A competitive mechanism for staphylococcal toxin SSL7 inhibiting the leukocyte IgA receptor, Fc alphaRI, is revealed by SSL7 binding at the C alpha2/C alpha3 interface of IgA

Leukocyte recruitment and effector functions like phagocytosis and respiratory burst are key elements of immunity to infection. Pathogen survival is dependent upon the ability to overwhelm, evade or inhibit the immune system. Pathogenic group A and group B streptococci are well known to produce virulence factors that block the binding of IgA to the leukocyte IgA receptor, Fc alphaRI, thereby inhibiting IgA-mediated immunity. Recently we found Staphylococcus aureus also interferes with IgA-mediated effector functions as the putative virulence factor SSL7 also binds IgA and blocks binding to Fc alphaRI. Herein we report that SSL7 and Fc alphaRI bind many of the same key residues in the Fc region of human IgA. Residues Leu-257 and Leu-258 in domain C alpha2 and residues 440-443 PLAF in C alpha3 of IgA lie at the C alpha2/C alpha3 interface and make major contributions to the binding of both the leukocyte receptor Fc alphaRI and SSL7. It is remarkable this S. aureus IgA binding factor and unrelated factors from streptococci are functionally convergent, all targeting a number of the same residues in the IgA Fc, which comprise the binding site for the leukocyte IgA receptor, Fc alphaRI.     

Functional basis for complement evasion by staphylococcal superantigen‐like 7

The human pathogen Staphylococcus aureus has a plethora of virulence factors that promote its colonization and survival in the host. Among such immune modulators are staphylococcal superantigen‐like (SSL) proteins, comprising a family of 14 small, secreted molecules that seem to interfere with the host innate immune system. SSL7 has been described to bind immunoglobulin A (IgA) and complement C5, thereby inhibiting IgA‐FcαRI binding and serum killing of Escherichia coli. As C5a generation, in contrast to C5b‐9‐mediated lysis, is crucial for immune defence against staphylococci, we investigated the impact of SSL7 on staphylococcal‐induced C5a‐mediated effects. Here, we show that SSL7 inhibits C5a generation induced by staphylococcal opsonization, slightly enhanced by its IgA‐binding capacity. Moreover, we demonstrate a strong protective activity of SSL7 against staphylococcal clearance in human whole blood. SSL7 strongly inhibited the C5a‐induced phagocytosis of S. aureus and oxidative burst in an in vitro whole‐blood inflammation model. Furthermore, we found that SSL7 affects all three pathways of complement activation and inhibits the cleavage of C5 by interference of its binding to C5 convertases. Finally, SSL7 effects were also demonstrated in vivo. In a murine model of immune complex peritonitis, SSL7 abrogated the C5a‐driven influx of neutrophils in mouse peritoneum.     

Pentraxins and IgA share a binding hot‐spot on FcαRI

The pentraxins, C‐reactive protein (CRP), and serum amyloid P component (SAP) have previously been shown to function as innate opsonins through interactions with Fcγ receptors. The molecular details of these interactions were elucidated by the crystal structure of SAP in complex with FcγRIIA. More recently, pentraxins were shown to bind and activate FcαRI (CD89), the receptor for IgA. Here, we used mutations of the receptor based on a docking model to further examine pentraxin recognition by FcαRI. The solution binding of pentraxins to six FcαRI alanine cluster mutants revealed that mutations Y35A and R82A, on the C‐and F‐strands of the D1 domain, respectively, markedly reduced receptor binding to CRP and SAP. These residues are in the IgA‐binding site of the receptor, and thus, significantly affected receptor binding to IgA. The shared pentraxin and IgA‐binding site on FcαRI is further supported by the results of a solution binding competition assay. In addition to the IgA‐binding site, pentraxins appear to interact with a broader region of the receptor as the mutation in the C′‐strand (R48A/E49A) enhanced pentraxin binding. Unlike Fcγ receptors, the H129A/I130A and R178A mutations on the BC‐ and FG‐loops of D2 domain, respectively, had little effect on FcαRI binding to the pentraxins. In conclusion, our data suggest that the pentraxins recognize a similar site on FcαRI as IgA.     

