3D Structures of IgA, IgM, and Components.

Immunoglobulin G (IgG) is currently the most studied immunoglobin class and is frequently used in antibody therapeutics in which its beneficial effector functions are exploited. IgG is composed of two heavy chains and two light chains, forming the basic antibody monomeric unit. In contrast, immunoglobulin A (IgA) and immunoglobulin M (IgM) are usually assembled into dimers or pentamers with the contribution of joining (J)-chains, which bind to the secretory component (SC) of the polymeric Ig receptor (pIgR) and are transported to the mucosal surface. IgA and IgM play a pivotal role in various immune responses, especially in mucosal immunity. Due to their structural complexity, 3D structural study of these molecules at atomic scale has been slow. With the emergence of cryo-EM and X-ray crystallographic techniques and the growing interest in the structure-function relationships of IgA and IgM, atomic-scale structural information on IgA-Fc and IgM-Fc has been accumulating. Here, we examine the 3D structures of IgA and IgM, including the J-chain and SC. Disulfide bridging and N-glycosylation on these molecules are also summarized. With the increasing information of structure-function relationships, IgA- and IgM-based monoclonal antibodies will be an effective option in the therapeutic field.

IgG4 and IgE transcripts in childhood allergic asthma reflect divergent antigen-driven selection.

The physiologic function of the "odd" Ab IgG4 remains enigmatic. IgG4 mediates immunotolerance, as, for example, during specific immunotherapy of allergies, but it mediates tissue damage in autoimmune pemphigus vulgaris and "IgG4-related disease." Approximately half of the circulating IgG4 molecules are bispecific owing to their unique ability to exchange half-molecules. Better understanding of the interrelation between IgG4 and IgE repertoires may yield insight into the pathogenesis of allergies and into potential novel therapies that modulate IgG4 responses. We aimed to compare the selective forces that forge the IgG4 and IgE repertoires in allergic asthma. Using an IgG4-specific RT-PCR, we amplified, cloned, and sequenced IgG4 H chain transcripts of PBMCs from 10 children with allergic asthma. We obtained 558 functional IgG4 sequences, of which 286 were unique. Compared with previously published unique IgE transcripts from the same blood samples, the somatic mutation rate was significantly enhanced in IgG4 transcripts (62 versus 83%; p < 0.001), whereas fewer IgG4 sequences displayed statistical evidence of Ag-driven selection (p < 0.001). On average, the hypervariable CDRH3 region was four nucleotides shorter in IgG4 than in IgE transcripts (p < 0.001). IgG4 transcripts in the circulation of children with allergic asthma reflect some characteristics of classical Ag-driven B2 immune responses but display less indication of Ag selection than do IgE transcripts. Although allergen-specific IgG4 can block IgE-mediated allergen presentation and degranulation of mast cells, key factors that influence the Ag-binding properties of the Ab differ between the overall repertoires of circulating IgG4- and IgE-expressing cells.

Human plasma-derived polymeric IgA and IgM antibodies associate with secretory component to yield biologically active secretory-like antibodies.

Immunotherapy with monoclonal and polyclonal immunoglobulin is successfully applied to improve many clinical conditions, including infection, autoimmune diseases, or immunodeficiency. Most immunoglobulin products, recombinant or plasma-derived, are based on IgG antibodies, whereas to date, the use of IgA for therapeutic application has remained anecdotal. In particular, purification or production of large quantities of secretory IgA (SIgA) for potential mucosal application has not been achieved. In this work, we sought to investigate whether polymeric IgA (pIgA) recovered from human plasma is able to associate with secretory component (SC) to generate SIgA-like molecules. We found that ∼15% of plasma pIgA carried J chain and displayed selective SC binding capacity either in a mixture with monomeric IgA (mIgA) or after purification. The recombinant SC associated covalently in a 1:1 stoichiometry with pIgA and with similar efficacy as colostrum-derived SC. In comparison with pIgA, the association with SC delayed degradation of SIgA by intestinal proteases. Similar results were obtained with plasma-derived IgM. In vitro, plasma-derived IgA and SIgA neutralized Shigella flexneri used as a model pathogen, resulting in a delay of bacteria-induced damage targeted to polarized Caco-2 cell monolayers. The sum of these novel data demonstrates that association of plasma-derived IgA or IgM with recombinant/colostrum-derived SC is feasible and yields SIgA- and SIgM-like molecules with similar biochemical and functional characteristics as mucosa-derived immunoglobulins.

