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

(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

In the accompanying study (Cho, M., and Cummings, R. D.(1995) J. Biol. Chem. 270, 5198-5206), we reported that Chinese hamster ovary (CHO) cells synthesize galectin-1. We have now used several approaches to define the subcellular location and biosynthesis of galectin-1 in these cells. Galectin-1 was present on the cell surface, as assessed by immunofluorescent staining with monospecific antibody to the protein. Quantitation of the surface-localized galectin-1 was achieved by metabolically radiolabeling cells with [S]Met/Cys and measuring the amount of lectin (i) sensitive to trypsin, (ii) accessible to biotinylating reagents, and (iii) accessible to the haptenic disaccharide lactose. By all three procedures, approx1/2 of the radiolabeled galectin-1 associated with cells was shown to be on the cell surface with the remainder intracellular. The kinetics of externalization of galectin-1 was monitored by pulse-chase radiolabeling, and it was shown that cells secrete the protein with a t approx 20 h. The cell surface form of galectin-1 in CHO cells was active and bound to surface glycoconjugates, but lectin accumulating in the culture media was inactive. Lectin synthesized by mutant Lec8 CHO cells, which are unable to galactosylate glycoproteins, was not found on the surface and quantitatively accumulated in the media in an inactive form. Taken together, our results demonstrate that galectin-1 is quantitatively externalized by CHO cells and can associate with surface glycoconjugates where the lectin activity is stabilized.


INTRODUCTION

Many vertebrate and invertebrate tissues contain soluble, so-called S-type beta-galactoside-binding lectins, which are now known to represent a class of related carbohydrate-binding proteins termed galectins (Gitt and Barondes, 1986; Barondes et al., 1994). Galectin-1 is an 14-kDa subunit protein originally found in electric eels by Teichberg et al.(1975) and subsequently in bovine heart and other tissues by DeWaard et al.(1976) and Nowak et al.(1976).

The proposed biological functions of galectin-1 in possibly mediating cellular interactions with extracellular matrix have been difficult to reconcile, however, with the original observation that in fibroblasts the lectin is found primarily in the cytoplasm and the isolated lectin is completely unstable in the absence of reducing agents (DeWaard et al., 1976). Galectin-1 lacks a recognizable signal sequence, contains free sulfhydryl residues, and lacks disulfide bonds (Hirabayashi et al., 1987; Clerch et al., 1988; Whitney et al., 1986; Tracey et al., 1992). Interestingly, in differentiating myoblasts, but not actively replicating myoblasts, galectin-1 appears to be externalized from the cells, and this externalization may involve a novel mechanism independent of the normal secretory process (Cooper and Barondes, 1990). In many cells and tissues, galectin-1 is found to be in both the cytoplasm of cells and localized in the extracellular matrix (Allen et al., 1990; Cooper et al., 1991; Harrison and Wilson, 1992). No studies have been reported on the biosynthesis of galectin-1 in fibroblasts nor in cells in which it may be secreted constitutively.

These observations prompted us to analyze the structure, cellular location, and biosynthesis of galectin-1 in a commonly studied cell line of Chinese hamster ovary (CHO) (^1)cells. We reported that galectin-1 from CHO cells occurs in an active monomeric form that can reversibly dimerize with a K approx 7 µM (Cho and Cummings, 1995). Moreover, we found that the lectin is stable in the absence of reducing agents when bound to ligands, but the lectin rapidly loses activity when not complexed with ligand. In the present report, we demonstrate that galectin-1 is constitutively and quantitatively externalized by CHO cells and binds to plasma membrane glycoconjugates where the lectin retains activity. In contrast, in the absence of surface ligands, secreted galectin-1 is inactive. The results are discussed in terms of the overall structure and biosynthesis of galectin-1 in animal cells.


