Correspondence to walter.nickel{at}urz.uni-heidelberg.de
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Abbreviations used in this paper: APC, allophycocyanin; Gal-1, galectin-1.
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Introduction |
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So far, it has been assumed that galectins do not interact with their counter receptors until they have been released into the extracellular space. In the current study, we provide direct evidence that these interactions are an integral part of the export mechanism itself. We report that both Gal-1 mutants deficient in ß-galactoside binding and mutant cell lines deficient in the biogenesis of galectin counter receptors are defective with regard to Gal-1 secretion. These data are further emphasized by the finding that CGL-2, a distant relative from the multicellular fungus Coprinopsis cinerea with only limited similarity to mammalian galectin-1 at the level of both primary and quaternary structure (Lobsanov et al., 1993; Walser et al., 2004) and, therefore, not being expected to be recognized as an export substrate in mammalian cells, is found to be secreted from CHO cells. Alike mammalian Gal-1, CGL-2 export from CHO cells is blocked under conditions that do not permit interactions with ß-galactosidecontaining counter receptors. The combined data presented in this study establish that the so far elusive targeting motif of Gal-1 is primarily defined by its ß-galactosidespecific carbohydrate recognition domain. Our findings suggest that galectin ligands such as ß-galactosidecontaining glycolipids act as cargo receptors. We hypothesize that galectin counter receptors either act at an intracellular level in order to recruit cytoplasmic galectins to the nonclassical export pathway or at an extracellular level by exerting a pulling force to promote directional transport of galectins across the plasma membrane.
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Results |
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Wild-type forms of both Gal-1 and CGL-2 fail to be exported from CHO cells lacking functional counter receptors for galectins
To conclusively address the question whether counter receptors play a direct role in the export mechanism of galectins, we generated cell lines expressing various forms of Gal-1 and CGL-2GFP fusion proteins based on a CHO mutant termed clone 13 (Briles et al., 1977; Deutscher and Hirschberg, 1986). In this mutant cell line, the Golgi apparatusresident transporter for UDP-galactose is defective and hence, these cells do not produce galectin counter receptors as galactosyltransferases in the Golgi lumen do not receive activated galactose residues as substrates (Deutscher and Hirschberg, 1986). As a result, both glycoproteins and glycolipids derived from clone 13 cells do not contain ß-galactosides in their sugar moieties and, therefore, do not bind galectins on their cell surface (unpublished data).
As demonstrated in the cell surface biotinylation experiments shown in Fig. 5, both Gal-1GFP (A, lanes 2 and 3) and CGL-2GFP (D, lanes 11 and 12) are efficiently exported from CHO wild-type cells (combined signals for cell surface and medium fractions). By contrast, when expressed in CHO clone 13 cells, the wild-type forms of Gal-1 and CGL-2GFP fusion proteins fail to get access to the extracellular space as the combined signals for cell surface and medium fractions (Fig. 5 G [lanes 2 and 3] and J [lanes 11 and 12], respectively) do not differ significantly from the negative control (GFP; Fig. 5, G, F, and L [lanes 17 and 18]) and are largely reduced as compared with those observed in CHO wild-type cells (A and D, respectively). As expected, export of ß-galactoside binding-deficient mutants (as exemplified by W69G and E72A for Gal-1 as well as W72G for CGL-2) is not only blocked in CHO wild-type cells (Fig. 2, B, C, and E; Fig. 5, B, C, and E) but also in CHO clone 13 cells (Fig. 5, H, I and K, respectively).
