The structure and dynamics of secretory component and its interactions with polymeric immunoglobulins.
ABSTRACT: As a first-line vertebrate immune defense, the polymeric immunoglobulin receptor (pIgR) transports polymeric IgA and IgM across epithelia to mucosal secretions, where the cleaved ectodomain (secretory component; SC) becomes a component of secretory antibodies, or when unliganded, binds and excludes bacteria. Here we report the 2.6Å crystal structure of unliganded human SC (hSC) and comparisons with a 1.7Å structure of teleost fish SC (tSC), an early pIgR ancestor. The hSC structure comprises five immunoglobulin-like domains (D1-D5) arranged as a triangle, with an interface between ligand-binding domains D1 and D5. Electron paramagnetic resonance measurements confirmed the D1-D5 interface in solution and revealed that it breaks upon ligand binding. Together with binding studies of mutant and chimeric SCs, which revealed domain contributions to secretory antibody formation, these results provide detailed models for SC structure, address pIgR evolution, and demonstrate that SC uses multiple conformations to protect mammals from pathogens.
Project description:The polymeric Ig receptor (pIgR) transports polymeric Abs across epithelia to the mucosa, where proteolytic cleavage releases the ectodomain (secretory component [SC]) as an integral component of secretory Abs, or as an unliganded protein that can mediate interactions with bacteria. SC is conserved among vertebrates, but domain organization is variable: mammalian SC has five domains (D1-D5), whereas avian, amphibian, and reptilian SC lack the D2 domain, and fish SC lacks domains D2-D4. In this study, we used double electron-electron resonance spectroscopy and surface plasmon resonance binding studies to characterize the structure, dynamics, and ligand binding properties of avian SC, avian SC domain variants, and a human SC (hSC) variant lacking the D2 domain. These experiments demonstrated that, unlike hSC, which adopts a compact or "closed" domain arrangement, unliganded avian SC is flexible and exists in both closed and open states, suggesting that the mammalian SC D2 domain stabilizes the closed conformation observed for hSC D1-D5. Experiments also demonstrated that avian and mammalian pIgR share related, but distinct, mechanisms of ligand binding. Together, our data reveal differences in the molecular recognition mechanisms associated with evolutionary changes in the pIgR protein.
Project description:The polymeric immunoglobulin receptor (pIgR) transports immunoglobulins from the basolateral to the apical surface of epithelial cells. PIgR was recently shown to be associated with kidney dysfunction. The immune defense is initiated at the apical surface of epithelial cells where the N-terminal domain of pIgR, termed secretory component (SC), is proteolytically cleaved and released either unbound (free SC) or bound to immunoglobulins. The aim of our study was to evaluate the association of pIgR peptides with the cardio-renal syndrome in a large cohort and to obtain information on how the SC is released. We investigated urinary peptides of 2964 individuals available in the Human Urine Proteome Database generated using capillary electrophoresis coupled to mass spectrometry. The mean amplitude of 23 different pIgR peptides correlated negatively with the estimated glomerular filtration rate (eGFR, rho?=?-0.309, p?<?0.0001). Furthermore, pIgR peptides were significantly increased in cardiovascular disease (coronary artery disease and heart failure) after adjustment for eGFR. We further predicted potential proteases involved in urinary peptide generation using the Proteasix algorithm. Peptide cleavage site analysis suggested that several, and not one, proteases are involved in the generation of the SC. In this large cohort, we could demonstrate that pIgR is associated with the cardio-renal syndrome and provided a more detailed insight on how pIgR can be potentially cleaved to release the SC.
Project description:The polymeric immunoglobulin receptor (pIgR) has been proposed as a therapeutic target, but its potential depends on the efficiency of uptake and trafficking of the receptor ligand. Mouse monoclonal antibodies (Mabs) directed against pIgR, selected for strong binding to secretory component (SC) and secretory IgA (sIgA), were tested in a transcytosis assay in 16HBEo--cells (human bronchial epithelial cell line) transfected with human pIgR. Intracellular trafficking was followed by confocal microscopy. Mabs fell into two classes. For two Mabs, transcytosis from basolateral to apical surface is rapid, unidirectional, and little Mab is retained in the cell. For three Mabs, basolateral to apical transcytosis occurs to a significantly lesser extent, reverse transcytosis is permitted, and some of the Mab is retained in the perinuclear region even after 24 h. When tested for their ability to recognize and immunoprecipitate pIgR with systematic truncations and deletions of the five immunoglobulin (Ig)-like domains, all Mabs bound to the fifth Ig-like domain, but three of them also bound to the C-terminal region of pIgR near the plasma membrane. Different binding sites probably account for the different trafficking of these Mabs and may predict differential therapeutic utility.
