolar range. The monomer-dimer equilibrium is both timedependent and reversible, and both forms of the lectin are able to bind carbohydrate. Lectin derived from the cytoplasm of CHO cells occurs in both monomer and dimer forms. Furthermore, we have found that the lectin is extremely stable in the absence of reducing agents when the protein is bound to carbohydrate ligands. These results are meaningful toward our understanding of this widely distributed lectin and provide new information that extends the findings of other groups.

Galectin-1 was first identified by its hemagglutinating activity and inhibition of agglutination by lactose, which indicated that the lectin is multivalent and binds to sugars on cell surfaces (Teichberg et al., 1975; de Waard et al., 1976). Two unusual features of the lectin were also found. The lectin required reducing conditions to maintain its activity, and much of the lectin was found to be intracellular (Briles et al., 1979; Barondes, 1984). Recent crystallographic analysis of bovine galectin-1 are consistent with other studies in demonstrating that the lectin is a homodimer with each subunit possessing one sugar binding site (Liao et al., 1994).

Galectin-1 from CHO cells, as from many other sources, contains 6 cysteine residues, and, in all cases examined, these occur as free sulfhydryls (Hirabayashi and Kasai, 1993). Selective mutagenesis of the cysteine residues in human galectin-1 has demonstrated that cysteine is not required for carbohydrate binding activity (Hirabayashi and Kasai, 1991; Abbott and Feizi, 1991). Rather, in the absence of reducing agent, the free cysteine residues lead to oxidative damage to the protein and perhaps to aberrant oligomerization by disulfide bond formation (Tracey et al., 1992). This requirement for reducing conditions to maintain activity led to the earlier description of galectins as sulfhydryl-type or S-type lectins. Another unusual feature of galectin-1 is that it lacks a typical secretory signal sequence and it is synthesized on free polysomes in the cytosol (Clerch et al., 1988; Wilson et al., 1989), and it is not secreted by the normal secretory pathway (Cooper and Barondes, 1990).

In terms of complex carbohydrate binding activity, galectin-1 binds to desialylated glycoconjugates containing clustered glycosides with terminal beta1,4-linked galactosyl residues and binds to N-acetyllactosamine better than to lactose (Briles et al., 1977; Barondes, 1984; Lee et al., 1990). The lectin also binds with high affinity to sialylated poly-N-acetyllactosamine sequences [3Galbeta1-4GlcNAcbeta1](n) of the type found in laminin and lysosome-associated membrane proteins 1 and 2 (Leffler and Barondes, 1986; Merkle and Cummings, 1988; Sparrow et al., 1987; Do, et al., 1990; Zhou and Cummings, 1993).

These observations taken together raise several interesting and complex questions about galectin-1. How could galectin-1 be efficient and functional in binding extracellular glycoconjugates if the protein requires reducing conditions to maintain activity? Does the lectin occur in a monomeric form and how is formation of the dimer regulated? What is the nature of the intracellular and extracellular forms of the protein? Is the stability of the lectin affected by its interaction with glycoconjugates? In our study we attempted to address both the mechanisms of how the protein becomes a dimer and the significance of reducing activity to maintain lectin activity.

Our results demonstrate that the binding of galectin-1 to appropriate glycoconjugates can greatly stabilize the lectin in the absence of a reducing environment. Moreover, the inactivation of the lectin not associated with ligand could provide a type of control mechanism to regulate the extracellular levels of the lectin in vivo and turnover the lectin. Conceivably, the regulation of galectin-1 activity by extracellular oxidation might be comparable to regulation of alpha-1-antitrypsin activity by its sensitivity to oxidation (Travis and Salvesen, 1983). There is a report that the C-terminal half-domain of galectin-3, which is homologous to galectin-1, is sufficient for saccharide binding (Agrwal et al., 1993). Furthermore, this folded polypeptide was resistant to thermal denaturation when it was bound to lactose. If galectin-1 is bound to a proper ligand i.e. poly-N-acetyllactosamine, the correct conformation of galectin-1 might be retained, which might prevent galectin-1 inactivation due to formation of intra- or intermolecular disulfide bonds.

The existence of galectin-1 as a homodimer is well established by many studies (Lobsanov et al., 1993; Liao et al., 1994), but the details of dimerization and its significance have not been clearly studied (Roff and Wang, 1983; Beyer et al., 1980). Our results demonstrate that dimerization is dependent on the concentration of lectin and the K(d) for the monomer-dimer is approx7 µM. In addition, this equilibrium is fully reversible and takes about 20 h to reach. It is interesting that the monomer-dimer equilibrium is reached at a relatively much slower rate than the binding kinetics of the lectin to laminin. In ELISA-type binding studies to laminin performed in a short time assay (2 h), most of the galectin-1 is in the form of a dimer, whereas in the longer assays (20 h), the protein is mostly monomeric. Although we do not have a precise explanation at this point, it seems likely that the anomalous binding behavior of the lectin observed in Fig. 4B is due to this monomer-dimer equilibrium. Simple ligands such as lactose clearly do not affect the monomer-dimer equilibrium. It is conceivable that glycoprotein ligands could alter the equilibrium between monomer and dimer, but this has not yet been investigated.

It has been reported by Wells and Mallucci(1991) that galectin-1 is an autocrine negative growth factor at concentrations in the subnanomolar range. At this concentration, the lectin would be predicted to be monomeric and raises the question of whether the lectin has a different biological function in the monomeric state than in the dimeric state. This possibility remains to be explored. One recent report suggested that galectin-1 can form an inactive tetramer at a subnanomolar concentration (Wells and Mallucci, 1992). In our study we could not detect such a tetramer; however, we could see a large aggregation product when we incubated rGal-1 (but not C2SrGal-1) in the absence of reducing agent for 30-40 h. The lectin in this aggregate was unable to bind laminin (data not shown).

In the accompanying report (Cho and Cummings, 1995), we describe our studies on the biosynthesis of galectin-1 in CHO cells. Our results reveal that the lectin secreted by cells is found both at the cell surface, where it is bound to surface glycoconjugates, and in the media in free form. As anticipated by the findings of the current study, the free form accumulating in the media of the cells is inactive whereas the form on the cell surface and in the cytoplasm of cells is functional. These studies reveal that galectin-1 has a complex regulation, involving equilibrium between monomer (non-cross-linking form) and dimer (cell-agglutinable form), inactive forms, and active forms bound to glycoconjugates. The overall biosynthesis and fate of galectin-1 in the extracellular space during biosynthesis is considered in more detail in the accompanying manuscript (Cho and Cummings, 1995).


FOOTNOTES

*
This work was supported by National Institutes of Health Grant CA37626 (to R. D. C.). A preliminary report of this study was presented at the Biochemical Society Meeting No. 649 at Imperial College, London in December 1993 and an abstracted version of the work was reported (Cho and Cummings, 1994). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) M96676[GenBank].

(^1)
The abbreviations used are: CHO, Chinese hamster ovary; rGal-1, recombinant galectin-1; C2SrGal-1, rGal-1 containing serine rather than cysteine at the second position; PBS-azide, phosphate-buffered saline (6.7 mM KH(2)PO(4), 150 mM NaCl (pH 7.4), 0.02% NaN(3)); SPB-azide, PBS-azide containing 14 mM 2-mercaptoethanol; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay; PCR, polymerase chain reaction.


ACKNOWLEDGEMENTS

We thank Dr. Kelley Moremen for advice in the cloning of the CHO-derived galectin-1 and Dr. Kwame Nyame and Dr. Rodger McEver for helpful discussions.


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