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ß2-microglobulin is important for cell surface expression and pH-dependent IgG binding of human FcRn

Asja Praetor and Walter Hunziker*

Institute of Molecular and Cell Biology, Epithelial Cell Biology Laboratory, 30 Medical Drive, Singapore 117609, Republic of Singapore



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  		Fig. 1. Characterization of FO-1 cells expressing the FcRn {alpha}-chain alone or in combination with ß2m. (A) Western blot analysis. Lysates of FO-1 cells (lane 1), FO-1 ß2m cells (lane 2), FO-1 cells expressing Myc-hFcRn (lane 3), and FO-1 ß2m cells expressing Myc-hFcRn (lane 4) were analyzed by SDS-PAGE and immunoblotting using polyclonal anti-FcRn peptide antibodies ({alpha}-FcRn; upper panel), monoclonal anti-Myc antibodies ({alpha}-Myc; middle panel), and polyclonal anti-ß2m antibodies ({alpha}-ß2m; lower panel). For reasons unknown, the anti-FcRn serum detected the {alpha}-chain less well in lysates from FO-1 ß2m than in FO-1 cells. (B) Co-localization of the FcRn {alpha}-chain and ß2m. FO-1 cells (a,c) and FO-1 ß2m cells expressing Myc-hFcRn (c,d,e) were fixed, permeabilized and stained with monoclonal anti-Myc ({alpha}-Myc; a,b) and polyclonal anti-ß2m antibodies ({alpha}-ß2-m; c,d). Panel e shows the merged staining for Myc-hFcRn (green) and ß2m (red). (C) Co-immunoprecipitation of the FcRn {alpha}-chain and ß2m. FO-1 cells (lanes 1,5), FO-1 cells expressing Myc-hFcRn (lanes 2,6), FO-1 ß2m cells (lanes 3,7) and FO-1 ß2m cells expressing Myc-hFcRn (lanes 4,8) were lyzed and immunoprecipitated with monoclonal anti-Myc antibodies ({alpha}-Myc IP; lanes 1-4) or polyclonal anti-ß2m antibodies ({alpha}-ß2m IP; lanes 5-8). Precipitated proteins were immunoblotted with polyclonal anti-Myc antibodies ({alpha}-Myc; top panel) and polyclonal anti-ß2m antibodies ({alpha}-ß2m; bottom panel). The data in Fig. 1 is representative of at least three independent experiments carried out using two different cell clones.

 


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  		Fig. 2. The pH-dependent internalization and binding of IgG is impaired in the absence of ß2m. (A) IgG internalization. FO-1 cells (a,e), FO-1 ß2m cells (b,f), FO-1 cells expressing Myc-hFcRn (c,g), and FO-1 ß2m cells expressing Myc-hFcRn (d,h) were incubated in medium at pH 6.5 (a-d) or pH 7.4 (e-h) in the presence of hIgG (1 µg/ml) for 30 minutes at 37°C. After cooling the cells on ice and washing off unbound IgG, cells were fixed, permeabilized and stained with labeled secondary antibodies to detect internalized hIgG. (B) Binding of FcRn to IgG-agarose. FO-1 cells (lanes 1,5), FO-1 cells expressing Myc-hFcRn (lanes 2,6), FO-1 ß2m cells (lanes 3,7) and FO-1 ß2m cells expressing Myc-hFcRn (lanes 4,8) were lyzed in CHAPS buffer at pH 6.5 (top panels; pH 6.5) or pH 7.4 (bottom panels; pH 7.4). Cell lysates were blotted using monoclonal anti-Myc ({alpha}-Myc) or polyclonal anti-ß2m ({alpha}-ß2m) antibodies (lanes 1-4; lysate). Alternatively, cell lysates were incubated with IgG-agarose and bound proteins then eluted and blotted (lanes 5-8; IgG binding). The data in Fig. 2 is representative for at least three independent experiments carried out using two different cell clones.

 


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  		Fig. 3. Subcellular distribution and internalization of FcRn. FO-1 cells (a,e,i), FO-1ß2m cells (b,f,j), FO-1 cells expressing Myc-hFcRn (c,g,k) and FO-1ß2m cells expressing Myc-hFcRn (d,h,l) were fixed, permeabilized and stained with anti-myc antibodies to visualize the subcellular distribution of Myc-hFcRn (a-d). Alternatively, cells were incubated with anti-Myc antibodies at 4°C, fixed and surface-bound anti-Myc was detected with labeled secondary antibodies to visualize Myc-hFcRn present on the cell surface (e-h). In panels i-l, cells were allowed to internalize anti-Myc antibodies for 60 minutes at 37°C, washed with acid to remove non-internalized antibodies bound to the cell surface and then fixed, permeabilized and stained with labeled secondary antibodies to detect anti-Myc antibodies that had been internalized. Panels show representative data of one of two clones analyzed.

 


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  		Fig. 4. Detection of cell surface FcRn. The cell surface of FO-1 cells (lane 3), FO-1ß2m cells (lane 5), FO-1 cells expressing Myc-hFcRn (lanes 1,4) and FO-1ß2m cells expressing Myc-hFcRn (lanes 2,6) were biotinylated on ice with a membrane-impermeable reagent. Cells were lysed and an aliquot of the lysates directly analyzed by immunoblotting (lysate; lanes 1,2) to detect the presence of Myc-hFcRn ({alpha}-Myc). The remaining lysate was precipitated with anti-Myc antibodies ({alpha}-Myc IP) and biotinylated Myc-hFcRn present in the precipitates detected by blotting with streptavidin-HRP (SA-HRP, lanes 3-6, top panel). Alternatively, biotinylated proteins were first precipitated with streptavidin-agarose (SA-P) and precipitates immunoblotted to detect Myc-hFcRn ({alpha}-Myc, lower panel). hc, heavy chain of the antibody used for immunoprecipitation. The data shown is representative of at least three independent experiments, each carried out using two different cell clones.

 


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  		Fig. 5. FcRn colocalizes with an ER marker in the absence of ß2m. FO-1 (a-c,g-i,m-o) or FO-1ß2m expressing Myc-hFcRn (panels d-f,j-l,p-r) were fixed, permeabilized and stained with monoclonal anti-EEA1 (a,d), anti-lamp1 (g,j) or anti-ribophorin II (m,p) antibodies to label endosomes, lysosomes or the ER, respectively, and polyclonal anti-Myc antibodies (b,e,h,k,n,q) to detect Myc-hFcRn. In c,f,i,l,o,r, the staining for endosomes, lysosomes or ER (green) was merged with that for Myc-hFcRn (red). The panels show representative data for one of two clones analyzed.

 


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  		Fig. 6. FcRn remains EndoH sensitive in the absence of ß2m. Lysates of FO-1 cells (lane 1), FO-1ß2m cells (lane 7), FO-1 cells expressing Myc-hFcRn (lanes 2-6) and FO-1ß2m cells expressing Myc-hFcRn (lanes 8-12) were either directly blotted (lanes 1,2,7,8) or first incubated with enzyme buffer alone (lanes 3,5,9,11) or with EndoH (lanes 4,10) or glycosidase F (lanes 6,12) for 3.5 hours at 37°C. Blots were probed with monoclonal anti-Myc ({alpha}-Myc; top panel) or polyclonal anti-ß2m ({alpha}-ß2m; bottom panel) antibodies. The data is representative for three independent experiments, each carried out with two different cell clones.

 

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