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Cell surface counter receptors are essential components of the unconventional export machinery of galectin-1.

Seelenmeyer C, Wegehingel S, Tews I, Künzler M, Aebi M, Nickel W - J. Cell Biol. (2005)

Bottom Line: Intriguingly, we also find that a distant relative of galectin-1, the fungal lectin CGL-2, is a substrate for nonclassical export from Chinese hamster ovary (CHO) cells.Alike mammalian galectin-1, a CGL-2 mutant defective in beta-galactoside binding, does not get exported from CHO cells.We conclude that the beta-galactoside binding site represents the primary targeting motif of galectins defining a galectin export machinery that makes use of beta-galactoside-containing surface molecules as export receptors for intracellular galectin-1.

View Article: PubMed Central - PubMed

Affiliation: Heidelberg University Biochemistry Center (BZH), 69120 Heidelberg, Germany.

ABSTRACT
Galectin-1 is a component of the extracellular matrix as well as a ligand of cell surface counter receptors such as beta-galactoside-containing glycolipids, however, the molecular mechanism of galectin-1 secretion has remained elusive. Based on a nonbiased screen for galectin-1 export mutants we have identified 26 single amino acid changes that cause a defect of both export and binding to counter receptors. When wild-type galectin-1 was analyzed in CHO clone 13 cells, a mutant cell line incapable of expressing functional galectin-1 counter receptors, secretion was blocked. Intriguingly, we also find that a distant relative of galectin-1, the fungal lectin CGL-2, is a substrate for nonclassical export from Chinese hamster ovary (CHO) cells. Alike mammalian galectin-1, a CGL-2 mutant defective in beta-galactoside binding, does not get exported from CHO cells. We conclude that the beta-galactoside binding site represents the primary targeting motif of galectins defining a galectin export machinery that makes use of beta-galactoside-containing surface molecules as export receptors for intracellular galectin-1.

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Quantitative analysis of export of various galectin–GFP fusion proteins from CHO cells using flow cytometry. CHO cells were grown on six-well plates and induced with doxycycline for 48 h at 37°C to express the fusion proteins indicated. After removal of the medium, cells were labeled with affinity-purified anti-GFP antibodies while they were still attached to the culture dishes. Primary antibodies were labeled with APC-conjugated secondary antibodies followed by detachment of the cells using PBS/EDTA. GFP (expression level; green) and APC-derived fluorescence (cell surface; blue) were quantified by flow cytometry using a FACSCalibur system (Becton Dickinson; n = 4). For further details see Materials and methods.
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fig3: Quantitative analysis of export of various galectin–GFP fusion proteins from CHO cells using flow cytometry. CHO cells were grown on six-well plates and induced with doxycycline for 48 h at 37°C to express the fusion proteins indicated. After removal of the medium, cells were labeled with affinity-purified anti-GFP antibodies while they were still attached to the culture dishes. Primary antibodies were labeled with APC-conjugated secondary antibodies followed by detachment of the cells using PBS/EDTA. GFP (expression level; green) and APC-derived fluorescence (cell surface; blue) were quantified by flow cytometry using a FACSCalibur system (Becton Dickinson; n = 4). For further details see Materials and methods.

