Asialoglycoprotein receptor 1 is a novel PCSK9-independent ligand of liver LDLRthat is shed by Furin
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 exhibited [~]34% lower risk of coronary artery disease and [~]10-14% non-HDL-cholesterol reduction. Since PCSK9 is a major degrader of LDLR, the regulation of LDLR and/or PCSK9 by ASGR1 was studied. Thus, we investigated the role of endogenous/overexpressed ASGR1 on LDLR degradation and functionality by Western-blot and immunofluorescence in HepG2 naive and HepG2-PCSK9-knockout cells. ASGR1, like PCSK9, targets LDLR and both interact with/enhance the degradation of the receptor independently. Such lack of cooperativity between PCSK9 and ASGR1 on LDLR expression was confirmed in livers of wild-type (WT) versus Pcsk9-/- mice. ASGR1-knockdown in HepG2 naive 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-LDL uptake, associated with [~]9-fold increased cell-surface LDLR. Overexpression of WT-ASGR1/2 reduced primarily the immature non-O-glycosylated LDLR ([~]110 kDa), whereas the triple Gln240/Trp244/Glu253 Ala-mutant (loss of carbohydrate-binding) reduced the mature form of the LDLR ([~]150 kDa), suggesting that ASGR1 binds the LDLR in sugar-dependent and -independent fashion. Furin sheds ASGR1 at RKMK103{downarrow} into a secreted form, likely resulting in a loss-of-function on LDLR. LDLR is the first example of a liver-receptor ligand of ASGR1. Additionally, we demonstrate that lack of ASGR1 enhances LDLR levels and DiI-LDL incorporation, independently of PCSK9. Overall, silencing of ASGR1 and PCSK9 may lead to higher LDL-uptake by hepatocytes, thereby providing a novel approach to further reduce LDL-cholesterol.