The staphylococcal superantigen-like protein 7 binds IgA and complement C5 and inhibits IgA-Fc alpha RI binding and serum killing of bacteria

The staphylococcal superantigen-like proteins (SSLs) are close relatives of the superantigens but are coded for by a separate gene cluster within a 19-kb region of the pathogenicity island SaPIn2. rSSL7 (formally known as SET1) bound with high affinity (K(D), 1.1 nM) to the monomeric form of human IgA1 and IgA2 plus serum IgA from primate, pig, rat, and horse. SSL7 also bound the secretory form of IgA found in milk from human, cow, and sheep, and inhibited IgA binding to cell surface FcalphaRI (CD89) and to a soluble form of the FcalphaRI protein. In addition to IgA, SSL7 bound complement factor C5 from human (K(D), 18 nM), primate, sheep, pig, and rabbit serum, and inhibited complement-mediated hemolysis and serum killing of a Gram-negative organism Escherichia coli. SSL7 is a superantigen-like protein secreted from Staphylococcus aureus that blocks IgA-FcR interactions and inhibits complement, leading to increased survival of a sensitive bacterium in blood.     

A common theme in interaction of bacterial immunoglobulin-binding proteins with immunoglobulins illustrated in the equine system

The M protein of Streptococcus equi subsp. equi known as fibrinogen-binding protein (FgBP) is a cell wall-associated protein with antiphagocytic activity that binds IgG. Recombinant versions of the seven equine IgG subclasses were used to investigate the subclass specificity of FgBP. FgBP bound predominantly to equine IgG4 and IgG7, with little or no binding to the other subclasses. Competitive binding experiments revealed that FgBP could inhibit the binding of staphylococcal protein A and streptococcal protein G to both IgG4 and IgG7, implicating the Fc interdomain region in binding to FgBP. To identify which of the two IgG Fc domains contributed to the interaction with FgBP, we tested two human IgG1/IgA1 domain swap mutants and found that both domains are required for full binding, with the CH3 domain playing a critical role. The binding site for FgBP was further localized using recombinant equine IgG7 antibodies with single or double point mutations to residues lying at the CH2-CH3 interface. We found that interaction of FgBP with equine IgG4 and IgG7 was able to disrupt C1q binding and antibody-mediated activation of the classical complement pathway, demonstrating an effective means by which S. equi may evade the immune response. The mode of interaction of FgBP with IgG fits a common theme for bacterial Ig-binding proteins. Remarkably, for those interactions studied in detail, it emerges that all the Ig-binding proteins target the CH2-CH3 domain interface, regardless of specificity for IgG or IgA, streptococcal or staphylococcal origin, or host species (equine or human).     

The crystal structure of staphylococcal superantigen-like protein 11 in complex with sialyl Lewis X reveals the mechanism for cell binding and immune inhibition

Staphylococcus aureus is a major pathogen that produces a family of 14 staphylococcal superantigen-like (SSL) proteins, which are structurally similar to superantigens but do not stimulate T cells. SSL11 is one member of the family that is found in all staphylococcal strains. Recombinant SSL11 bound to granulocytes and monocytes through a sialic acid-dependent mechanism and was rapidly internalized. SSL11 also bound to sialic acid-containing glycoproteins, such as the Fc receptor for IgA (FcalphaRI) and P-selectin glycoprotein ligand-1 (PSGL-1), and inhibited neutrophil attachment to a P-selectin-coated surface. Biosensor analysis of two SSL11 alleles binding to sialyl Lewis X [sLe(x)- Neu5Acalpha2-3Galbeta1-4(Fuc1-3)GlcNAc] coupled to bovine serum albumin gave dissociation constants of 0.7 and 7 mum respectively. Binding of SSL11 to a glycan array revealed specificity for glycans containing the trisaccharide sialyllactosamine (sLacNac - Neu5Acalpha2-3Galbeta1-4GlcNAc). A 1.6 A resolution crystal structure of SSL11 complexed with sLe(x) revealed a discrete binding site in the C-terminal beta-grasp domain, with predominant interactions with the sialic acid and galactose residues. A single amino acid mutation in the carbohydrate binding site abolished all SSL11 binding. Thus, SSL11 is a staphylococcal protein that targets myeloid cells by binding sialyllactosamine-containing glycoproteins.     

The IgG Fc contains distinct Fc receptor (FcR) binding sites: the leukocyte receptors Fc gamma RI and Fc gamma RIIa bind to a region in the Fc distinct from that recognized by neonatal FcR and protein A

