Soluble NSF attachment protein receptor molecular mimicry by a Legionella pneumophila Dot/Icm effector

Upon infection, Legionella pneumophila uses the Dot/Icm type IV secretion system to translocate effector proteins from the Legionella-containing vacuole (LCV) into the host cell cytoplasm. The effectors target a wide array of host cellular processes that aid LCV biogenesis, including the manipulation of membrane trafficking. In this study, we used a hidden Markov model screen to identify two novel, non-eukaryotic soluble NSF attachment protein receptor (SNARE) homologs: the bacterial Legionella SNARE effector A (LseA) and viral SNARE homolog A proteins. We characterized LseA as a Dot/Icm effector of L. pneumophila, which has close homology to the Qc-SNARE subfamily. The lseA gene was present in multiple sequenced L. pneumophila strains including Corby and was well distributed among L. pneumophila clinical and environmental isolates. Employing a variety of biochemical, cell biological and microbiological techniques, we found that farnesylated LseA localized to membranes associated with the Golgi complex in mammalian cells and LseA interacted with a subset of Qa-, Qb- and R-SNAREs in host cells. Our results suggested that LseA acts as a SNARE protein and has the potential to regulate or mediate membrane fusion events in Golgi-associated pathways.

Two monoclonal antibodies to precisely the same epitope of type ii collagen select non-crossreactive phage clones by phage display: Implications for autoimmunity and molecular mimicry

Two monoclonal antibodies (mAb) CB268 and CII-C1 to type II collagen (CII) react with precisely the same conformational epitope constituted by the residues ARGLT on the three chains of the CII triple helix. The antibodies share structural similarity, with most differences in the complementarity determining region 3 of the heavy chain (HCDR3). The fine reactivity of these mAbs was investigated by screening two nonameric phage-displayed random peptide libraries. For each mAb, there were phage clones (phagotopes) that reacted strongly by ELISA only with the selecting mAb, and inhibited binding to CII only for that mAb, not the alternate mAb. Nonetheless, a synthetic peptide RRLPFGSQM corresponding to an insert from a highly reactive CII-C1-selected phagotope, which was unreactive (and non-inhibitory) with CB268, inhibited the reactivity of CB268 with CII. Most phage-displayed peptides contained a motif in the first part of the molecule that consisted of two basic residues adjacent to at least one hydrophobic residue (e.g. RRL or LRR), but the second portion of the peptides differed for the two mAbs. We predict that conserved CDR sequences interact with the basic-basic-hydrophobic motif, whereas non-conserved amino acids in the binding sites (especially HCDR3) interact with unique peptide sequences and limit cross-reactivity. The observation that two mAbs can react identically with a single epitope on one antigen (CII), but show no cross-reactivity when tested against a second (phagotope) indicates that microorganisms could exhibit mimics capable of initiating autoimmunity without this being evident from conventional assays.

Molecular mimicry: Can epitope mimicry induce autoimmune disease?

Mimicry of host antigens by infectious agents may induce cross-reactive autoimmune responses to epitopes within host proteins which, in susceptible individuals, may tip the balance of immunological response versus tolerance toward response and subsequently lead to autoimmune disease. Epitope mimicry may indeed be involved in the pathogenesis of several diseases such as post-viral myocarditis or Chagas disease, but for many other diseases in which it has been implicated, such as insulin-dependent diabetes mellitis or rheumatoid arthritis, convincing evidence is still lacking. Even if an epitope mimic can support a cross-reactive T or B cell response in vitro, its ability to induce an autoimmune disease in vivo will depend upon the appropriate presentation of the mimicked host antigen in the target tissue and, in the case of T cell mimics, the ability of the mimicking epitope to induce a proliferative rather than anergizing response upon engagement of the MHC-peptide complex with the T cell receptor. B cell presentation of mimicking foreign antigen to T cells is a possible mechanism for instigating an autoimmune response to self antigens that in turn can lead to autoimmune disease under particular conditions of antigen presentation, secondary signalling and effector cell repertoire. In this review evidence in support of epitope mimicry is examined in the light of the necessary immunological considerations of the theory.

