Structure and function relationships in IgA.

Immunoglobulin A (IgA) has a critical role in immune defense particularly at the mucosal surfaces, and is equipped to do so by the unique structural attributes of its heavy chain and by its ability to polymerize. Here, we provide an overview of human IgA structure, describing the distinguishing features of the IgA1 and IgA2 subclasses and mapping the sites of interaction with host receptors important for IgA's functional repertoire. Remarkably, these same interaction sites are targeted by binding proteins and proteases produced by various pathogens as a means to subvert the protective IgA response. As interest in the prospect of therapeutic IgA-based monoclonal antibodies grows, the emerging understanding of the relationship between IgA structure and function will be invaluable for maximizing the potential of these novel reagents.

IgA in the horse: cloning of equine polymeric Ig receptor and J chain and characterization of recombinant forms of equine IgA.

As in other mammals, immunoglobulin A (IgA) in the horse has a key role in immune defense. To better dissect equine IgA function, we isolated complementary DNA (cDNA) clones for equine J chain and polymeric Ig receptor (pIgR). When coexpressed with equine IgA, equine J chain promoted efficient IgA polymerization. A truncated version of equine pIgR, equivalent to secretory component, bound with nanomolar affinity to recombinant equine and human dimeric IgA but not with monomeric IgA from either species. Searches of the equine genome localized equine J chain and pIgR to chromosomes 3 and 5, respectively, with J chain and pIgR coding sequence distributed across 4 and 11 exons, respectively. Comparisons of transcriptional regulatory sequences suggest that horse and human pIgR expression is controlled through common regulatory mechanisms that are less conserved in rodents. These studies pave the way for full dissection of equine IgA function and open up possibilities for immune-based treatment of equine diseases.

Functional and clinical consequences of Fc receptor polymorphic and copy number variants.

Receptors for immunoglobulins (Fc receptors) play a central role during an immune response, as they mediate the specific recognition of antigens of almost infinite diversity by leucocytes, thereby linking the humoral and cellular components of immunity. Indeed, engagement of Fc receptors by immunoglobulins initiates a range of immunoregulatory processes that might also play a role in disease pathogenesis. In the circulation, five main types of immunoglobulins (Ig) exist - namely IgG, IgA, IgE, IgM and IgD and receptors with the ability to recognize and bind to IgG (Fc gamma receptor family), IgE (Fc epsilon RI and CD23), IgA (CD89; Fc alpha/microR) and IgM (Fc alpha/microR) have been identified and characterized. However, it is astonishing that nearly all the known human Fc receptors display extensive genetic variation with clear implications for their function, thus representing a substantial genetic risk factor for the pathogenesis of a range of chronic inflammatory disorders.

The human IgA-Fc alpha receptor interaction and its blockade by streptococcal IgA-binding proteins.

IgA plays a key role in immune defence of the mucosal surfaces. IgA can trigger elimination mechanisms against pathogens through the interaction of its Fc region with Fc alpha Rs (receptors specific for the Fc region of IgA) present on neutrophils, macrophages, monocytes and eosinophils. The human Fc alpha R (CD89) shares homology with receptors specific for the Fc region of IgG (Fc gamma Rs) and IgE (Fc epsilon RIs), but is a more distantly related member of the receptor family. CD89 interacts with residues lying at the interface of the two domains of IgA Fc, a site quite distinct from the homologous regions at the top of IgG and IgE Fc recognized by Fc gamma R and Fc epsilon RI respectively. Certain pathogenic bacteria express surface proteins that bind to human IgA Fc. Experiments with domain-swap antibodies and mutant IgAs indicate that binding of three such proteins (Sir22 and Arp4 of Streptococcus pyogenes and beta protein of group B streptococci) depend on sites in the Fc interdomain region of IgA, the binding region also used by CD89. Further, we have found that the streptococcal proteins can inhibit interaction of IgA with CD89, and have thereby identified a mechanism by which a bacterial IgA-binding protein may modulate IgA effector function.