The CH2-CH3 interface of the IgG Fc domain contains the binding sites for a number of Fc receptors including Staphylococcal protein A and the neonatal Fc receptor (FcRn). It has recently been proposed that the CH2-CH3 interface also contains the principal binding site for an isoform of the low affinity IgG Fc receptor II (Fc gamma RIIb). The Fc gamma RI and Fc gamma RII binding sites have previously been mapped to the lower hinge and the adjacent surface of the CH2 domain although contributions of the CH2-CH3 interface to binding have been suggested. This study addresses the question whether the CH2-CH3 interface plays a role in the interaction of IgG with Fc gamma RI and Fc gamma RIIa. We demonstrate that recombinant soluble murine Fc gamma RI and human Fc gamma RIIa did not compete with protein A and FcRn for binding to IgG, and that the CH2-CH3 interface therefore appears not to be involved in Fc gamma RI and Fc gamma RIIa binding. The importance of the lower hinge was confirmed by introducing mutations in the proposed binding site (LL234,235AA) which abrogated binding of recombinant soluble Fc gamma RIIa to human IgG1. We conclude that the lower hinge and the adjacent region of the CH2 domain of IgG Fc is critical for the interaction between Fc gamma RIIa and human IgG, whereas contributions of the CH2-CH3 interface appear to be insignificant.     

Protein glycosylation in infectious disease pathobiology and treatment

A host of bacteria and viruses are dependent on O-linked and N-linked glycosylation to perform vital biological functions. Pathogens often have integral proteins that participate in host-cell interactions such as receptor binding and fusion with host membrane. Fusion proteins from a broad range of disparate viruses, such as paramyxovirus, HIV, ebola, and the influenza viruses share a variety of common features that are augmented by glycosylation. Each of these viruses contain multiple glycosylation sites that must be processed and modified by the host post-translational machinery to be fusogenically active. In most viruses, glycosylation plays a role in biogenesis, stability, antigenicity and infectivity. In bacteria, glycosylation events play an important role in the formation of flagellin and pili and are vitally important to adherence, attachment, infectivity and immune evasion. With the importance of glycosylation to pathogen survival, it is clear that a better understanding of the processes is needed to understand the pathogen requirement for glycosylation and to capitalize on this requirement for the development of novel therapeutics.     

Episodes of natural selection shaped the interactions of IgA-Fc with FcalphaRI and bacterial decoy proteins

FcalphaRI, a receptor for IgA-Fc, recruits myeloid cells to attack IgA-coated pathogens. By competing with FcalphaRI for IgA, bacterial decoys, like SSL7 of Staphylococcus aureus, subvert this defense. We examined how pathogen selection has driven the diversification and coevolution of IgA and FcalphaRI. In higher primates, the IgA binding site of FcalphaRI diversified under positive selection, a strong episode occurring in hominoid ancestors about the time of the IgA gene duplication. The differential binding of SSL7 to IgA-Fc of different species correlates with substitution at seven positions in IgA-Fc, two of which were positively selected in higher primates. Two others, which reduce SSL7 binding, emerged during episodes of positive selection in the rabbit and rodent lineages. The FcalphaRI-IgA interaction evolves episodically under two types of positive selection: pressure from pathogen decoys selects for IgA escape variants which, in turn, selects for FcalphaRI variants to keep up with the novel IgA. When FcalphaRI cannot keep up, its function is lost and the gene becomes susceptible to elimination, as occurred in the mouse genome, either by chance or selection on one of the many linked, variable immune system genes. A cluster of positively selected residues presents a putative binding site for unknown IgA-binding factors.     

IgA-specific proteins of pathogenic bacteria

Data on structure and specificity of bacterial IgA receptors (IgA-binding M-like proteins Arp4 and Sir22 from hemolytic streptococci of serogroup A, β-antigen from hemolytic streptococci of serogroup B, and SSL family proteins from Staphylococcus aureus) are surveyed in this review. The principal conclusion derived from comparison is the fact that all bacterial receptors bind the same site in the IgA molecule overlapping with the binding site of endogenous human IgA receptor CD89. We assume that this site, consisting of spatially close amino acid strands Leu257-Gly259 in domain Cα2 and Pro440-Phe443 in domain Cα3, is subject to conformational rearrangement induced by the binding of antigen in the IgA active site.     

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.     

Cytoplasmic domain of human Fcalpha/mu receptor is required for ligand internalization

The Fcalpha/mu receptor (Fcα/μR), a type I transmembrane protein, is an immunoglobulin Fc receptor for both IgA and IgM. Its functions in immune defense are not clear at present. In this work, human Fcα/μR was expressed in CHO, 293T, and COS-7 cells to study its biochemical functions. Fcα/μR expressed by CHO and 293T was only in monomer form in cytoplasma and the monomeric receptor could not bind IgA or IgM. In comparison, Fcα/μR expressed by COS-7 cells had both monomer and dimer forms. The binding assay showed that Fcα/μR expressed by COS-7 cells could bind IgM strongly and IgA weakly, implying that dimeric receptor could be expressed on cell membrane and functioned. The bound IgM could be internalized and the internalization was abolished when the cytoplasmic domain of Fcα/μR was truncated. Therefore, the cytoplasmic portion of human Fcα/μR is required in the internalization.     