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Discussion |
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In this study we identify galectin counter receptors (i.e., ß-galactosidecontaining cell surface glyolipids and/or glycoproteins) as essential components of the overall process of Gal-1 secretion. Based on an unbiased screen, we have identified 26 single-site mutations in Gal-1 that cause both binding deficiency to counter receptors and export deficiency. In fact, we could not identify a single Gal-1 ß-galactoside binding mutant that retained export competence. To put this finding one step further we analyzed Gal-1 secretion in a somatic CHO mutant cell line defective in a Golgi apparatusresident transporter that is required for translocation of activated galactose from the cytoplasm into the lumen of the Golgi apparatus, a process essential for the generation of ß-galactosidecontaining glyolipids and glycoproteins (Deutscher and Hirschberg, 1986). Intriguingly, we found that wild-type Gal-1 fails to get exported from this mutant cell line demonstrating that indeed secretion of Gal-1 from mammalian cells strictly depends on functional interactions between Gal-1 and its counter receptors. These data are emphasized by our findings on CGL-2, a distant relative of Gal-1 from the multicellular fungus C. cinerea. Even though similarities between CGL-2 and human Gal-1 are very weak at both the level of the primary and the quaternary structure (Lobsanov et al., 1993; Walser et al., 2004), CGL-2 is recognized by mammalian cells as an export substrate. Strikingly, a single-site mutation (W72G) that is known to cause CGL-2 binding deficiency to ß-galactosides (Walser et al., 2004), results in a block of export from CHO cells of CGL-2, again consistent with our finding that functional interactions with counter receptors are essential for the overall export process.
Regarding the molecular mechanism of Gal-1 export from mammalian cells, there are two possible scenarios that would be consistent with the data presented in this study. On the one hand, galectin counter receptors on the cell surface might be part of a molecular trap through which secreted Gal-1 molecules would be removed from an equilibrium between an intracellular and an extracellular pool of Gal-1. In principle, the extracellular galectin trap could be necessary for sustained Gal-1 export of the cytoplasmic pool. However, in the absence of functional interactions between Gal-1 and its counter receptors, Gal-1 export is apparently fully blocked (Fig. 2). Therefore, the trapping mechanism does not satisfactorily explain our observations as Gal-1 transport should be observable at least to some extent until a certain equilibrium between intra- and extracellular pools is reached. Therefore, in a variation of this model, ß-galactosidecontaining cell surface molecules might be tightly coupled to the translocation machinery. In this way, counter receptors might function by exerting a pulling force at the extracellular side of the putative translocation pore required for directional transport of galectin-1 across the plasma membrane.
An alternative explanation of our results would be that galectin counter receptors act as export receptors for Gal-1. It is indeed tempting to speculate that Gal-1 interactions with counter receptors are not restricted to the extracellular space but rather already occur at the cytoplasmic side of the plasma membrane. This assumption does, of course, not comply with the established view, namely that for example glycolipids are exclusively localized to the extracellular leaflet of the plasma membrane with the glycan moieties being exposed to the extracellular space. However, it appears possible that a limited number of, for example, ß-galactosidecontaining glycolipids gets translocated to the inner leaflet of the plasma membrane catalyzed by a plasma membrane-resident flippase. This subpopulation probably would not be detectable under steady-state conditions. In this model, retrotranslocation of counter receptors occupied by Gal-1 would mediate export to the extracellular space.
Although both models are certainly speculative at this point, they are consistent with several observations that have been made previously. First, Gal-1 membrane translocation has been reported to occur at the level of the plasma membrane (Cooper and Barondes, 1990; Mehul and Hughes, 1997; Hughes, 1999; Nickel, 2003; Schäfer et al., 2004). Second, Huet and colleagues recently reported pharmacological evidence that galectin-4 secretion is impaired in epithelial cells after treatment with 1-benzyl-2-acetamido-2-deoxy--D-galactopyranoside, an inhibitor of glycosylation (Delacour et al., 2005). Third, it has been reported that membrane translocation of both FGF-2 and Gal-1 occurs in a folded state (Backhaus et al., 2004). In the context of the current study, this finding is of particular interest as both models described above can only be true if the ß-galactoside binding site of Gal-1 remains functional during membrane translocation. In this regard, the putative translocation machinery might be functionally related to the bacterial twin-arginine translocation system through which proteins are secreted in a fully folded state (Robinson and Bolhuis, 2004; Nickel, 2005).
