The joining (J) chain is a small polypeptide, expressed by mucosal and glandular plasma cells, which regulates polymer formation of immunoglobulin (Ig)A and IgM. J-chain incorporation into polymeric IgA (pIgA, mainly dimers) and pentameric IgM endows these antibodies with several salient features. First, a high valency of antigen-binding sites, which makes them suitable for agglutinating bacteria and viruses; little or no complement-activating potential, which allows them to operate in a noninflammatory fashion; and, most importantly, only J-chain-containing polymers show high affinity for the polymeric Ig receptor (pIgR), also known as transmembrane secretory component (SC). This epithelial glycoprotein mediates active external transfer of pIgA and pentameric IgM to exocrine secretions. Thus, secretory IgA (SIgA) and SIgM, as well as free SC, are generated by endoproteolytic cleavage of the pIgR extracellular domain. The secretory antibodies form the 'first line' of defence against pathogens and noxious substances that favour the mucosae as their portal of entry. The J chain is involved in creating the binding site for pIgR/SC in the Ig polymers, not only by determining the polymeric quaternary structure but apparently also by interacting directly with the receptor protein. Therefore, both the J chain and the pIgR/SC are key proteins in secretory immunity.

Local production of secretory (S)IgA provides adaptive immunologic protection of mucosal surfaces, but SIgA is also protective when administered passively, such as in breast milk. Therefore, SIgA is a potential candidate for therapeutic administration, but its complex structure with four different polypeptide chains produced by two distinct cell types complicates recombinant production. The J chain is critical in the structure of SIgA because it is required for efficient polymerization of IgA and for the affinity of such polymers to the secretory component (SC)/polymeric (p)IgR. To better understand the role of the J chain in SIgA production, we have generated various mutant forms of the human J chain and analyzed the function of these mutants when coexpressed with IgA. We found that the C terminus of the J chain was not required for the formation of IgA polymers, but was essential for the binding of pIgA to SC. Likewise, we found that two of the intrachain disulfide bridges (Cys13:Cys101 and Cys109:Cys134) were also required for the binding of pIgA to SC but, interestingly, not for IgA polymerization. Conversely, the last intrachain disulfide bridge (Cys72:Cys92) was not essential for either of these two J chain functions. Finally, we demonstrated that the presence of only Cys15 or Cys69 was sufficient to support polymerization of IgA, but that these polymers were mostly noncovalently stabilized. Nevertheless, these polymers bound free SC with nearly the same affinity as pIgA containing wild-type J chain, but were transcytosed by pIgR-expressing polarized epithelial cells at a reduced efficiency.

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The binding of non-specific human IgM to the surface of infected erythrocytes is important in rosetting, a major virulence factor in the pathogenesis of severe malaria due to Plasmodium falciparum, and IgM binding has also been implicated in placental malaria. Here we have identified the IgM-binding parasite ligand from a virulent P. falciparum strain as PfEMP1 (TM284var1 variant), and localized the region within this PfEMP1 variant that binds IgM (DBL4β domain). We have used this parasite IgM-binding protein to investigate the interaction with human IgM. Interaction studies with domain-swapped antibodies, IgM mutants and anti-IgM mAbs showed that PfEMP1 binds to the Fc portion of the human IgM heavy chain and requires the IgM Cμ4 domain. Polymerization of IgM was shown to be crucial for the interaction because PfEMP1 binding did not occur with mutant monomeric IgM molecules. These results with PfEMP1 protein have physiological relevance because infected erythrocytes from strain TM284 and four other IgM-binding P. falciparum strains showed analogous results to those seen with the DBL4β domain. Detailed investigation of the PfEMP1 binding site on IgM showed that some of the critical amino acids in the IgM Cμ4 domain are equivalent to those regions of IgG and IgA recognised by Fc-binding proteins from bacteria, suggesting that this region of immunoglobulin molecules may be of major functional significance in host-microbe interactions. We have therefore shown that PfEMP1 is an Fc-binding protein of malaria parasites specific for polymeric human IgM, and shows functional similarities with Fc-binding proteins from pathogenic bacteria.

Abs of the secretory Ig (SIg) system reinforce numerous innate defense mechanisms to protect the mucosal surfaces against microbial penetration. SIgs are generated by a unique cooperation between two distinct cell types: plasma cells that produce polymers of IgA or IgM (collectively called pIgs) and polymeric Ig receptor (pIgR)-expressing secretory epithelial cells that mediate export of the pIgs to the lumen. Apical delivery of SIgs occurs by cleavage of the pIgR to release its extracellular part as a pIg-bound secretory component, whereas free secretory components are derived from an unoccupied receptor. The joining chain (J chain) is crucial in pIg/SIg formation because it serves to polymerize Igs and endows them with a binding site for the pIgR. In this study, we show that the J chain from divergent tetrapods including mammals, birds, and amphibians efficiently induced polymerization of human IgA, whereas the J chain from nurse shark (a lower vertebrate) did not. Correctly assembled polymers showed high affinity to human pIgR. Sequence analysis of the J chain identified two regions, conserved only in tetrapods, which by mutational analysis were found essential for pIgA-pIgR complexing. Furthermore, we isolated and characterized pIgR from the amphibian Xenopus laevis and demonstrated that its pIg binding domain showed high affinity to human pIgA. These results showed that the functional site of interaction between pIgR, J chain and Ig H chains is conserved in these species and suggests that SIgs originated in an ancestor common to tetrapods.

