Cell surface counter receptors are essential components of the unconventional export machinery of galectin-1

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.

Figure 1.

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.

Figure 2.

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.

Figure 3.

Quantitative analysis of export of various galectin–GFP fusion proteins from CHO cells using flow cytometry. CHO cells were grown on six-well plates and induced with doxycycline for 48 h at 37°C to express the fusion proteins indicated. After removal of the medium, cells were labeled with affinity-purified anti-GFP antibodies while they were still attached to the culture dishes. Primary antibodies were labeled with APC-conjugated secondary antibodies followed by detachment of the cells using PBS/EDTA. GFP (expression level; green) and APC-derived fluorescence (cell surface; blue) were quantified by flow cytometry using a FACSCalibur system (Becton Dickinson; n = 4). For further details see Materials and methods.

Figure 4.

Stability of galectin–GFP fusion proteins in conditioned media derived from CHO cells. The fusion proteins indicated were expressed in CHO cells for 48 h at 37°C (six-well plates; 70% confluency). From each cell line, a cell-free supernatant was prepared. Normalized amounts (GFP fluorescence) were incubated in conditioned medium derived from CHO cells for the times indicated followed by immunoprecipitation (lanes 2–4) using affinity-purified anti-GFP antibodies. The samples were analyzed by SDS-PAGE and Western blotting using antibodies directed against GFP. Lane 1, input (no IP, 10%); lane 2, no incubation (IP, 10%); lane 3, incubation for 48 h at 4°C (IP, 10%); lane 4, incubation for 48 h at 37°C (IP, 10%). For further details see Materials and methods.

Figure 5.

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.

Figure 6.

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.

Figure 7.

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–GFP_W69G; third row, Gal-1–GFP_E72A; forth row, CGL-2–GFP; fifth row, CGL-2–GFP_W72G; 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.