Submitted on January 28, 2008
Revised on March 12, 2008
Accepted on March 18, 2008
Functional analysis of sites within PCSK9 responsible for hypercholesterolemia
Shilpa Pandit, Doug Wisniewski, Joseph C. Santoro, Sookhee Ha, Vijayalakshmi Ramakrishnan, Rose M. Cubbon, Richard T. Cummings, Samuel D. Wright, Carl P. Sparrow, Ayesha Sitlani, and Timothy S. Fisher
Cardiovascular Diseases, Merck, Rahway, NJ 07065
Corresponding Author: timothy_fisher{at}merck.com
Mutations within proprotein convertase subtilisin/kexin type 9 (PCSK9) are associated with dominant forms of familial hypercholesterolemia. PCSK9 binds the low density lipoprotein receptor (LDLR) and addition of PCSK9 to cells promotes degradation of LDLR. PCSK9 mutant proteins associated with hypercholesterolemia (S127R and D374Y) are more potent in decreasing LDL uptake than wild-type PCSK9. To better understand the mechanism by which mutations at the Ser127 and Asp374 residues of PCSK9 influence PCSK9 function, a limited vertical scanning mutagenesis was performed at both sites. S127R and S127K proteins were more potent in decreasing LDL uptake than wild-type PCSK9, while each D374 mutant tested was more potent in reducing LDL uptake when the proteins were added exogenously to cells. The potencies of D374 mutants in lowering LDL-uptake correlated with their ability to interact with LDLR in vitro. Combining S127R and D374Y was also found to have an additive effect in enhancing PCSK9's ability to reduce LDL uptake. Modeling of PCSK9 S127 and D374 mutations indicates that mutations which enhance PCSK9 function stabilize or destabilize the protein, respectively. In conclusion, these results suggest a model in which mutations at Ser127 and Asp374 residues modulate PCSK9's ability to regulate LDLR function through distinct mechanisms.
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