Molecular mapping of epitopes on melanocyte-specific protein Pmel17 which are recognized by autoantibodies in patients with vitiligo.
Previously, we reported the identification of Pmel17 autoantibodies in some patients with vitiligo. Here, we have determined the B cell epitopes on Pmel17 which are recognized by these autoantibodies. Deletion derivatives of Pmel17 cDNA were constructed using either subcloning of specific cDNA fragments or polymerase chain reaction amplification. Full-length Pmel17 cDNA and its truncated derivatives were then translated in vitro to produce [(35)S]-labelled proteins. The radiolabelled ligands were used subsequently in radiobinding assays to investigate the reactivity of sera from vitiligo patients. Two epitope regions were identified: one located at the C-terminal end of Pmel17 between amino acids 634--644 and one in a central region of the protein between amino acids 326--341. Computer analysis of the potential B cell epitopes on Pmel17 revealed that the epitope domain encompassing amino acids 326--341 was located in an area of the protein which was predicted to be highly antigenic. In contrast, the epitope identified at the C-terminal of Pmel17 (amino acids 634--644) was located in a region of the protein predicted to have low antigenicity. The amino acid sequences of the identified Pmel17 epitopes were compared to the amino acid sequences of the related melanogenic enzymes tyrosinase, tyrosinase-related protein-1 and tyrosinase-related protein-2. However, no sequence homology was found between either of the Pmel17 epitopes and the aforementioned proteins. This finding is consistent with our previous study in which we were unable to show the presence of Pmel17 antibodies which were cross-reactive with either tyrosinase, tyrosinase-related protein-1 or tyrosinase-related protein-2. It also suggests that the IgG response to Pmel17 is distinct from the antibody response to the other melanocyte-specific antigens.
Kemp
E H
EH
Division of Clinical Sciences, Northern General Hospital, University of Sheffield, Sheffield, UK. e.h.kemp@sheffield.ac.uk
Waterman
E A
EA
Gawkrodger
D J
DJ
Watson
P F
PF
Weetman
A P
AP
D000328:Adult; D000368:Aged; D001323:Autoantibodies / Q000097:blood / Q000276:immunology*; D018604:Epitope Mapping; D018985:Epitopes, B-Lymphocyte / Q000235:genetics / Q000276:immunology*; D005260:Female; D006801:Humans; D008297:Male; D008544:Melanocytes / Q000276:immunology*; D008562:Membrane Glycoproteins; D008875:Middle Aged; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics / Q000276:immunology*; D011863:Radioimmunoassay; D014820:Vitiligo / Q000097:blood / Q000276:immunology*; D058951:gp100 Melanoma Antigen
D001323:Autoantibodies; D018985:Epitopes, B-Lymphocyte; D008562:Membrane Glycoproteins; C548637:PMEL protein, human; D011506:Proteins; D058951:gp100 Melanoma Antigen