Mapping of human autoantibody binding sites on the calcium-sensing receptor.
Previously, we have demonstrated the presence of anti-calcium-sensing receptor (CaSR) antibodies in patients with autoimmune polyglandular syndrome type 1 (APS1), a disease that is characterized in part by hypoparathyroidism involving hypocalcemia, hyperphosphatemia, and low serum levels of parathyroid hormone. The aim of this study was to define the binding domains on the CaSR of anti-CaSR antibodies found in APS1 patients and in one patient suspected of having autoimmune hypocalciuric hypercalcemia (AHH). A phage-display library of CaSR peptides was constructed and used in biopanning experiments with patient sera. Selectively enriched IgG-binding peptides were identified by DNA sequencing, and subsequently, immunoreactivity to these peptides was confirmed in ELISA. Anti-CaSR antibody binding sites were mapped to amino acid residues 41-69, 114-126, and 171-195 at the N-terminal of the extracellular domain of the receptor. The major autoepitope was localized in the 41-69 amino acid sequence of the CaSR with antibody reactivity demonstrated in 12 of 12 (100%) APS1 patients with anti-CaSR antibodies and in 1 AHH patient with anti-CaSR antibodies. Minor epitopes were located in the 114-126 and 171-195 amino acid domains, with antibody reactivity shown in 5 of 12 (42%) and 4 of 12 (33%) APS1 patients, respectively. The results indicate that epitopes for anti-CaSR antibodies in the AHH patient and in the APS1 patients who were studied are localized in the N-terminal of the extracellular domain of the receptor. The present work has demonstrated the successful use of phage-display technology in the discovery of CaSR-specific epitopes targeted by human anti-CaSR antibodies.
Kemp
E Helen
EH
Department of Human Metabolism, School of Medicine, University of Sheffield, Sheffield S10 2JF, United Kingdom. e.h.kemp@sheffield.ac.uk
Gavalas
Nikos G
NG
Akhtar
Samia
S
Krohn
Kai J E
KJ
Pallais
J Carl
JC
Brown
Edward M
EM
Watson
Philip F
PF
Weetman
Anthony P
AP
D000293:Adolescent; D000328:Adult; D000595:Amino Acid Sequence; D001323:Autoantibodies / Q000276:immunology*; D001665:Binding Sites; D016022:Case-Control Studies; D002648:Child; D016384:Consensus Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000276:immunology; D016884:Polyendocrinopathies, Autoimmune / Q000097:blood / Q000276:immunology; D025941:Protein Interaction Mapping / Q000379:methods*; D044169:Receptors, Calcium-Sensing / Q000737:chemistry* / Q000276:immunology*; D055815:Young Adult
D001323:Autoantibodies; C095550:CASR protein, human; D000939:Epitopes; D019151:Peptide Library; D010455:Peptides; D044169:Receptors, Calcium-Sensing