Project description:Animals are continuously threatened by pathogens entering the body through natural openings. Here we show that in chicken ( Gallus gallus ), secretory IgA (sIgA) protects the epithelia lining these natural cavities. A gene encoding a chicken polymeric Ig receptor ( GG-pIgR ), a key component of sIgA, was identified, and shown to be expressed in the liver, intestine and bursa of Fabricius. All motifs involved in pIgR function are present, with a highly conserved Ig-binding motif in the first Ig-like domain. Physical association of GG-pIgR with pIgA in bile and intestine demonstrates that this protein is a functional receptor. Thus, as shown for mammals, this receptor interacts with J-chain-containing polymeric IgA (pIgA) at the basolateral epithelial cell surface resulting in transcytosis and subsequent cleavage of the pIgR, releasing sIgA in the mucosal lumen. Interestingly, the extracellular portion of GG-pIgR protein comprises only four Ig-like domains, in contrast with the five domain structure found in mammalian pIgR genes. The second Ig-like domain of mammalian pIgR does not have an orthologous domain in the chicken gene. The presence of pIgR in chicken suggests that this gene has evolved before the divergence of birds and reptiles, indicating that secretory Igs may have a prominent role in first line defence in various non-mammalian species.
Project description:Polymeric Ig receptor (pIgR) is a central player in mucosal immunity that mediates the delivery of polymeric IgA and IgM to the apical surface of epithelial cells via transcytosis. Emerging evidence suggests that Th17 cells not only mediate autoimmunity but also play key roles in mucosal host defense against pathogens. We demonstrate that OVA-specific CD4(+) Th17 cells, in addition to causing neutrophilic inflammation in mice, mediated a pronounced influx of CD19(+) B cells into the lungs following Ag inhalation. Coincident with this recruitment was a striking induction in pIgR expression by the bronchial epithelium and a subsequent increase in airway IgM and secretory IgA levels. Intranasal administration of IL-17 revealed a crucial role for this cytokine in inducing pIgR expression by the epithelium. These findings support a key role for Th17 cells in pulmonary immune defense against respiratory pathogens by promoting pIgR-mediated transport of secretory IgA and IgM into the airway.
Project description:The polymeric immunoglobulin receptor (pIgR) mediates transcytosis of dimeric immunoglobulin A (dIgA) and its release into mucosal secretions. The present study reveals the complexity of the trafficking of pIgR to the apical plasma membrane in epithelial cells with exocrine secretory functions; in rabbit lacrimal gland acinar cells (LGACs), trafficking of pIgR involves both the transcytotic pathway and one arm of the regulated secretory pathway. By specifically tracking pIgR endocytosed from the basolateral membrane, we show here that the Rab11a-regulated transcytotic pathway mediates the basal-to-apical transport of pIgR, and that pIgR sorted into the transcytotic pathway does not access the regulated secretory pathway. However, previous work in LGACs expanded in the present study has shown that some pIgR is localized to Rab3D-enriched mature secretory vesicles (SVs). Myosin Vb and myosin Vc motors modulate release of proteins from the Rab11a-regulated transcytotic pathway and the Rab3D-enriched secretory pathway in LGACs, respectively. Confocal fluorescence microscopy and biochemical assays showed that inhibition of myosin Vb and myosin Vc activity by overexpression of their dominant-negative mutants each significantly but differentially impaired aspects of apically targeted pIgR trafficking and secretory component release, suggesting that these motors function to regulate pIgR trafficking in both the transcytotic and exocytotic pathways. Intriguingly, a second mature SV population enriched in Rab27b was devoid of pIgR cargo, suggesting the specialization of Rab3D-enriched mature SVs to carry a particular subset of cargo proteins from the trans-Golgi network to the apical plasma membrane.
