Genetic studies have demonstrated an important role for
proprotein convertase subtilisin/
kexin type 9 (
PCSK9) as a determinant of
plasma cholesterol levels. However, the underlying molecular mechanism is not completely understood. To this end, we have generated a mammalian cell
expression system for human
PCSK9 and its
mutants and produced
transgenic mice expressing human
PCSK9. HEK293T cells transfected with the human
PCSK9 DNA construct expressed and
secreted PCSK9 and displayed decreased
LDLR levels; functional
PCSK9 protein was purified from the conditioned medium. In vitro studies showed that
PCSK9 self-associated in a concentration-, temperature-, and pH-dependent manner. A mixture of
PCSK9 monomers, dimers, and trimers displayed an enhanced
LDLR degrading activity compared to
monomeric PCSK9. A
gain-of-function mutant, D374Y, displayed greatly increased self-association compared to
wild-type PCSK9. Moreover, we demonstrated that the
catalytic domain of
PCSK9 is responsible for the self-association. Self-association of
PCSK9 was enhanced by incubation with
mouse apoE-/-
VLDL and inhibited by incubation with both human and
mouse HDL. When
PCSK9 protein was incubated with total serum, it partially associated with
LDL and HDL but not with
VLDL. In
transgenic mice,
PCSK9 also associated with
LDL and HDL but not with
VLDL. We conclude that self-association is an intrinsic property of
PCSK9,
correlated to its LDLR-degrading activity and affected by plasma
lipoproteins. These results provide a basis for developing strategies to manipulate
PCSK9 activity in the circulation for the treatment of
hypercholesterolemia.
