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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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Analysis of β-galactoside binding efficiency of various galectin–GFP fusion proteins based on binding to CHO cells and binding to lactose beads. In A, the various fusion proteins indicated were expressed in CHO cells. Cell-free supernatants were prepared and normalized by GFP fluorescence. The various supernatants were then incubated with CHO cells for 1 h at 4°C to allow cell surface binding. After treatment with affinity-purified anti-GFP antibodies and APC-conjugated secondary antibodies, cell surface binding was quantified by flow cytometry. In B, detergent lysates normalized by GFP fluorescence were incubated with lactose beads for 1 h at 4°C. The nonbound fraction was separated and, after extensive washing, bound material was eluted with SDS sample buffer. Input (lane 1; 5%), nonbound material (lane 2; 5%), and bound material (lane 3; 5%) 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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fig1: Analysis of β-galactoside binding efficiency of various galectin–GFP fusion proteins based on binding to CHO cells and binding to lactose beads. In A, the various fusion proteins indicated were expressed in CHO cells. Cell-free supernatants were prepared and normalized by GFP fluorescence. The various supernatants were then incubated with CHO cells for 1 h at 4°C to allow cell surface binding. After treatment with affinity-purified anti-GFP antibodies and APC-conjugated secondary antibodies, cell surface binding was quantified by flow cytometry. In B, detergent lysates normalized by GFP fluorescence were incubated with lactose beads for 1 h at 4°C. The nonbound fraction was separated and, after extensive washing, bound material was eluted with SDS sample buffer. Input (lane 1; 5%), nonbound material (lane 2; 5%), and bound material (lane 3; 5%) were analyzed by SDS-PAGE and Western blotting using affinity-purified anti-GFP antibodies. For further details see Materials and methods.

Mentions: To elucidate the export targeting motif we generated more than 100 single amino acid mutants of human Gal-1. The individual mutants were selected in three different ways: (1) a random mutagenesis using a low fidelity PCR; (2) targeted mutagenesis of surface residues based on the crystal structure of galectin-1 (Lobsanov et al., 1993); and (3) targeted mutagenesis of residues conserved between human Gal-1 and CGL-2 from C. cinerea. All mutants were stably expressed as GFP fusion proteins in CHO cells using a doxycycline-dependent transactivator system (Engling et al., 2002). All mutant galectin proteins were analyzed for their capability to interact with counter receptors based on binding to both CHO cells (Fig. 1 A) and lactose coupled to beads (Fig. 1 B). Using the wild-type forms of Gal-1 and CGL-2 as positive controls and GFP alone as a negative control, we identified a total of 26 Gal-1 mutants as being deficient for binding to β-galactoside–containing counter receptors (Table I), some of which have been reported previously to be impaired in terms of binding to β-galactosides (Hirabayashi and Kasai, 1991; Scott and Zhang, 2002; Ford et al., 2003; Lopez-Lucendo et al., 2004). All mutants were clearly defective in binding to β-galactosides as they did not significantly differ from the GFP negative control in both assays (Fig. 1, A and B, lane 3).


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)

Analysis of β-galactoside binding efficiency of various galectin–GFP fusion proteins based on binding to CHO cells and binding to lactose beads. In A, the various fusion proteins indicated were expressed in CHO cells. Cell-free supernatants were prepared and normalized by GFP fluorescence. The various supernatants were then incubated with CHO cells for 1 h at 4°C to allow cell surface binding. After treatment with affinity-purified anti-GFP antibodies and APC-conjugated secondary antibodies, cell surface binding was quantified by flow cytometry. In B, detergent lysates normalized by GFP fluorescence were incubated with lactose beads for 1 h at 4°C. The nonbound fraction was separated and, after extensive washing, bound material was eluted with SDS sample buffer. Input (lane 1; 5%), nonbound material (lane 2; 5%), and bound material (lane 3; 5%) 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

Show All Figures
getmorefigures.php?uid=PMC2171196&req=5

fig1: Analysis of β-galactoside binding efficiency of various galectin–GFP fusion proteins based on binding to CHO cells and binding to lactose beads. In A, the various fusion proteins indicated were expressed in CHO cells. Cell-free supernatants were prepared and normalized by GFP fluorescence. The various supernatants were then incubated with CHO cells for 1 h at 4°C to allow cell surface binding. After treatment with affinity-purified anti-GFP antibodies and APC-conjugated secondary antibodies, cell surface binding was quantified by flow cytometry. In B, detergent lysates normalized by GFP fluorescence were incubated with lactose beads for 1 h at 4°C. The nonbound fraction was separated and, after extensive washing, bound material was eluted with SDS sample buffer. Input (lane 1; 5%), nonbound material (lane 2; 5%), and bound material (lane 3; 5%) were analyzed by SDS-PAGE and Western blotting using affinity-purified anti-GFP antibodies. For further details see Materials and methods.
Mentions: To elucidate the export targeting motif we generated more than 100 single amino acid mutants of human Gal-1. The individual mutants were selected in three different ways: (1) a random mutagenesis using a low fidelity PCR; (2) targeted mutagenesis of surface residues based on the crystal structure of galectin-1 (Lobsanov et al., 1993); and (3) targeted mutagenesis of residues conserved between human Gal-1 and CGL-2 from C. cinerea. All mutants were stably expressed as GFP fusion proteins in CHO cells using a doxycycline-dependent transactivator system (Engling et al., 2002). All mutant galectin proteins were analyzed for their capability to interact with counter receptors based on binding to both CHO cells (Fig. 1 A) and lactose coupled to beads (Fig. 1 B). Using the wild-type forms of Gal-1 and CGL-2 as positive controls and GFP alone as a negative control, we identified a total of 26 Gal-1 mutants as being deficient for binding to β-galactoside–containing counter receptors (Table I), some of which have been reported previously to be impaired in terms of binding to β-galactosides (Hirabayashi and Kasai, 1991; Scott and Zhang, 2002; Ford et al., 2003; Lopez-Lucendo et al., 2004). All mutants were clearly defective in binding to β-galactosides as they did not significantly differ from the GFP negative control in both assays (Fig. 1, A and B, lane 3).

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