Preferential V beta gene usage and lack of junctional sequence conservation among human T cell receptors specific for a tetanus toxin-derived peptide: evidence for a dominant role of a germline-encoded V region in antigen/major histocompatibility complex recognition.

To investigate the structural and genetic basis of the T cell response to defined peptide/major histocompatibility (MHC) class II complexes in humans, we established a large panel of T cell clones (61) from donors of different HLA-DR haplotypes and reactive with a tetanus toxin-derived peptide (tt830-844) recognized in association with most DR molecules (universal peptide). By using a bacterial enterotoxin-based proliferation assay and cDNA sequencing, we found preferential use of a particular V beta region gene segment, V beta 2.1, in three of the individuals studied (64%, n = 58), irrespective of whether the peptide was presented by the DR6wcI, DR4w4, or DRw11.1 and DRw11.2 alleles, demonstrating that shared MHC class II antigens are not required for shared V beta gene use by T cell receptors (TCRs) specific for this peptide. V alpha gene use was more heterogeneous, with at least seven different V alpha segments derived from five distinct families encoding alpha chains able to pair with V beta 2.1 chains to form a tt830-844/DR-specific binding site. Several cases were found of clones restricted to different DR alleles that expressed identical V beta and (or very closely related) V alpha gene segments and that differed only in their junctional sequences. Thus, changes in the putative complementary determining region 3 (CDR3) of the TCR may, in certain cases, alter MHC specificity and maintain peptide reactivity. Finally, in contrast to what has been observed in other defined peptide/MHC systems, a striking heterogeneity was found in the junctional regions of both alpha and beta chains, even for TCRs with identical V alpha and/or V beta gene segments and the same restriction. Among 14 anti-tt830-844 clones using the V beta 2.1 gene segment, 14 unique V beta-D-J beta junctions were found, with no evident conservation in length and/or amino acid composition. One interpretation for this apparent lack of coselection of specific junctional sequences in the context of a common V element, V beta 2.1, is that this V region plays a dominant role in the recognition of the tt830-844/DR complex.

Structure of the secretory immunoglobulin A core.

Secretory immunoglobulin A (sIgA) represents the immune system's first line of defense against mucosal pathogens. IgAs are transported across the epithelium, as dimers and higher-order polymers, by the polymeric immunoglobulin receptor (pIgR). Upon reaching the luminal side, sIgAs mediate host protection and pathogen neutralization. In recent years, an increasing amount of attention has been given to IgA as a novel therapeutic antibody. However, despite extensive studies, sIgA structures have remained elusive. Here, we determine the atomic resolution structures of dimeric, tetrameric, and pentameric IgA-Fc linked by the joining chain (JC) and in complex with the secretory component of the pIgR. We suggest a mechanism in which the JC templates IgA oligomerization and imparts asymmetry for pIgR binding and transcytosis. This framework will inform the design of future IgA-based therapeutics.

Structural insights into immunoglobulin M.

Immunoglobulin M (IgM) plays a pivotal role in both humoral and mucosal immunity. Its assembly and transport depend on the joining chain (J-chain) and the polymeric immunoglobulin receptor (pIgR), but the underlying molecular mechanisms of these processes are unclear. We report a cryo-electron microscopy structure of the Fc region of human IgM in complex with the J-chain and pIgR ectodomain. The IgM-Fc pentamer is formed asymmetrically, resembling a hexagon with a missing triangle. The tailpieces of IgM-Fc pack into an amyloid-like structure to stabilize the pentamer. The J-chain caps the tailpiece assembly and bridges the interaction between IgM-Fc and the polymeric immunoglobulin receptor, which undergoes a large conformational change to engage the IgM-J complex. These results provide a structural basis for the function of IgM.