Mucosal surfaces are protected by polymeric immunoglobulins that are transported across the epithelium by the polymeric immunoglobulin receptor (pIgR). Only polymeric IgA and IgM containing a small polypeptide called the "joining" (J) chain can bind to the pIgR. J chain-positive IgA consists of dimers, and some larger polymers, whereas only IgM pentamers incorporate the J chain. We made domain swap chimeras between human IgA1 and IgM and found that the COOH-terminal domains of the heavy chains (C␣3 and C4, respectively) dictated the size of the polymers formed and also which polymers incorporated the J chain. We also showed that chimeric IgM molecules engineered to contain C␣3 were able to bind the rabbit pIgR. Since the rabbit pIgR normally does not bind IgM, these results suggest that the COOH-terminal domain of the polymeric immunoglobulins is primarily responsible for interaction with the pIgR. Finally, we made a novel chimeric IgA immunoglobulin, containing the terminal domain from IgM. This recombinant molecule formed J chain-containing pentamers that could, like IgA, efficiently form covalent complexes with the human pIgR ectodomain, known as secretory component.

Upon APC-targeted DNA vaccination, transfected cells secrete fusion proteins with targeting units specific for surface molecules on APC. In this study, we have tested several different targeting units for their ability to influence the magnitude and subclass of Ab responses to hemagglutinin from influenza A virus. The experiments employed bivalent homodimeric Ig-based molecules (vaccibodies). The overall efficiency in BALB/c mice depended on the targeting units in the following order: αMHC class II > αCD11c > αCD40 > Xcl-1 = MIP-1α > FliC > GM-CSF > Flt-3L > αDEC205. GM-CSF induced mainly IgG1, whereas Xcl1, MIP-1α, αCD40, and αDEC205 induced predominantly IgG2a. A more balanced mixture of IgG1 and IgG2a was observed with αCD11c, αMHC class II, Flt-3L, and FliC. Similar results of IgG subclass–skewing were obtained in Th1-prone C57BL/6 mice with a more limited panel of vaccines. IgG1 responses in BALB/c occurred early after immunization but declined relatively rapidly over time. IgG2a responses appeared later but lasted longer (>252 d) than IgG1 responses. The most efficient targeting units elicited short- and long-term protection against PR8 influenza (H1N1) virus in BALB/c mice. The results suggest that targeting of Xcr1+ conventional type 1 dendritic cells preferentially induces IgG2a responses, whereas simultaneous targeting of several dendritic cell subtypes also induces IgG1 responses. The induction of distinct subclass profiles by different surface molecules supports the APC–B cell synapse hypothesis. The results may contribute to generation of more potent DNA vaccines that elicit high levels of Abs with desired biologic effector functions.

Here we unravel the structural features of human IgM and IgA that govern their interaction with the human Fca/l receptor (hFca/lR). Ligand polymerization status was crucial for the interaction, because hFca/lR binding did not occur with monomeric Ab of either class. hFca/lR bound IgM with an affinity in the nanomolar range, whereas the affinity for dimeric IgA (dIgA) was tenfold lower. Panels of mutant IgM and dIgA were used to identify regions critical for hFca/lR binding. IgM binding required contributions from both Cl3 and Cl4 Fc domains, whereas for dIgA, an exposed loop in the Ca3 domain was crucial. This loop, comprising residues Pro440-Phe443, lies at the Fc domain interface and has been implicated in the binding of host receptors FcaRI and polymeric Ig receptor (pIgR), as well as IgA-binding proteins produced by certain pathogenic bacteria. Substitutions within the Pro440-Phe443 loop resulted in loss of hFca/lR binding. Furthermore, secretory component (SC, the extracellular portion of pIgR) and bacterial IgA-binding proteins were shown to inhibit the dIgA-hFca/lR interaction. Therefore, we have identified a motif in the IgA-Fc inter-domain region critical for hFca/lR interaction, and highlighted the multi-functional nature of a key site for protein-protein interaction at the IgA Fc domain interface.Key words: Human Fca/m receptor . IgA . IgM IntroductionA receptor for IgM and IgA, termed Fca/m receptor (Fca/mR), was first described in the mouse where it is constitutively expressed on the majority of B cells and macrophages [1]. Mouse Fca/mR (mFca/mR) can mediate endocytosis of IgM-coated targets, and may contribute to the primary stages of the immune response to microbes. It has been suggested that mFca/mR, due to its high affinity for IgM and intermediate affinity for IgA, may assist also in the maintenance of plasma concentrations of IgM and IgA [2]. Although there is no significant homology between Fca/mR and other known proteins, the N-terminal Ig-like domain of Fca/mR Ã These authors contributed equally to this work. Certain mAb raised against mFca/mR have been found to bind to a peptide encompassing the region from mFca/mR's Ig-like domain that corresponds to the Ig-binding motif in domain 1 of pIgR [6]. Interestingly, these mAb can block binding of IgA and IgM to mFca/mR. The fact that these same mAb cross-react with hFca/mR, which carries the conserved motif in its Ig-like domain, suggests that the site for IgM and IgA on hFca/mR lies in this same region. In contrast, the site(s) on human IgM and IgA that interacts with hFca/mR is unknown. To gain a better understanding of the structural requirements for the interaction of human IgA and IgM with hFca/mR, we have produced recombinant dIgA and pentameric IgM and have adopted a mutational approach to identify individual residues on the Fc involved in interaction with hFca/mR. We demonstrate that both the Cm3 and Cm4 domains of IgM are required for binding to the receptor, and that residues at the Fc inter-domain region of dIgA are critical for hFca...