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First published online 12 February 2003
doi: 10.1242/jcs.00312

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The cancer antigen CA125 represents a novel counter receptor for galectin-1

Claudia Seelenmeyer^*, Sabine Wegehingel^*, Johannes Lechner and Walter Nickel

Biochemie-Zentrum Heidelberg (BZH), Im Neuenheimer Feld 328, 69120 Heidelberg, Germany

View larger version (88K): [in a new window]   Fig. 1. Identification of CA125 as a counter receptor of galectin-1. (A) Affinity purification of galectin-1-interacting proteins. Both soluble (lanes 1, 2, 5, 6) and membrane (lanes 3, 4, 7, 8) fractions of HeLa cells were incubated with either GST—galectin-1 beads (lanes 2, 4, 6, 8) or GST beads as a control (lanes 1, 3, 5, 7). Bound proteins were eluted sequentially with lactose (lanes 1-4) and glutathione (lanes 5-8), followed by separation on Novex NuPage 10% Bis-Tris gels. Protein bands were visualized using SilverQuest. (B) Immunoblot analysis of the proteins eluted from the GST—galectin-1 and GST matrices, respectively. The fractions were loaded in the same order as shown in panel A. The anti-CA125 antibody OC125 was used as primary antibody followed by detection by electrochemiluminescence. (C) Immunoblot analysis as shown in panel B employing an anti-CA125-C-TERM1-356 antiserum for the detection of CA125-derived fragments. (D) Amino acid sequence of CA125-C-TERM. Boxed sequences indicate tryptic peptides derived from band 1 (panel A, lane 2) as identified by mass spectrometry.

View larger version (17K): [in a new window]   Fig. 2. CA125 displays differential binding efficiency towards galectin-1 when compared with galectin-3. (A) Soluble (lanes 1-4) and membrane fractions (lanes 5-8) were prepared from HeLa cells followed by the incubation with either GST—galectin-1 (lanes 1, 3, 5, 7) or GST—galectin-3 (lanes 2, 4, 6, 8) beads. The amounts of GST—galectin-1 and GST—galectin-3 fusion proteins, respectively, used for affinity purification of CA125-derived fragments were shown to be comparable by western blotting employing affinity-purified anti-GST antibodies (lanes 9 and 10). Following extensive washing, bound proteins were eluted sequentially with lactose (lanes 1, 2, 5, 6) and glutathione (lanes 3, 4, 7, 8). Eluted proteins were separated on 10% Novex NuPage Bis-Tris gels and transferred to a blotting membrane. Electrochemiluminescence detection of CA125-derived fragments was performed employing the mAb OC125. (B) Quantitative analysis of CA125-derived fragments in the fractions shown in panel A employing Bio-Rad® QuantityOne® Software. (C) Total protein pattern of lactose-eluted proteins derived from the galectin-1 matrix (lane 1) and the galectin-3 matrix (lane 2). Eluted proteins were separated on NuPage Bis-Tris gels followed by silver staining according to standard procedures. Labels indicate examples for proteins that preferentially bind to galectin-1 (•), galectin-3 () or proteins that equally bind galectin-1 and galectin-3 ().

View larger version (26K): [in a new window]   Fig. 3. A 1148 amino acid, C-terminal fragment of CA125, CA125-C-TERM, retains the ability of CA125 to bind specifically to galectin-1. CHO and HeLa cells were induced to express CA125-C-TERM by retroviral transduction. For comparison, CHO and HeLa cells were included that were treated with retroviral control particles. A detergent lysate of the cells was prepared and incubated with either GST—galectin-1-, GST—galectin-3 or GST beads. Following extensive washing, the beads were treated with lactose. Eluted proteins were separated on 10% Novex NuPage Bis-Tris gels followed by transfer to a blotting membrane. CA125-C-TERM was then detected by OC125 staining using electrochemiluminescence (A). For comparison, the pattern of CA125-derived fragments isolated from HeLa cells is shown in the leftmost lane (control). (B) The intensity of CA125-C-TERM-derived bands was quantified using Bio-Rad® QuantityOne® software. (C) A crosslinking experiment is shown employing disuccinimidyl glutarate (DSG). CA125-C-TERM-expressing CHO cells were lysed with detergent followed by incubation of the cell-free supernatant with GST—galectin-1 beads. After extensive washing, DSG was added at a final concentration of 0.5 mM. Crosslinking products were eluted with SDS sample buffer and analysed by SDS-PAGE and western blotting employing affinity-purified anti-galectin-1 and monoclonal anti-CA125 antibodies. The square bracket indicates crosslinking products with an apparent molecular mass of about 160-180 kDa positive for galectin-1 and CA125. In the range of 120-130 kDa, other galectin-1-containing crosslinking products are observed.