Design of novel antimicrobial peptide dimer analogues with enhanced antimicrobial activity in vitro and in vivo by intermolecular triazole bridge strategy.

Currently, antimicrobial peptides have attracted considerable attention because of their broad-sprectum activity and low prognostic to induce antibiotic resistance. In our study, for the first time, a series of side-chain hybrid dimer peptides J-AA (Anoplin-Anoplin), J-RR (RW-RW), and J-AR (Anoplin-RW) based on the wasp peptide Anoplin and the arginine- and tryptophan-rich hexapeptide RW were designed and synthesized by click chemistry, with the intent to improve the antimicrobial efficacy of peptides against bacterial pathogens. The results showed that all dimer analogues exhibited up to a 4-16 fold increase in antimicrobial activity compared to the parental peptides against bacterial strains. Furthermore, the antimicrobial activity was confirmed by time-killing kinetics assay with two strains which showed that these dimer analogues at 1, 2×MIC were rapidly bactericidal and reduced the initial inoculum significantly during the first 2-6h. Notably, dimer peptides showed synergy and additivity effects when used in combination with conventional antibiotics rifampin or penicillin respectively against the multidrug-resistant strains. In the Escherichia coli-infected mouse model, all of hybrid dimer analogues had significantly lower degree of bacterial load than the untreated control group when injected once i.p. at 5mg/kg. In addition, the infected mice by methicillin-resistant (MRSA) strain could be effectively treated with J-RR. All of dimer analogues had membrane-active action mode. And the membrane-dependent mode of action signifies that peptides functions freely and without regard to conventional resistant mechanisms. Circular dichroism analyses of all dimer analogues showed a general predominance of α-helix conformation in 50% trifluoroethanol (TFE). Additionally, the acute toxicities study indicated that J-RR or J-AR did not show the signs of toxicity when adult mice exposed to concentration up to 120mg/kg. The 50% lethal dose (LD50) of J-AA was 53.6mg/kg. In conclusion, to design and synthesize side chain-hybrid dimer analogues via click chemistry may offer a new strategy for antibacterial therapeutic option.

Recombinant human J-chain: fix the protein aggregations and yield maximize.

Polymeric immunoglobulin (dimeric IgA and pentameric IgM) molecules can assembly by using the immunoglobulin J (joining) chain and across the epithelial cell layers. Based on its amino acid and gene sequences data, disulfide bond (2 bonds) assignment secondary structure predictions, and chemical properties, a model for J-chain folding has been proposed. However, the crystal structure of the J-chain protein is still far from obtained, because the J-chain expression and its protein downstream has a permanent aggregation problems, due to its two free thiol groups. Our work focused on the chemical blocking of free cysteines-SH or to mutate these cysteines into serine residues. The chemical blocking yielded partially soluble proteins with new structures (carboxyamidomethyl cysteine and carboxyamidomethyl methionine) at cysteine and methionine residues. While mutate the cysteines into serine has been yielded a complete soluble (11.5 mg/l) J-chain protein which migrate (SDS-PAGE) at 27 KDa. We were used pET22b expression vector and E. coli BL21 (DE3) to produce the J-chain protein. For maximization the production yield of j-chain foreign protein, the batch culture was developed. We described the scaling-up production in term of kinetic behavior to the recombinant E.coli and optimization of cultivation parameters in 3-L bench-top bioreactor. The process was automated through a computer aided data bioprocessing system AFS-BioCommand multi-process management program to regulate the cell growth rate, temperature, pH and agitation speed based on dissolved oxygen. The results showed an obvious increasing in biomass by 5.98 g/L after about 27 h [corrected]

Late events in assembly determine the polymeric structure and biological activity of secretory IgM.

