First in vivo evidence of a disulfide‑linked parathyroid hormone dimer caused by the p.Arg25Cys mutation  — ASN Events
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First in vivo evidence of a disulfide‑linked parathyroid hormone dimer caused by the p.Arg25Cys mutation  (#358)

Sihoon Lee 1 , Hunsang Lee 2 , Stine Linding Andersen 3 , Je-Hyun Baek 4
  1. Department of Internal Medicine, Gachon University Gil Medical Center, Namdong-gu, Incheon, Korea
  2. Department of Life Sciences, Korea University, Seoul, Korea
  3. Department of Clinical Medicine, Aalborg University, Aalborg, Denmark
  4. R&D Center for Clinical Mass Spectrometry, Seegene Medical Foundation, Seoul, Korea

BACKGROUNDS The p.Arg25Cys mutation in the parathyroid hormone (PTH) gene has been associated with autosomal recessive hypoparathyroidism, and previous biochemical studies indicated that the mutant monomer displays impaired receptor activation while the substituted cysteine residue permits formation of intermolecular disulfide bonds. Whether such disulfide‑linked PTH dimers are present under physiological conditions, however, has remained unknown.

METHODS To determine whether the p.Arg25Cys variant forms a stable circulating PTH dimer in vivo, serum samples from patients carrying the p.Arg25Cys mutation and from a corresponding knock-in mouse model recapitulating the human variant were supplemented with stable isotope-labeled PTH peptides and subjected to weak cation exchange extraction, controlled proteolysis, and high-resolution LC-MS/MS analysis.

RESULTS A tryptic fragment uniquely corresponding to the disulfide-linked VEWLCK-VEWLCK species was unequivocally detected and quantified in both patients and knock-in mice, providing the first direct molecular evidence of a circulating PTH dimer in vivo. All three homozygous patients exhibited markedly elevated total PTH concentrations predominantly in the dimeric form, demonstrating that the dimer is highly stable within the physiological redox environment and represents the major circulating circulating species. The same predominance of the dimeric species was observed in homozygous knock-in mice, indicating that the humanized mouse model faithfully recapitulates the molecular phenotype observed in patients. In contrast, heterozygous carriers and heterozygous knock-in mice exhibited both wild-type monomeric PTH and mutant dimeric PTH, whereas no dimeric species were detected in wild-type humans or mice. Together, these findings establish that the p.Arg25Cys mutation produces a physiologically stable disulfide-linked PTH dimer in vivo across species and confirm that this species is not an analytical artifact but a bona fide hormone variant with mechanistic and translational significance.

CONCLUSION The dominance of the dimeric form further supports the rationale for investigating engineered dimeric PTH analogs as potential therapeutic agents for osteoporosis.