Cryptic Antigenic Determinants on the Extracellular Pyruvate Dehydrogenase Complex/Mimeotope Found in Primary Biliary Cirrhosis

Abstract

Affinity mass spectrometry (AMS) was used to evaluate the structural diversity of the E2 component of pyruvate dehydrogenase complex (PDC) in normal and diseased liver cells, including those from patients with the autoimmune disease primary biliary cirrhosis (PBC). Two different antibodies to PDC-E2, the immunodominant mitochondrial autoantigen in patients with PBC, were used. AMS was performed directly on frozen liver sections and purified bile duct epithelial cells. Mass spectrometric signals associated with the molecular recognition of PBC-specific antigenic determinants were enhanced by an in situ enzyme-linked signal amplification process. Samples from patients with PBC gave strong positive signals for the antigen(s) recognized by the monoclonal antibody C355.1. Conversely, tissues from normal and disease controls showed only a minimal signal. AMS was used to identify specific antigenic determinants within the E2 component of PDC for comparison with unknown antigenic determinants observed by affinity capture with C355.1 monoclonal antibody from PBC samples. PDC components bound to C355.1 were mapped and identified by mass before dissociation from the E2 component. A similar approach was used to identify unknown antigenic determinants associated with PBC. We believe AMS may be an important new approach with wide application to the identification of molecules associated with a number of disease states.

Heterogeneous response of antimitochondrial autoantibodies and bile duct apical staining monoclonal antibodies to pyruvate dehydrogenase complex E2: The molecule versus the mimic

“…The identity of the antigen at the apical surface has still not been established. 3,4,33 However, we have previously reported that the apical staining is also seen with mAbs against the branched chain ketoacid dehydrogenase complex-E2 and the 2-oxoglutarate dehydrogenase complex-E2 antigens. 6 These mAbs do not cross-react with PDC-E2.…”

Lysine Acetylation Is a Highly Abundant and Evolutionarily Conserved Modification in Escherichia Coli

“…Peptides released from immunoisolated complexes have been analyzed by both MALDI-TOF mass spectrometry and electrospray tandem mass spectrometry for epitope mapping (39,40), sequencing histocompatibility complex-binding peptides (41), and analysis of disease-related peptides (42). In addition to sequence-specific antibodies, pan-antibodies, such as anti-phosphotyrosine antibody, have also been used to isolate and to identify tyrosine-phosphorylated proteins on a global scale in response to extracellular stimulation (43,44).…”

Presence of disease specific autoantibodies against liver sinusoidal cells in primary biliary cirrhosis

“…A probable diagnosis requires the presence of two criteria, and a definite diagnosis requires all three [2] . The major mitochondrial autoantigens recognized by AMAs are members of the 2-oxoacid dehydrogenase complex (ODC) family [3][4][5] . Studies based on immunohistochemistry and affinity mass spectrometry have suggested that either PDC-E2 (a member of the ODC family) or a cross-reactive molecule is present in greatly increased amounts at the apical surface of biliary epithelial cells (BECs) from patients with AMA-positive or AMA-negative PBC, but not from normal individuals or patients with other liver diseases [4,5] .…”

“…The major mitochondrial autoantigens recognized by AMAs are members of the 2-oxoacid dehydrogenase complex (ODC) family [3][4][5] . Studies based on immunohistochemistry and affinity mass spectrometry have suggested that either PDC-E2 (a member of the ODC family) or a cross-reactive molecule is present in greatly increased amounts at the apical surface of biliary epithelial cells (BECs) from patients with AMA-positive or AMA-negative PBC, but not from normal individuals or patients with other liver diseases [4,5] . Additionally, in other studies, in addition to the intense staining of the apical surface of BECs, positive staining was observed in a subset of macrophages in portal lymph nodes [6] and in hepatocytes [7] .…”