EXPERIMENTAL PROCEDURES

Materials

Molecular mass marker (14-200 kDa) for SDS-polyacrylamide gel electrophoresis, Immunoprecipitin, and tissue culture reagents were purchased from Life Technologies, Inc. Bovine serum albumin (fraction V), asialofetuin, alkaline phosphatase, conjugated mouse anti-rabbit IgG, fluorescein isothiocyanate-mouse anti-rabbit IgG, Sephadex G-25, 2-mercaptoethanol, leupeptin, trifluoroacetic acid, phenylmethylsulfonyl fluoride, pepstatin, aprotinin, ethanolamine, and Protein A-Sepharose were obtained from Sigma. Lactose, acetone, EDTA (disodium salt), and sodium bicarbonate were obtained from J. T. Baker. S-Protein Labeling Mix (1200 Ci/mmol) was purchased from DuPont NEN. NHS-biotin and streptavidin-Sepharose were obtained from Pierce Chemical. R-PE-conjugated F(ab)(2) goat anti-rabbit antibody was purchased from Zymed. Alkaline phosphatase color developing kit was obtained from Bio-Rad.

Cell Culture, Metabolic Radiolabeling, and Biotinylation

Chinese hamster ovary (CHO-K1) cells were cultured as described previously (Smith et al., 1990). To metabolically radiolabel proteins, CHO cells were cultured in 100-mm dishes, incubated for 30 min at 37 °C in methionine-free media, pulse-labeled for 40 min at 37 °C in the same media with 250 µCi of S-Protein Labeling Mix, and chased with 15 ml of normal growth media. At each time point, the culture medium was gently collected, and the cell layers were washed with 2 ml of ice-cold PBS (150 mM NaCl, 6.7 mM KH(2)PO(4), pH 7.4). They were then incubated with 100 mM lactose/PBS for 5 min for lactose-washed cells or incubated with 100 mM maltose/PBS for control cells. After all the wash solutions were collected, the cells were scraped from the dish into 1 ml of PBS containing 1% Nonidet P-40, 50 µg/ml phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, 0.5 µg/ml aprotinin, 0.7 µg/ml pepstatin, and 5 mg/ml BSA. The dishes were washed with another 1 ml of PBS and pooled with the first milliliter collected. Media, wash solutions, and cell samples were immediately frozen in liquid nitrogen. For biotinylation experiments, cells were cultured overnight in media containing S-Protein Labeling Mix (250 µCi), washed twice with PBS, and 2 ml of 10 mg/ml sulfo-NHS-biotin in PBS was added. After a 30-min incubation at 4 °C, another 2 ml of sulfo-NHS-biotin was added and incubated for an additional 30 min.

Immunoprecipitation and Western Blotting

For each 1 ml of sample, 50 µl of immunoprecipitin was added and incubated for 1 h at 4 °C and then centrifuged to remove the immunoprecipitin and cell debris. One hundred microliters of affinity-purified monospecific antigalectin-1 antibody (80 µg/ml) (Cho and Cummings, 1995) was added to the supernatant, and this mixture was rotated for 1 h at 4 °C. After a 1-h incubation with Protein A-Sepharose, the mixture was centrifuged to pellet immunoprecipitated complexes. The galectin-1-antibody complexes immobilized on the Protein A-Sepharose were washed five times with washing buffer (0.5% Nonidet P-40, 0.5% BSA in PBS), and galectin-1 was eluted with Laemmli sample buffer (Laemmli, 1970) by boiling for 5 min. In the case of biotinylated cells, immunoprecipitated galectin-1 was eluted with 0.1 M glycine buffer (pH 2.5) and separated from Protein A-Sepharose by centrifugation. The pH of the galectin-1-containing supernatant was adjusted by adding one-tenth volume of 1 M Tris-HCl (pH 8.0), and biotinylated galectin-1 was precipitated from the supernatant with streptavidin-Sepharose. The streptavidin-Sepharose was separated from the supernatant and washed twice with PBS, and the wash solutions were combined with the supernatant and precipitated by trichloroacetic acid. The galectin-1 bound to streptavidin-Sepharose was eluted with Laemmli buffer. This eluent and the trichloroacetic acid-precipitated material were analyzed by SDS-PAGE on 13.5% acrylamide under reducing conditions. The gels were stained with Coomassie Blue (50% MeOH, 20% acetic acid, 0.1% Coomassie blue) and destained (30% MeOH, 7% acetic acid). For fluorography, the gels were agitated in the En^3Hancer (DuPont NEN) according to the manufacturer's instructions, then dried and exposed to x-ray film at 70 °C using Kodak X-OMAT AR. After development, exposed areas of film were quantified by densitometry (Molecular Dynamics Model 3008 computing densitometer). For Western blotting, proteins were electrotransferred overnight at 4 °C onto nitrocellulose membranes, as described (Cho and Cummings, 1995). Membranes were blocked for 2 h with PBS containing 5% nonfat dry milk (Carnation). All additional immunostaining steps were performed in PBS with 0.05% Tween 20 (TPBS) at room temperature. Membranes were incubated with primary antibody for 1 h. After three washes with TPBS, membranes were incubated with 1:3,000 diluted alkaline phosphatase conjugated secondary antibody (anti-rabbit IgG mouse antibody) for 1 h. Membranes were washed in TPBS five times for 5 min and were developed with alkaline phosphatase color developing solution.

