The Role of Peptide Libraries in the Identification of Novel Autoantigen Targets in Autoimmune Diseases
Abstract: Identification of pathogenetically relevant autoantigen targets is a major goal in the study of autoimmune diseases. Indeed it may allow the development of new specific diagnostic tools and facilitate the understanding of the pathogenesis of a disease in order to individualize possible novel treatments. The random peptide library is a molecular biology method that consists of the display of random peptides on live microorganisms. The screening of the library with pooled immunoglobulins obtained from patients affected by an autoimmune disease may lead to the identification of novel autoantigens and of antibodies that are able to bind such antigens with high affinity. Testing patients' sera for the presence of these antibodies may be helpful in the diagnosis of the disease. Moreover such antibodies can be investigated for their functional activity and therefore provide new insights into the pathogenesis of the disease.
The incidence of autoimmune disorders is increasing worldwide. Autoimmune disorders are generally classified according to the organs or tissues that are affected: indeed an autoimmune disease can be characterized by the aggression of a particular tissue and sometimes of a single cell population. However in the prototypic example of autoimmune diseases, i.e., systemic lupus erythematosus (SLE), the immune response is mainly directed against widely expressed antigens (e.g., nucleic acids and other nuclear antigens). Patients affected by autoimmune disorders are characterized by the production of several organ- and non-organ specific autoantibodies (Shoenfeld et al., 2007). In SLE the autoantibodies with distinct specificities identified so far have increased to the incredible number of more than 150. Little is known on the molecular and cellular mechanisms that underline the pathogenesis of such autoimmune aggression: clinical and experimental evidence suggests that the interplay between a particular genetic background and different environmental factors may be responsible for the breaking of immune tolerance towards self-antigens and for the maintenance of self-directed aggression. Genetic susceptibility to autoimmunity is widely studied and many recent reports have identified genes, such as STAT4 and IRF5, that are responsible for an enhanced inflammatory response, associated to different autoimmune diseases (Scofield, 2009; Dieudè et al., 2009). On the contrary, the search for environmental agents that may be causative of the disease has given controversial results and the link is still under debate and investigation. Among the environmental factors, infectious agents have been proposed as the best candidate triggers in the pathogenesis of autoimmunity. Infections may contribute to the development of autoimmunity at different levels: (i) promoting immune dysregulation and chronic inflammation, and (ii) triggering adaptive immune responses cross-reactive with self antigens (antigenic mimicry). The observation that a long period of immune dysfunction often precedes the clinical onset of disease (Kokkonen et al., 2010) suggests that exogenous and endogenous viruses may be responsible for organ- and non-organ specific autoimmune disorders (Denman and Rager-Zisman, 2004).
Autoimmunity may follow the infection with a pathogen that exhibits similarities in some of its epitopes with self-proteins of the host, a mechanism known as molecular mimicry (Fierabracci, 2009a). As a consequence, antibodies produced against the pathogen behave indeed as autoantibodies. Another possible explanation is that the chronic state of disease is due to the enhanced challenge over time of autoreactive T cells by an increased number of autoantigenic peptide determinants (epitope spreading). Proteins to which the immune system is self-tolerant (self-proteins) may evoke autoimmune responses if their expression becomes altered as they undergo post-translational modifications, denaturation, and misfolding. In conclusion, the likelihood for an infection to trigger an autoimmune disease depends on the complex individual host-pathogen interaction and its qualitative and quantitative impact on the innate and adaptive immune responses.
The identification of pathogenetically relevant autoantigen targets is a crucial issue in autoimmunity. Over the last decades several techniques have been proposed to address this issue. Peptide libraries represent a new molecular biology technique that is able to identify novel antigens towards which an immune response is specifically present in a given autoimmune disease and absent in others (Fierabracci, 2009b). Moreover this technique allows the identification of antigens derived from infectious agents that possibly share similarity with self-antigens, thus allowing the study of the presence of a cross-reactive humoral or cellular immune response through the mentioned mechanism of molecular mimicry.
We have used this technique to screen the sera of patients affected by different autoimmune conditions and recently the peptide library approach has allowed the identification of a novel antibody in autoimmune pancreatitis, a rare, recently described autoimmune disorder (Frulloni et al., 2009).