Mapping of the binding site for FcμR in human IgM-Fc

FcμR is a high-affinity receptor for the Fc portion of human IgM. It participates in B cell activation, cell survival and proliferation, but the full range of its functions remains to be elucidated. The receptor has an extracellular immunoglobulin (Ig)-like domain homologous to those in Fcα/μR and pIgR, but unlike these two other IgM receptors which also bind IgA, FcμR exhibits a binding specificity for only IgM-Fc. Previous studies have suggested that the IgM/FcμR interaction mainly involves the Cμ4 domains with possible contributions from either Cμ3 or Cμ2. To define the binding site more precisely, we generated three recombinant IgM-Fc proteins with specific mutations in the Cμ3 and Cμ4 domains, as well as a construct lacking the Cμ2 domains, and analyzed their interaction with the extracellular Ig-like domain of FcμR using surface plasmon resonance analysis. There is a binding site for FcμR in each IgM heavy chain. Neither the absence of the Cμ2 domains nor the quadruple mutant D340S/Q341G/D342S/T343S (in Cμ3 adjacent to Cμ2) affected FcμR binding, whereas double mutant K361D/D416R (in Cμ3 at the Cμ4 interface) substantially decreased binding, and a single mutation Q510R (in Cμ4) completely abolished FcμR binding. We conclude that glutamine at position 510 in Cμ4 is critical for IgM binding to FcμR. This will facilitate discrimination between the distinct effects of FcμR interactions with soluble IgM and with the IgM BCR.     

Characterization of the ligand binding site of the bovine IgA Fc receptor (bFc alpha R)

Recently, we identified a bovine IgA Fc receptor (bFc alpha R), which shows high homology to the human myeloid Fc alpha R, CD89. IgA binding has previously been shown to depend on several specific residues located in the B-C and F-G loops of the membrane-distal extracellular domain 1 of CD89. To compare the ligand binding properties of these two Fc alpha Rs, we have mapped the IgA binding site of bFc alpha R. We show that, in common with CD89, Tyr-35 in the B-C loop is essential for IgA binding. However, in contrast to earlier observations on CD89, mutation of residues in the F-G loop did not significantly inhibit IgA binding.     

Dual roles of PspC, a surface protein of Streptococcus pneumoniae, in binding human secretory IgA and factor H

Streptococcus pneumoniae, also known as the pneumococcus, contains several surface proteins that along with the polysaccharide capsule function in antiphagocytic activities and evasion of the host immune system. These pneumococcal proteins interact with the host immune system in various ways and possess a wide range of biological activities that suggests that they may be involved at different stages of pneumococcal infection. PspC, also known as CbpA and SpsA, is one of several pneumococcal surface proteins that binds host proteins, including factor H (FH) and secretory IgA (sIgA) via the secretory component. Previous work by our laboratory has demonstrated that PspC on the surface of live pneumococcal cells binds FH. This paper provides evidence that FH activity is maintained in the presence of PspC and that the PspC binding site is located in the short consensus repeat 6-10 region of FH. We also report for the first time that although both FH and sIgA binding has been localized to the alpha-helical domain of PspC, the binding of FH to PspC is not inhibited by sIgA. ELISA, surface plasmon resonance, and flow cytometry indicate that the two host proteins do not compete for binding with PspC and likely do not share the same binding sites. We confirmed by Western analysis that the binding sites are separate using recombinant PspC proteins. These PspC variants bind FH yet fail to bind sIgA. Thus, we conclude that FH and sIgA can bind concurrently to the alpha-helical region of PspC.     

The specificity of the IgA receptor purified from human neutrophils.

A receptor for IgA was purified from human polymorphonuclear neutrophils (PMN) by affinity chromatography on human serum IgA-Sepharose. The receptor appeared on SDS/polyacrylamide gels as a diffuse band with an apparent molecular mass of 50-70 kDa, whether reduced or non-reduced. During purification, the protein showed remarkable stability to proteolytic digestion by endogenous PMN proteinases. Purified radioiodinated receptor re-bound to IgA-Sepharose, but not to IgG-Sepharose or BSA-Sepharose. The binding of the receptor to IgA-Sepharose was inhibited in a dose-dependent manner by human serum IgA1 or IgA2 or secretory IgA1 or IgA2, but not by IgG or IgM. Binding of receptor to IgA-Sepharose was also inhibited by the Fc fragment of IgA, but not by the Fab fragment. An IgA fragment produced by digestion with pepsin which lacks the CH3 domain also inhibited binding, but to a more limited extent than did the whole IgA molecule.     