Amino acid sequence requirements in the human IgA1 hinge for cleavage by streptococcal IgA1 proteases.

All the IgA1 proteases of the different pathogenic species of Streptococcus cleave the hinge of the alpha chain of human IgA1 only at one proline-threonine peptide bond. In order to study the importance of these amino acids for cleavage, several hinge mutant recombinant IgA1 antibodies were constructed. The mutations were found to be without major effect upon the structure or functional abilities of the antibodies. However, they had a major effect upon their sensitivity to cleavage by some of the IgA1 proteases.

Streptococcal IgA-binding proteins bind in the Calpha 2-Calpha 3 interdomain region and inhibit binding of IgA to human CD89.

Certain pathogenic bacteria express surface proteins that bind to the Fc part of human IgA or IgG. These bacterial proteins are important as immunochemical tools and model systems, but their biological function is still unclear. Here, we describe studies of three streptococcal proteins that bind IgA: the Sir22 and Arp4 proteins of Streptococcus pyogenes and the unrelated beta protein of group B streptococcus. Analysis of IgA domain swap and point mutants indicated that two loops at the Calpha2/Calpha3 domain interface are critical for binding of the streptococcal proteins. This region is also used in binding the human IgA receptor CD89, an important mediator of IgA effector function. In agreement with this finding, the three IgA-binding proteins and a 50-residue IgA-binding peptide derived from Sir22 blocked the ability of IgA to bind CD89. Further, the Arp4 protein inhibited the ability of IgA to trigger a neutrophil respiratory burst via CD89. Thus, we have identified residues on IgA-Fc that play a key role in binding of different streptococcal IgA-binding proteins, and we have identified a mechanism by which a bacterial IgA-binding protein may interfere with IgA effector function.

Fc receptors and immunity to parasites.

Fc receptors (FcRs) are crucial in the immune system; they mediate a plethora of biological functions as diverse as antigen presentation, phagocytosis, cytotoxicity, induction of inflammatory cascades and modulation of immune responses. Parasites, in order to survive in the immunocompetent host, have devised ingenious methods to subvert this important aspect of the immune response. This article discusses the current thinking on FcRs, their role in immunity to parasites, and immune evasion strategies employed by parasites in their attempt to neutralize the important immune defense mechanisms mediated by these molecules.

Cleavage of human IgE mediated by Schistosoma mansoni.

BACKGROUND: IgE-mediated mechanisms are important in protection against helminth parasites. However, schistosomes are long-lived in mammalian hosts, presumably as a result of immune evasion strategies. We sought evidence for one such strategy, namely specific cleavage of host IgE. METHODS: Human IgE, IgA and IgG were incubated with extracts from cercarial and schistosomular stages of Schistosoma mansoni or with schistosomular culture supernatants. The resulting products were analysed by Western blotting with Ig-specific antibodies. Numerous protease inhibitors were assessed for ability to inhibit the observed cleavage of IgE by the extracts. Partial purification of the IgE-proteolytic activity from cercarial extract was achieved by gel filtration. To test IgE function, we compared the abilities of untreated and schistosomular-treated IgE to mediate rosette formation through interaction with Fcepsilon receptors. RESULTS: Cercarial and schistosomular extracts were found to cleave human, mouse and rat IgE but not human IgA1, IgA2 or IgG1. Schistosomular culture supernatants displayed similar proteolytic activity towards IgE. Immunoblotting suggested that cleavage occurred close to the Cepsilon2/Cepsilon3 domain interface of the IgE heavy chain. PMSF and elastatinal inhibited cleavage, suggesting that the protease involved is an elastase-like serine protease, particularly since porcine pancreatic elastase also cleaved IgE to give similar-sized products. Further, the chloromethyl ketone derivatized peptide MeO-Suc-Ala-Ala-Pro-Leu- CMK, known to specifically inhibit the schistosome elastase, prevented IgE cleavage. Cleavage of human IgE rendered the antibody molecule unable to interact with U937 cells expressing FcepsilonRII. CONCLUSIONS: An elastase-like protease in S. mansoni is able to render IgE non-functional.

