Catalytic domain of PDC-E2 contains epitopes recognized by antimitochondrial antibodies in primary biliary cirrhosis

Abstract

Aim: To search for further immunodominant peptides of the pyruvate dehydrogenase complex E2-component (PDC-E2) recognized by antimitochondrial antibodies (AMA) in primary biliary cirrhosis (PBC).

Methods: Sera from 95 patients with PBC were tested by enzyme-linked immunosorbent assay against 33 synthetic overlapping peptides (25 amino acids; aa) covering the entire length of the E2-subunit of PDC-E2. Furthermore, the inner lipoyl peptide 167-184 was used in an unlipoylated and a lipoylated form as well as coupled to ovalbumin. Sera from 11 AMA negative/ANA positive PBC patients, 63 patients with other liver disorders and 22 healthy blood donors served as controls.

Results: Of the 95 PBC-sera, 74% reacted with the peptide 475-499 and 58% with the peptide 407-431 located within the catalytic domain of PDC-E2. Patients with other disorders or healthy controls were positive in only up to 18%. Antibodies to the unlipoylated and lipoylated peptide 167-184 within the inner lipoyl domain were found in only 5% and 11% of the PBC sera, respectively; using ovalbumin-coupled peptides, the incidence increased up to 57% (unlipoylated form).

Conclusion: Peptides within the catalytic site of PDC-E2 rather than the previously reported lipoyl binding peptide 167-184 may represent major immunodominant epitopes recognized by AMA in PBC.

Figure 1

Amino acid (aa) sequence of the human pyruvate dehydrogenase complex E2-component (PDC-E2). For epitope mapping 25 mer peptides with 8 overlapping amino acids were constructed (see also Table 2). Overlapping amino acids are printed in bold; amino acid sequences of the immunodominant lipoyl binding epitopes in the outer (aa 41-53) and inner lipoyl domain (aa 167-183) are underlined. *Lipoyl binding lysine in the outer and inner lipoyl domains; **Active sites within the catalytic domain (S480 and H534).

Figure 2

Box plots showing the reactivity of sera from 95 primary biliary cirrhosis (PBC) patients (grey bars) and 22 blood donors (white bars) with 33 overlapping peptides spanning the whole PDC-E2 sequence. IgG antibody reactivities. A: Peptide 1-16; B: Peptide 17-33; IgM antibody reactivities. C: Peptide 1-16; D: Peptide 17-33. Solid bars extend from the 25th to 75th perzentile. The line in the middle is the median. The whiskers extend down to the lowest value and up to the highest value. Significantly higher antibody reactivity with PBC sera than with sera from healthy individuals: ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. Significantly lower antibody reactivity with PBC sera than with sera from healthy individuals: ^dP < 0.001. ELISA: Enzyme-linked immunosorbent assay.

Figure 3

IgG- and IgM-reactivity of sera from 95 PBC patients with the OVA coupled unlipoylated and lipoylated peptide 167-184 (OVA-167-184 and OVC-167-184-LA) (without subtraction of the OVA-values) and OVA alone.