Proparathyroid hormone is preferentially cleaved to parathyroid hormone by the prohormone convertase furin. A mass spectrometric study.
Parathyroid hormone (PTH), an 84-amino acid peptide, is the major regulator of blood calcium homeostasis. Its mRNA, in addition to encoding the mature peptide, also encodes a "pre" sequence of 25 amino acids and a basic "pro" hexapeptide. To assess which of the subtilisin-like prohormone convertases can process proPTH to PTH we coinfected cells with a vaccinia virus construct expressing human preproPTH and vaccinia virus constructs expressing furin, PC1 or PC2. BSC-40 cells, having a constitutive secretory pathway, and GH4C1 cells, having a regulated secretory pathway, were used. PTH biosynthetic products in cell extracts and media were purified by high performance liquid chromatography, identified by radioimmunoassay, and unambiguously defined as either proPTH or PTH by ion-spray mass spectrometry. In both cell types, furin was the most effective in processing proPTH to PTH. In all cases only PTH was released into the medium. In addition, partially purified furin and PC1 were tested for their ability to appropriately cleave a tridecapeptide spanning the prohormone cleavage site found in proPTH. Here too furin was much more effective at cleaving at the correct site. Northern blot analysis and in situ hybridization showed that furin and preproPTH mRNA are co-expressed in the parathyroid, whereas PC1, PC2, and PC5 are not and PACE4 is expressed only at very low levels. Taken together these studies strongly suggest that furin is the enzyme responsible for the physiological processing of proPTH to PTH.
Hendy
G N
GN
Department of Medicine, McGill University, Montréal, Québec, Canada.
Bennett
H P
HP
Gibbs
B F
BF
Lazure
C
C
Day
R
R
Seidah
N G
NG
D000595:Amino Acid Sequence; D000818:Animals; D002522:Chlorocebus aethiops; D045683:Furin; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010281:Parathyroid Hormone / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D013381:Subtilisins / Q000235:genetics / Q000502:physiology*
D010281:Parathyroid Hormone; D011498:Protein Precursors; D012333:RNA, Messenger; C003219:proparathormone; D013381:Subtilisins; D045683:Furin