(Received for publication, October 27, 1994; and in revised form, January 9, 1995)
From the
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,
1/2 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
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.
Many vertebrate and invertebrate tissues contain soluble,
so-called S-type -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) ()cells. We reported that galectin-1 from CHO cells
occurs in an active monomeric form that can reversibly dimerize with a K
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.
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.
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.
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 1/2 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.
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.
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.
, galectin-1 remaining in maltose-washed
cells;
, galectin-1 remaining in lactose-washed cells;
,
galectin-1 occurring in media;
, 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.
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.
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.