Helicobacter pylori and autoimmune pancreatitis - Wiley Online Library

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J. Cell. Mol. Med. Vol 9, No 3, 2005 pp. 741-744

Medical Hypothesis

Helicobacter pylori and autoimmune pancreatitis: role of carbonic anhydrase via molecular mimicry? F. Guarneri a *, C. Guarneri a, S. Benvenga b c a

b

Istituto di Dermatologia, Policlinico "Gaetano Martino", Università di Messina, Messina, Italy Sezione di Endocrinologia del Dipartimento Clinico Sperimentale di Medicina e Farmacologia, Università di Messina, Messina, Italy c Programma Infradipartimentale di Endocrinologia Molecolare Clinica, Azienda Ospedaliera Universitaria Policlinico Gaetano Martino, Messina, Italy

Received: August 5, 2005; Accepted: August 26, 2005

Abstract Autoimmune pancreatitis is a recently defined nosological entity, which accounts for 4.6-6% of all forms of chronic pancreatitis and is often associated with other autoimmune diseases, particularly Sjögren's syndrome. Possession of the HLA DRB1*0405-DQB1*0401 genotype confers a risk for the development of autoimmune pancreatitis. Autoantibodies against carbonic anhydrase II and lactoferrin are frequently present in affected subjects and are suspected to have a pathogenic role. A link between gastric infection by Helicobacter pylori and autoimmune pancreatitis has been hypothesized. We used in silico protein analysis and search for HLA binding motifs to verify this hypothesis. We found a significant homology between human carbonic anhydrase II and α-carbonic anhydrase of Helicobacter pylori, an enzyme which is fundamental for the survival and proliferation of the bacterium in the gastric environment. Moreover, the homologous segments contain the binding motif of the HLA molecule DRB1*0405. Our data strengthen the hypothesis that gastric Helicobacter pylori infection can trigger autoimmune pancreatitis in genetically predisposed subjects.

Keywords: autoimmune pancreatitis • Helicobacter pylori • molecular mimicry • carbonic anhydrase • amino acid sequence analysis • in silico protein analysis

Introduction The term "autoimmune pancreatitis" (AIP) was proposed in 1995 by Yoshida K et al. [1], who observed "a patient with pancreatitis who had * Correspondence to: Fabrizio GUARNERI, MD, Viale Annunziata - Residence dei Fiori - villa 7 I-98168 Messina, Italy. Tel.: +39 090 357070 Fax: +39 090 2927691 E-mail: [email protected]

hyperglobulinemia, was autoantibody-positive, and responded to steroid therapy" but "failed to show any evidence of association with Sjogren's syndrome or other collagen diseases". In the same paper, the authors also reviewed the literature and identified several cases suspicious for AIP. According to Etemad and Whitcomb [2], autoimmune pancreatitis is "an inflammatory process of the pancreas due to an autoimmune mecha-

nism establishing etiology of chronic pancreatitis". AIP accounts for 4.6–6% of all forms of chronic pancreatitis and is often associated with other autoimmune diseases, particularly Sjögren's syndrome. Affected subjects are more frequently males (male:female ratio = 2:1) and have a mean age of 59.1–59.4 years, according to different authors [3–8]. Antibodies against human carbonic anhydrase II (CA-II), an enzyme located in the pancreatic ductal epithelium, and lactoferrin are frequently present in the serum of patients affected by AIP, thus suggesting a role for these proteins as autoantigens in the disease [3, 4]. In particular, the importance of anti-CA-II autoantibodies in the diagnosis of AIP has been recently confirmed [8]. Possession of the HLA DRB1*0405-DQB1*0401 genotype confers a risk for the development of AIP [9]. Based on an extensive review of literature, a recent paper appeared in this Journal [10] hypothesized that gastric Helicobacter pylori infection could trigger autoimmune pancreatitis through several different and probably cooperating mechanisms, including molecular mimicry between human and bacterial antigens. Prompted by this interesting theory, we aimed at identifying the potentially cross-reactive human and bacterial protein(s) using amino acid sequence comparison. This technique has been already used in the field of immune-related diseases, yielding significant results [11–15].

