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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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Subcellular distribution of Gal-1–GFP and CGL-2–GFP reporter molecules in CHO wild-type and CHO clone 13 cells as revealed by confocal microscopy. First row, Gal-1–GFP; second row, Gal-1–GFPW69G; third row, Gal-1–GFPE72A; forth row, CGL-2–GFP; fifth row, CGL-2–GFPW72G; sixth row, GFP. First column, GFP live imaging; second column, GFP imaging of fixed CHO wild-type cells; third column, cell surface staining of fixed CHO wild-type cells using affinity-purified anti-GFP antibodies; fourth column, GFP imaging of fixed CHO clone 13 cells; fifth column, cell surface staining of fixed CHO clone 13 cells using affinity-purified anti-GFP antibodies.
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fig7: Subcellular distribution of Gal-1–GFP and CGL-2–GFP reporter molecules in CHO wild-type and CHO clone 13 cells as revealed by confocal microscopy. First row, Gal-1–GFP; second row, Gal-1–GFPW69G; third row, Gal-1–GFPE72A; forth row, CGL-2–GFP; fifth row, CGL-2–GFPW72G; sixth row, GFP. First column, GFP live imaging; second column, GFP imaging of fixed CHO wild-type cells; third column, cell surface staining of fixed CHO wild-type cells using affinity-purified anti-GFP antibodies; fourth column, GFP imaging of fixed CHO clone 13 cells; fifth column, cell surface staining of fixed CHO clone 13 cells using affinity-purified anti-GFP antibodies.

Mentions: To compare the subcellular distribution of Gal-1 and CGL-2 mutant forms with the corresponding wild-type proteins, we analyzed the cell lines described above by confocal microscopy (Fig. 7). When living CHO wild-type cells were imaged, all reporters were found in the cytoplasm as well as to some extent in the nucleus (Fig. 7, first column). When CHO wild-type and CHO clone 13 (unpublished data) were compared, no differences in live cell imaging could be observed for any of the reporter molecules being analyzed. In general, a similar picture was observed after fixation both for CHO wild-type and CHO clone 13 cells (Fig. 7, second and fourth column, respectively), however, in some cases aggregates or particulate structures were observed that apparently represent fixation artifacts. Consistent with the FACS experiments shown in Fig. 3, cell surface staining of all cell lines using affinity-purified anti-GFP antibodies revealed an extracellular population in CHO wild-type cells only for the wild-type forms of Gal-1–GFP and CGL2–GFP (Fig. 7, third column). In CHO clone 13 cells, cell surface staining could not be detected for any of the reporter molecules including the wild-type forms of Gal-1 and CGL-2 (Fig. 7, fifth column).


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)

Subcellular distribution of Gal-1–GFP and CGL-2–GFP reporter molecules in CHO wild-type and CHO clone 13 cells as revealed by confocal microscopy. First row, Gal-1–GFP; second row, Gal-1–GFPW69G; third row, Gal-1–GFPE72A; forth row, CGL-2–GFP; fifth row, CGL-2–GFPW72G; sixth row, GFP. First column, GFP live imaging; second column, GFP imaging of fixed CHO wild-type cells; third column, cell surface staining of fixed CHO wild-type cells using affinity-purified anti-GFP antibodies; fourth column, GFP imaging of fixed CHO clone 13 cells; fifth column, cell surface staining of fixed CHO clone 13 cells using affinity-purified anti-GFP antibodies.
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Related In: Results  -  Collection

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fig7: Subcellular distribution of Gal-1–GFP and CGL-2–GFP reporter molecules in CHO wild-type and CHO clone 13 cells as revealed by confocal microscopy. First row, Gal-1–GFP; second row, Gal-1–GFPW69G; third row, Gal-1–GFPE72A; forth row, CGL-2–GFP; fifth row, CGL-2–GFPW72G; sixth row, GFP. First column, GFP live imaging; second column, GFP imaging of fixed CHO wild-type cells; third column, cell surface staining of fixed CHO wild-type cells using affinity-purified anti-GFP antibodies; fourth column, GFP imaging of fixed CHO clone 13 cells; fifth column, cell surface staining of fixed CHO clone 13 cells using affinity-purified anti-GFP antibodies.
Mentions: To compare the subcellular distribution of Gal-1 and CGL-2 mutant forms with the corresponding wild-type proteins, we analyzed the cell lines described above by confocal microscopy (Fig. 7). When living CHO wild-type cells were imaged, all reporters were found in the cytoplasm as well as to some extent in the nucleus (Fig. 7, first column). When CHO wild-type and CHO clone 13 (unpublished data) were compared, no differences in live cell imaging could be observed for any of the reporter molecules being analyzed. In general, a similar picture was observed after fixation both for CHO wild-type and CHO clone 13 cells (Fig. 7, second and fourth column, respectively), however, in some cases aggregates or particulate structures were observed that apparently represent fixation artifacts. Consistent with the FACS experiments shown in Fig. 3, cell surface staining of all cell lines using affinity-purified anti-GFP antibodies revealed an extracellular population in CHO wild-type cells only for the wild-type forms of Gal-1–GFP and CGL2–GFP (Fig. 7, third column). In CHO clone 13 cells, cell surface staining could not be detected for any of the reporter molecules including the wild-type forms of Gal-1 and CGL-2 (Fig. 7, fifth column).

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