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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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Quantitation of export of galectin–GFP fusion proteins from CHO wild-type and CHO clone 13 cells using cell surface biotinylation and immunoprecipitation from cell culture supernatants. The fusion proteins indicated were expressed in both CHO wild-type and CHO clone 13 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 employing streptavidin beads. Aliquots from the input material (0.25%), the biotinylated fraction (25%) and the immunoprecipitate from the cell culture medium fraction (25%) were analyzed by SDS-PAGE and Western blotting using affinity-purified anti-GFP antibodies. Primary antibodies were detected with Alexa 680–coupled anti–rabbit secondary antibodies. Signals for galectin–GFP fusion proteins and GFP were quantified using a Odyssey imaging system (LI-COR Biotechnology). The combined signals for the cell medium and the material associated with the cell surface were calculated as a percentage of the total amount of galectin–GFP fusion protein expressed in each case. These data were corrected for unspecific release as monitored by GFP present in the medium of the cells. The extracellular population of Gal-1–GFP secreted from CHO wild-type cells was set to 100%. For further details see Materials and methods.
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fig6: Quantitation of export of galectin–GFP fusion proteins from CHO wild-type and CHO clone 13 cells using cell surface biotinylation and immunoprecipitation from cell culture supernatants. The fusion proteins indicated were expressed in both CHO wild-type and CHO clone 13 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 employing streptavidin beads. Aliquots from the input material (0.25%), the biotinylated fraction (25%) and the immunoprecipitate from the cell culture medium fraction (25%) were analyzed by SDS-PAGE and Western blotting using affinity-purified anti-GFP antibodies. Primary antibodies were detected with Alexa 680–coupled anti–rabbit secondary antibodies. Signals for galectin–GFP fusion proteins and GFP were quantified using a Odyssey imaging system (LI-COR Biotechnology). The combined signals for the cell medium and the material associated with the cell surface were calculated as a percentage of the total amount of galectin–GFP fusion protein expressed in each case. These data were corrected for unspecific release as monitored by GFP present in the medium of the cells. The extracellular population of Gal-1–GFP secreted from CHO wild-type cells was set to 100%. For further details see Materials and methods.

Mentions: In Fig. 6, export from both CHO wild-type and CHO clone 13 cells of the various galectin–GFP fusion proteins (as measured by cell surface biotinylation and immunoprecipitation from the cell culture supernatants) was quantified using fluorescent secondary antibodies combined with a LI-COR Odyssey imaging system. For each fusion protein, the combined signals derived from cell surface biotinylation and material from the cell culture supernatant were expressed as a percentage of the overall expression level of a given fusion protein. To compare export of the various fusion proteins, secretion of Gal-1–GFP from CHO wild-type cells was set to 100%. As demonstrated in Fig. 6, the wild-type forms of both Gal-1–GFP and CGL-2–GFP are secreted from CHO wild-type cells, however, export is largely reduced when Gal-1–GFP and CGL-2–GFP are expressed in CHO clone 13 cells. The various mutant forms of Gal-1 and CGL-2 are neither exported from CHO wild-type nor from CHO clone 13 cells to a significant extent.


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)

Quantitation of export of galectin–GFP fusion proteins from CHO wild-type and CHO clone 13 cells using cell surface biotinylation and immunoprecipitation from cell culture supernatants. The fusion proteins indicated were expressed in both CHO wild-type and CHO clone 13 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 employing streptavidin beads. Aliquots from the input material (0.25%), the biotinylated fraction (25%) and the immunoprecipitate from the cell culture medium fraction (25%) were analyzed by SDS-PAGE and Western blotting using affinity-purified anti-GFP antibodies. Primary antibodies were detected with Alexa 680–coupled anti–rabbit secondary antibodies. Signals for galectin–GFP fusion proteins and GFP were quantified using a Odyssey imaging system (LI-COR Biotechnology). The combined signals for the cell medium and the material associated with the cell surface were calculated as a percentage of the total amount of galectin–GFP fusion protein expressed in each case. These data were corrected for unspecific release as monitored by GFP present in the medium of the cells. The extracellular population of Gal-1–GFP secreted from CHO wild-type cells was set to 100%. For further details see Materials and methods.
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Related In: Results  -  Collection

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fig6: Quantitation of export of galectin–GFP fusion proteins from CHO wild-type and CHO clone 13 cells using cell surface biotinylation and immunoprecipitation from cell culture supernatants. The fusion proteins indicated were expressed in both CHO wild-type and CHO clone 13 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 employing streptavidin beads. Aliquots from the input material (0.25%), the biotinylated fraction (25%) and the immunoprecipitate from the cell culture medium fraction (25%) were analyzed by SDS-PAGE and Western blotting using affinity-purified anti-GFP antibodies. Primary antibodies were detected with Alexa 680–coupled anti–rabbit secondary antibodies. Signals for galectin–GFP fusion proteins and GFP were quantified using a Odyssey imaging system (LI-COR Biotechnology). The combined signals for the cell medium and the material associated with the cell surface were calculated as a percentage of the total amount of galectin–GFP fusion protein expressed in each case. These data were corrected for unspecific release as monitored by GFP present in the medium of the cells. The extracellular population of Gal-1–GFP secreted from CHO wild-type cells was set to 100%. For further details see Materials and methods.
Mentions: In Fig. 6, export from both CHO wild-type and CHO clone 13 cells of the various galectin–GFP fusion proteins (as measured by cell surface biotinylation and immunoprecipitation from the cell culture supernatants) was quantified using fluorescent secondary antibodies combined with a LI-COR Odyssey imaging system. For each fusion protein, the combined signals derived from cell surface biotinylation and material from the cell culture supernatant were expressed as a percentage of the overall expression level of a given fusion protein. To compare export of the various fusion proteins, secretion of Gal-1–GFP from CHO wild-type cells was set to 100%. As demonstrated in Fig. 6, the wild-type forms of both Gal-1–GFP and CGL-2–GFP are secreted from CHO wild-type cells, however, export is largely reduced when Gal-1–GFP and CGL-2–GFP are expressed in CHO clone 13 cells. The various mutant forms of Gal-1 and CGL-2 are neither exported from CHO wild-type nor from CHO clone 13 cells to a significant extent.

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