Mentions: In previous studies, we established both a cell surface biotinylation assay and a FACS-based assay system designed to quantitatively assess secretion of Gal-1 and other unconventional secretory proteins from CHO cells (Seelenmeyer et al., 2003; Stegmayer et al., 2005). To quantitatively study export of Gal-1 mutants deficient in binding to β-galactosides, we combined these assays with immunoprecipitation of Gal-1–GFP fusion proteins from the medium of the expressing cells using affinity-purified anti-GFP antibodies. As shown in Fig. 2 A, most of the extracellular Gal-1–GFP population was found to be associated with the cell surface of CHO cells (lane 2) with only a minor portion being found in the medium (lane 3). As controls, both GFP without Gal-1 tag (Fig. 2 F) and endogenous Gal-1 (Fig. 2 G) were compared with Gal-1–GFP. As expected, GFP could not be detected on the cell surface (Fig. 2 F, lane 2) and only small amounts were detectable in the medium (Fig. 2 F, lane 3). By contrast, endogenous Gal-1 was found both on the cell surface and in the medium of CHO cells (Fig. 2 G). The overall efficiency of Gal-1 versus Gal-1–GFP secretion was similar with ∼10% of each reporter molecule being localized to the extracellular space under steady-state conditions. These data indicate that tagging of Gal-1 with GFP does not interfere with its secretion. Consistent with the biochemical data, the wild-type form of Gal-1–GFP was detected on the cell surface using the FACS assay as shown in Fig. 3. As expected, mutant forms of Gal-1–GFP such as W69G and E72A, which are defective with regard to binding to β-galactosides could not be detected on the cell surface using both the biotinylation (Fig. 2, B and C, lane 2) and the FACS assay (Fig. 3). Strikingly, however, Gal-1–GFPW69G and Gal-1–GFPE72A were also absent from the medium suggesting that they are not secreted from CHO cells (Fig. 2, B and C, lane 3). We then tested whether these Gal-1 mutants are stable in conditioned medium derived from CHO cells in order to analyze whether protein degradation causes their absence from the supernatants of Gal-1–GFPW69G and Gal-1–GFPE72A-expressing cells. As demonstrated in Fig. 4, both the wild-type form of Gal-1–GFP (A) and the β-galactoside-binding mutants (B and C, respectively) are not degraded when incubated in conditioned CHO medium at 37°C for 48 h (compare lanes 2 and 4). Thus, the absence of the reporters from both the cell surface and the medium of Gal-1–GFPW69G– and Gal-1–GFPE72A–expressing CHO cells demonstrates that they are no substrates for the Gal-1 export machinery. Intriguingly, the combined phenotypes for Gal-1–GFPW69G and Gal-1–GFPE72A shown in Figs. 2–4 were also found for all other β-galactoside–binding mutants of Gal-1 listed in Table I (unpublished data). These findings imply that binding to β-galactosides of Gal-1 is a prerequisite to enter the export pathway.


Cell surface counter receptors are essential components of the unconventional export machinery of galectin-1.

Seelenmeyer C, Wegehingel S, Tews I, Künzler M, Aebi M, Nickel W - J. Cell Biol. (2005)

Quantitative analysis of export of various galectin–GFP fusion proteins from CHO cells using flow cytometry. CHO cells were grown on six-well plates and induced with doxycycline for 48 h at 37°C to express the fusion proteins indicated. After removal of the medium, cells were labeled with affinity-purified anti-GFP antibodies while they were still attached to the culture dishes. Primary antibodies were labeled with APC-conjugated secondary antibodies followed by detachment of the cells using PBS/EDTA. GFP (expression level; green) and APC-derived fluorescence (cell surface; blue) were quantified by flow cytometry using a FACSCalibur system (Becton Dickinson; n = 4). For further details see Materials and methods.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171196&req=5

