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
| Autor: | Joseph C. Santoro, Sookhee Ha, Paola Lo Surdo, Rose M. Cubbon, Anka G. Ehrhardt, Xun Shen, Dale Lewis, Richard T. Cummings, Douglas Wisniewski, Yan G. Ni, Brian K. Hubbard, Lionello Ruggeri, Matthew J. Bottomley, Jon H. Condra, Shilpa Pandit, Leila Njimoluh, Cinzia Volpari, Stefania Di Marco, Laura Orsatti, A. Noto, Holly A. Hammond, Timothy S. Fisher, Dana D. Wood, Ayesha Sitlani, Andrea Carfi |
|---|---|
| Rok vydání: | 2010 |
| Předmět: |
Low-density lipoprotein receptor gene family
media_common.quotation_subject Hypercholesterolemia Biology Biochemistry Protein Structure Secondary Fragment antigen-binding chemistry.chemical_compound Immunoglobulin Fab Fragments Humans Internalization Molecular Biology media_common PCSK9 Serine Endopeptidases Antibodies Monoclonal Hep G2 Cells Cell Biology Proprotein convertase Protein Structure Tertiary Lipoproteins LDL chemistry Amino Acid Substitution Receptors LDL Low-density lipoprotein LDL receptor Mutagenesis Site-Directed Kexin lipids (amino acids peptides and proteins) Proprotein Convertases Proprotein Convertase 9 |
| Zdroj: | The Journal of biological chemistry. 285(17) |
| ISSN: | 1083-351X |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|