Abstract
The hepatic carbohydrate-recognizing asialoglycoprotein receptor (ASGR1) mediates the endocytosis/lysosomal degradation of desialylated glycoproteins following binding to terminal galactose/N-acetylgalactosamine. Human heterozygote carriers of ASGR1 deletions exhibit ∼34% lower risk of coronary artery disease and ∼10% to 14% reduction of non-HDL cholesterol. Since the proprotein convertase PCSK9 is a major degrader of the low-density lipoprotein receptor (LDLR), we investigated the degradation and functionality of LDLR and/or PCSK9 by endogenous/overexpressed ASGR1 using Western blot and immunofluorescence in HepG2-naïve and HepG2-PCSK9-knockout cells. ASGR1, like PCSK9, targets LDLR, and both independently interact with/enhance the degradation of the receptor. This lack of cooperativity between PCSK9 and ASGR1 was confirmed in livers of wildtype (WT) and Pcsk9−/− mice. ASGR1 knockdown in HepG2-naïve cells significantly increased total (∼1.2-fold) and cell-surface (∼4-fold) LDLR protein. In HepG2-PCSK9-knockout cells, ASGR1 silencing led to ∼2-fold higher levels of LDLR protein and DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate)-LDL uptake associated with ∼9-fold increased cell-surface LDLR. Overexpression of WT-ASGR1/2 primarily reduced levels of immature non-O-glycosylated LDLR (∼110 kDa), whereas the triple Ala-mutant of Gln240/Trp244/Glu253 (characterized by loss of carbohydrate binding) reduced expression of the mature form of LDLR (∼150 kDa), suggesting that ASGR1 binds the LDLR in both a sugar-dependent and -independent fashion. The protease furin cleaves ASGR1 at the RKMK103↓ motif into a secreted form, likely resulting in a loss of function on LDLR. Altogether, we demonstrate that LDLR is the first example of a liver-receptor ligand of ASGR1. We conclude that silencing of ASGR1 and PCSK9 may lead to higher LDL uptake by hepatocytes, thereby providing a novel approach to further reduce LDL cholesterol levels.
Highlights
Atherosclerosis, the underlying cause of cardiovascular disease (CVD) [1]
Whole-body in situ hybridization histochemistry of ASGR1 and ASGR2 mRNA expression during development and in the adult revealed that both transcripts are mainly expressed in liver starting at embryonic days 17 and 15, respectively, and showed that their expression increases until adulthood (Fig. 1B)
A Tabula Muris compendium of single-cell RNA-Seq transcriptome data from 20 mouse organs and tissues [14] revealed that ASGR1 transcripts are exclusively expressed in liver, in hepatocytes (Fig. 1C)
Summary
Atherosclerosis, the underlying cause of cardiovascular disease (CVD) [1]. The removal of plasma LDLc is primarily mediated by the low-density lipoprotein receptor (LDLR) located on the surface of hepatocytes. The development of inhibitory human monoclonal antibodies against PCSK9 represents a powerful treatment strategy for the management of CVD, which has been shown to reduce the risk of cardiovascular events in clinical populations [9] Despite these hallmark studies and their utility for lipid lowering to prevent CVD, other surface proteins on hepatocytes that bind to and modulate their interaction with PCSK9 and/or LDLR as well as LDLc uptake have not been thoroughly characterized [6]. Comparison of plasma LDLc decrease in LOF PCSK9 R46L (−17 mg/dl; 30% reduction in CVD risk) with that in LOF ASGR1 del mutation (−11 mg/dl) illustrates that reduction in CVD risk observed in this ASGR1 mutant (34%) was 2-fold greater than would have been predicted (i.e., 18%) from the associated modest reduction of LDLc [12]. The purpose of the present study was to investigate the possible regulation of the LDLR by ASGR1 and the role of PCSK9 in this process
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