Cleavage of a recombinant human immunoglobulin A2 (IgA2)-IgA1 hybrid antibody by certain bacterial IgA1 proteases.

To understand more about the factors influencing the cleavage of immunoglobulin A1 (IgA1) by microbial IgA1 proteases, a recombinant human IgA2/IgA1 hybrid molecule was generated. In the hybrid, termed IgA2/A1 half hinge, a seven-amino-acid sequence corresponding to one half of the duplicated sequence making up the IgA1 hinge was incorporated into the equivalent site in IgA2. Insertion of the IgA1 half hinge into IgA2 did not affect antigen binding capacity or the functional activity of the hybrid molecule, as judged by its ability to bind to IgA Fcalpha receptors and trigger respiratory bursts in neutrophils. Although the IgA2/A1 hybrid contained only half of the IgA1 hinge, it was found to be cleaved by a variety of different bacterial IgA1 proteases, including representatives of those that cleave IgA1 in the different duplicated halves of the hinge, namely, those of Prevotella melaninogenica, Streptococcus pneumoniae, S. sanguis, Neisseria meningitidis types 1 and 2, N. gonorrhoeae types 1 and 2, and Haemophilus influenzae type 2. Thus, for these enzymes the recognition site for IgA1 cleavage is contained within half of the IgA1 hinge region; additional distal elements, if required, are provided by either an IgA1 or an IgA2 framework. In contrast, the IgA2/A1 hybrid appeared to be resistant to cleavage with S. oralis and some H. influenzae type 1 IgA1 proteases, suggesting these enzymes require additional determinants for efficient substrate recognition.

Identification of residues in the CH2/CH3 domain interface of IgA essential for interaction with the human fcalpha receptor (FcalphaR) CD89.

Cellular receptors for IgA (FcalphaR) mediate important protective functions. An extensive panel of site-directed mutant IgAs was used to identify IgA residues critical for FcalphaR (CD89) binding and triggering. Although a tailpiece-deleted IgA1 was able to bind and trigger CD89, antibodies featuring CH3 domain exchanges between human IgA1 and IgG1 could not, indicating that both domains but not the tailpiece are required for FcalphaR recognition. To further investigate the role of the interdomain region, numerous IgA1s, each with a point substitution in either of two interdomain loops (Leu-257-Gly-259 in Calpha2; Pro-440-Phe-443 in Calpha3), were generated. With only one exception (G259R), substitutions produced either ablation (L257R, P440A, A442R, F443R) or marked reduction (P440R) in CD89 binding and triggering. Further support for involvement of these interdomain loops was provided by interspecies comparisons of IgA. Thus a human IgA1 mutant, LA441-442MN, which mimicked the mouse IgA loop sequence through substitution of two adjacent residues in the Calpha3 loop, was found, like mouse IgA, not to bind CD89. In contrast, bovine IgA1, identical to human IgA1 within these interdomain loops despite numerous differences elsewhere in the Fc region, did bind CD89. We have thus identified motifs in the interdomain region of IgA Fc critical for FcalphaR binding and triggering, significantly enhancing present understanding of the molecular basis of the IgA-FcalphaR interaction.

The Fab and Fc fragments of IgA1 exhibit a different arrangement from that in IgG: a study by X-ray and neutron solution scattering and homology modelling.