Material and methods We first extracted the amino acid sequences of human carbonic anhydrase II and lactoferrin from the public domain database Entrez Protein, and compared each of them with all known Helicobacter pylori proteins (n=15,919 as of July 17, 2005) using the software BLAST (Basic Local Alignment Search Tool) [16]. Next, we searched the HLA ligand/motif Online Database [17] for binding motifs corresponding to HLA DRB1*0405 and DQB1*0401. Motifs for HLA DRB1*0405 are "F/Y/W/V/I/L/M, X, X, V/I/L/M/D/E, X, N/S/T/Q/K/D, X, X, D/E/Q" and "Y, X, X, X, X, V/T, X, X, D"; no motif was found for HLA DQB1*0401. Occurrences of the above motifs were searched in the homologous proteins. Finally, we used the software SWISS-MODEL [18] (available through Web interface at http://swissmodel

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.expasy.org/SWISS-MODEL.html) to obtain a threedimensional model of the homologous proteins, and the RasMol program (version 2.6, which is freely available at http://www.umass.edu/microbio/rasmol/getras.htm) to visualize and compare the models obtained.

Results We found a significant (E= 9x10-17) homology only for human CA-II: segment 5-255 of this protein is 27% identical and 41% similar to segment 23-239 of carbonic anhydrase of Helicobacter pylori (HpCA). Similarity refers to identical plus chemically similar amino acids. The homologous tract spanned a large part of the entire proteins, since CA-II and HpCA are 260 and 247 amino acid long, respectively. The HLA DRB1*0405 binding motif "F/Y/W/V/I/L/M, X, X, V/I/L/M/D/E, X, N/S/T/Q/K/D, X, X, D/E/Q" occurred twice in each protein (positions 66–74 and 156–164 in CA-II, 16–24 and 35–43 in HpCA), and in three cases out of a total of 4 it was located within the shared segment. The other binding motif of the same HLA haplotype was not present in the above proteins. Concerning three-dimensional structures, SWISS-MODEL was able to build an almost complete model (259 out of 260 amino acids) of CAII, and a partial model (aa 6-227) of HpCA (which, however, includes the vast majority of the aforementioned part homologous to CA-II). The models are shown in Fig. 1.

Discussion The theory of microbial triggering of autoimmunity was formulated several years ago, based on the epidemiological data showing an increased incidence of some autoimmune diseases during, or soon after, specific infections [11]. Only recently, however, it has been definitively shown that molecular mimicry between human and microbial antigens can be, in genetically predisposed subjects, a trigger for the onset of a large number of autoimmune diseases [11, 12].

J. Cell. Mol. Med. Vol 9, No 3, 2005

Fig. 1 Molecular models of segment 2-260 of human carbonic anhydrase II (CA-II) and segment 6-227 of α-carbonic anhydrase of Helicobacter pylori (HpCA). Green, yellow and gray indicate identical, chemically similar or chemically dissimilar (non homologous) amino acids, respectively.

Molecular mimicry between human pancreatic autoantigens and proteins produced by Helicobacter pylori has been recently hypothesized [10], but the molecules involved have not been identified. We found that CA-II and HpCA are significantly homologous, and that the shared segments contain the binding motifs of the HLA molecule DRB1*0405, reported as a risk factor for AIP [9]. Data from literature suggest a potentially high relevance of our findings. Indeed, Helicobacter pylori produces two different forms of HpCA, α and β, which have distinct localizations within the bacterial cell [19]. Interestingly, αHpCA, the isoform which is significantly similar to CA-II, is attached to the surface of the bacterium [19], and thus it is an ideal target for host immunity, as a surface antigen. From a functional point of view, α-HpCA is essential for gastric colonization and acid acclimation in the gastric environment [20], and consequently for survival and proliferation of Helicobacter pylori.

In fact, experimental data show a significant decrease (~3 log 10) in acid survival for alpha-HpCA knockout mutants of Helicobacter pylori. Similar results can be obtained when normal strains are cultured in the presence of acetazolamide, a potent carbonic anhydrase inhibitor [17]. The development of host immune response against this protein is very likely to happen, since blocking the enzymatic function of carbonic anhydrase could reduce bacterial vitality and significantly help fighting the infection. These data, combined with our in silico results, suggest a possible molecular mechanism for the triggering of AIP by Helicobacter pylori. The host immune response developed against α-HpCA could, in genetically predisposed subjects, turn against the autoantigen CA-II because of molecular mimicry, thus causing autoimmune pancreatitis. Our hypothesis is strengthened by the occurrence, in the shared segments of both the human protein and 743

its homologous counterpart, of binding motifs corresponding to HLA DRB1*0405. Although the major histocompatibility complex is not the sole hereditary factor involved in the development of autoimmune diseases, this haplotype has a role because it is significantly more frequent in subjects with AIP than in the normal population. In vivo and/or in vitro experiments clearly remain the only definitive way to test a hypothesis. However, in silico research can be a valuable tool for the research in the field of immune-mediated diseases, by at least narrowing the list of candidate antigens which are possibly involved.

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