IgM antibodies can be secreted in at least two functional polymeric forms that can be distinguished according to subunit composition. While IgM hexamers comprise six H2L2 monomeric subunits, pentamers contain an additional polypeptide, the J chain. In the presence of high abundance J chain protein, IgM pentamers are preferentially assembled at the expense of hexamers. To determine the mechanism by which J chain regulates the assembly process, we defined the point at which J chain is added to assembling polymers. We found no evidence for the presence of J chain in small IgM assembly intermediates of IgM, suggesting that it was not stably associated with these complexes. However, J chain was found associated with large polymeric IgM complexes exhibiting sedimentation properties of intracellular pentameric structures. These complexes were frequently not completely covalently assembled; however, complete covalent assembly of J chain-containing pentameric complexes did occur prior to their maturation in the Golgi. These data argue that pentameric structures are the substrate for J chain incorporation into assembling IgM and suggest that the incorporation of J chain is thermodynamically favored over the addition of a sixth monomeric subunit into an assembling polymer. We conclude that late events in IgM polymer assembly, specifically the insertion of J chain, the exclusion of an additional monomeric subunit, and the covalent closure of the pentameric IgM molecule, determine the polymeric structure and, consequently, the biological activity of secreted IgM.

ERdj5 is required as a disulfide reductase for degradation of misfolded proteins in the ER.

Membrane and secretory proteins cotranslationally enter and are folded in the endoplasmic reticulum (ER). Misfolded or unassembled proteins are discarded by a process known as ER-associated degradation (ERAD), which involves their retrotranslocation into the cytosol. ERAD substrates frequently contain disulfide bonds that must be cleaved before their retrotranslocation. Here, we found that an ER-resident protein ERdj5 had a reductase activity, cleaved the disulfide bonds of misfolded proteins, and accelerated ERAD through its physical and functional associations with EDEM (ER degradation-enhancing alpha-mannosidase-like protein) and an ER-resident chaperone BiP. Thus, ERdj5 is a member of a supramolecular ERAD complex that recognizes and unfolds misfolded proteins for their efficient retrotranslocation.

Human colostrum: identification of minor proteins in the aqueous phase by proteomics.

Human colostrum is an important source of protective, nutritional and developmental factors for the newborn. We have investigated the low abundance proteins in the aqueous phase of human colostrum, after depletion of the major proteins secretory IgA, lactoferrin, alpha-lactalbumin and HSA by immunoabsorption, using 2-D LC and gel-based proteomic methods. One hundred and fifty-one proteins were identified, 83 of which have not been previously reported in human colostrum, or milk. This is the first comprehensive proteomic analysis of human colostrum produced during the first 48 h of lactation.

IgA receptors in health and disease.

The varied interaction of the Fc region of IgA with receptors confers this antibody class with many of its unique properties. The epithelial polymeric Ig receptor on mucosal epithelial cells transports polymeric immunoglobulin A (pIgA) produced by mucosal B cells to the mucosal surface where, in complex with the secretory component (SC), this secretory immunoglobulin A (SIgA) excludes the multitude of dietary, environmental, and microbial antigens that continuously bombard the mucosae. In health, this IgA-mediated exclusion not only forms the initial defence against infection, it also spares the systemic immune system from potentially deleterious responses to innocuous antigens which can otherwise culminate in inflammatory bowel disease or asthma. Beyond antigen exclusion, in closer encounters with antigens, IgA receptors play roles in protective immunity and disease. FcaRI is the principal myeloid IgA receptor and is responsible for differing IgA-mediated effector responses such as respiratory burst, degranulation, and phagocytosis variously by granulyoctes, monocytes, and macrophages. Furthermore an unknown IgA receptor specific for the secretory component (SC) elicits powerful effector responses from eosinophils. On dendritic cells, FcaRI participates in antigen presentation while on microfold cells, key cells in mucosal antigen presentation, another unknown IgA receptor functions in the transport of antigens across the mucosal epithelial barrier. The activity of another uncharacterized IgA1/IgD receptor on T cells may affect autoimmune disorders. The interplay of different IgA receptors affects immune complex deposition in the common renal disease immunoglobulin A nephropathy (IgAN). Finally, the therapeutic application of various IgA receptors has been sought in the areas of infectious disease, vaccines, and cancer.

The J chain is essential for polymeric Ig receptor-mediated epithelial transport of IgA.