Immunohistochemistry

Cells were cultured as above on chamber slides (Nunc Inc.) for immunohistochemistry. Cells were washed with PBS and fixed for 30 min at 4 °C with PBS containing 2% paraformaldehyde. Cells were permeabilized by treatment for 1 h with PBS containing 0.05% saponin at 37 °C (from this point on, all solutions for permeabilized cells contain 0.025% saponin). Cells were stained with monospecific anti-galectin-1 antibody diluted to 8 µg/ml in PBS containing 1% BSA for 1 h, washed with three times with PBS, and incubated for 1 h with anti-rabbit IgG conjugated with fluorescein isothiocyanate (Sigma) diluted 1:200 in PBS, 1% BSA. The samples were washed three times for 5 min each with PBS, mounted in mounting medium (KPL Inc., Gaithersburg, MD), and analyzed in a fluorescence microscope.

Trypsin Sensitivity

One 10-cm dish of CHO cells was labeled overnight with 250 µCi of S-Protein Labeling Mix. The radiolabeled cells were treated with 2 ml of trypsin-EDTA (0.05% trypsin and 0.53 mM EDTA) for 20 min at 37 °C, followed by addition of trypsin inhibitor 10 times the concentration of trypsin to stop the reaction. The cells were lysed in 2 ml of ice cold PBS containing 1% Triton X-100 and protease inhibitors (50 µg/ml phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, 0.5 µg/ml aprotinin, 0.7 µg/ml pepstatin). The soluble lysate was analyzed by Western blotting and immunoprecipitation.

Asialofetuin-Sepharose Chromatography

Asialofetuin (5 mg/ml) was 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 (Pharmacia Biotech Inc.). The coupling efficiency was estimated to be 5 mg/ml by determining the amount of uncoupled protein by BCA assay. The media collected in pulse-chase experiments were applied to this asialofetuin-affinity chromatography column. The column was washed with 10 column volumes of SPB (PBS containing 14 mM 2-mercaptoethanol) followed by elution with 0.1 M lactose in SPB. The unbound and lactose-eluted materials were collected for immunoprecipitation.


RESULTS

Immunohistochemical Localization of Galectin-1 in CHO Cells

In the accompanying study (Cho and Cummings, 1995), we described the preparation of monospecific polyclonal antisera to galectin-1 and demonstrated that by Western blot the purified antibody reacted with only galectin-1. In studies described below, we also found that the purified antibody immunoprecipitated only galectin-1 from metabolically radiolabeled cells. This purified antibody was used to probe the subcellular distribution of the lectin in wild-type CHO cells and a mutant cell line termed Lec8 CHO. Lec8 CHO cells are unable to galactosylate their glycolipids or glycoproteins due to a defective transport of UDPGal into the Golgi apparatus (Briles et al., 1977; Stanley, 1984; Deutscher and Hirschberg, 1986). Lec8 CHO cells do not contain glycoconjugate ligands for the galectin-1 (Zhou and Cummings, 1993).

To study the distribution of galectin-1, CHO cells were cultured on glass chamber slides to 50-70% confluence. The cultured cells were fixed and incubated with the antibody to galectin-1 without permeabilization. Both intracellular and extracellular galectin-1 were stained by initially fixing the cells, permeabilizing with 0.05% saponin, and incubating with antibody. Preimmune IgG did not react with permeabilized CHO cells (Fig. 1A). Galectin-1 was widely distributed throughout the cytoplasm, but it was absent from nuclei in both CHO and Lec8 CHO cells (Fig. 1, B and C), which is characteristic of cytoplasmic proteins. However, in nonpermeabilized CHO cells, the edges of the cells were strongly stained, which is typical for cell surface protein (Fig. 1D). We hypothesized that galectin-1 on the surface of CHO cells might be present there because of its interaction with surface glycoconjugates.


