Biomedical subjects

M Scealy

Publications and source records attributed to M Scealy.

5 recordsLinked to original sources

PubMed2001

A comparative study of antibody expressions in primary biliary cirrhosis and autoimmune cholangitis using phage display.

Primary biliary cirrhosis (PBC) and autoimmune cholangitis (AIC) are serologic expressions of an autoimmune liver disease affecting biliary ductular cells. Previously we screened a phage-displayed random peptide library with polyclonal IgG from 2 Australian patients with PBC and derived peptides that identified a single conformational (discontinuous) epitope in the inner lipoyl domain of the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), the characteristic autoantigen in PBC. Here we have used phage display to investigate the reactivity of PBC sera from 2 ethnically and geographically distinct populations, Japanese and Australian, and the 2 serologic expressions, PBC and AIC. Random 7-mer and 12-mer peptide libraries were biopanned with IgG from 3 Japanese patients with PBC and 3 with AIC who did not have anti-PDC-E2. The phage clones (phagotopes) obtained were tested by capture enzyme-linked immunosorbent assay (ELISA) for reactivity with affinity-purified anti-PDC-E2, and compared with those obtained from Australian patients with PBC. Peptide sequences of the derived phagotopes and sequences derived by biopanning with irrelevant antisera were aligned to develop a guide tree based on physicochemical similarity. Both Australian and Japanese PBC-derived phagotopes were distributed in branches of the guide tree that contained the peptide sequences MH and FV previously identified as part of an immunodominant conformational epitope of PDC-E2, indicating that epitope selection was not influenced by the racial origin of the PBC sera. Biopanning with either PBC or AIC-derived IgG yielded phagotopes that reacted with anti-PDC-E2 by capture ELISA, further establishing that there is a similar autoimmune targeting in PBC and AIC.

Adult↗

PubMed2000

Conformational epitopes on the diabetes autoantigen GAD65 identified by peptide phage display and molecular modeling.

The major diabetes autoantigen, glutamic acid decarboxylase (GAD65), contains a region of sequence similarity, including six identical residues PEVKEK, to the P2C protein of coxsackie B virus, suggesting that cross-reactivity between coxsackie B virus and GAD65 can initiate autoimmune diabetes. We used the human islet cell mAbs MICA3 and MICA4 to identify the Ab epitopes of GAD65 by screening phage-displayed random peptide libraries. The identified peptide sequences could be mapped to a homology model of the pyridoxal phosphate (PLP) binding domain of GAD65. For MICA3, a surface loop containing the sequence PEVKEK and two adjacent exposed helixes were identified in the PLP binding domain as well as a region of the C terminus of GAD65 that has previously been identified as critical for MICA3 binding. To confirm that the loop containing the PEVKEK sequence contributes to the MICA3 epitope, this loop was deleted by mutagenesis. This reduced binding of MICA3 by 70%. Peptide sequences selected using MICA4 were rich in basic or hydroxyl-containing amino acids, and the surface of the GAD65 PLP-binding domain surrounding Lys358, which is known to be critical for MICA4 binding, was likewise rich in these amino acids. Also, the two phage most reactive with MICA4 encoded the motif VALxG, and the reverse of this sequence, LAV, was located in this same region. Thus, we have defined the MICA3 and MICA4 epitopes on GAD65 using the combination of phage display, molecular modeling, and mutagenesis and have provided compelling evidence for the involvement of the PEVKEK loop in the MICA3 epitope.

Amino Acid Sequence↗

PubMed2000

Prediction of the immunodominant epitope of the pyruvate dehydrogenase complex E2 in primary biliary cirrhosis using phage display.

Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by autoantibodies reactive with the pyruvate dehydrogenase complex. A conformational epitope has been mapped to aa 91-227 within the inner lipoyl domain of the E2 subunit (pyruvate dehydrogenase complex E2 (PDC-E2)). We have used phage display to further localize this epitope. A random heptapeptide library was screened using IgG from two patients with PBC, with negative selection using pooled normal IgG. Phage that contained peptide inserts (phagotopes) selected using PBC sera differed from those selected using IgG from patients with RA or polychondritis. Two motifs occurred only among the PBC-selected phagotopes; these were MH (13 sequences, 16 phagotopes) and FV (FVEHTRW, FVEIYSP, FVLPWRI). The phagotopes selected were tested for reactivity with anti-PDC-E2 affinity purified from four patients with PBC. Phagotopes that contained 1 of 15 different peptide sequences were reactive with one or more of these four anti-PDC-E2 preparations, whereas phagotopes that contained 1of the remaining 28 sequences were negative. The peptides (FVLPWRI, MHLNTPP, MHLTQSP) encoded by three phagotopes that were strongly reactive with all four preparations of anti-PDC-E2 were synthesized. Each of the selected peptides, but not an irrelevant peptide, inhibited the reactivity by ELISA of PBC serum with recombinant PDC-E2 and reduced the inhibition of the enzyme activity of PDC by a PBC serum. The peptide sequences, along with the known NMR structure of the inner lipoyl domain of PDC-E2, allow the prediction of nonsequential residues 131HM132 and 178FEV180 that contribute to a conformational epitope.