View larger version (37K): [in a new window]   Fig. 4. Galectin-1 binding to CA125-C-TERM largely depends on O-linked ß-galactose-terminated oligosaccharide chains. CHOMCAT-TAM2 (wild-type background with regard to galactosylation of both proteins and lipids) and CHOclone 13 cells (deficient with regard to galactosylation of both proteins and lipids; (Deutscher and Hirschberg, 1986) stably expressing CA125-C-TERM were used to prepare cell-free detergent lysates followed by incubation with GST and GST—galectin-1 beads, respectively. Where indicated, CHOMCAT-TAM2 were treated with 10 µg/ml tunicamycin for 18 hours at 37°C prior to cell lysis. In each experiment, the CA125-C-TERM signal derived from 0.2% of the input was compared with 4% of the material bound to either GST or GST—galectin-1 beads. Protein samples were separated on NuPage Bis-Tris gels followed by CA125-C-TERM immunoblotting employing the mAb OC125. (A) Lysates derived from CA125-C-TERM-expressing CHOMCAT-TAM2 cells (lanes 1-3), CA125-C-TERM-expressing CHOMCAT-TAM2 cells treated with tunicamycin (lanes 4-6), CA125-C-TERM-deficient CHOMCAT-TAM2 cells (lanes 7-9), CA125-C-TERM-deficient CHOMCAT-TAM2 cells treated with tunicamycin (lanes 10-12), CA125-C-TERM-expressing CHOclone 13 cells (lanes 14-16) and CA125-C-TERM-deficient CHOclone 13 cells (lanes 17-19). In lane 13, HeLa-derived CA125 eluted from GST—galectin-1 beads is shown as a control. (B) Quantification of the results shown in panel A. On the basis of the input signal (0.2% of starting material; panel A, lane 1), about 40% of CA125-C-TERM present in the cell lysate is recovered on GST—galectin-1 beads under the conditions used (panel A, lane 3, 4% of eluate), based on quantification employing Bio-Rad® QuantityOne® software. This value was set to 100% binding efficiency and compared with CA125-C-TERM—galectin-1 binding efficiencies measured with lysates either derived from tunicamycin-treated CHOMCAT-TAM2 cells or from CHOclone 13 cells. The results shown represent mean values of two independent experiments.

View larger version (27K): [in a new window]   Fig. 5. CA125-C-TERM cell-surface expression in CHOclone 13 cells does not result in increased binding capacity for exogenously added galectin-1. CA125-C-TERM-expressing and CA125-C-TERM-deficient CHOMCAT-TAM2 and CHOclone 13 cells were grown to 70% confluency. Where indicated, cells were treated with 10 µg/ml tunicamycin for 18 hours at 37°C. Cells were then dissociated from the culture plates followed by incubation with 40 µg/ml recombinant GST—galectin-1 for 30 minutes at room temperature. Following labeling with affinity-purified anti-galectin-1 antibodies under native conditions, the various samples were analyzed for cell-surface-bound recombinant galectin-1 using FACS. Autofluorescence (filled light-blue curve: CHOMCAT-TAM2; filled gray curve: CHOclone 13) was determined based on cells not treated with antibodies. Untreated CHOMCAT-TAM2 cells are shown in dark blue. Tunicamycin-treated CHOMCAT-TAM2 cells are shown in red. CHOclone 13 cells are shown in dark green (CA125-C-TERM-expressing) and light green (CA125-C-TERM-deficient), respectively.

View larger version (19K): [in a new window]   Fig. 6. CA125-C-TERM is transported to the cell surface of both CHO and HeLa cells as determined by FACS. CHOMCAT-TAM2 (A) and HeLaMCAT-TAM2 (B) cells, respectively, were transduced with retroviral particles encoding CA125-C-TERM. Following 3 days of incubation at 37°C, cells were dissociated from the culture plates using a protease-free buffer and processed with anti-CA125 antibodies (OC125). Primary antibodies were detected with anti-mouse antibodies coupled to Alexa-488. CA125 cell-surface localization was analyzed by FACS. Autofluorescence was determined with trypsin-treated cells (red curves). Non-transduced cells prepared in the absence of trypsin are indicated by green curves. CA125-C-TERM-transduced cells prepared in the absence of trypsin are indicated by blue curves.

