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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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Comparative analysis 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 (A–F) and CHO clone 13 cells (G–L) 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; 0.25%), the biotinylated fraction (lane 2; 25%) and the immunoprecipitate from the cell culture medium fraction (lane 3; 25%) were analyzed by SDS-PAGE and Western blotting using affinity-purified anti-GFP antibodies. For further details see Materials and methods.
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fig5: Comparative analysis 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 (A–F) and CHO clone 13 cells (G–L) 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; 0.25%), the biotinylated fraction (lane 2; 25%) and the immunoprecipitate from the cell culture medium fraction (lane 3; 25%) were analyzed by SDS-PAGE and Western blotting using affinity-purified anti-GFP antibodies. For further details see Materials and methods.

Mentions: As demonstrated in the cell surface biotinylation experiments shown in Fig. 5, both Gal-1–GFP (A, lanes 2 and 3) and CGL-2–GFP (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-2–GFP 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 β-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).


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)

Comparative analysis 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 (A–F) and CHO clone 13 cells (G–L) 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; 0.25%), the biotinylated fraction (lane 2; 25%) and the immunoprecipitate from the cell culture medium fraction (lane 3; 25%) were analyzed by SDS-PAGE and Western blotting using affinity-purified anti-GFP antibodies. 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

fig5: Comparative analysis 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 (A–F) and CHO clone 13 cells (G–L) 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; 0.25%), the biotinylated fraction (lane 2; 25%) and the immunoprecipitate from the cell culture medium fraction (lane 3; 25%) were analyzed by SDS-PAGE and Western blotting using affinity-purified anti-GFP antibodies. For further details see Materials and methods.
Mentions: As demonstrated in the cell surface biotinylation experiments shown in Fig. 5, both Gal-1–GFP (A, lanes 2 and 3) and CGL-2–GFP (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-2–GFP 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 β-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).

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