Amino Acid Sequence↗

PubMed2000

Monoclonal antibody screening of a phage-displayed random peptide library reveals mimotopes of chemokine receptor CCR5: implications for the tertiary structure of the receptor and for an N-terminal binding site for HIV-1 gp120.

The chemokine receptor CCR5 contains seven transmembrane-spanning domains. It binds chemokines and acts as co-receptor for macrophage (m)-tropic (or R5) strains of HIV-1. Monoclonal antibodies (mAb) to CCR5, 3A9 and 5C7, were used for biopanning a nonapeptide cysteine (C)-constrained phage-displayed random peptide library to ascertain contact residues and define tertiary structures of possible epitopes on CCR5. Reactivity of antibodies with phagotopes was established by enzyme-linked immunosorbent assay (ELISA). mAb 3A9 identified a phagotope C-HASIYDFGS-C (3A9 / 1), and 5C7 most frequently identified C-PHWLRDLRV-C (5C7 / 1). Corresponding peptides were synthesized. Phagotopes and synthetic peptides reacted in ELISA with corresponding antibodies and synthetic peptides inhibited antibody binding to the phagotopes. Reactivity by immunofluorescence of 3A9 with CCR5 was strongly inhibited by the corresponding peptide. Both mAb 3A9 and 5C7 reacted similarly with phagotopes and the corresponding peptide selected by the alternative mAb. The sequences of peptide inserts of phagotopes could be aligned as mimotopes of the sequence of CCR5. For phage 3A9 / 1, the motif SIYD aligned to residues at the N terminus and FG to residues on the first extracellular loop; for 5C7 / 1, residues at the N terminus, first extracellular loop, and possibly the third extracellular loop could be aligned and so would contribute to the mimotope. The synthetic peptides corresponding to the isolated phagotopes showed a CD4-dependent reactivity with gp120 of a primary, m-tropic HIV-1 isolate. Thus reactivity of antibodies raised to CCR5 against phage-displayed peptides defined mimotopes that reflect binding sites for these antibodies and reveal a part of the gp120 binding sites on CCR5.

Amino Acid Sequence↗

PubMed1999

Multiple alignment and sorting of peptides derived from phage-displayed random peptide libraries with polyclonal sera allows discrimination of relevant phagotopes.

Biopanning of phage-displayed random peptide libraries is a powerful technique for identifying peptides that mimic epitopes (mimotopes) for monoclonal antibodies (mAbs). However, peptides derived using polyclonal antisera may represent epitopes for a diverse range of antibodies. Hence following screening of phage libraries with polyclonal antisera, including autoimmune disease sera, a procedure is required to distinguish relevant from irrelevant phagotopes. We therefore applied the multiple sequence alignment algorithm PILEUP together with a matrix for scoring amino acid substitutions based on physicochemical properties to generate guide trees depicting relatedness of selected peptides. A random heptapeptide library was biopanned nine times using no selecting antibodies, immunoglobulin G (IgG) from sera of subjects with autoimmune diseases (primary biliary cirrhosis (PBC) and type 1 diabetes) and three murine ascites fluids that contained mAbs to overlapping epitope(s) on the Ross River Virus envelope protein 2. Peptides randomly sampled from the library were distributed throughout the guide tree of the total set of peptides whilst many of the peptides derived in the absence of selecting antibody aligned to a single cluster. Moreover peptides selected by different sources of IgG aligned to separate clusters, each with a different amino acid motif. These alignments were validated by testing all of the 53 phagotopes derived using IgG from PBC sera for reactivity by capture ELISA with antibodies affinity purified on the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), the major autoantigen in PBC: only those phagotopes that aligned to PBC-associated clusters were reactive. Hence the multiple sequence alignment procedure discriminates relevant from irrelevant phagotopes and thus a major difficulty with biopanning phage-displayed random peptide libraries with polyclonal antibodies is surmounted.

Algorithms↗