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 (Cys(13):Cys(101) and Cys(109):Cys(134)) were also required for the binding of pIgA to SC but, interestingly, not for IgA polymerization. Conversely, the last intrachain disulfide bridge (Cys(72):Cys(92)) was not essential for either of these two J chain functions. Finally, we demonstrated that the presence of only Cys(15) or Cys(69) 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.

The joining (J) chain is present in invertebrates that do not express immunoglobulins.

Joining (J) chain is a component of polymeric, but not monomeric, immunoglobulin (Ig) molecules and may play a role in their polymerization and transport across epithelial cells. To date, study of the J chain has been confined to vertebrates that produce Ig and in which the J chain displays a considerable degree of structural homology. The role of the J chain in Ig polymerization has been questioned and, since the J chain can be expressed in lymphoid cells that do not produce Ig, it is possible that the J chain may have other functions. To explore this possibility, we have surveyed J-chain gene, mRNA, and protein expression by using reverse transcriptase-coupled PCR, Northern blot analysis, and immunoblot analysis in invertebrate species that do not produce Ig. We report that the J-chain gene is expressed in invertebrates (Mollusca, Annelida, Arthropoda, Echinodermata, and Holothuroidea), as well as in representative vertebrates (Mammalia, Teleostei, Amphibia). Furthermore, J-chain cDNA from the earthworm has a high degree of homology (68-76%) to human, mouse, and bovine J chains. Immunohistochemical studies reveal that the J chain is localized in the mucous cells of body surfaces, intestinal epithelial cells, and macrophage-like cells of the earthworm and slug. This study suggests that the J chain is a primitive polypeptide that arose before the evolution of Ig molecules and remains highly conserved in extent invertebrates and vertebrates.

Interplay of J chain and disulfide bonding in assembly of polymeric IgM.

Normal mouse IgM is synthesized as hexamers in the absence of J chain and as pentamers in its presence. Previous work has suggested that polymer size is also closely related to formation of the inter-mu chain disulfide bond mediated by cysteine 414, one of three cysteines involved in inter-mu chain bonding. This correlation in turn suggested that formation of C414-C414 might be required for J chain to influence how IgM assembles and that formation of C414-C414 might affect the J chain/IgM stoichiometry. To test such hypotheses we have used cell lines which either expressed or did not express J chain to produce IgM in which serine was substituted for C414. In contrast to the case of IgM assembled from normal mu chains, IgM-S414 was secreted mostly as pentamers and tetramers but not as hexamers, irrespective of J chain synthesis. These results indicate that the role of J chain as modulator of IgM structure and function requires C414. Moreover, a more detailed analysis of the structure of J-plus and J-minus IgM-S414 revealed that J chain, in fact, influenced the nature of secreted IgM-S414: In the absence of J chain, some IgM-S414 was secreted as dimers and trimers, while in the presence of J chain, some IgM was secreted as non-covalently assembled pentamers. These results imply that disulfide bonding can occur differently from the pattern depicted in conventional models of IgM structure.

Rheumatoid factor idiotypic and antigenic specificity is strongly influenced by the light chain VJ junction.

The aim of this study was to define the structural basis for rheumatoid factor (RF) specificity and for the expression of the RF light chain-associated Ids, 4C9 and 6B6.6, by determining the reactivity of recombined heavy and light chains of Ig derived from monoclonal B cell lines of patients with rheumatoid arthritis and of light chains with site-directed mutations. We found that expression of the 4C9 and 6B6.6 Ids resulted from use of the VkIIIa genes Humkv 328 and Vg, but only in the presence of a permissive VJ junction. Expression of the Ids was independent of heavy chain use for the Humkv328-encoded light chains, but was highly dependent on the associated heavy chain for the Vg-encoded light chains. The RF specificity of the Abs was primarily heavy chain dependent, but the light chain VJ junction was critical in determining the relative avidity of the Abs for Fc. Our study points to the critical contribution of the somatically generated VJ junction to RF autoantibody specificity and to the expression of the two RF-associated Ids studied.