Processing and trafficking of a prohormone convertase 2 active site mutant
Sang-Nam Lee1, Magdalena M Kacprzak, Robert Day
1Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center/Research Institute for Children, New Orleans, Louisiana 70118, USA.
Biochemical and Biophysical Research Communications
|February 27, 2007
Summary
The active site serine mutant of proprotein convertase 2 (PC2-S383A) is efficiently secreted and trafficked, unlike other mutants. This suggests its propeptide aids proper folding and secretion, even with an altered active site.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Proprotein convertases (PCs) are essential serine proteases involved in processing zymogens.
- Proper folding and secretory pathway transit of PCs are crucial; active site mutations typically lead to ER retention and degradation.
Purpose of the Study:
- To investigate the processing, folding, and secretory pathway trafficking of a specific active site serine mutant of PC2 (PC2-S383A).
- To determine if the propeptide of PC2 can facilitate proper folding and secretion despite an inactive catalytic site.
Main Methods:
- Utilized Chinese Hamster Ovary (CHO-K1) and AtT-20 cells to express the PC2-S383A mutant.
- Analyzed protein secretion, cleavage site accessibility, pH-dependence of cleavage, and inhibition by proprotein convertase inhibitors.
- Performed in vitro digestion assays with various convertases.
- Detected immunoreactive PC2 in secretory granules.
Main Results:
- PC2-S383A was efficiently secreted as an intact zymogen from CHO-K1 cells, indicating successful folding.
- In AtT-20 cells, PC2-S383A underwent pH-dependent cleavage at a secondary site within the propeptide, inhibitable by a PC inhibitor.
- In vitro studies confirmed the accessibility of this secondary cleavage site to exogenous convertases.
- Significant amounts of PC2-S383A were localized to secretory granules, confirming efficient pathway transit.
Conclusions:
- The propeptide of PC2 can ensure proper folding and efficient transport through the secretory pathway, even when the active site serine is mutated.
- PC2-S383A exhibits distinct processing characteristics compared to other active site mutants, highlighting the role of the propeptide in secretion.
- These findings provide insights into the mechanisms governing PC2 maturation and trafficking.
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