Abstract
PCSK9 enhances the cellular degradation of the LDL receptor (LDLR), leading to increased plasma LDL cholesterol. This multidomain protein contains a prosegment, a catalytic domain, a hinge region, and a cysteine-histidine rich domain (CHRD) composed of three tightly packed modules named M1, M2, and M3. The CHRD is required for the activity of PCSK9, but the mechanism behind this remains obscure. To define the contribution of each module to the function of PCSK9, we dissected the CHRD structure. Six PCSK9 deletants were generated by mutagenesis, corresponding to the deletion of only one (ΔM1, ΔM2, ΔM3) or two (ΔM12, ΔM13, ΔM23) modules. Transfection of HEK293 cells showed that all deletants were well processed and expressed compared with the parent PCSK9 but that only those lacking the M2 module were secreted. HepG2 cells lacking endogenous PCSK9 (HepG2/shPCSK9) were used for the functional analysis of the extracellular or intracellular activity of PCSK9 and its deletants. To analyze the ability of the deletants to enhance the LDLR degradation by the intracellular pathway, cellular expressions revealed that only the ΔM2 deletant retains a comparable total LDLR-degrading activity to full-length PCSK9. To probe the extracellular pathway, HepG2/shPCSK9 cells were incubated with conditioned media from transfected HEK293 or HepG2/shPCSK9 cells, and cell surface LDLR levels were analyzed by FACS. The results showed no activity of any secreted deletant compared with PCSK9. Thus, although M2 is dispensable for secretion, its presence is required for the extracellular activity of PCSK9 on cell surface LDLR.
Highlights
Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates low-density lipoprotein receptor levels in the liver
Expression, Zymogen Processing, and Secretion of cysteine-histidine rich domain (CHRD) Module Deletants—The reported crystal structures of PCSK9 revealed that its C-terminal CHRD contains three distinct Cys/ His-rich modules named M1, M2, and M3 [7,8,9]
The CHRD was shown to be critical for targeting the PCSK9'LDL receptor (LDLR) complex to endosomes/lysosomes for degradation [13, 29]
Summary
PCSK9 regulates low-density lipoprotein receptor levels in the liver. The importance of the M1, M2, and M3 modules within the C terminus of PCSK9 is unknown. PCSK9 enhances the cellular degradation of the LDL receptor (LDLR), leading to increased plasma LDL cholesterol. This multidomain protein contains a prosegment, a catalytic domain, a hinge region, and a cysteine-histidine rich domain (CHRD) composed of three tightly packed modules named M1, M2, and M3. Transfection of HEK293 cells showed that all deletants were well processed and expressed compared with the parent PCSK9 but that only those lacking the M2 module were secreted. To analyze the ability of the deletants to enhance the LDLR degradation by the intracellular pathway, cellular expressions revealed that only the ⌬M2 deletant retains a comparable total LDLR-degrading activity to fulllength PCSK9. M2 is dispensable for secretion, its presence is required for the extracellular activity of PCSK9 on cell surface LDLR
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