CLEAVAGE-ASSOCIATED ENHANCEMENT OF AN ANTIGENIC SITE IN THE BIOLOGICALLY-ACTIVE NH2-TERMINAL REGION OF PARATHYROID-HORMONE

Immunological properties of an antiserum prepared to a synthetic peptide of bovine parathyroid hormone (PTH) consisting of the first 12 amino acids of the NH2-terminal region, bovine PTH-(1–12) [bPTH-(1–12)], were examined in a competitive-binding RIA. With a radioiodinated fragment of 13 amino acids [125I-labeled bPTH-(1–12)-Tyr13] as tracer and bPTH and various synthetic NH2-terminal fragments of the hormone as competing antigens, the reactivities, on a molar basis, of the intact hormone and the peptides bPTH-(1–34), bPTH-(1–28), bPTH-(1–26), bPTH-(2–34), and bPTH-(3′34) were found to be 40–200 times weaker than the reactivity of the immunogen bPTH-(1–12). Certain minor structural changes were associated with considerable variation in immunological reactivity. The cross-reactivity of bPTH-(1–26) relative to bPTH-(1–12) was 2 times greater than that of the larger peptides [bPTH-(1–28), bPTH-(1–34), or bPTH-(l-84)]. Cross-reactivity of bPTH-(1–34) fell by 80–90% with shortening of the NH2- terminus by either one [bPTH-(2–34)] or two [bPTH-(3′34)] amino acids. A major alteration in the cross-reactivity of the larger peptides occurred with treatment of solutions of either bPTH-(1–84) or bPTH-(1–34) with pancreatic trypsin, a procedure that cleaves the hormones at residues 13′14, liberating the NH2-terminal fragment bPTH-(1–13). There was a marked increase in immunologic potency resulting from the cleavages; the molar ratio of the amount of peptide required to produce a given competitive inhibition in the immunoassay decreased from 80:1 to 1:1; i.e. bPTH-(1–34) or bPTH-(1–84) :bPTH-(1–12). These observations indicate that certain antigenic determinants expressed in the amino-terminal peptide of bPTH-(l-12) are not as readily available for antibody recognition when incorporated into the larger peptides [bPTH-(1–84) and bPTH-U-34)] as they are when present in the smaller peptide [bPTH-(1–12)]. The results are consistent with the proposal that the biologically active amino-terminal region of bPTH exists in a higher order conformation, resulting in the shielding of antigenic sites from interaction with antibodies. The findings also suggest that, in RIAs, antibodies prepared against fragments of PTH may recognize these fragments better than they recognize the intact hormone. © 1979 by The Endocrine Society.