A small domain (6.5 kDa) of bacterial protein G inhibits C3 covalent binding to the Fc region of IgG immune complexes

Attachment of the complement component C3 to antigen-antibody (Ag-Ab) complexes (immune complexes, IC) is the key molecular event responsible for the elimination of many Ag in the form of Ag-Ab-C3b. The CH1 domain and the Fc region of the Ab, which have previously been involved in the binding of C3b, are also the targets of several bacterial IgG-binding proteins, particularly proteins G and A. Here we describe the ability of a small recombinant protein G domain (B2; 6.5 kDa) to inhibit the covalent binding of C3b to the Fc portion of IgG without affecting the binding to the Fab part. Protein G (B2 domain) produced a remarkable inhibition of covalent binding of C3b to IC formed with rabbit IgG, but none with the F(ab ′ )₂ fragment, indicating that B2 interferes with the C3b binding to the Fc region. A weak inhibition was observed with IC formed with mouse IgG2b which preferentially binds B2 domain on the CH1 domain of the Fab. To confirm these data, recombinant single-chain Ab devoid of CH1 domains (scAb), and including the rabbit or human Fc portion (hinge-CH2-CH3), were produced and used to form IC. Protein G-B2 domain inhibited C3b binding to IC formed with scAb of either human or rabbit constant regions, supporting the view of a specific blockade of C3b binding to the Fc region. A similar inhibition of C3b binding was observed using protein A instead of protein G B2 domain and the same set of IC. On the CH1 domain, C3b and B2 bind on opposite faces, and therefore do not interfere with each other in their binding. However, B2 domain bound to the inter-CH2-CH3 region impedes the C3b binding to the Fc. This inhibition clarifies the specificity of C3b for the different regions of IgG and explains how bacterial IgG-binding proteins provide the bacteria with a mechanism of evasion from the opsonizing action of complement and contribute to the virulence. This could be a general mechanism of escape because protein G binds the majority of mammalian Ig.     

Functional interplay between the B-box 2 and the B30.2(SPRY) domains of TRIM5alpha

The retroviral restriction factors, TRIM5alpha and TRIMCyp, consist of RING and B-box 2 domains separated by a coiled coil from carboxy-terminal domains. These carboxy-terminal domains (the B30.2(SPRY) domain in TRIM5alpha and the cyclophilin A domain in TRIMCyp) recognize the retroviral capsid. Here we show that some B-box 2 changes in TRIM5alpha, but not in TRIMCyp, resulted in decreased human immunodeficiency virus (HIV-1) capsid binding. The phenotypic effects of these B-box 2 changes on the restriction of retroviral infection depended on the potency of restriction and the affinity of the TRIM5alpha interaction with the viral capsid, two properties specified by the B30.2(SPRY) domain. Thus, some alterations in the TRIM5alpha B-box 2 domain apparently affect the orientation or conformation of the B30.2(SPRY) domain, influencing capsid recognition.     

The N-terminal end of the CH2 domain of chimeric human IgG1 anti-HLA-DR is necessary for C1q, Fc gamma RI and Fc gamma RIII binding.

We have found that amino acid residues necessary for C1q and Fc gamma R binding of human IgG1 are located in the N-terminal region of the CH2 domain, residues 231-238, using a matched set of engineered antibodies based on the anti-HLA-DR antibody L243. Changing the leucine 235 in the CH2 region of IgG3 and IgG4 to glutamic acid was already known to abolish Fc gamma RI binding. We have confirmed this for IgG1 and also found a concomitant abolition of human complement lysis with retention of Fc gamma RIII-mediated function. Changing the glycine at 237 to alanine of IgG1 also abolished Fc gamma RI binding and reduced human complement lysis and Fc gamma RIII-mediated function. Exchanging the whole region 233-236 with the sequence found in human IgG2, abolished Fc gamma RI binding and human complement lysis and reduced Fc gamma RIII-mediated function of IgG1. In contrast, a change in the previously described C1q-binding motif, from lysine at 320 to alanine, had no effect on IgG1-mediated complement lysis.     