Figure 1: Immunolocalization of galectin-1 in CHO cells. CHO cells were fixed with paraformaldehyde and incubated with anti-galectin-1 with or without prior permeabilization by saponin treatment, followed by R-PE-conjugated mouse anti-rabbit antibodies. The cells were mounted and observed by fluorescence microscopy. A, permeabilized CHO cells stained with preimmune sera; B, permeabilized CHO cells stained with monospecific galectin-1 antibody; C, permeabilized Lec8 CHO cells stained with monospecific galectin-1 antibody; D, nonpermeabilized CHO cells stained with monospecific galectin-1 antibody; and E, nonpermeabilized Lec8 CHO cells stained with monospecific galectin-1 antibody.



To test this possibility, nonpermeabilized Lec8 CHO cells were immunostained with the same antibody. In the nonpermeabilized Lec8 CHO cells there was no significant surface antigen detectable (Fig. 1E). These results demonstrate that wild-type CHO cells contain surface-bound galectin-1 and that it is present there as a complex with membrane glycoconjugates containing galactose.

Cell Surface Galectin-1 Is Trypsin-sensitive

To further investigate the presence and amount of galectin-1 associated with the cell surface, we assessed the susceptibility of surface lectin to trypsin. In one approach, wild-type CHO cells attached to Petri dishes were exposed to trypsin-EDTA for 20 min, followed by solubilization, SDS-PAGE, and Western blotting with antibody to galectin-1 (Fig. 2A). Galectin-1 was readily detectable in total cell extracts, and approximately one-half of the lectin was lost upon treatment of the cells with trypsin. The trypsin-resistant lectin was intracellular, as shown by the fact that when 1% Triton X-100 was added prior to trypsin treatment to solubilize them and expose the cytosolic lectin, most of the galectin-1 was destroyed by the protease (Fig. 2A). In another approach, total proteins in CHO cells were metabolically radiolabeled with [S]Met/Cys. The radiolabeled cells attached to Petri dishes were exposed to trypsin-EDTA for 20 min, followed by solubilization, immunoprecipitation with antibody to galectin-1, SDS-PAGE, and fluorography. Compared to cells not treated with trypsin, the trypsin-treated cells showed a reduction of galectin-1 band intensity (Fig. 2B). Densitometric analysis of the lanes in Fig. 2B indicated that about one-half of the lectin was lost upon trypsin treatment of intact cells. As a measurement of cell integrity in this experiment, we determined both the activity of lactate dehydrogenase in the cell extract and media and trypan blue staining of the cells. Both approaches demonstrated that over 90% of the cells were intact after a 3-h incubation in the trypsin-EDTA solution (data not shown). These data demonstrate that about one-half of the galectin-1 associated with CHO cells is located on the cell surface and is sensitive to trypsin.


Figure 2: Trypsin sensitivity of galectin-1 in intact CHO cells. One 10-cm dish of CHO cells were labeled overnight with 250 µCi of S-Protein Labeling Mix and then treated, as indicated, with trypsin-EDTA (0.05% trypsin and 0.53 mM EDTA) for 20 min at 37 °C. Trypsin was inactivated by addition of a 10-fold excess of trypsin inhibitor over the enzyme. The cells were lysed in 2 ml of PBS containing 1% of Nonidet P-40 and protease inhibitors. The soluble lysate was analyzed by Western blotting and immunoprecipitation, as described under ``Experimental Procedures.'' A, immunoblots after SDS-PAGE with monospecific anti-galectin-1 antibody. B, immunoprecipitates of S-labeled galectin-1 with monospecific anti-galectin-1 antibody.



