title: Furin-cleaved proprotein convertase subtilisin/kexin type 9 (PCSK9) is active and modulates low density lipoprotein receptor and serum cholesterol levels.
abstract: Proprotein convertase subtilisin/kexin 9 (PCSK9) regulates plasma LDL cholesterol levels by regulating the degradation of LDL receptors. Another proprotein convertase, furin, cleaves PCSK9 at Arg(218)-Gln(219) in the surface-exposed "218 loop." This cleaved form circulates in blood along with the intact form, albeit at lower concentrations. To gain a better understanding of how cleavage affects PCSK9 function, we produced recombinant furin-cleaved PCSK9 using antibody Ab-3D5, which binds the intact but not the cleaved 218 loop. Using Ab-3D5, we also produced highly purified hepsin-cleaved PCSK9. Hepsin cleaves PCSK9 at Arg(218)-Gln(219) more efficiently than furin but also cleaves at Arg(215)-Phe(216). Further analysis by size exclusion chromatography and mass spectrometry indicated that furin and hepsin produced an internal cleavage in the 218 loop without the loss of the N-terminal segment (Ser(153)-Arg(218)), which remained attached to the catalytic domain. Both furin- and hepsin-cleaved PCSK9 bound to LDL receptor with only 2-fold reduced affinity compared with intact PCSK9. Moreover, they reduced LDL receptor levels in HepG2 cells and in mouse liver with only moderately lower activity than intact PCSK9, consistent with the binding data. Single injection into mice of furin-cleaved PCSK9 resulted in significantly increased serum cholesterol levels, approaching the increase by intact PCSK9. These findings indicate that circulating furin-cleaved PCSK9 is able to regulate LDL receptor and serum cholesterol levels, although somewhat less efficiently than intact PCSK9. Therapeutic anti-PCSK9 approaches that neutralize both forms should be the most effective in preserving LDL receptors and in lowering plasma LDL cholesterol.
authors.affiliation: Department of Early Discovery Biochemistry, Genentech, Inc., South San Francisco, California 94080, USA.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Lipari
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.forename: Paul
authors.initials: P
authors.lastname: Moran
authors.forename: Monica
authors.initials: M
authors.lastname: Kong-Beltran
authors.forename: Tao
authors.initials: T
authors.lastname: Sai
authors.forename: Joyce
authors.initials: J
authors.lastname: Lai
authors.forename: S Jack
authors.initials: SJ
authors.lastname: Lin
authors.forename: Ganesh
authors.initials: G
authors.lastname: Kolumam
authors.forename: Jose
authors.initials: J
authors.lastname: Zavala-Solorio
authors.forename: Anita
authors.initials: A
authors.lastname: Izrael-Tomasevic
authors.forename: David
authors.initials: D
authors.lastname: Arnott
authors.forename: Jianyong
authors.initials: J
authors.lastname: Wang
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Peterson
authors.forename: Daniel
authors.initials: D
authors.lastname: Kirchhofer
mesh_terms: D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry; D002784:Cholesterol / Q000097:blood* / Q000235:genetics; D045683:Furin / Q000235:genetics / Q000378:metabolism*; D056945:Hep G2 Cells; D006801:Humans; D008099:Liver / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D059748:Proteolysis; D011973:Receptors, LDL / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; C573412:LDLR protein, human; D011973:Receptors, LDL; D002784:Cholesterol; C472125:PCSK9 protein, human; C472126:Pcsk9 protein, mouse; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D012697:Serine Endopeptidases; C468731:FURIN protein, human; D045683:Furin