Molecular mimicry by antiidiotypic monoclonal antibody to gonadotropin releasing hormone

Antipeptide and antiidiotypic antibodies to several receptors are known to mimic their respective ligands in transducing signals on binding their receptors. In our attempts to study gonadotropin releasing hormone receptor, antipeptide and antiidiotypic monoclonal antibodies specific to the receptor were established earlier. The antipeptide mAb F1G4 was to a synthetic peptide corresponding to the extracellular domain of human GnRH receptor and the antiidiotypic mAb 4D10C1 was to the idiotype of a GnRH specific mAb. Here we report the physiological effects of the two mAbs on binding the receptor, as investigated using in vitro cultures of(a) human term placental villi and (b) rat pituitaries. The mAb 4D10C1 exerted a dose-dependent release of human chorionic gonadonopin in cultures of human term placental villi as well as luteinising and follicle stimulating hormones in cultures of rat pituitaries.

A Family of Helminth Molecules that Modulate Innate Cell Responses via Molecular Mimicry of Host Antimicrobial Peptides

Over the last decade a significant number of studies have highlighted the central role of host antimicrobial (or defence) peptides in modulating the response of innate immune cells to pathogen-associated ligands. In humans, the most widely studied antimicrobial peptide is LL-37, a 37-residue peptide containing an amphipathic helix that is released via proteolytic cleavage of the precursor protein CAP18. Owing to its ability to protect against lethal endotoxaemia and clinically-relevant bacterial infections, LL-37 and its derivatives are seen as attractive candidates for anti-sepsis therapies. We have identified a novel family of molecules secreted by parasitic helminths (helminth defence molecules; HDMs) that exhibit similar biochemical and functional characteristics to human defence peptides, particularly CAP18. The HDM secreted by Fasciola hepatica (FhHDM-1) adopts a predominantly alpha-helical structure in solution. Processing of FhHDM-1 by F. hepatica cathepsin L1 releases a 34-residue C-terminal fragment containing a conserved amphipathic helix. This is analogous to the proteolytic processing of CAP18 to release LL-37, which modulates innate cell activation by classical toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS). We show that full-length recombinant FhHDM-1 and a peptide analogue of the amphipathic C-terminus bind directly to LPS in a concentration-dependent manner, reducing its interaction with both LPS-binding protein (LBP) and the surface of macrophages. Furthermore, FhHDM-1 and the amphipathic C-terminal peptide protect mice against LPS-induced inflammation by significantly reducing the release of inflammatory mediators from macrophages. We propose that HDMs, by mimicking the function of host defence peptides, represent a novel family of innate cell modulators with therapeutic potential in anti-sepsis treatments and prevention of inflammation.

Genomic analysis of ERVWE2 locus in patients with multiple sclerosis: absence of genetic association but potential role of human endogenous retrovirus type W elements in molecular mimicry with myelin antigen

Human endogenous retroviruses (HERVs) arise from ancient infections of the host germline cells by exogenous retroviruses, constituting 8% of the human genome. Elevated level of envelope transcripts from HERVs-W has been detected in CSF, plasma and brain tissues from patients with Multiple Sclerosis (MS), most of them from Xq22.3, 15q21.3, and 6q21 chromosomes. However, since the locus Xq22.3 (ERVWE2) lack the 5' LTR promoter and the putative protein should be truncated due to a stop codon, we investigated the ERVWE2 genomic loci from 84 individuals, including MS patients with active HERV-W expression detected in PBMC. In addition, an automated search for promoter sequences in 20 kb nearby region of ERVWE2 reference sequence was performed. Several putative binding sites for cellular cofactors and enhancers were found, suggesting that transcription may occur via alternative promoters. However, ERVWE2 DNA sequencing of MS and healthy individuals revealed that all of them harbor a stop codon at site 39, undermining the expression of a full-length protein. Finally, since plaque formation in central nervous system (CNS) of MS patients is attributed to immunological mechanisms triggered by autoimmune attack against myelin, we also investigated the level of similarity between envelope protein and myelin oligodendrocyte glycoprotein (MOG). Comparison of the MOG to the envelope identified five retroviral regions similar to the Ig-like domain of MOG. Interestingly, one of them includes T and B cell epitopes, capable to induce T effector functions and circulating Abs in rats. In sum, although no DNA substitutions that would link ERVWE2 to the MS pathogeny was found, the similarity between the envelope protein to MOG extends the idea that ERVEW2 may be involved on the immunopathogenesis of MS, maybe facilitating the MOG recognizing by the immune system. Although awaiting experimental evidences, the data presented here may expand the scope of the endogenous retroviruses involvement on MS pathogenesis

