Abstract text (incl. figure legends and references)

Mucosal immune system is the largest component of immune system and mediates interactions with a diverse range of antigens from toxin molecules to pathogen organisms of dietary or environmental source. Moreover, it comprises physiologically diverse compartments such as ocular, oral, gastrointestinal, respiratory etc mucosae. Secretory Immunoglobulin A (SIgA) is the major antibody of mammalian mucosa. It is a polymeric antibody and interacts with a large and diverse set of antigens. Mode of action for SIgA seems to be mostly non-inflammatory and involves coating, clumping, cross-linking (Kathrin Moor, et al. 2017). Structure of SIgA was recently solved (Kumar Bharathkar, Parker, et al. 2020) and comprises two copies of IgA with one Joining-Chain (JC). The full molecular assembly furthermore comprises Secretory Component (SC). However, the Fab fragments were not resolved likely due to high flexibility.

We characterized the SIgA using cryo electron microscopy (cryoEM) in presence of its substrate, the O-antigen. One SIgA and one O-antigen molecule comprise four Fabs and 50-60 repeating units, respectively. The molar ratio of SIgA/O-antigen is 2/1, which implies that two tetravalent SIgA molecules are bound to one molecule O-antigen comprising 50-60 antigen repeating units (corresponding to 1 Fab /6.25-7.5 antigen repeating units). Freshly glow discharged Quantifoil Cu R 2/2 grids were optimized for 0.5 mg/ml SIgA sample or its complex with O-antigen at a ratio of 2:1 using Vitrobot (3.5 µl, 22°C, 2-4 s blotting time). Data was collected using Titan Krios (Thermo Fisher Scientific) and GIF/K3 (Gatan) by applying 63 e/Å2 at 0.65 pix/Å. The collected 8060 movies were processed in cryoSPARC to solve the structure.

Few conclusions can be drawn from a comparison of the published SIgA structure with our SIgA structure or the SIgA-O-antigen complex. The obtained structure of the SIgA shows no change in the overall structure upon binding of the O-antigen. However, addition of the O-antigen markedly improves the resolution of the complex (currently ca. 3.2 Å) possibly resulting from a higher stability of complex. Furthermore, the obtained structure is similar to the published structure referred to earlier, but differences are observed especially for parts of the SC where the polypeptide chain is better resolved, and a different atomic model can be built. Besides, we are working currently to resolve the Fabs and Fab-antigen interaction using cryoEM and additional techniques such X-ray crystallization.

References:

Kumar Bharathkar, Parker, et al. eLife 2020;9:e56098. DOI: https://doi.org/10.7554/eLife.56098

Kathrin Moor, et al. Nature 2017; DOI:10.1038/nature22058