The localization of the binding site(s) on human IgG1 for the Fc receptors on homologous monocytes and heterologous mouse macrophages.

Two different methods, a rosette assay and a direct binding assay, have been employed in an examination of the binding of human IgG1 to mouse macrophages. In both cases, inhibition of IgG binding was demonstrated by Fc (CH2 + CH3 domains) and pFc' (CH3 domains) fragments of human IgG. In a homologous system, the binding of 125I-human IgG to human peripheral-blood monocytes was inhibited by the Fc fragment whereas the pFc' fragment was inactive. Scatchard plot analysis of the binding data from both the heterologous and homologous systems allowed association constants and numbers of receptors per cell to be calculated. A more thorough examination of the possible location of IgG Fc-receptor binding sites was made using less orthodox proteolytic cleavage fragments of IgG. The site on human IgG1 responsible for binding to mouse macrophage Fc receptors was confirmed to be within the CH3 domains. Human IgG1 binding to homologous monocytes was shown, using a dimeric C gamma 2 domain fragment, to be via the CH2 domains, and was dependent on the integrity of the covalent interaction between the C gamma 2 domains at the hinge region.     

Identification of residues within the L2 region of rhesus TRIM5α that are required for retroviral restriction and cytoplasmic body localization

The intracellular restriction factor TRIM5α, inhibits infection by numerous retroviruses in a species-specific manner. The best characterized example of this restriction is the TRIM5α protein from rhesus macaques (rhTRIM5α), which potently inhibits HIV-1 infection. TRIM5α localizes to cytoplasmic assemblies of protein referred to as cytoplasmic bodies, though the role that these bodies play in retroviral restriction is unclear. We employed a series of truncation mutants to identify a discrete region, located within the Linker2 region connecting the coiled-coil and B30.2/PRYSPRY domains of TRIM5α, which is required for cytoplasmic body localization. Deletion of this region in the context of full-length rhTRIM5α abrogates cytoplasmic body localization. Alanine mutagenesis of the residues in this region identifies two stretches of amino acids that are required for both cytoplasmic body localization and retroviral restriction. This work suggests that the determinants that mediate TRIM5α localization to cytoplasmic bodies play a requisite role in retroviral restriction.     

Immunoglobulin Binding Specificities of the Homology Regions (Domains) of Protein A

Protein A binds immunoglobulins and it has two target structures, one in Feγ (C_H) and the other in selected V_H regions. The protein has five homology regions (domains). A, B, C, D, and E. Fc-binding and V_H-binding have been reported to be non-competitive, suggesting that different domains are responsible for the binding of the two ligands. On the other hand, all five domains have been reported to bind Fc. I studied binding of different immunoglobulins by protein A or its domain B (rBB). The results show that separate domains bind V_H and Fc. If all five domains are capable of binding Fc, the ones that bind V_h have low affinity for Fc. Furthermore, the number of Fc-binding domains varies depending on the type of the IgG being bound. Human IgG1 or lgG2 or rabbit IgG (Fc) seem to be bound by several domains (possibly four), and domain B is one of them. Mouse IgG1 or lgG2b are bound by fewer domains not including B. Murine lgG2a is also bound by fewer domains but B is one of them.     

A dominant-negative effect of cynomolgus monkey tripartite motif protein TRIM5alpha on anti-simian immunodeficiency virus SIVmac activity of an African green monkey orthologue

African green monkey (AGM) tripartite motif protein (TRIM) 5alpha can inhibit both human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus SIVmac, whereas cynomolgus monkey (CM) TRIM5alpha can inhibit HIV-1, but not SIVmac. We previously reported that the 17-amino-acid region and an adjacent 20-amino-acid duplication in the SPRY(B30.2) domain of AGM TRIM5alpha determined the species specificity. In the present study, we demonstrated that CM TRIM5alpha had a dominant-negative effect on the anti-SIVmac activity of AGM TRIM5alpha. In contrast, mutant TRIM5alphas lacking the 20-amino-acid duplication did not have the dominant-negative effect, even though they failed to restrict SIVmac. These results indicated that oligomerization of the SPRY domain is required for anti-SIVmac activity and suggest that tight interaction between the viral capsid and all three molecules in one TRIM5alpha trimer may not be necessary for restriction activity.     