Crossreactive recognition of viral, self, and bacterial peptide ligands by human class I-restricted cytotoxic T lymphocyte clonotypes: Implications for molecular mimicry in autoimmune disease

The immunodominant, CD8+ cytotoxic T lymphocyte (CTL) response to the HLA-B8-restricted peptide, RAKFKQLL, located in the Epstein–Barr virus immediate-early antigen, BZLF1, is characterized by a diverse T cell receptor (TCR) repertoire. Here, we show that this diversity can be partitioned on the basis of crossreactive cytotoxicity patterns involving the recognition of a self peptide—RSKFRQIV—located in a serine/threonine kinase and a bacterial peptide—RRKYKQII—located in Staphylococcus aureus replication initiation protein. Thus CTL clones that recognized the viral, self, and bacterial peptides expressed a highly restricted αβ TCR phenotype. The CTL clones that recognized viral and self peptides were more oligoclonal, whereas clones that strictly recognized the viral peptide displayed a diverse TCR profile. Interestingly, the self and bacterial peptides equally were substantially less effective than the cognate viral peptide in sensitizing target cell lysis, and also resulted only in a weak reactivation of memory CTLs in limiting dilution assays, whereas the cognate peptide was highly immunogenic. The described crossreactions show that human antiviral, CD8+ CTL responses can be shaped by peptide ligands derived from autoantigens and environmental bacterial antigens, thereby providing a firm structural basis for molecular mimicry involving class I-restricted CTLs in the pathogenesis of autoimmune disease.

Similar antigenic surfaces, rather than sequence homology, dictate T-cell epitope molecular mimicry.

Molecular mimicry, normally defined by the level of primary-sequence similarities between self and foreign antigens, has been considered a key element in the pathogenesis of autoimmunity. Here we describe an example of molecular mimicry between two overlapping peptides within a single self-antigen, both of which are recognized by the same human self-reactive T-cell clone. Two intervening peptides did not stimulate the T-cell clone, even though they share nine amino acids with the stimulatory peptides. Molecular modeling of major histocompatibility complex class II-peptide complexes suggests that both of the recognized peptides generate similar antigenic surfaces, although these are composed of different sets of amino acids. The molecular modeling of a peptide shifted one residue from the stimulatory peptide, which was recognized in the context of the same HLA molecule by another T-cell clone, generated a completely different antigenic surface. Functional studies using truncated peptides confirmed that the anchor residues of the two "mimicking" epitopes in the HLA groove differ. Our results show, for two natural epitopes, how molecular mimicry can occur and suggest that studies of potential antigenic surfaces, rather than sequence similarity, are necessary for analyzing suspected peptide mimicry.

Introduction: Epitope mimicry as a component cause of autoimmune disease

The causes of autoimmune diseases have yet to be fully elucidated. Autoantibodies, autoreactive T cell responses, the presence of a predisposing major histocompatibility complex (MHC) haplotype and responsiveness to corticosteroids are features, and some are possibly contributory causes of autoimmune disease. The most challenging question is how autoimmune diseases are triggered. Molecular mimicry of host cell determinants by epitopes of infectious agents with ensuing cross-reactivity is one of the most popular yet still controversial theories for the initiation of autoimmune diseases [1]. Throughout the 1990s, hundreds of research articles focusing to various extents on epitope mimicry, as it is more accurately described in an immunological context, were published annually. Many of these articles presented data that were consistent with the hypothesis of mimicry but that did not actually prove the theory. Other equally convincing reports indicated that epitope mimicry was not the cause of the autoimmune disease despite sequence similarity between molecules of infectious agents and the host. Some 20 years ago, Rothman [2] proposed a model for disease causation and I have used this as a framework to examine the role of epitope mimicry in the development of autoimmune disease. The thesis of Rothman’s model is that an effect, in this instance autoimmune disease, arises as a result of a cause. In most cases, multiple-component causes contribute synergistically to yield the effect, and each of these components alone is insufficient as a cause. Logically, some component causes, such as the presence of a particular autoimmune response, are also necessary causes.