Random peptide libraries (RPLs) represent a large collection of molecular structures that can be efficiently displayed on the surface of bacteriophages and/or bacteria. RPLs have been widely utilized to select peptides that bind to a selector molecule. The peptide library approach is an efficient technique for the characterization of ligands with no prior information regarding antibody specificity. This would allow the recognition of candidate antigens involved in initiation or perpetuation of autoimmune diseases. RPLs represent a powerful tool for the study of antigen/antibody interactions. Each peptide is displayed on the surface of a microorganism and is encoded by a specific region of the genome. Antibodies and other binding proteins are used to select specifically for peptide ligands. Relatively high-affinity peptides for a variety of peptide- and non-peptide-binding ligates have been affinity-isolated from epitope libraries. This technology has been used to map epitopes on proteins and to find peptide mimics (mimotope) for non-peptide-binding structures. The current challenge lies in developing epitope library technology so that tight-binding peptide ligands can be detected for a wider variety of ligates, including those that recognize folded proteins. Should this be accomplished, many powerful applications can be envisioned in the areas of drug design and the development of diagnostic markers and vaccines (Scott, 1992).
The Use of Escherichia coli in Peptide Libraries
Various methods have been designed to study protein-protein interactions including displaying proteins and peptides on live microorganisms. The most studied is Escherichia coli for its ability to display conformation-constrained random peptides on its surface as functional fusions between flagellin and thioredoxin. The flagellin, the most abundant structural protein of flagella, is used to carry thioredoxin, a cytoplasmic protein, on the cell surface. Thioredoxin has an active site loop in which the peptide sequences and their derivatives are inserted and, in this way, a defined scaffold is created to be displayed in a stable secondary and tertiary structure. The scaffold is inserted into a dispensable region, in the central part of flagellin, to form the fused, chimeric protein. After the induction, the chimeric protein is exported and it is assembled on the bacterial surface. Using this technique, the random dodecapeptide library is created and used to devise a bio-panning procedure for a prototype application mapping antibody epitopes or to study protein-protein interactions involved in receptor-ligand binding, enzyme-substrate specificity, and antigen-antibody reaction.
The Use of Peptide Library in Autoimmune Diseases
By using pooled immunoglobulin (Ig) G purified from sera obtained from patients affected by the disease of interest, this approach can provide new specific reagents that can be helpful in the diagnosis of an autoimmune disease and/or lead to the identification of novel autoantigen targets.
We screened RPLs with IgG derived from patients to identify autoantigens in Systemic Sclerosis (Lunardi et al., 2000), Cogan’s Syndrome (Lunardi et al., 2002), chronic idiopathic urticaria (Puccetti et al., 2005), Sjögren’s syndrome (Navone et al., 2005), Celiac disease (Zanoni et al., 2006), Crohn’s disease (Lunardi et al., 2009), and very recently in autoimmune pancreatitis (Frulloni et al., 2009).
Using this procedure we identified novel antigen targets in the above mentioned autoimmune diseases and, in some cases, we could for the first time suggest a link between individual infectious agents and the pathogenesis of autoimmune aggression through a mechanism of molecular mimicry. Indeed, despite intensive research, the etiopathogenesis of most autoimmune diseases remains ill-defined. Infections are considered main environmental factors contributing to the development of systemic autoimmune inflammatory disorders. Therefore RPLs have shown to be of great utility in attempts to clarify this difficult and controversial field of autoimmunity.
The Use of Peptide Library in Autoimmune Pancreatitis
We have applied the peptide library approach to a recently described autoimmune disorder called autoimmune pancreatitis (AIP). AIP is a newly recognized type of pancreatitis that is characterized by diffuse or focal swelling of the pancreas due to lymphoplasmacytic infiltration and fibrosis of the pancreatic parenchyma. Various morphologic descriptions have been proposed to characterize this disease: nonalcoholic duct-destructive chronic pancreatitis, lymphoplasmacytic sclerosing pancreatitis with cholangitis, chronic sclerosing pancreatitis, pseudotumorous pancreatitis, and duct-narrowing chronic pancreatitis. Recently, the term “autoimmune pancreatitis” has become widely accepted, although it is apparent that autoimmune pancreatitis is a heterogeneous disease (Finkelberg et al., 2006).
The Japan Pancreas Society has proposed diagnostic criteria for autoimmune pancreatitis such as the presence of high serum IgG4 antibody level (Hamano et al., 2001), pancreas enlargement and pancreatic duct narrowing, lymphoplasmacytic infiltration, response to corticosteroid therapy, and association with other autoimmune diseases such as autoimmune hepatitis, sclerosing cholangitis, primary biliary cirrhosis, sialadenitis, inflammatory bowel disease, and Sjögren syndrome (Nakazawa et al., 2006). AIP cases are difficult to diagnose because of atypical imaging findings, and the main problem is the differential diagnosis from the more frequent pancreatic cancer. It has been reported that up to 10% of the patients who undergo pancreatic resection for a suspected pancreatic cancer have a final diagnosis of pancreatitis (Wolfson et al., 2005). Since the criteria for the diagnosis of AIP are not yet completely defined, the identification of serological markers is of great impact in clinical practice.