The findings presented in this study also provide a potential explanation for the apparent nonexistence of a linear targeting motif in Gal-1. Our data conclusively point to a direct role of counter receptors as export adaptors and the ß-galactoside binding motif of Gal-1 as the primary targeting element. In this context, it is interesting to note that secretion from Saccharomyces cerevisiae of both rat galectin-1 and C. cinerea CGL-2 has been reported (Cleves et al., 1996; Boulianne et al., 2000). This is because this organism does not contain endogenous galectins and the existence of glycolipids and glycoproteins containing ß-galactosides has not been clarified. It, therefore, remains to be investigated whether secretion of galectins from S. cerevisiae occurs by a molecular mechanism similar to that of mammalian cells. Finally, it is of note that the secretory mechanism being postulated in this study provides a functional basis for quality control in the overall process of Gal-1 secretion. As the ß-galactoside binding motif of Gal-1 is shown to be the primary targeting element for secretion, quality control is in place since only properly folded Gal-1 will be recognized by the export machinery (Nickel, 2005).
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Materials and methods |
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Generation of stable cell lines expressing both wild-type and mutant forms of galectinGFP fusion proteins in a doxycycline-dependent manner
Mutant forms of Gal-1 and CGL-2 were generated by PCR-based site-directed mutagenesis and cloned as GFP fusion constructs into the retroviral vector pREV-TRE2. CHO cell lines expressing the various fusion proteins in a doxycycline-dependent manner were generated as described previously (Engling et al., 2002; Seelenmeyer et al., 2003).
Quantitative analysis of galectin binding to cell surfaces using flow cytometry
Both wild-type and mutant forms of Gal-1GFP and CGL-2GFP fusion proteins were expressed in CHO cells by incubating the cells in the presence of doxycycline (1 µg/ml) for 48 h at 37°C. Cell-free supernatants were prepared by homogenization combining freezethaw cycles with sonication. Membranes were removed in two steps by centrifugation at 1,000 gav (10 min at 4°C) and 100,000 gav (1 h at 4°C). The resulting supernatants were analyzed for the amounts of fusion protein based on GFP fluorescence as measured with a fluorescence plate reader (SpectraMax Gemini XS; Molecular Devices Corp.). Normalized amounts of cell-free supernatants (150 GFP units corresponding to 1.5 µg GFP) were then incubated with CHO cells not expressing galectinGFP fusion proteins (CHOMCAT-TAM2; Engling et al., 2002) for 1 h at 4°C to allow cell surface binding. After treatment with affinity-purified anti-GFP antibodies and APC-conjugated secondary antibodies, cell surface binding was quantified by flow cytometry as described before (Engling et al., 2002; Seelenmeyer et al., 2003).
Biochemical analysis of galectin binding to counter receptors using lactose beads
Both wild-type and mutant forms of Gal-1GFP and CGL-2GFP fusion proteins were expressed in CHO cells by incubating the cells in the presence of doxycycline (1 µg/ml) for 48 h at 37°C. After detachment of cells from the culture dishes using PBS/EDTA, cells were sedimented and solubilized in PBS/TX-100. Normalized amounts of the detergent lysates (50 GFP units corresponding to 0.5 µg GFP; SpectraMax Gemini XS; Molecular Devices Corp.) were then incubated with lactose beads (Sigma-Aldrich) for 1 h at 4°C. After extensive washing in TX-100containing buffer, bound material was eluted using SDS sample buffer. Both the flow-through fraction and the SDS eluates were analyzed by SDS-PAGE and Western blotting using anti-GFP antibodies as described in the legend of Fig. 1.