Mimicry of 21. 5 KDa myelin basic protein peptide by a Maedi Visna virus polymerase peptide does not contribute to the pathogenesis of encephalitis in sheep

Epitope mimicry is the theory that an infectious agent such as a virus causes pathological effects via mimicry of host proteins and thus elicits a cross-reactive immune response to host tissues. Weise and Carnegie (1988) found a region of sequence similarity between the pol gene of the Maedi Visna virus (MVV), which induces demyelinating encephalitis in sheep, and myelin basic protein (MBP), which is known to induce experimental allergic encephalitis (EAE) in laboratory animals. In this study, cross-reactions between sera raised in sheep against synthetic peptides of MVV (TGKIPWILLPGR) and 21.5 kDa MBP (SGKVPWLKRPGR) were demonstrated using enzyme-linked immunosorbant assay (ELISA) and thin layer chromatography (TLC) immunoprobing. The antibody responses of MVV-infected sheep were investigated using ELISA against the peptides, and MBP protein, immunoprobing of the peptides on TPC plates and Western blotting against MBP. Slight significant reactions to the 21.5 kDa MBP peptide (P < 0.001) and to a lesser extent sheep MBP (P < 0.004) were detected in ELISA. The MBP peptide evoked stronger responses from more sera than the MVV peptide on immunoprobed TLC plates. On the Western blots, eight of the 23 sheep with Visna had serum reactivity to MBP. This slight reaction to MBP in MVV-infected sheep is of interest because of the immune responses to MBP evident in multiple sclerosis and EAE, but its relevance in Visna is limited since no correlation with disease severity was observed. The cell-mediated immune responses of MVV-infected sheep against similar peptides was assessed. The peptides did not stimulate proliferation of peripheral blood lymphocytes of MVV-infected sheep. Since the MVV peptide was not recognised by antibodies or T lymphocytes from MVV-infected and encephalic sheep, it was concluded that epitope mimicry of this 21.5 kDa MBP peptide by the similar MVV pol peptide was not contributing to the immunopathogenesis of Visna. The slight antibody response to MBP and the MBP peptide can be attributed to by-stander effects of the immunopathology of MVV-induced encephalitis.

Mimicry between HTLV-I and myelin basic protein: No response in HTLV-I-associated myelopathy patients

The reactivity to a peptide from the HTLV-I polyprotein (FKLPGLNSR) and a similar sequence from myelin basic protein (MBP) (FKLGGRDSR) was examined in relation to the proposal that mimicry of MBP by HTLV-I could be involved in autoimmune responses in HTLV-I-associated myelopathy (HAM). It was found that rabbit antibodies raised against the HTLV-I peptide recognised both peptides, with a titre of 1/10240 to the HTLV-I peptide and 1/5220 to the MBP peptide. Human sera from HAM patients and a HTLV-I carrier without HAM showed slightly higher responses to the HTLV-I peptide compared to the responses from uninfected human sera. HAM patients had greater responses to the HTLV-I peptide than to the similar MBP peptide and an unrelated bovine MBP peptide. There was no recognition of the peptides by peripheral blood lymphocytes from HAM patients or a HTLV-I carrier without HAM. It was concluded that although cross-reactivity was demonstrated in rabbits and the HTLV-I peptide was recognised by sera from HAM patients, the epitope does not appear to evoke a mimicking response to the similar region in MBP. Hence it is not likely to be involved in the pathogenesis of HAM through molecular mimicry.

Molecular structure of antibodies against small ligands

Antibodies which bind bioactive ligands can serve as a template for the generation of a second antibody which may react with the physiological receptor. This phenomenon of molecular mimicry by antibodies has been described in a variety of systems. In order to understand the chemical and molecular mechanisms involved in these interactions, monoclonal antibodies directed against two pharmacologically active alkaloids, morphine and nicotine, were carefully studied using experimental and theoretical molecular modeling techniques. The molecular characterization of these antibodies involved binding studies with ligand analogs and determination of the variable region amino acid sequence. A three-dimensional model of the anti-morphine binding site was constructed using computational and graphics display techniques. The antibody response in BALB/c mice to morphine appears relatively restricted, in that all of the antibodies examined in this study contained a $\lambda$ light chain, which is normally found in only 5% of mouse immunoglobulins. This study represents the first use of theoretical and experimental modeling techniques to describe the antigen binding site of a mouse Fv region containing a $\lambda$ light chain. The binding site model indicates that a charged glutamic acid residue and aromatic side chains are key features in ionic and hydrophobic interactions with the ligand morphine. A glutamic acid residue is found in the identical position in the anti-nicotine antibody and may play a role in binding nicotine. ^

Molecular basis for the recognition of two structurally different major histocompatibility complex/peptide complexes by a single T-cell receptor

