Heparan sulfate proteoglycans present PCSK9 to the LDL receptor

Abstract

Coronary artery disease is the main cause of death worldwide and accelerated by increased plasma levels of cholesterol-rich low-density lipoprotein particles (LDL). Circulating PCSK9 contributes to coronary artery disease by inducing lysosomal degradation of the LDL receptor (LDLR) in the liver and thereby reducing LDL clearance. Here, we show that liver heparan sulfate proteoglycans are PCSK9 receptors and essential for PCSK9-induced LDLR degradation. The heparan sulfate-binding site is located in the PCSK9 prodomain and formed by surface-exposed basic residues interacting with trisulfated heparan sulfate disaccharide repeats. Accordingly, heparan sulfate mimetics and monoclonal antibodies directed against the heparan sulfate-binding site are potent PCSK9 inhibitors. We propose that heparan sulfate proteoglycans lining the hepatocyte surface capture PCSK9 and facilitates subsequent PCSK9:LDLR complex formation. Our findings provide new insights into LDL biology and show that targeting PCSK9 using heparan sulfate mimetics is a potential therapeutic strategy in coronary artery disease.

Cell-associated, Heparin-like Molecules Modulate the Ability of LDL to Regulate PCSK9 Uptake

“…First, we treated HepG2 cells with heparinase to remove cell surface heparin-like molecules (including HSPGs) prior to evaluating the effect of LDL on our PCSK9-NLuc uptake assay. Unsurprisingly, and consistent with prior results (31), heparinase treatment in the absence of LDL reduced PCSK9-NLuc uptake ( Fig. 2A, orange), with its absolute effect similar to that of pre-incubation of PCSK9 with LDL.…”

“…Our initial results suggested that the cell-based factor might be directly competing with LDL to bind PCSK9 on a relatively slow kinetic timescale. We hypothesized that HSPGs on the HepG2 cells may be this cell-based factor, given the recent description of their affinity for the PCSK9 prodomain and involvement in PCSK9 uptake (31). To test this, we evaluated PCSK9 uptake with reagents expected to modify HSPG activity.…”

“…Deletion of these residues also improves PCSK9 binding to soluble LDLR (18), and increases the ability of PCSK9 to downregulate LDLR on cells (19). By contrast, the binding site of HSPGs to the PCSK9 prodomain encompasses an arginine-rich motif spanning residues 93 to 139 (31). To ask whether the N-terminal prodomain residues were sufficient to mediate the inhibitory effect of LDL on PCSK9 uptake (and therefore non-overlapping with the HSPG binding site), we created secreted PCSK9 prodomains to use in competition experiments.…”

“…Despite the general requirement for self-cleavage and secretion for full PCSK9 function, the initial GOF PCSK9 mutant identified, S127R, has long been known to have paradoxically reduced self-processing, suggesting additional mechanisms can overcome this functional restriction (26,30). Recently, heparan sulfate proteoglycans (HSPGs) have been identified as co-receptors for PCSK9 on the hepatocyte cell surface, with a specific arginine-rich motif encompassing residues 93-139 in the PCSK9 prodomain as the likely binding site mediating this interaction (31).…”

A transient amphipathic helix in PCSK9’s prodomain facilitates low-density lipoprotein binding

“…Indeed, LDL association may stabilize an autoinhibitory prodomain-CM1 interaction in PCSK9 that otherwise can be released by activating factors. Two such factors are known to improve the LDLR binding function of PCSK9, namely acidic pH (11,16,22) and its association with heparin-sulfate proteoglycans (HSPGs) at the surface of hepatocytes (59). HSPGs were recently shown to modulate the ability of LDL to inhibit PCSK9 activity, suggesting an interplay between these regulatory factors (23).…”