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Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli
Published on: February 5, 2019
Modulation of prohormone convertase 1/3 properties using site-directed mutagenesis
Akihiko Ozawa1, Juan R Peinado, Iris Lindberg
1Department of Anatomy and Neurobiology, University of Maryland, Baltimore, 20 Penn Street, Health Sciences Facility II Room S251, Baltimore, Maryland 21201, USA.
Endocrinology
|July 9, 2010
Summary
Prohormone convertase (PC)1/3 has low activity compared to PC2. Introducing PC2-specific mutations into PC1/3 altered its pH optimum and activity, but cellular factors still control its function.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Prohormone convertases (PCs), including PC1/3 and PC2, are essential serine proteases responsible for cleaving peptide hormone and neuropeptide precursors.
- Recombinant PC1/3 displays significantly lower specific activity than PC2, despite high sequence identity in their catalytic domains.
Purpose of the Study:
- To investigate the molecular basis for PC1/3's lower catalytic activity compared to PC2 by introducing PC2-specific mutations into the PC1/3 catalytic domain.
- To elucidate the roles of specific residues and domain interactions in determining the unique properties and activity of PC1/3.
Main Methods:
- Site-directed mutagenesis was used to introduce PC2-specific amino acid sequences into the catalytic domain of human PC1/3.
- Enzyme activity assays were performed using fluorogenic substrates and recombinant precursor proteins.
- Stable transfection of mutant PC1/3 into PC12 cells was utilized to assess activity against endogenous substrates like proneurotensin.
- Analysis of zymogen expression, propeptide cleavage, and secretion was conducted for specific mutants.
Main Results:
- Mutation of RQG(314) to SY in PC1/3 shifted its pH optimum towards neutral pH and increased the activity of the 66-kDa form by two- to four-fold, although full-length mutants showed no enhanced activity.
- Stable expression of the RQG(314)SY mutant in PC12 cells did not lead to increased activity against proneurotensin, indicating cellular regulation.
- Mutation of the 7B2 binding site (GIVTDA(243-248) to QPFMTDI) resulted in increased zymogen expression but impaired propeptide cleavage and secretion, suggesting ER retention.
Conclusions:
- Specific residues within the catalytic cleft contribute to convertase-specific properties, but domain interactions may play a more significant role in defining PC1/3's unique characteristics.
- Cellular factors and intracellular trafficking mechanisms impose regulatory controls on PC1/3 activity beyond its intrinsic enzymatic properties.
- Understanding these molecular and cellular determinants is crucial for elucidating the precise roles of PC1/3 in neuropeptide processing and secretion.
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