2C is a typical alloreactive cytotoxic T lymphocyte clone that recognizes two different ligands. These ligands are adducts of the allo-major histocompatibility complex (MHC) molecule H-2Ld and an endogenous octapeptide, and of the self-MHC molecule H-2Kb and another peptide. MHC-binding and T-cell assays with synthetic peptides in combination with molecular modeling studies were employed to analyze the structural basis for this crossreactivity. The molecular surfaces of the two complexes differ greatly in densities and distributions of positive and negative charges. However, modifications of the peptides that increase similarity decrease the capacities of the resulting MHC peptide complexes to induce T-cell responses. Moreover, the roles of the peptides in ligand recognition are different for self- and allo-MHC-restricted T-cell responses. The self-MHC-restricted T-cell responses were finely tuned to recognition of the peptide. The allo-MHC-restricted responses, on the other hand, largely ignore modifications of the peptide. The results strongly suggest that adaptation of the T-cell receptor to the different ligand structures, rather than molecular mimicry by the ligands, is the basis for the crossreactivity of 2C. This conclusion has important implications for T-cell immunology and for the understanding of immunological disorders.

Monoclonal antibody screening of a phage-displayed random peptide library reveals mimotopes of chemokine receptor CCR5: Implications for the tertiary structure of the receptor and for an n-terminal binding site for HIV-1 gp120

The chemokine receptor CCR5 contains seven transmembrane-spanning domains. It binds chemokines and acts as co-receptor for macrophage (m)-tropic (or R5) strains of HIV-1. Monoclonal antibodies (mAb) to CCR5, 3A9 and 5C7, were used for biopanning a nonapeptide cysteine (C)-constrained phage-displayed random peptide library to ascertain contact residues and define tertiary structures of possible epitopes on CCR5. Reactivity of antibodies with phagotopes was established by enzyme-linked immunosorbent assay (ELISA). mAb 3A9 identified a phagotope C-HASIYDFGS-C (3A9/1), and 5C7 most frequently identified C-PHWLRDLRV-C (5C7/1). Corresponding peptides were synthesized. Phagotopes and synthetic peptides reacted in ELISA with corresponding antibodies and synthetic peptides inhibited antibody binding to the phagotopes. Reactivity by immunofluorescence of 3A9 with CCR5 was strongly inhibited by the corresponding peptide. Both mAb 3A9 and 5C7 reacted similarly with phagotopes and the corresponding peptide selected by the alternative mAb. The sequences of peptide inserts of phagotopes could be aligned as mimotopes of the sequence of CCR5. For phage 3A9/1, the motif SIYD aligned to residues at the N terminus and FG to residues on the first extracellular loop; for 5C7/1, residues at the N terminus, first extracellular loop, and possibly the third extracellular loop could be aligned and so would contribute to the mimotope. The synthetic peptides corresponding to the isolated phagotopes showed a CD4-dependent reactivity with gp120 of a primary, m-tropic HIV-1 isolate. Thus reactivity of antibodies raised to CCR5 against phage-displayed peptides defined mimotopes that reflect binding sites for these antibodies and reveal a part of the gp120 binding sites on CCR5.

Rheumatoid arthritis sera react with a phage-displayed peptide selected by a monoclonal antibody to type II collagen that has homology to EBNA-1

Antibodies to type II collagen, and to Epstein Barr virus nuclear antigen-1 (EBNA-1) have been associated with rheumatoid arthritis (RA). In studies involving probing of phage-displayed random peptide libraries with an antibody to type II collagen, CII-C1, we observed that among 17 phagotopes selected 5 expressed peptides with homology with the sequence of EBNA-1. The residues in common were RLPFG. Hence we tested sera from 50 patients with RA, of whom 26 had antibodies to native type II collagen, and 43 healthy controls, for reactivity by ELISA with a phagotope selected 4 times, which expressed the peptide RRLPFGSQM. Eight RA sera (16%) but no normal sera reacted with the phagotope (p = 0.025). This reactivity could not be correlated with reactivity of RA sera with EBNA-1 by semi-quantitative western blot, with which reactivity occurred in 78% of RA patients and 81% of controls. Evidence for molecular mimicry was not found insofar as the phagotope did not inhibit reactivity of RA sera with EBNA-1 and CII-C1 was not reactive with EBNA-1. We conclude that the reactivity of the RA sera with the phagotope is most likely due to the phagotope being a mimic of an epitope of type II collagen for a proportion of RA sera.