Project description:The natural immune response to Helicobacter pylori neither clears infection nor prevents reinfection. However, the ability of secretory antibodies to influence the course of H. pylori infection has not been determined. We compared the natural progression of H. pylori infection in wild-type C57BL/6 mice with that in mice lacking the polymeric immunoglobulin receptor (pIgR) that is essential for the secretion of polymeric antibody across mucosal surfaces. H. pylori SS1-infected wild-type and pIgR knockout (KO) mice were sampled longitudinally for gastrointestinal bacterial load, antibody response, and histological changes. The gastric bacterial loads of wild-type and pIgR KO mice remained constant and comparable at up to 3 months postinfection (mpi) despite SS1-reactive secretory IgA in the intestinal contents of wild-type mice at that time. Conversely, abundant duodenal colonization of pIgR KO animals contrasted with the near-total eradication of H. pylori from the intestine of wild-type animals by 3 mpi. H. pylori was cultured only from the duodenum of those animals in which colonization in the distal gastric antrum was of sufficient density for immunohistological detection. By 6 mpi, the gastric load of H. pylori in wild-type mice was significantly lower than in pIgR KO animals. While there was no corresponding difference between the two mouse strains in gastric pathology results at 6 mpi, reductions in gastric bacterial load correlated with increased gastric inflammation together with an intestinal secretory antibody response in wild-type mice. Together, these results suggest that naturally produced secretory antibodies can modulate the progress of H. pylori infection, particularly in the duodenum.
Project description:Commensal bacteria co-exist on the mucosal surfaces of all vertebrates. The host's mucosal immune system must tolerate commensals while fighting pathogens. One of the mechanisms used by the mucosal immune system to maintain homeostasis is the secretion of immunoglobulins (Igs) across epithelial barriers, which is achieved via the polymeric immunoglobulin receptor (pIgR). Rainbow trout pIgR is known to transport IgT and IgM across epithelia. However, other biological functions for trout pIgR or trout secretory component (tSC) remain unknown. This study investigates the interaction of tSC with commensal bacteria, pathogenic bacteria and a fungal pathogen. Our results show that the majority of trout skin and gut bacteria are coated in vivo by tSC. In vitro, tSC present in mucus coats trout commensal isolates such as Microbacterium sp., Staphylococcus warneri, Flectobacillus major, Arthrobacter stackebrantii, and Flavobacterium sp. and the pathogens Vibrio anguillarum and Edwardsiella ictaluri with coating levels ranging from 8% to 70%. Moreover, we found that the majority of tSC is in free form in trout mucus and free tSC is able to directly bind bacteria. We propose that binding of free SC to commensal bacteria is a key and conserved mechanism for maintenance of microbial communities in vertebrate mucosal surfaces.
Project description:Mucosal immunity is dominated by secretory IgA and IgM, although these are less favorable compared to IgG molecules for therapeutic development. Polymeric IgA and IgM are actively transported across the epithelial barrier via engagement of the polymeric Ig receptor (pIgR), but IgG molecules lack a lumen-targeted active transport mechanism, resulting in poor biodistribution of IgG therapeutics in mucosal tissues. In this work, we describe the discovery and characterization of single-domain antibodies (VHH) that engage pIgR and undergo transepithelial transport across the mucosal epithelium. The anti-pIgR VHH panel displayed a broad range of biophysical characteristics, epitope diversity, IgA competition profiles and transcytosis activity in cell and human primary lung tissue models. Making use of this diverse VHH panel, we studied the relationship between biophysical and functional properties of anti-pIgR binders targeting different domains and epitopes of pIgR. These VHH molecules will serve as excellent tools for studying pIgR-mediated transport of biologics and for delivering multispecific IgG antibodies into mucosal lumen, where they can target and neutralize mucosal antigens.
Project description:The mammalian gut is inhabited by a large and complex microbial community that lives in a mutualistic relationship with its host. Innate and adaptive mucosal defense mechanisms ensure a homeostatic relationship with this commensal microbiota. Secretory antibodies are generated from the active polymeric Ig receptor (pIgR)-mediated transport of IgA and IgM antibodies to the gut lumen and form the first line of adaptive immune defense of the intestinal mucosa. We probed mucosal homeostasis in pIgR knockout (KO) mice, which lack secretory antibodies. We found that in pIgR KO mice, colonic epithelial cells, the cell type most closely in contact with intestinal microbes, differentially expressed (>2-fold change) more than 200 genes compared with wild type mice, and upregulated the expression of anti-microbial peptides in a commensal-dependent manner. Detailed profiling of microbial communities based on 16S rRNA genes revealed differences in the commensal microbiota between pIgR KO and wild type mice. Furthermore, we found that pIgR KO mice showed increased susceptibility to dextran sulfate sodium (DSS)-induced colitis, and that this was driven by their conventional intestinal microbiota. In conclusion, secretory antibodies or the pIgR itself are required to maintain a stable commensal microbiota. In the absence of these humoral effector components, gut homeostasis is disturbed and the outcome of colitis significantly worsened. 4 groups: wild type mice treated with antibiotic (5 replicates), wild type mice left untreated (5 replicates), pIgR KO mice treated with antibiotic (6 replicates), and pIgR KO mice left untreated (6 replicates).