Human immunoglobulin A (IgA) is an abundant antibody that mediates immune protection at mucosal surfaces as well as in plasma. The IgA1 isotype contains two four-domain Fab fragments and a four-domain Fc fragment analogous to that in immunoglobulin G (IgG), linked by a glycosylated hinge region made up of 23 amino acid residues from each of the heavy chains. IgA1 also has two 18 residue tailpieces at the C terminus of each heavy chain in the Fc fragment. X-ray scattering using H2O buffers and neutron scattering using 100 % 2H2O buffers were performed on monomeric IgA1 and a recombinant IgA1 that lacks the tailpiece (PTerm455). The radii of gyration RG from Guinier analyses were similar at 6.11-6.20 nm for IgA1 and 5.84-6.16 nm for PTerm455, and their cross-sectional radii of gyration RXS were also similar. The similarity of the RG and RXS values suggests that the tailpiece of IgA1 is not extended outwards in solution. The IgA1 RG values are higher than those for IgG, and the distance distribution function P(r) showed two distinct peaks, whereas a single peak was observed for IgG. Both results show that the hinge of IgA1 results in an extended Fab and Fc arrangement that is different from that in IgG. Automated curve-fit searches constrained by homology models for the Fab and Fc fragments were used to model the experimental IgA1 scattering curves. A translational search to optimise the relative arrangement of the Fab and Fc fragments held in a fixed orientation resembling that in IgG was not successful in fitting the scattering data. A new molecular dynamics curve-fit search method generated IgA1 hinge structures to which the Fab and Fc fragments could be connected in any orientation. A search based on these identified a limited family of IgA1 structures that gave good curve fits to the experimental data. These contained extended hinges of length about 7 nm that positioned the Fab-to-Fab centre-to-centre separation 17 nm apart while keeping the corresponding Fab-to-Fc separation at 9 nm. The resulting extended T-shaped IgA1 structures are distinct from IgG structures previously determined by scattering and crystallography which have Fab-to-Fab and Fab-to-Fc centre-to-centre separations of 7-9 nm and 6-8 nm, respectively. It was concluded that the IgA1 hinge is structurally distinct from that in IgG, and this results in a markedly different antibody structure that may account for a unique immune role of monomeric IgA1 in plasma and mucosa.

The glycosylation and structure of human serum IgA1, Fab, and Fc regions and the role of N-glycosylation on Fcα receptor interactions.

The human serum immunoglobulins IgG and IgA1 are produced in bone marrow and both interact with specific cellular receptors that mediate biological events. In contrast to IgA1, the glycosylation of IgG has been well characterized, and its interaction with various Fc receptors (Fc Rs) has been well studied. In this paper, we have analyzed the glycosylation of IgA1 and IgA1 Fab and Fc as well as three recombinant IgA1 molecules, including two N-glycosylation mutants. Amino acid sequencing data of the IgA1 Fc O-glycosylated hinge region indicated that O-glycans are located at Thr228, Ser230, and Ser232, while O-glycan sites at Thr225 and Thr236 are partially occupied. Over 90% of the N-glycans in IgA1 were sialylated, in contrast to IgG, where < 10% contain sialic acid. This paper contains the first report of Fab glycosylation in IgA1, and (in contrast to IgG Fab, which contains only N-linked glycans) both N- and O-linked oligosaccharides were identified. Analysis of the N-glycans attached to recombinant IgA1 indicated that the Cα 2 N-glycosylation site contained mostly biantennary glycans, while the tailpiece site, absent in IgG, contained mostly triantennary structures. Further analysis of these data suggested that processing at one Fc N-glycosylation site affects the other. Neutrophil Fcα R binding studies, using recombinant IgA1, indicated that neither the tailpiece region nor the N-glycans in the C alpha 2 domain contribute to IgA1-neutrophil Fcα R binding. This contrasts with IgG, where removal of the Fc N-glycans reduces binding to the Fcγ R. The primary sequence and disulfide bond pattern of IgA1, together with the crystal structures of IgG1 Fc and mouse IgA Fab and the glycan sequencing data, were used to generate a molecular model of IgA1. As a consequence of both the primary sequence and S-S bond pattern, the N-glycans in IgA1 Fc are not confined within the inter-α-chain space. The accessibility of the Cα 2 N-glycans provides an explanation for the increased sialylation and galactosylation of IgA1 Fc over that of IgG Fc N-glycans, which are confined in the space between the two Cγ 2 domains. This also suggests why in contrast to IgG Fc, the IgA1 N-glycans are not undergalactosylated in rheumatoid arthritis.