Surface-localized Galectin-1 Can Be Biotinylated

To confirm the cell surface distribution of the galectin-1, wild-type CHO cells were metabolically radiolabeled with [S]Met/Cys, and the cells were treated with sulfo-NHS-biotin to biotinylate surface proteins. Because sulfo-NHS-biotin cannot penetrate cell membranes (Orr, 1981), it only can react with amino groups on proteins exposed extracellularly.

An excess amount of sulfo-NHS-biotin was used to quantitatively biotinylate CHO cell surface proteins. After biotinylation, cells were harvested and immunoprecipitated. The immunoprecipitated antibody-galectin-1-Protein A-Sepharose complexes were dissociated by adding low pH elution buffer, and Protein A-Sepharose beads were removed by centrifugation. After adjusting the pH to neutral, the supernatant was incubated with streptavidin-Sepharose to isolate the biotinylated galectin-1 from the total immunoprecipitated galectin-1. Samples were analyzed by SDS-PAGE fluorography, and the bands of galectin-1 on the fluorograms were quantified by densitometric scanning. As shown in Fig. 3A, 45% of galectin-1 was biotinylated on intact cells and subsequently bound to streptavidin-Sepharose, with the remainder occurring in the supernatant. These results are consistent with those obtained through trypsinization above and indicate that approx1/2 of galectin-1 is surface-localized in wild-type CHO cells.


Figure 3: Surface localization of galectin-1 by biotinylation. CHO cells (A) and Lec8 CHO cells (B) were labeled overnight with 250 µCi of S-Protein Labeling Mix. One set of each cell type was reserved, and the total galectin-1 was isolated by immunoprecipitation. A second set of cells was biotinylated with 10 mg/ml of the membrane-impermeable reagent sulfo-NHS-biotin by two treatments for 1 h at 4 °C. The biotinylated cells were lysed, and galectin-1 was immunoprecipitated. The immunoprecipitates were dissociated by low pH treatment, and the biotinylated galectin-1 was recovered by absorption to streptavidin-Sepharose. Material unbound and bound to streptavidin-Sepharose was trichloroacetic acid-precipitated and analyzed by SDS-PAGE and fluorography. The lanes are designated as follows: Total imm.ppt, total immunoprecipitated surface plus intracellular galectin-1; Avidin unbound, intracellular galectin-1; Avidin bound, cell surface galectin-1.



Biotinylation experiments were also performed using Lec8 CHO cells. Interestingly, galectin-1 from Lec8 CHO cells was found quantitatively in the streptavidin-Sepharose unbound material, proving that galectin-1 was located inside the cells, but not at the cell surface (Fig. 3B). Furthermore, this result demonstrates that intracellular galectin-1 is inaccessible to sulfo-NHS-biotin. In experiments described below, it is shown that Lec8 CHO cells do secrete galectin-1 to the outside.

Galectin-1 Binds to the Cell Surface through Carbohydrate-Lectin Interactions

To further demonstrate that galectin-1 on the surface of wild-type CHO cells is bound to carbohydrate determinants, CHO cells in Petri dishes were washed with 0.1 M lactose in PBS or PBS for 5 min. The lactose wash solution and the lactose-washed cells were analyzed for galectin-1 by Western blot analysis. Galectin-1 was present in the lactose wash (Fig. 4, lane 1), and there was a decrease of galectin-1 in the lactose-washed cells compared to control treated cells (Fig. 4, lanes 2 and 3). These results, together with those above using Lec8 CHO cells, confirm that galectin-1 is present on the surface of wild-type CHO cells and is bound in a lactose-inhibitable fashion to surface glycoconjugates.


Figure 4: Western blot analysis of total galectin-1 in CHO cells before and after washing cells with lactose. CHO cells were grown to 80% confluency, and the culture medium was gently collected. The cell layer was washed with 2 ml of ice-cold PBS, and surface galectin-1 was eluted by treatment with 100 mM lactose/PBS for 5 min. The treated cells were lysed. One-tenth of the total lysate and two-tenths of the trichloroacetic acid-precipitable material from the lactose eluted sample were analyzed by SDS-PAGE and Western blotting. The lanes are designated as follows: lane 1, galectin-1 eluted from cell surfaces by lactose washing; lane 2, intracellular form of galectin-1 following lactose washing of intact cells; lane 3, total cell-associated galectin-1 prior to lactose washing.