The cause of AIP is unknown. Its autoimmune origin has been suggested but never proven, and little is known about the pathogenesis of this condition.
To identify pathogenetically relevant autoantigen targets, we screened a random peptide library with pooled IgG obtained from 20 patients with autoimmune pancreatitis (Frulloni et al., 2009). Different peptides were recognized by the pooled IgG purified from patients’ sera and, among the detected peptides, AIP1-7 peptide was recognized by 18 out of 20 AIP patients (90%) and by 4 out of 40 (10%) patients with pancreatic cancer, demonstrating that the AIP1-7 peptide sequence contains an epitope recognized by the vast majority of the sera from patients with AIP, but not by the sera of healthy controls and of patients with other autoimmune disorders. The results were validated in another series of patients with autoimmune pancreatitis or pancreatic cancer: 14 of 15 patients with AIP (93%) and 1 of 70 patients with pancreatic cancer (1%) had a positive test for antibodies against the AIP1-7 peptide. When the training (first group of patients studied) and validation groups were combined, the test for AIP1-7 was positive in 33 of 35 patients with AIP (94%) and in 5 of 110 patients with pancreatic cancer (5%).
The AIP1-7 peptide showed homology with an amino acid sequence segment of plasminogen binding protein (PBP) of Helicobacter pylori and with ubiquitin-protein ligase E3 component n-recognin 2 (UBR2), an enzyme highly expressed in acinar cells of the pancreas. Antibodies against the PBP peptide were detected in 19 of 20 patients with AIP (95%) and in 4 of 40 patients with pancreatic cancer (10%) of the training group. Such reactivity was not detected in patients with alcohol-induced chronic pancreatitis or intraductal papillary mucinous neoplasm.
These findings may have important implication in the understanding of the disease pathogenesis. Indeed some reports have recently suggested that Helicobacter pylori infection may be implicated in the pathogenesis of AIP. However, a direct link between AIP and Helicobacter pylori has never been proven. In most cases the association has been postulated on the structural homology between carbonic anhydrase and Helicobacter pylori derived proteins (Guarneri et al., 2005). However, it is important to note that antibodies against carbonic anhydrase are not an exclusive marker of AIP since they can be found also in other autoimmune diseases such as Sjögren’s syndrome.
We found that the antibodies directed against the AIP1-7 peptide recognize the Helicobacter pylori derived protein PBP that shares homology with the AIP1-7 peptide. This protein is associated with the ability of the bacterium to degrade host components and therefore is potentially associated with the pathogenicity of Helicobacter pylori. Antibodies against the Helicobacter pylori derived PBP peptide are present in nearly all the patients with AIP, but not in other pancreatic diseases, suggesting a potential etiological link between Helicobacter pylori infection and the pathogenesis of AIP. Indeed, the Helicobacter pylori-derived PBP peptide shares homology with the human protein UBR2, which is highly expressed in the pancreatic acinar cells, and most interestingly, antibodies purified from patients with AIP that are specific for the Helicobacter peptide recognize the self protein UBR2, indicating that UBR2 can be considered a novel and yet unidentified autoantigen target in AIP. Therefore we provide evidence that Helicobacter pylori infection may be linked to the pathogenesis of AIP through a mechanism of molecular mimicry.
In conclusion, the peptide library approach is a powerful tool in the identification of novel autoantigen targets in autoimmune diseases and plays an important role in studying the relationship between infectious agents and autoimmunity.
Using this approach we have recently reported the identification of an antigenic peptide that is relevant in the diagnosis of AIP and it puts forth new insights in the etiopathogenesis of the disease.
This finding is of particular interest, since no specific serologic marker has been so far identified for AIP. The test to detect this marker is therefore of great clinical impact since it provides a useful tool to aid the identification of patients with AIP and to differentiate them from patients with pancreatic cancer. Moreover the combination of this test with the detection of IgG4 levels can increase the ability to discriminate the two diseases.
(Corresponding author: Claudio Lunardi, M.D., Department of Clinical and Experimental Medicine, University Hospital, University of Verona, 37134 Verona, Italy.)
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[Discovery Medicine, 9(46):224-228, March 2010.]