Multiple binding sites on the CH2 domain of IgG for mouse Fc gamma R11.

Important mammalian defensive functions such as phagocytosis are triggered in leukocytes by the interaction of the Fc region of IgG with cell surface receptors (Fc gamma R). The CH2 domain of IgG has been implicated previously as the site of interaction with human and mouse Fc gamma R. This domain was mapped for interaction with mouse Fc gamma R11 expressed by the macrophage-like cell line P388D1, using two panels of a total of 32 site-directed mutants of mouse IgG2b and chimeric human IgG3 monoclonal antibodies. Two potential binding sites have been identified: one in or within the vicinity of the lower hinge site on IgG for human Fc gamma R1, and one within the binding site on IgG for Clq. The three mutant IgGs (Gly 237----Ala, Asn 297----Ala, and Glu 318----Ala) which do not interact in complexed form also fail to bind as monomers. A 1H NMR study of the three non-binding monomeric mutants suggests that the mutations are largely site-specific, indicating that IgG interacts with mouse Fc gamma R11 at two regions within the CH2 domain. This interaction dictates phagocytosis mediated by Fc gamma R11 of the P388D1 cell line.     

Molecular aspects of human FcgammaR interactions with IgG: functional and therapeutic consequences

The binding of IgG antibodies to receptors for the Fc region of IgG (FcgammaR) is a critical step for the initiation and/or the control of effector immune functions once immune complexes have been formed. Site-directed and random mutagenesis as well as domain-swapping, NMR and X-ray cristallography have made it possible to get detailed insights in the molecular mechanisms that govern IgG/FcgammaR interactions and to define some of the structural determinants that impact IgG binding to the various FcgammaR. It has demonstrated the role of particular stretches and individual residues located in the lower hinge region of the CH2 domain and in the CH2 and CH3 domains of the Fc region. The importance of the sugar components linked to asparagine 297 in the binding properties of IgG1, the human IgG isotype the most widely used in antibody-based therapies, has been also highlighted. These data have led to the engineering of a new generation of monoclonal antibodies for therapeutic use with optimized effector functions.     

Characterization of TRIM5alpha trimerization and its contribution to human immunodeficiency virus capsid binding

The coiled-coil domain of the tripartite motif (TRIM) family protein TRIM5alpha is required for trimerization and function as an antiretroviral restriction factor. Unlike the coiled-coil regions of other related TRIM proteins, the coiled coil of TRIM5alpha is not sufficient for multimerization. The linker region between the coiled-coil and B30.2 domains is necessary for efficient TRIM5alpha trimerization. Most of the hydrophilic residues predicted to be located on the surface-exposed face of the coiled coil can be altered without compromising TRIM5alpha antiviral activity against human immunodeficiency virus (HIV-1). However, changes that disrupt TRIM5alpha trimerization proportionately affect the ability of TRIM5alpha to bind HIV-1 capsid complexes. Therefore, TRIM5alpha trimerization makes a major contribution to its avidity for the retroviral capsid, and to the ability to restrict virus infection.     

Localization of the site of the murine IgG1 molecule that is involved in binding to the murine intestinal Fc receptor

