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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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Biochemical analysis of export of various galectin–GFP fusion proteins from CHO cells using cell surface biotinylation and immunoprecipitation from cell culture supernatants. The fusion proteins indicated were expressed in CHO cells for 48 h at 37°C (six-well plates; 70% confluency). The medium was removed and subjected to immunoprecipitation using affinity-purified anti-GFP antibodies. Cell surfaces were treated with a membrane-impermeable biotinylation reagent. After detergent-mediated cell lysis biotinylated and nonbiotinylated proteins were separated using streptavidin beads. Aliquots from the input material (lane 1; 1%), the biotinylated fraction (lane 2; 10%) and the immunoprecipitate from the cell culture medium fraction (lane 3; 50%) were analyzed by SDS-PAGE and Western blotting using affinity-purified anti-GFP antibodies. In G, affinity-purified anti–Gal-1 antibodies were used to detect endogenous Gal-1. For further details see Materials and methods.
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fig2: Biochemical analysis of export of various galectin–GFP fusion proteins from CHO cells using cell surface biotinylation and immunoprecipitation from cell culture supernatants. The fusion proteins indicated were expressed in CHO cells for 48 h at 37°C (six-well plates; 70% confluency). The medium was removed and subjected to immunoprecipitation using affinity-purified anti-GFP antibodies. Cell surfaces were treated with a membrane-impermeable biotinylation reagent. After detergent-mediated cell lysis biotinylated and nonbiotinylated proteins were separated using streptavidin beads. Aliquots from the input material (lane 1; 1%), the biotinylated fraction (lane 2; 10%) and the immunoprecipitate from the cell culture medium fraction (lane 3; 50%) were analyzed by SDS-PAGE and Western blotting using affinity-purified anti-GFP antibodies. In G, affinity-purified anti–Gal-1 antibodies were used to detect endogenous Gal-1. 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)

Biochemical analysis of export of various galectin–GFP fusion proteins from CHO cells using cell surface biotinylation and immunoprecipitation from cell culture supernatants. The fusion proteins indicated were expressed in CHO cells for 48 h at 37°C (six-well plates; 70% confluency). The medium was removed and subjected to immunoprecipitation using affinity-purified anti-GFP antibodies. Cell surfaces were treated with a membrane-impermeable biotinylation reagent. After detergent-mediated cell lysis biotinylated and nonbiotinylated proteins were separated using streptavidin beads. Aliquots from the input material (lane 1; 1%), the biotinylated fraction (lane 2; 10%) and the immunoprecipitate from the cell culture medium fraction (lane 3; 50%) were analyzed by SDS-PAGE and Western blotting using affinity-purified anti-GFP antibodies. In G, affinity-purified anti–Gal-1 antibodies were used to detect endogenous Gal-1. For further details see Materials and methods.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171196&req=5

fig2: Biochemical analysis of export of various galectin–GFP fusion proteins from CHO cells using cell surface biotinylation and immunoprecipitation from cell culture supernatants. The fusion proteins indicated were expressed in CHO cells for 48 h at 37°C (six-well plates; 70% confluency). The medium was removed and subjected to immunoprecipitation using affinity-purified anti-GFP antibodies. Cell surfaces were treated with a membrane-impermeable biotinylation reagent. After detergent-mediated cell lysis biotinylated and nonbiotinylated proteins were separated using streptavidin beads. Aliquots from the input material (lane 1; 1%), the biotinylated fraction (lane 2; 10%) and the immunoprecipitate from the cell culture medium fraction (lane 3; 50%) were analyzed by SDS-PAGE and Western blotting using affinity-purified anti-GFP antibodies. In G, affinity-purified anti–Gal-1 antibodies were used to detect endogenous Gal-1. 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