We seek to elucidate recognition and transcytosis mechanisms for a transcytotic immunoglobulin Fc receptor (FcRs): the polymeric Ig receptor (pIgR), which transports polymeric IgA (pIgA) across mucosal epithelia into mucosal secretions. pIgR represents a new direction that is complementary to our previous studies of the neonatal Fc receptor (FcRn), a transcytotic FcR that transfers maternal immunoglobulin G (IgG) across epithelia to the fetus or suckling newborn. In the previous funding cycle, we characterized FcRn and other FcRs using biochemical/biophysical and structural approaches (X-ray crystallography and single particle electron microscopy). We also developed methods to visualize vesicles transporting FcRn-IgG complexes inside epithelial cells by electron tomography (ET), deriving high resolution 3-D snapshots of dynamic events during transcytosis. We will apply these methods to study IgA, the predominant immunoglobulin in mucosal secretions where it exists as a polymer (pIgA) in complex with pIgR, a multi-domain receptor that mediates basolateral-to-apical transcytosis of pIgA across polarized epithelial cells to deliver pIgR-pIgA complexes to mucosal secretions where they bind host and pathogen proteins. While most interactions protect the host epithelial barrier, binding to some pathogen proteins (e.g., from S. pneumoniae bacteria) enhances disease virulence through reverse transcytosis that can facilitate bacterial invasion. Despite well-established, fundamental roles in immune system function, the molecular mechanisms and architecture governing pIgA and pIgR remain largely uncharacterized. We propose to characterize pIgR complexes with pIgA and pathogen proteins in solution and inside cells using X-ray crystallography, complimentary biochemical experiments, and electron microscopy: both single particle EM to derive structures of large protein complexes, and ET to delineate transcytotic pathways in 3-D in epithelial cells. Resulting structural models of individual proteins, complexes, and transporting cells will reveal the architecture of pIgA, pIgR, and their complexes with pathogen proteins as well as the molecular mechanisms governing their interactions, function, and transport during health and disease. Models resulting from these studies will provide insights into fundamental transcytotic receptor-mediated pIgA processes, which are necessary to understand mucosal and humoral immunity as well as disease pathology, and to develop existing and novel immunotherapies. Our results will also be of general cell biological interest, since they address a molecular mechanism by which recognition and proper sorting of internalized cargo is performed by intracellular trafficking machinery.
Fc receptor (FcR)-mediated transcytosis across polarized epithelial cells is a fundamental process for moving immunoglobulins (Igs) to essential sites of action. We will derive structural models of receptor-Ig complexes in isolation and during transcytosis inside epithelial cells;resulting models and trafficking studies of pIgR and IgA interactions with pathogen receptors will provide insight into pathogenesis and molecular detail describing binding interfaces that could be exploited directly in therapeutic design. Comparisons with previous studies of FcRn-mediated IgG transport will establish molecular mechanisms by which recognition and proper sorting of internalized cargo is performed by intracellular traffickin machinery.
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