Site-directed mutagenesis of a recombinant Fc hinge fragment has recently been used to localize the site of the murine IgG1 molecule that is involved in the control of catabolism (the “catabolic site”). In the current study, the effects of these CH2 and CH3 domain mutations (Ile 253 to Ala 253, His 310 to Ala 310, Gln 311 to Asn 311, His 433 to Ala 433 and Asn 434 to G1n 434) on intestinal transfer of Fc hinge fragments in neonatal mice have been analyzed. Studies using direct transfer and competition assays demonstrate that the mutations affect the transmission from intestinal lumen into serum in a way that correlates closely with the effects of the mutations on pharmacokinetics. Binding studies of several of the Fc hinge fragments to isolated neonatal brush borders have been used to confirm the in vivo transmission data. These analyses have resulted in the localization of the binding site for the intestinal transfer receptor, FcRn, to specific residues of the murine Fc hinge fragment. These residues are located at the CH2-CH3 domain interface and overlap with both the catabolic site and staphylococcal protein A (SpA) binding site. The pH dependence of IgG1 or Fc fragment binding to FcRn is consistent with the localization of the FcRn interaction site to a region of the Fc that encompasses two histidine residues (His 310 and His 433). To assess whether one or two FcRn binding sites per Fc hinge are required for intestinal transfer, a hybrid Fc hinge fragment comprising a heterodimer of one Fc hinge with the wild-type IgG1 sequence and a mutant Fc hinge with a defective catabolic site (mutated at His 310, G1n 311, His 433 and Asn 434) has been analyzed in direct and competition transmission assays. The studies demonstrate that the Fc hybrid is transferred with significantly reduced efficiency compared to the wild type Fc hinge homodimer and indicate that the binding to FcRn, and possibly subsequent transfer, is enhanced by the presence of two FcRn binding sites per Fc hinge fragment.     

The herpes simplex virus type 1 Fc receptor discriminates between IgG1 allotypes

Herpes simplex virus type 1 (HSV-1) expresses a complex of two virally encoded glycoproteins, gE and gl, which is capable of binding nonimmune human IgG. The gE-gl complex has thus become known as an Fc receptor (FcR), which reportedly binds human IgG subclasses in the order IgG4 > IgG1 > or = IgG2 and does not bind IgG3 from many individuals. There is, however, allelic variation in the genes encoding the human IgG1 heavy chain constant region and this gives rise to allotypes of IgG1. Using recombinant monoclonal IgG molecules of known isotype and mutants thereof we have unexpectedly discovered that the HSV-1 FcR discriminates between IgG1 allotypes. This is evidence of functional differences between IgG1 allotypes that may account for their distribution in populations. Furthermore, these findings suggest HSV-1 FcR binding sites on the IgG molecule some distance from the proposed binding site in the CH2-CH3 domain interface.     

Anti‐retroviral activity of TRIM5α

Human immunodeficiency virus type 1 (HIV‐1) shows a very narrow host range limited to humans and chimpanzees. Experimentally, HIV‐1 does not infect Old World monkeys, such as rhesus (Rh) and cynomolgus (CM) monkeys, and fails to replicate in activated CD4 positive T lymphocytes obtained from these monkeys. In contrast, simian immunodeficiency virus isolated from a macaque monkey (SIVmac) can replicate well in both Rh and CM. In 2004, tripartite motif 5α (TRIM5α) was identified as a host factor which plays an important role in the restricted host range of HIV‐1. Rh and CM TRIM5α restrict HIV‐1 infection but not SIVmac, while in comparison, anti‐viral activity of human TRIM5α against those viruses is very weak. TRIM5α consists of the RING, B‐box 2, coiled‐coil and SPRY (B30.2) domains. The RING domain is frequently found in E3 ubiquitin ligase and TRIM5α is degraded via the ubiquitin‐proteasome pathway during HIV‐1 restriction. TRIM5α recognises the multimerised capsid (viral core) of an incoming virus by its α‐isoform specific SPRY domain and is believed to be involved in innate immunity to control retroviral infection. Differences in amino acid sequences in the SPRY domain of TRIM5α of different monkey species were found to affect species‐specific restriction of retrovirus infection, while differences in amino acid sequences in the viral capsid protein determine viral sensitivity to restriction. Accurate structural analysis of the binding surface between the viral capsid protein and TRIM5α SPRY is thus required for the development of new antiretroviral drugs that enhance anti‐HIV‐1 activity of human TRIM5α. Copyright © 2009 John Wiley & Sons, Ltd.     

