Serum antibodies to carbonic anhydrase IV in patients with autoimmune pancreatitis

Abstract

Background and aims: Serum antibodies to carbonic anhydrase (CA) II have been reported in patients with autoimmune pancreatitis (AIP) and Sjogren's syndrome (SjS). However, their significance in the pathogenesis of these diseases is controversial. The aim of this study was to identify serum antibodies to CA isozymes, which are expressed in ductal cells of the pancreas.

Methods: Recombinant proteins of human CAs IV, IX, and XII were obtained using a bacterial expression system, and five CA IV peptides with theoretically high antigenicity were synthesised. Western blotting and enzyme linked immunosorbent assay (ELISA) were used to detect serum antibodies to the CA isozymes.

Results: The first screening analysis by western blot showed serum antibodies to CA IV among three CA isozymes in patients with idiopathic chronic pancreatitis, including AIP patients. Further analysis by ELISA showed a significantly increased prevalence of serum antibodies to the truncated CA IV protein and the CA IV synthetic peptide (LGS LTT PTC DEK VVW TVF REP I) in patients with definite AIP (4/15 and 6/20, respectively; p<0.01), probable AIP (6/14 and 3/14; p<0.02), and SjS (9/20 and 8/40; p<0.001) compared with normal controls (0/26). There was no significant difference in the antibody prevalence rates between normal controls and patients with alcoholic chronic pancreatitis (2/15 in each) or pancreatic cancer (2/14 and 1/14, respectively). The presence of serum antibodies to the CA IV peptide showed significant correlations with serum gamma-globulin and IgG levels in AIP patients.

Conclusions: These findings suggest that CA IV may be a target antigen that is commonly expressed in epithelial cells of specific tissues involved in AIP and its related diseases.

Figure 1

Schematic presentation of the truncated carbonic anhydrase (CA) IV proteins and synthetic peptides. (A) Precursor CA IV is 312 amino acids long. N⁻-CA IV lacks the N terminal signal sequence (18 amino acid residues), C⁻-CA IV lacks the C terminal glycosyl phosphatidylinositol (GPI) anchored sequence (28 residues), and N⁻/C⁻-CA IV lacks both terminal sequences. (B) Theoretical antigenicity on the amino acid sequence of human CA IV was analysed using the computer. Five sequences representing high antigenicity were selected (indicated by bars with numbers 1–5) and the peptides with these sequences were synthesised.

Figure 2

Western blotting representing serum antibodies to carbonic anhydrase (CA) IV in patients with AIP (case 1) and ICP (case 2). Protein staining profiles of the blots are shown on the left panel. Both patients showed significant signals for serum antibodies to CA IV-glutathione S-transferase (GST) fusion protein (arrows) but no detectable signal was observed for serum antibodies to GST alone (arrowhead). In case 1, several bands were concomitantly seen on the blots with both CA IV-GST and GST, which were probably derived by reactions to bacterial endogenous proteins acquired in this patient.

Figure 3

Molecular properties of three truncated carbonic anhydrase (CA) IV proteins. (A) A silver stained sodium dodecyl sulphate-polyacrylamide gel electrophoresis profile with three types of truncated CA IV after denaturing and refolding. Along with several faint bands, a strongly expressed protein was seen in each lane, corresponding to N⁻/C⁻-CA IV (30.3 kDa; lane 1), N⁻-CA IV (33.2 kDa; lane 2), and C⁻-CA IV (32.2 kDa; lane 3). (B) A silver stained sodium dodecyl sulphate-polyacrylamide gel electrophoresis profile with the N⁻/C⁻-CA IV protein, which was further purified with the use of an N-hydroxysuccinimide activated affinity column. A single band was observed and was then considered as an antigen for ELISA. (C) Catalytic activity of three truncated CA proteins: T-T₀/T, time to reach equilibration in the CO₂ hydration reaction in the absence of CA (T₀) and in the presence of a sample (T). Only the N⁻/C⁻-CA IV protein showed CA activity (82% of T-T₀/T). CA activity was completely inhibited by acetazolamide (AZ).

Figure 4

Serum antibodies to carbonic anhydrase (CA) IV peptide 4 by ELISA in patients with definite autoimmune pancreatitis (definite AIP, n = 15), probable AIP (n = 14), alcoholic chronic pancreatitis (ACP, n = 15), pancreatic cancer (PC, n = 14), Sjögren’s syndrome (SjS, n = 20), and normal controls (NC, n = 26). The broken line indicates a cut off value defined by the mean (2 SD) in NC.

Figure 5

Correlation between titres of serum antibodies to carbonic anhydrase (CA) IV peptide 4 and to those of the N⁻/C⁻-CA IV protein in patients with definite and probable autoimmune pancreatitis.