Biochemical analysis of galectin export from CHO cells using cell surface biotinylation and immunoprecipitation from cell culture supernatants
GalectinGFP fusion proteins were expressed in the corresponding CHO cell lines by incubation in the presence of doxycycline (1 µg/ml) for 48 h at 37°C (six-well plates; 70% confluency). The medium was removed and the cells washed once with PBS. Medium and PBS wash buffer were combined and subjected to immunoprecipitation using affinity-purified anti-GFP antibodies. To analyze secretion of endogenous Gal-1, affinity-purified antiGal-1 antibodies (Seelenmeyer et al., 2003) were used for immunoprecipitation experiments. Cell surfaces were treated with a membrane-impermeable biotinylation reagent (EZ-Link Sulfo-NHS-SS-Biotin; Pierce Chemical Co.). After detergent-mediated cell lysis, the cells were scraped off the culture plates, insoluble material was then removed by low speed centrifugation. Biotinylated and nonbiotinylated proteins were separated using streptavidin beads (UltraLink immobilized streptavidin; Pierce Chemical Co.). Material bound to streptavidin beads was eluted with SDS sample buffer. Samples from the input material, the nonbound fraction and the streptavidin-bound fraction were analyzed by SDS-PAGE and Western blotting as indicated in the legends to Figs. 2 and 5. For the biotinylation and immunoprecipitation experiments depicted in Fig. 6, secondary antibodies coupled to a fluorophor (Alexa 680) were used to allow quantitation of the signal using a Odyssey imaging system (LI-COR Biotechnology).
Quantitative analysis of galectin export from CHO cells using flow cytometry
GalectinGFP fusion proteins as well as GFP were expressed in the corresponding CHO cell lines by incubation in the presence of doxycycline (1 µg/ml) for 48 h at 37°C (six-well plates; 70% confluency). The medium was removed and the cells washed once with PBS. Both primary (anti-GFP; 1 h) and secondary (APC-conjugated antirabbit; 30 min) antibody labeling was conducted at 4°C with the cells being attached to the culture plates. The cells were then detached from the culture plates using cell dissociation buffer (Life Technologies). Before the flow cytometry analysis, propidium iodide (1 µg/ml) was added in order to detect damaged cells. GFP (expression level)- and APC-derived fluorescence (cell surface population) were analyzed using a FACSCalibur flow cytometer (Becton Dickinson). Background fluorescence was determined by measuring CHOMACT-TAM2 cells (Engling et al., 2002), which were treated with both primary and secondary APC-conjugated secondary antibodies. GFP- and APC-derived fluorescence were measured simultaneously without compensation.
Stability analysis of both wild-type and mutant forms of galectinGFP fusion proteins in conditioned media derived from CHO cells
Cell-free supernatants of both wild-type and mutant forms of Gal-1GFP and CGL-2GFP fusion proteins as well as GFP as a control were obtained as described above. Normalized amounts (50 GFP units corresponding to 0.5 µg GFP; SpectraMax Gemini XS; Molecular Devices Corp.) were then diluted 1:10 in conditioned medium derived from CHO cells. Samples were then either directly subjected to immunoprecipitation using anti-GFP antibodies (Fig. 4, lane 2), incubated for 48 h at 4°C followed by immunoprecipitation (Fig. 4, lane 3) or incubated for 48 h at 37°C followed by immunoprecipitation (Fig. 4, lane 4). In each case, bound material was eluted with SDS sample buffer. The samples were then analyzed by SDS-PAGE and Western blotting using anti-GFP antibodies and antirabbit secondary antibodies (clone RG-16; see above) coupled to HRP (ECL detection).
Confocal microscopy
CHO cells transduced with the reporter constructs indicated were grown on glass coverslips for 36 h at 37°C in the presence of 1 µg/ml doxycycline (70% confluency). The cells were then either processed for live imaging or subjected to paraformaldehyde fixation (3% wt/vol, 20 min at 4°C) without permeabilization followed by antibody processing as indicated. Alexa546-coupled secondary antibodies were used for cell surface staining experiments. The specimens were mounted in Fluoromount G (Southern Biotechnology Associates, Inc.) and viewed with a confocal microscope (LSM 510; Carl Zeiss MicroImaging, Inc.).
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Acknowledgments |
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This work was supported by a grant from the German Research Council (SFB 638, project A8).
Submitted: 6 June 2005
Accepted: 14 September 2005
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References |
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