Structure-function analysis of the prosegment of the proprotein convertase PC5A

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Proprotein convertase subtilisin/kexin type 5 A (PC5A) is a serine protease involved in processing various protein substrates.
• The prosegment of PC5A plays a role in its activation and function, but specific residues critical for activity remain to be fully elucidated.

Purpose of the Study:

• To investigate the role of specific residues within the PC5A prosegment in its proteolytic function.
• To characterize the inhibitory potential of PC5A prosegment peptides and their mutants against PC5A and other related proteases.

Main Methods:

• Cellular expression of wild-type and mutant PC5A.
• In vitro enzymatic assays using pro-vascular endothelial growth factor C (pro-VEGF-C) as a substrate.
• In vitro inhibition assays using synthetic peptides mimicking the PC5A prosegment and its mutants.
• Cellular inhibition assays in Chinese hamster ovary FD11 cells overexpressing pro-VEGF-C.

Main Results:

• PC5A mutants lacking the prosegment or with mutations at the primary (R116A) and secondary (R84A) autocatalytic cleavage sites were inactive.
• Synthetic peptides mimicking the PC5A prosegment (pPC5) demonstrated nanomolar inhibitory potency against PC5A.
• The preprosegment of PC5 (ppPC5) showed potent inhibition of PC5A in cellular assays, comparable to known inhibitors, but with modest selectivity over Furin.

Conclusions:

• Cleavage of the PC5A prosegment at Arg(116) and Arg(84) is essential for its cellular activity.
• The preprosegment of PC5 (ppPC5) is a highly potent inhibitor of PC5A, offering potential as a therapeutic agent with moderate selectivity.