Biosynthesis of Galectin-1

We considered that the surface localization of galectin-1 could arise by lectin either leaking from damaged cells or secreted through a biosynthetic process. To evaluate these possibilities, wild-type CHO cells were pulse-labeled for 40 min with [S]Met/Cys in low methionine media and chased for up to 30 h in normal media lacking [S]Met/Cys. At each time point, one set of cells were washed with lactose (50 mM) just before immunoprecipitation with antibody to galectin-1, and the other set was washed with either maltose or PBS as controls. Radiolabeled galectin-1 was immunoprecipitated from culture media and cells and resolved by SDS-PAGE. The amount of galectin-1 immunoprecipitated was quantified by densitometric scanning of the fluorogram. This experiment was repeated three separate times, and the results were similar in each case. One of these experiments is summarized in Fig. 5.


Figure 5: Pulse-chase kinetics of galectin-1 biosynthesis in CHO cells. CHO cells were grown in 10-cm dishes and were pulsed with 250 µCi of S-Protein Labeling Mix for 40 min followed by a chase period extending up to 30 h. At each time point, the culture medium was gently collected and the cell layer was washed with 2 ml of ice-cold PBS. The cells were then incubated with 100 mM lactose/PBS for 5 min for lactose-washed cell or incubated with 100 mM maltose/PBS as a control. Galectin-1 was immunoprecipitated from lactose-washed cells, control cells (maltose-washed), and media. The immunoprecipitates were analyzed by SDS-PAGE and fluorography (bottom panel). The top panel shows the amount of lectin (as a percentage of the total amount) at each time point during the chase period. Lactate dehydrogenase activity in the media versus total lactate dehydrogenase activity in the cells was monitored as a function of time to assess the integrity of cells. bullet, galectin-1 remaining in maltose-washed cells; box, galectin-1 remaining in lactose-washed cells; circle, galectin-1 occurring in media; up triangle, lactate dehydrogenase activity in media as a percent of total activity.



Most of the newly synthesized galectin-1 was externalized and found in the media after 30 h (Fig. 5, bottom panel), and this secretion is quantified in the graph (Fig. 5, top panel). However, up to 11 h, most of the galectin-1 was found in the cell extract (Fig. 5). Over the same time period, differences were observed in lectin recovered between PBS- or maltose-washed cells and lactose-washed cells (Fig. 5). More lectin was eluted from intact cells using lactose versus maltose washing conditions in the times before 11 h. Both the maltose-wash and PBS-wash gave similar results. Neither maltose nor PBS would be expected to elute lectin specifically bound to glycoconjugates, since maltose does not inhibit hemagglutination by galectin-1.

These results demonstrate that newly synthesized galectin-1 is externalized, attaches to cell surface glycoconjugates, and remains there for several hours before being released into the media. Furthermore, secretion of galectin-1 by CHO cells is quantitative and occurs at a rate of about 3-4% per h. By 30 h of chase, at least 80% of galectin-1 was secreted by the cells into the media. During the chase time, the intactness of the cells was determined by measuring the activity of lactate dehydrogenase in the media and plotted as a relative percentage to that present in the extract of cells at the 30-h time point (the 0 h extract set to 100%) (Fig. 5). Throughout the chase time, less then 2% of total lactate dehydrogenase activity could be found in the media. These results demonstrate that galectin-1 is quantitatively secreted by CHO cells and does not gain access to the extracellular space through cell lysis.

Galectin-1 Found in Media Is Inactive

To investigate lectin activity of galectin-1 secreted into media, 30-h chase media from CHO cells, as in Fig. 5, was collected and applied to a column containing asialofetuin-Sepharose. Galectin-1 was immunoprecipitated from the unbound fractions and lactose-eluted fractions. Galectin-1 found in CHO culture media after a 30-h chase was inactive in binding to asialofetuin-Sepharose (Fig. 6A).


Figure 6: Inactivity of galectin-1 in the media in Lec8 CHO cells. CHO and Lec8 CHO cells were grown on a 10-cm dish until 90% confluency and radiolabeled with 250 µCi of S-Protein Labeling Mix for 30 min and chased for 3 h. Each medium was collected and applied to an asialofetuin column. Galectin-1 was immunoprecipitated from asialofetuin-Sepharose (ASF-SEP) bound and unbound fractions.