Comparative requirements for the restriction of retrovirus infection by TRIM5alpha and TRIMCyp

The restriction factors, TRIM5alpha in most primates and TRIMCyp in owl monkeys, block infection of various retroviruses soon after virus entry into the host cell. Rhesus monkey TRIM5alpha (TRIM5alpha rh) inhibits human immunodeficiency virus (HIV-1) and feline immunodeficiency virus (FIV) more potently than human TRIM5alpha (TRIM5alpha hu). TRIMCyp restricts infection of HIV-1, simian immunodeficiency virus of African green monkeys (SIV agm) and FIV. Early after infection, TRIMCyp, like TRIM5alpha rh and TRIM5alpha hu, decreased the amount of particulate viral capsid in the cytosol of infected cells. The requirements for the TRIMCyp and TRIM5alpha domains in restricting different retroviruses were investigated. Potent restriction of FIV by TRIMCyp occurred in the complete absence of RING and B-box 2 domains; by contrast, efficient FIV restriction by TRIM5alpha rh required these domains. Variable region 1 of the TRIM5alpha rh B30.2 domain contributed to the potency of HIV-1, FIV and equine infectious anemia virus restriction. Thus, although differences exist in the requirements of TRIMCyp and TRIM5alpha for RING/B-box 2 domains, both restriction factors exhibit mechanistic similarities.     

Investigation of the binding site of mouse IgG subclasses to homologous peritoneal macrophages.

The binding of mouse myeloma IgG1, IgG2a, IgG2b, IgG1 Fc, IgG2b Fc and a pepsin produced C-terminal subfragment of IgG1 Fc and IgG2b Fc (provisionally identified as pFc') to mouse peritoneal macrophages was investigated. The high affinity cytophilic antibodies belonged to IgG2 subclasses and the binding site of these antibodies was located in the CH3 homology region.     

Relationship between SPRY and B30.2 protein domains. Evolution of a component of immune defence?

SPRY and B30.2 are homologous domains which can be identified in 11 protein families encoded in the human genome. These include cell surface receptors of the immunoglobulin super-family (BTNs), negative regulators of the JAK/STAT pathway (SOCS-box SSB1-4) and proteins encoded by the numerous TRIM genes. Collectively, proteins containing SPRY and B30.2 domains cover a wide range of functions, including regulation of cytokine signalling (SOCS), RNA metabolism (DDX1, hnRNPs), intracellular calcium release (RyR receptors), immunity to retroviruses (TRIM5alpha) as well as regulatory and developmental processes (HERC1, Ash2L). In order to clarify the evolutionary relationship between the two domains, we compiled a curated database of SPRY and B30.2-domain sequences. We show that while SPRY domains are evolutionarily ancient, B30.2 domains, found in BTN and TRIM proteins, are a more recent evolutionary adaptation, comprising the combination of SPRY with an additional domain, PRY. The combination of SPRY and PRY to produce B30.2 domains may have been selected and maintained as a component of immune defence.     

Effect of zinc on human IgG1 and its FcγR interactions

In the present study, we show that histidines 310 and 435 at the CH2-CH3 interface of the Fc portion of human IgG1 can coordinate a Zn2+ and participate in the control of the CH2-CH2 interdomain opening. Structures obtained in the absence of Zn2+ have a reduced interdomain gap that likely hamper FcγR binding. This closed conformation of the Fc is stabilized by inter-CH2 domain sugar contacts. Zinc appears to counteract the sugar mediated constriction, suggesting that zinc could be an important control factor in IgG1/FcγR interactions. The results of binding studies performed in the presence of EDTA on FcγR expressing cells supports this hypothesis. When a mutated Fc fragment, in which histidines 310 and 435 have been substituted by lysines (Fc H/K), was compared with the wild-type Fc in crystallographic studies, we found that the mutations leave the interface unaltered but have a long-range effect on the CH2 interdomain separation. Moreover, these substitutions have a differential effect on the binding of IgG1 to Fcγ receptors and their functions. Interaction with the inhibitory FcγRIIB is strongly perturbed by the mutations and mutant IgG1 H/K only weakly engages this receptor. By contrast, higher affinity FcγR are mostly unaffected.     

