Annexin A2 on lung epithelial cell surface is recognized by severe acute respiratory syndrome-associated coronavirus spike domain 2 antibodies

Abstract

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infection causes lung failure characterized by atypical pneumonia. We previously showed that antibodies against SARS-CoV spike domain 2 (S2) in the patient sera can cross-react with human lung epithelial cells; however, the autoantigen is not yet identified. In this study, we performed proteomic studies and identified several candidate autoantigens recognized by SARS patient sera in human lung type II epithelial cell A549. Among the candidate proteins, annexin A2, which was identified by mass spectrometry analysis and had the highest score by Mascot data search, was further characterized and investigated for its role as an autoantigen. By confocal microscopic observation, SARS patient sera and anti-S2 antibodies were co-localized on A549 cells and both of them were co-localized with anti-annexin A2 antibodies. Anti-annexin A2 antibodies bound to purified S2 proteins, and anti-S2 bound to immunoprecipitated annexin A2 from A549 cell lysate in a dose-dependent manner. Furthermore, an increased surface expression and raft-structure distribution of annexin A2 was present in A549 cells after stimulation with SARS-induced cytokines interleukin-6 and interferon-gamma. Cytokine stimulation increased the binding capability of anti-S2 antibodies to human lung epithelial cells. Together, the upregulated expression of annexin A2 by SARS-associated cytokines and the cross-reactivity of anti-SARS-CoV S2 antibodies to annexin A2 may have implications in SARS disease pathogenesis.

Fig. 1

Epithelial cell self-antigens recognized by SARS patient sera. (A) Human A549 epithelial cell membrane fraction was separated by SDS-PAGE and immunoblotted with SARS patient sera (from a pool of five patients) or normal control sera. (B) Membrane fraction was analyzed by 2-DGE and silver stain (top panel) followed by blotting with SARS patient sera (bottom panel). Seven protein spots (a–g) were detected and protein identification is shown in Table 1.

Fig. 2

Mass spectrometry identification of annexin A2. The spot c on the 2D-gel as shown in Fig. 1B was excised and subjected to in-gel digestion, mass spectrometry analysis, and Mascot database search. (A) A summary of the Mascot search result is shown. Annexin A2 scored 308, the highest score among the significant hits (those above the shaded area). (B) Amino acid sequence of annexin A2. The peptide sequences in bold red were those matched in the database search. (C) Two examples, as underlined in (B) of tandem mass spectrometry data leading to positive identification of annexin A2. Using Mascot database search, these two product ion scan spectra were matched to the sequences QDIAFAYQR (left panel) and GLGTDEDSLIEIICSR (right panel), respectively. Most of the peaks were assigned as b and y series ions. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 3

Co-localization of SARS patient sera and anti-S2 antibodies with annexin A2 expressed on human epithelial cells. Cell binding was detected using immunostaining followed by confocal microscopic observation. (A and B) A549 cells were incubated with a 1:20 dilution of sera from SARS patients, followed by FITC-conjugated anti-human IgG (SARS patient, green). After washing, cells were then stained with anti-S2 (anti-S2, red) or anti-annexin A2 (anti-annexin A2, red) followed by TRITC-conjugated anti-mouse IgG. (C) Cells were incubated with antibodies against S2 followed by TRITC-conjugated anti-mouse IgG (anti-S2, red), and then FITC-conjugated anti-annexin A2 (anti-annexin A2, green). The signals of co-localization are shown (Merge, yellow). Bar: 20 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 4

Anti-annexin A2 antibodies bind to SARS-CoV S2 protein and anti-S2 antibodies bind to immunoprecipitated annexin A2. (A) Recombinant S2 protein was coated on ELISA plate with different doses as indicated, then incubated with control IgG or serial doses of anti-annexin A2 antibodies followed by HRP-conjugated anti-rabbit IgG. Cultures were developed using specific substrate ABTS and detected using an ELISA reader. Experiments were carried out in triplicate, and the averages of the relative OD as mean ± standard deviation (SD) are shown. (B) Annexin A2 was immunoprecipitated from various doses of A549 cell lysate using anti-annexin A2 antibodies. Cultures were then incubated with 5 μg/ml of control IgG or anti-S2 followed by HRP-conjugated anti-mouse IgG. Experiments were carried out in triplicate, and the averages of the relative OD as mean ± SD are shown. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 5

IL-6 and IFN-γ upregulate epithelial cell surface expression of annexin A2. (A) Surface (Sur) expression of annexin A2 in A549 cells pretreated with various doses of IL-6 or IFN-γ for 48 h was determined by Western blotting. The expression of intracellular annexin A2 is also shown. α-Tubulin was used as the control. (B) The protein expression and distribution of annexin A2 in A549 cells pretreated with 25 ng/ml of IL-6 or IFN-γ for 24 h was determined using confocal microscopic observation. Cells were stained with anti-annexin A2 followed by FITC-conjugated anti-mouse IgG (green). A cytosolic counterstaining was performed by mitochondrial labeling using MitoTracker (red). Bar: 16 μm. (C) The formation of lipid raft-like structure in A549 cells after IL-6 and IFN-γ treatment was demonstrated by caveolin-1 staining. Cells were incubated with FITC-conjugated anti-annexin A2 (green) and TRITC-conjugated anti-caveolin-1 (red). Co-localization of annexin A2 and caveolin-1 is shown (Merge, yellow). Bar: 16 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 6

IL-6 and IFN-γ enhance the epithelial cell-binding ability of anti-S2 antibodies. Human lung epithelial cells A549 and HL were treated with or without 25 ng/ml of IL-6 or IFN-γ for 24 h. Cells were stained with 1 μg of control IgG or anti-S2 followed by FITC-conjugated anti-mouse IgG and analyzed using flow cytometry. Data are presented as mean ± SD of triplicate cultures. **p < 0.01; ***p < 0.001.