We performed a similar experiment using both wild-type CHO cells and Lec8 CHO cells, except that the chase time was reduced from 30 to 3 h to enhance the possibility of finding active galectin-1. This was based on the accompanying study (Cho and Cummings, 1995) in which we found that galectin-1 in solution lacking either reducing agent or glycoconjugate ligand had a t of 6-10 h. The galectin-1 secreted into the media of wild-type CHO cells during a 3-h chase was unable to bind asialofetuin-Sepharose (Fig. 6B). In media from Lec8 cells, following the 3-h chase, more galectin-1 could be recovered as expected, but it too was unable to bind asialofetuin-Sepharose (Fig. 6B). These results demonstrate that galectin-1 secreted into the media by either wild-type CHO or Lec8 CHO cells is unstable and inactive.


DISCUSSION

We have shown that galectin-1 is synthesized in CHO cells in the cytosol and can be externalized and remains at the cell surface through carbohydrate-lectin interactions. We chose to study galectin-1 in CHO cells based on preliminary screening using Western blot analysis of various cell lines. The results demonstrated that CHO cells, compared to other common cell lines, produced high amounts of galectin-1. Furthermore, our results demonstrate that the Lec8 mutant of CHO cells generates and secretes galectin-1 as do normal CHO cells, but the lectin is not bound to the Lec8 CHO cell surface and accumulates in the media in an inactive form. The combined results of trypsin sensitivity, biotinylation, and haptenic sugar release demonstrate that about one-half of the total cell-associated galectin-1 in CHO cells is on the cell surface and the remainder is in the cytoplasm. Furthermore, the results show that galectin-1 is quantitatively secreted to the outside of the cell.

Previous studies on the localization of galectin-1 in cultured cells have provided conflicting evidence. In the original studies by Briles et al.(1979), it was found that galectin-1 in chick embryo fibroblast cells was cytosolic, and, in nonpermeabilized cells, no lectin was detectable on the cell surface. While we have not retested these cells for the presence of surface galectin-1, we have noticed that extensive washing of cells and the fixation process can result in loss of surface galectin-1. Galectin-1 on the surface of CHO cells is bound only by its reversible interactions with carbohydrate ligands. Thus, extensive washing of cells before fixation can cause significant losses of surface lectin. The pulse-chase experiments demonstrate that the surface-localized galectin-1 does not arise through release from the cytoplasm of damaged cells and binding of the lectin to surface glycoconjugates.

Cooper and Barondes(1990) reported that galectin-1 is neither secreted nor is present on the cell surface of actively replicating mouse myoblasts. They did find, however, that galectin-1 is secreted by differentiated mouse myoblasts, and that, during secretion, the lectin is observed by immunofluorescence to be within vesiculated structures closely underlying the plasma membrane (Cooper and Barondes, 1990). In contrast to this study, Harrison and Wilson(1992) reported that an undefined amount of galectin-1 was present on the cell surface of migrating myoblasts using immunofluorescent techniques and was concentrated in the ruffled edge. However, in that case, the lectin was neither susceptible to trypsin nor was it eluted from the cells by treatment with lactose, thus raising questions about the interpretation that the lectin is truly surface-localized. In our experiments, galectin-1 in CHO cells appears diffusely distributed throughout the cytoplasm, and there was no evidence for a vesiculated form of the lectin and lectin was constitutively secreted. Furthermore, the lectin was sensitive to trypsinization and eluted from the cell surface with lactose. In the case of mouse myoblasts, the vesiculated forms of galectin-1 were seen upon induced differentiation of the cells and thus may represent a special secretory mechanism for these cells (Cooper and Barondes, 1990).

Our studies strongly suggest that the carbohydrate-lectin interactions are the primary mechanism to both localize galectin-1 on the surface of cells and allow generation of an active form of the extracellular lectin. Galectin-1 is unstable in oxidizing conditions such as those likely to exist in the extracellular matrix. In the accompanying manuscript (Cho and Cummings, 1995), we demonstrated that galectin-1 was highly stable in the absence of reducing agent when the lectin is bound to a glycoconjugate ligand.