fig3: Quantitative analysis of export of various galectin–GFP fusion proteins from CHO cells using flow cytometry. CHO cells were grown on six-well plates and induced with doxycycline for 48 h at 37°C to express the fusion proteins indicated. After removal of the medium, cells were labeled with affinity-purified anti-GFP antibodies while they were still attached to the culture dishes. Primary antibodies were labeled with APC-conjugated secondary antibodies followed by detachment of the cells using PBS/EDTA. GFP (expression level; green) and APC-derived fluorescence (cell surface; blue) were quantified by flow cytometry using a FACSCalibur system (Becton Dickinson; n = 4). For further details see Materials and methods.
Mentions: In previous studies, we established both a cell surface biotinylation assay and a FACS-based assay system designed to quantitatively assess secretion of Gal-1 and other unconventional secretory proteins from CHO cells (Seelenmeyer et al., 2003; Stegmayer et al., 2005). To quantitatively study export of Gal-1 mutants deficient in binding to β-galactosides, we combined these assays with immunoprecipitation of Gal-1–GFP fusion proteins from the medium of the expressing cells using affinity-purified anti-GFP antibodies. As shown in Fig. 2 A, most of the extracellular Gal-1–GFP population was found to be associated with the cell surface of CHO cells (lane 2) with only a minor portion being found in the medium (lane 3). As controls, both GFP without Gal-1 tag (Fig. 2 F) and endogenous Gal-1 (Fig. 2 G) were compared with Gal-1–GFP. As expected, GFP could not be detected on the cell surface (Fig. 2 F, lane 2) and only small amounts were detectable in the medium (Fig. 2 F, lane 3). By contrast, endogenous Gal-1 was found both on the cell surface and in the medium of CHO cells (Fig. 2 G). The overall efficiency of Gal-1 versus Gal-1–GFP secretion was similar with ∼10% of each reporter molecule being localized to the extracellular space under steady-state conditions. These data indicate that tagging of Gal-1 with GFP does not interfere with its secretion. Consistent with the biochemical data, the wild-type form of Gal-1–GFP was detected on the cell surface using the FACS assay as shown in Fig. 3. As expected, mutant forms of Gal-1–GFP such as W69G and E72A, which are defective with regard to binding to β-galactosides could not be detected on the cell surface using both the biotinylation (Fig. 2, B and C, lane 2) and the FACS assay (Fig. 3). Strikingly, however, Gal-1–GFPW69G and Gal-1–GFPE72A were also absent from the medium suggesting that they are not secreted from CHO cells (Fig. 2, B and C, lane 3). We then tested whether these Gal-1 mutants are stable in conditioned medium derived from CHO cells in order to analyze whether protein degradation causes their absence from the supernatants of Gal-1–GFPW69G and Gal-1–GFPE72A-expressing cells. As demonstrated in Fig. 4, both the wild-type form of Gal-1–GFP (A) and the β-galactoside-binding mutants (B and C, respectively) are not degraded when incubated in conditioned CHO medium at 37°C for 48 h (compare lanes 2 and 4). Thus, the absence of the reporters from both the cell surface and the medium of Gal-1–GFPW69G– and Gal-1–GFPE72A–expressing CHO cells demonstrates that they are no substrates for the Gal-1 export machinery. Intriguingly, the combined phenotypes for Gal-1–GFPW69G and Gal-1–GFPE72A shown in Figs. 2–4 were also found for all other β-galactoside–binding mutants of Gal-1 listed in Table I (unpublished data). These findings imply that binding to β-galactosides of Gal-1 is a prerequisite to enter the export pathway.

Bottom Line: Intriguingly, we also find that a distant relative of galectin-1, the fungal lectin CGL-2, is a substrate for nonclassical export from Chinese hamster ovary (CHO) cells.Alike mammalian galectin-1, a CGL-2 mutant defective in beta-galactoside binding, does not get exported from CHO cells.We conclude that the beta-galactoside binding site represents the primary targeting motif of galectins defining a galectin export machinery that makes use of beta-galactoside-containing surface molecules as export receptors for intracellular galectin-1.

View Article: PubMed Central - PubMed

Affiliation: Heidelberg University Biochemistry Center (BZH), 69120 Heidelberg, Germany.

ABSTRACT
Galectin-1 is a component of the extracellular matrix as well as a ligand of cell surface counter receptors such as beta-galactoside-containing glycolipids, however, the molecular mechanism of galectin-1 secretion has remained elusive. Based on a nonbiased screen for galectin-1 export mutants we have identified 26 single amino acid changes that cause a defect of both export and binding to counter receptors. When wild-type galectin-1 was analyzed in CHO clone 13 cells, a mutant cell line incapable of expressing functional galectin-1 counter receptors, secretion was blocked. Intriguingly, we also find that a distant relative of galectin-1, the fungal lectin CGL-2, is a substrate for nonclassical export from Chinese hamster ovary (CHO) cells. Alike mammalian galectin-1, a CGL-2 mutant defective in beta-galactoside binding, does not get exported from CHO cells. We conclude that the beta-galactoside binding site represents the primary targeting motif of galectins defining a galectin export machinery that makes use of beta-galactoside-containing surface molecules as export receptors for intracellular galectin-1.

Show MeSH
Related in: MedlinePlus