Histamine modulates in vitro IgG production by pokeweed mitogen-stimulated human mononuclear cells

The specificity of the receptor for IgA (RFcα) on human peripheral blood monocytes and polymorphonuclear (PMN) cells was evaluated by the ability of various human IgA preparations to inhibit rosette formation between these cells and IgA-sensitized ox erythrocytes. RFc?α on PMNS and monocytes were blocked by both monomer and dimer IgA preparations indicating that multivalent expression of Fc regions does not play a major role in receptor binding and that neither secretory component nor J chain significantly influences the binding of RFcα to IgA. Immunoglobulins of both the IgA1 and IgA2 subclasses inhibited IgA rosette formation and were in fact quite similar in their efficiency of blocking of RFcα. An IgA paraprotein without a Cα₃ domain was an even better inhibitor of IgA rosette formation than the IgA1 or IgA2 immunoglobulins. This implicated the Cα₂ domain as the site on IgA which interacts with RFcα. Furthermore, that this Cα₃-deficient IgA, which exists as a half molecule, was very efficient at blocking RFcα also demonstrated that multivalent Fc expression is not important to binding of RFcα and moreover that the site on IgA which interacts with RFcα is not dependent on H-chain pairing. RFcα on both PMN cells and monocytes were susceptible to proteolysis by pronase at concentrations which did not affect the receptor for IgG on these cells. Within 18 hr after removal of RFcα these cells had resynthesized and displayed this receptor. Although it is unclear whether IgA alone can mediate the effector functions of PMNs and monocytes in mucosal areas, the present studies define more clearly the specificity and regenerative capacity of RFcα and provide a basis for understanding the role of receptors for IgA and the cells with which they are associated in immune defense especially on the mucosal surfaces.     

Production,characterization, and in vivo half-life extension of polymeric IgA molecules in mice

ABSTRACT

IgA antibodies have broad potential as a novel therapeutic platform based on their superior receptor-mediated cytotoxic activity, potent neutralization of pathogens, and ability to transcytose across mucosal barriers via polymeric immunoglobulin receptor (pIgR)-mediated transport, compared to traditional IgG-based drugs. However, the transition of IgA into clinical development has been challenged by complex expression and characterization, as well as rapid serum clearance that is thought to be mediated by glycan receptor scavenging of recombinantly produced IgA monomer bearing incompletely sialylated N-linked glycans. Here, we present a comprehensive biochemical, biophysical, and structural characterization of recombinantly produced monomeric, dimeric and polymeric human IgA. We further explore two strategies to overcome the rapid serum clearance of polymeric IgA: removal of all N-linked glycosylation sites creating an aglycosylated polymeric IgA and engineering in FcRn binding with the generation of a polymeric IgG-IgA Fc fusion. While previous reports and the results presented in this study indicate that glycan-mediated clearance plays a major role for monomeric IgA, systemic clearance of polymeric IgA in mice is predominantly controlled by mechanisms other than glycan receptor clearance, such as pIgR-mediated transcytosis. The developed IgA platform now provides the potential to specifically target pIgR expressing tissues, while maintaining low systemic exposure.     

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.     

Staphylococcal superantigen-like protein 10 binds to phosphatidylserine and apoptotic cells

Staphylococcal superantigen-like proteins (SSLs) are a family of exoproteins that have structural similarities to staphylococcal superantigens. Although SSLs do not have superantigenic activity, some of them have been reported to bind to host immune related molecules and they have been implicated in immune evasion by S. aureus. In this study, we showed that SSL10 is capable of binding to phospholipids. SSL10 bound to phosphatidylserine (PS) containing liposome, but not to phosphatidylcholine liposome. SSL10, but not SSL7, bound to PS containing liposome, suggesting that SSL10 specifically binds to PS. Analysis of PS binding ability among recombinant truncated SSL10 fragments revealed that the β-barrel in the N-terminal oligonucleotide/oligosaccharide-binding (OB)-fold domain contributes to PS binding capacity. Fluorescein isothiocyanate labeled OB-fold of SSL10 stained hydrogen peroxide treated Jurkat cells. Annexin V is widely utilized for detection of apoptosis. Unlike annexin V, the OB-fold domain of SSL10 also bound to apoptotic cells in the presence of EDTA, suggesting that the OB-fold of SSL10 recognizes PS and apoptotic cells in a Ca(2+) independent manner. These findings suggest SSL10 and its derived peptides may be a novel detection tool for apoptotic cells.