Pulse-chase experiments coupled with the lactose wash experiments showed that newly synthesized galectin-1 is externalized slowly (about 3% per h), and galectin-1 stays associated at the surface of plasma membrane for a period (about 10 h) through carbohydrate-lectin interactions. However, the amount of time galectin-1 remains bound to the cell surface could be longer in vivo than in vitro, due to the potentially large artificial dilution of the lectin in the media of in vitro cultured cells. During biosynthesis and secretion of the lectin, the surface-bound galectin-1 may be displaced by newly synthesized lectin and move to the media where it rapidly loses activity. These possibilities are summarized in Fig. 7, which illustrates some of the possible fates of galectin-1 during its biosynthesis and movement to the cell surface and media. This pathway incorporates also the findings of the accompanying manuscript (Cho and Cummings, 1995) in which galactin-1 is shown to occur in a reversible and active monomer/dimer form.


Figure 7: Proposed biosynthetic pathway for galectin-1 in CHO cells. This illustration depicts the biosynthesis of galectin-1 in CHO cells and some of the possible equilibria of the galectin-1 between monomer and dimer forms and complexation with carbohydrate ligands. Lectin in the cytosol is in both monomer and dimer forms. Following secretion, a metastable intermediate form of the lectin is depicted, which must either associate with carbohydrate ligands or become inactivated.



Our observation that the galectin-1 secreted by Lec8 CHO cells in a 3-h chase experiment is inactive may also have implications in regard to the mechanism of secretion. It is possible that galectin-1 is secreted in a metastable form (the postulated intermediate in Fig. 7) that requires immediate association with glycoconjugate ligand to stabilize the protein and allow correct protein folding. Possibly, this metastable form of galectin-1 synthesized in Lec8 CHO cells is rapidly inactivated by the oxidizing conditions which may lead to incorrect inter- or intradisulfide bonds. In wild-type CHO cells, this problem is averted by the association of secreted lectin with poly-N-acetyllactosamine-containing glycoproteins (Merkle and Cummings, 1988), such as lysosome-associated membrane proteins 1 and 2 on the cell surface (Do et al., 1990). In some cell types that synthesize basement glycoproteins, such as laminin, secreted galectin-1 may associate with those glycoconjugates and also be stabilized in the extracellular environment. In the accompanying report (Cho and Cummings, 1995), we found that galectin-1 binding to laminin highly stabilizes the activity of the lectin. Laminin has been found to be a high affinity ligand for galectin-1 (Zhou and Cummings, 1990, 1993), and several reports demonstrate that galectin1 can be found by immunohistochemical methods to be colocalized with laminin (Cooper and Barondes, 1990; Cooper et al., 1991).

Our studies reveal a potentially unique mechanism of regulating the activity of galectin-1. The activity of the protein may be regulated by its binding to available surface or extracellular matrix glycoconjugates. When none are available, or when the lectin is dissociated from glycoconjugates and cannot rebind readily, it is rapidly inactivated in the nonreducing extracellular environment. In addition, our finding of monomer-dimer forms of the lectin may also indicate that a complex process occurs whereby monomers bind to glycoconjugates and serve as sites for other monomers to bind, thereby facilitating multivalent activity of the lectin. The binding affinity of lectin to laminin is approximately 1-2 µM (Zhou and Cummings, 1990, 1993) and is in the same range as the binding affinity of monomers to each other (Cho and Cummings, 1995). It will be interesting in future studies to determine whether the monomer or dimer forms are secreted from cells and the mechanism(s) by which these forms pass through the plasma membrane and associate with extracellular ligands.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant CA37626 (to R. D. C.). 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.

(^1)
The abbreviations used are: CHO, Chinese hamster ovary; PBS, phosphate-buffered saline (6.7 mM KH(2)PO(4), 140 mM NaCl, pH 7.2); TPBS, phosphate-buffered saline containing 0.05% Tween 20; SPB, PBS containing 14 mM 2-mercaptoethanol; PAGE, polyacrylamide gel electrophoresis; NHS, N-hydroxysulfosuccinimide; BSA, bovine serum albumin.


ACKNOWLEDGEMENTS

We thank Dr. Kwame Nyame and Dr. Rodger McEver for helpful discussions.


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