title: A proprotein convertase subtilisin-like/kexin type 9 (PCSK9) C-terminal domain antibody antigen-binding fragment inhibits PCSK9 internalization and restores low density lipoprotein uptake.
abstract: PCSK9 binds to the low density lipoprotein receptor (LDLR) and leads to LDLR degradation and inhibition of plasma LDL cholesterol clearance. Consequently, the role of PCSK9 in modulating circulating LDL makes it a promising therapeutic target for treating hypercholesterolemia and coronary heart disease. Although the C-terminal domain of PCSK9 is not involved in LDLR binding, the location of several naturally occurring mutations within this region suggests that it has an important role for PCSK9 function. Using a phage display library, we identified an anti-PCSK9 Fab (fragment antigen binding), 1G08, with subnanomolar affinity for PCSK9. In an assay measuring LDL uptake in HEK293 and HepG2 cells, 1G08 Fab reduced 50% the PCSK9-dependent inhibitory effects on LDL uptake. Importantly, we found that 1G08 did not affect the PCSK9-LDLR interaction but inhibited the internalization of PCSK9 in these cells. Furthermore, proteolysis and site-directed mutagenesis studies demonstrated that 1G08 Fab binds a region of beta-strands encompassing Arg-549, Arg-580, Arg-582, Glu-607, Lys-609, and Glu-612 in the PCSK9 C-terminal domain. Consistent with these results, 1G08 fails to bind PCSK9DeltaC, a truncated form of PCSK9 lacking the C-terminal domain. Additional studies revealed that lack of the C-terminal domain compromised the ability of PCSK9 to internalize into cells, and to inhibit LDL uptake. Together, the present study demonstrate that the PCSK9 C-terminal domain contribute to its inhibition of LDLR function mainly through its role in the cellular uptake of PCSK9 and LDLR complex. 1G08 Fab represents a useful new tool for delineating the mechanism of PCSK9 uptake and LDLR degradation.
authors.affiliation: Department of Cardiovascular Diseases, Merck Research Laboratories, Rahway, New Jersey 07065, USA. yan_ni@merck.com
authors.forename: Yan G
authors.initials: YG
authors.lastname: Ni
authors.forename: Jon H
authors.initials: JH
authors.lastname: Condra
authors.forename: Laura
authors.initials: L
authors.lastname: Orsatti
authors.forename: Xun
authors.initials: X
authors.lastname: Shen
authors.forename: Stefania
authors.initials: S
authors.lastname: Di Marco
authors.forename: Shilpa
authors.initials: S
authors.lastname: Pandit
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Bottomley
authors.forename: Lionello
authors.initials: L
authors.lastname: Ruggeri
authors.forename: Richard T
authors.initials: RT
authors.lastname: Cummings
authors.forename: Rose M
authors.initials: RM
authors.lastname: Cubbon
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Santoro
authors.forename: Anka
authors.initials: A
authors.lastname: Ehrhardt
authors.forename: Dale
authors.initials: D
authors.lastname: Lewis
authors.forename: Timothy S
authors.initials: TS
authors.lastname: Fisher
authors.forename: Sookhee
authors.initials: S
authors.lastname: Ha
authors.forename: Leila
authors.initials: L
authors.lastname: Njimoluh
authors.forename: Dana D
authors.initials: DD
authors.lastname: Wood
authors.forename: Holly A
authors.initials: HA
authors.lastname: Hammond
authors.forename: Douglas
authors.initials: D
authors.lastname: Wisniewski
authors.forename: Cinzia
authors.initials: C
authors.lastname: Volpari
authors.forename: Alessia
authors.initials: A
authors.lastname: Noto
authors.forename: Paola
authors.initials: P
authors.lastname: Lo Surdo
authors.forename: Brian
authors.initials: B
authors.lastname: Hubbard
authors.forename: Andrea
authors.initials: A
authors.lastname: Carfí
authors.forename: Ayesha
authors.initials: A
authors.lastname: Sitlani
mesh_terms: D019943:Amino Acid Substitution; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D056945:Hep G2 Cells; D006801:Humans; D006937:Hypercholesterolemia / Q000188:drug therapy / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D008077:Lipoproteins, LDL / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011973:Receptors, LDL / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D008077:Lipoproteins, LDL; D011973:Receptors, LDL; C472125:PCSK9 protein, human; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D012697:Serine Endopeptidases