Related Experiment Videos
Proparathyroid hormone is preferentially cleaved to parathyroid hormone by the prohormone convertase furin. A mass
G N Hendy1, H P Bennett, B F Gibbs
1Department of Medicine, McGill University, Montréal, Québec, Canada.
Abstract:
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.
Insights
Furin is the primary enzyme that converts proparathyroid hormone (proPTH) into active parathyroid hormone (PTH), crucial for calcium regulation. This discovery clarifies a key step in PTH biosynthesis and calcium homeostasis.
Area of Science:
- Biochemistry
- Molecular Biology
- Endocrinology
Background:
- Parathyroid hormone (PTH) regulates blood calcium homeostasis.
- PTH is synthesized as a precursor, proPTH, requiring enzymatic processing.
- Subtilisin-like prohormone convertases are candidates for this processing.
Purpose of the Study:
- To identify the specific prohormone convertase responsible for processing proPTH to PTH.
- To investigate the role of furin, PC1, and PC2 in proPTH maturation.
Main Methods:
- Co-infection of cells (BSC-40 and GH4C1) with vaccinia virus constructs expressing preproPTH and convertases (furin, PC1, PC2).
- Purification and identification of PTH biosynthetic products using HPLC, radioimmunoassay, and ion-spray mass spectrometry.
- Analysis of mRNA expression using Northern blot and in situ hybridization.
Main Results:
- Furin demonstrated the highest efficiency in processing proPTH to PTH in both cell types.
- Only mature PTH was released into the medium, regardless of the convertase used.
- Furin effectively cleaved a synthetic peptide mimicking the proPTH cleavage site.
- Furin and preproPTH mRNA are co-expressed in the parathyroid, unlike PC1, PC2, and PC5.
Conclusions:
- Furin is strongly suggested to be the primary enzyme responsible for the physiological processing of proPTH to PTH.
- This finding elucidates a critical step in maintaining calcium homeostasis.