Structural requirements for assembly of dimeric IgA probed by site-directed mutagenesis of J chain and a cysteine residue of the alpha-chain CH2 domain.

The structural features of J chain required for interaction with IgA in IgA dimer assembly were investigated by coexpression of wild-type and mutant forms of J chain with IgA1 in CHO cells. With wild-type J chain, a mixture of J chain-containing dimers and monomers was secreted. Substitution of Cys14 of J chain with Ser resulted in expression of only monomer IgA covalently associated with J chain. Similarly, mutation of Cys68 to Ser also resulted in expression predominantly of a monomer IgA-J chain species. These results suggest that Cys14 and Cys68 play critical roles in formation of J chain-containing IgA dimers, with each forming a disulfide bridge to an IgA monomer. Substitution of Asn48 with Ala, to prevent attachment of N-linked carbohydrate to J chain, also resulted in markedly reduced dimer assembly, suggesting a requirement for the sugar moiety in J chain function. We also mutated Cys311 on the C alpha2 domain of the IgA heavy chain to Ser. When coexpressed with wild-type J chain, this mutant was still capable of forming dimers, indicating that this residue was not involved in dimerization. Taken together, our results are consistent with an arrangement in which IgA monomers are linked end-to-end with J chain interposed.

Alternative splicing of the human IgA Fc receptor CD89 in neutrophils and eosinophils.

Receptors for the Fc portion of IgA (Fc alpha R) trigger important immunological elimination processes against IgA-coated targets. Investigation of human Fc alpha R (CD89) transcripts in neutrophils, eosinophils and a monocyte-like cell line, THP-1, with the use of reverse transcriptase PCR, Northern blotting and RNase protection analysis, has provided evidence in these cell types for at least two distinct transcripts generated by alternative splicing. The cDNAs derived from the two major transcripts of both neutrophils and eosinophils have been cloned and sequenced. For both cell types, the larger clone represents the previously described full-length receptor, whereas the second, shorter, splice variant lacks the entire second, membrane-proximal, Ig-like domain. Stable CHO-K1 transfectants have been obtained for both full-length and truncated variant neutrophil receptors. Whereas the full-length receptor is recognized by a panel of five anti-Fc alpha R monoclonal antibodies (mAbs), the shorter variant is bound weakly by only two of the antibodies, suggesting that the epitopes recognized by the majority of the mAbs lie at least in part in the second Ig-like domain of Fc alpha R. Both full-length and splice variant forms of the receptor bind secretory IgA, but the weak binding to serum IgA seen with the full-length receptor is not evident with the shorter variant. Alternative splicing might therefore serve as a means of diversifying Fc alpha R structure and function.

Mutagenesis of the human IgA1 heavy chain tailpiece that prevents dimer assembly.

The structural features of the human IgA1 tailpiece required for interaction with J chain in IgA dimer assembly were investigated using a protein engineering approach. Wild-type and mutant forms of IgA1 were expressed in the mouse myeloma cell line, J558L, which endogenously expresses J chain. Wild-type IgA1 was secreted as a mixture of dimers and monomers. Deletion of the entire tailpiece by stop codon introduction completely prevented dimer formation. Similarly, substitution of the penultimate residue of the tailpiece, Cys471, with serine resulted in the secretion of IgA monomers alone. Substitution of Asn459 with alanine to prevent attachment of N-linked carbohydrate to the tailpiece also resulted in markedly reduced dimer assembly. These results indicate the critical role played by the tailpiece, and Cys471 in particular, in IgA dimerization. In addition, we found tailpiece-deleted IgA1 and the Cys to Ser471 mutant IgA1 were secreted as mixtures of covalently associated monomers (alpha 2L2) and alpha L half-molecules. The tailpiece may thus play some role in promoting the association of alpha-chains required for IgA monomer assembly.