Chimeric Fc receptors identify immunoglobulin-binding regions in human FcγRII and FcϵRI

FcγRII and Fc?RI are functionally distinct cell surface receptors for immunoglobulin (Ig); FcγRII binds IgG with low affinity, whereas Fc?RI binds IgE with high affinity, yet they are homologous in structure and sequence having extracellular regions containing two Ig-like domains with 38% amino acid identity. Chimeric receptors derived from human FcγRII and FcγRI were produced by exchanging homologous regions of the two receptors to define binding region(s) for IgG in FcγRII and IgE in Fc?RI. Firstly, a chimeric form of the Fc?RI α chain was produced by replacing the transmembrane region and cytoplasmic tail with that of FcγRII. This mutant α chain could be expressed on the cell surface independently of associated β and γ subunits, and retained high-affinity IgE binding, indicating that the extracellular region of the FcγRI α chain is sufficient for high-affinity IgE binding. Secondly, to identify the role of the individual domains in Fc binding of both FcγRII and FcγRI, chimeric receptors were generated by exchanging the first extracellular domains between FcγRII and the α chain mutant and used to demonstrate that the second extracellular domain of both receptors contains region(s) directly involved in Ig binding. Additional chimeric receptors were constructed to localize the Ig interactive regions in domain two of FcγRII and FcγRI; these identified a single region of IgG binding in FcγRII located between residues Ser¹³⁶ to Val¹⁶⁹, and at least three independent IgE binding regions in the FcγRI α chain, between residues Trp⁸⁷ to Lys¹²⁸, Tyr¹²⁹ to Asp¹⁴⁵, and Ser¹⁴⁶ to Val¹⁶⁹.     

The heterogeneity of bovine IgG2--V. Differences in the primary structure of bovine IgG2 allotypes.

The partial amino acid sequences of the gamma chains of the bovine IgG2a(A1) and IgG2a(A2) allotypes were determined. Sequence differences were found in the CH1 domain, the hinge region, and the CH3 domain. The hinge regions displayed only 71.4% similarity and all of the differences were of a radical nature. The A2 hinge has isoleucine instead of serine at 229, histidine for asparagine at 235, proline for histidine at 238, and cysteine instead of proline in position 234; the latter has the potential for forming an additional interheavy chain disulphide bridge. The occurrence of such a bridge could explain the presence of a pepsin fragment consisting of the hinge region and the Fc. A corresponding fragment is not obtained with the A1 allotype. Both allotypes have a shortened hinge region and a truncated CH2 domain. This feature is characteristic of all reported sequences of IgG2 proteins but not IgG1 in cattle and the goat. This structural feature may be important in subclass-specific recognition by Fc gamma receptors in ruminants. A surprising discovery was the occurrence of five substitutions in the CH3 domain of the IgG2a(A2) in comparison with the A1, which are shared with the CH3 of IgG1. These permit the occurrence of isoallotypic determinants and can explain the difficulty encountered in preparing A2-specific antisera during which adsorption with IgG1 is a routine procedure. The primary sequence data we report confirm the presence of major structural differences between the A allotypes of cattle that was suggested by previous work. The sequence of the A1 allotype most closely agrees with the two IgG2 sequences deduced from their nucleotide sequences whereas the sequence differences in the hinge and C-terminal CH3 make IgG2a(A2) unique. The structural differences between allotypes could have major consequences for such biological activities as phagocytosis, transepithelial transport, lymphocyte and complement activation.     

TRIM21: a cytosolic Fc receptor with broad antibody isotype specificity

Antibodies are key molecules in the fight against infections. Although previously thought to mediate protection solely in the extracellular environment, recent research has revealed that antibody-mediated protection extends to the cytosolic compartment of cells. This postentry viral defense mechanism requires binding of the antibody to a cytosolic Fc receptor named tripartite motif containing 21 (TRIM21). In contrast to other Fc receptors, TRIM21 shows remarkably broad isotype specificity as it does not only bind IgG but also IgM and IgA. When viral pathogens coated with these antibody isotypes enter the cytosol, TRIM21 is rapidly recruited and efficient neutralization occurs before the virus has had the time to replicate. In addition, inflammatory signaling is induced. As such, TRIM21 acts as a cytosolic sensor that engages antibodies that have failed to protect against infection in the extracellular environment. Here, we summarize our current understanding of how TRIM21 orchestrates humoral immunity in the cytosolic environment.