View larger version (43K): [in a new window]   Fig. 7. CA125-C-TERM is transported to the cell surface of both CHO and HeLa cells as determined by confocal microscopy. CHOMCAT-TAM2 and HeLaMCAT-TAM2 cells, respectively, were grown on glass cover slips followed by transduction with retroviral particles encoding CA125-C-TERM or with retroviral control particles that lack a cDNA insert in the viral genome. After 3 days of incubation at 37°C, the cells were fixed with paraform aldehyde. Specimens shown in A-D represent CHOMCAT-TAM2 cells that were not permeabilized to visualize exclusively cell-surface-localized CA125-C-TERM. Specimens shown in E-H represent Triton X-100-permeabilized HeLaMCAT-TAM2 cells to detect intracellular CA125-C-TERM. CA125-C-TERM was visualized with the mAb OC125 (A, B, E-H). The Golgi marker p27 was detected with a polyclonal rabbit antiserum directed against a synthetic peptide that corresponds to the cytoplasmic tail of p27 (C and D) (Jenne et al., 2002). Double staining was performed using secondary antibodies coupled to Alexa-488 and Alexa-546, respectively. Specimens were analyzed with a Zeiss LSM510 confocal microscope.

View larger version (102K): [in a new window]   Fig. 8. Intracellular CA125-C-TERM is localized to organelles of the classical ER/Golgi-dependent secretory pathway. HeLaMCAT-TAM2 cells stably expressing CA125-C-TERM were grown on glass cover slips. At about 70% confluency, cells were treated with brefeldin A (5 µg/ml) for 60 minutes or were left untreated as a control. Following fixation with paraform aldehyde, cells were treated with Triton X-100 to allow intracellular staining of antigens using antibodies directed against calreticulin, the KDEL receptor and CA125. Double staining was performed using secondary antibodies coupled to Alexa-488 and Alexa-546, respectively. Specimens were analyzed with a Zeiss LSM510 confocal microscope. (A) Anti-calreticulin, not treated with brefeldin A; (B) anti-CA125, not treated with brefeldin A; (C) anti-KDEL receptor, not treated with brefeldin A; (D) anti-CA125, not treated with brefeldin A; (E) anti-KDEL receptor, treated with brefeldin A; (F) anti-CA125, treated with brefeldin A.

View larger version (29K): [in a new window]   Fig. 9. CA125-C-TERM is transported to the cell surface by the classical ER/Golgi-dependent secretory pathway. HeLaMCAT-TAM2 cells stably expressing CA125-C-TERM were grown to 70% confluency. Where indicated, brefeldin A was added to the medium at 5 µg/ml. Following incubation for 90 minutes at 37°C, cells were trypsinized to remove cell-surface CA125-C-TERM and spread onto new culture plates at 70% confluency. The culture was then continued for 4 hours at 37°C in the presence or absence of brefeldin A, as indicated. CA125-C-TERM transported to the cell surface within this time period was quantified by FACS using the mAb OC125. (A) FACS histograms. Autofluorescence was determined by analyzing cells that were not treated with antibodies (light blue curve, filled). The red curve represents cells under steady-state conditions. The light green curve represents cells that were not treated with brefeldin A. Cells that were grown for 90 minutes in the presence of brefeldin A, followed by incubation for 4 hours in its absence, are shown in dark green. Cells that were incubated with brefeldin A over the whole course of the experiment are shown in dark blue. (B) Statistical analysis of four independent experiments. The colors of the bars correspond to the conditions detailed above.

View larger version (17K): [in a new window]   Fig. 10. Correlation of endogenous CA125 expression with cell-surface expression of endogenous galectin-1 in CHO and HeLa cells. CHO (A) and HeLa cells (B) were dissociated from culture plates using a protease-free buffer. Native cells were labeled with affinity-purified anti-galectin antibodies derived from a polyclonal rabbit antiserum. Cell-surface staining was analyzed by FACS using anti-rabbit secondary antibodies coupled to allophycocyanine to detect primary antibodies. Autofluorescence levels (red curves in A and B) were determined with cells treated with trypsin prior to the FACS analysis. Galectin-1 cell-surface levels are indicated in green (CHO; panel A) and blue (HeLa; panel B), respectively. Total galectin-1 expression levels in CHO and HeLa cells, respectively, were analyzed by quantifying galectin-1 in total SDS cell lysates based on a western blot analysis (C). Lanes 1 and 4 represent the material of 20,000 cells, lanes 2 and 5 represent the material of 50,000 cells and lanes 3 and 6 represent the material of 150,000 cells. The results from CHO cells are shown in lanes 1-3, the results from HeLa cells are shown in lanes 4-6. Galectin-1 was detected with an affinity-purified rabbit antiserum directed against recombinant full-length galectin-1.

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