The asialoglycoprotein receptor 1 (ASGR1): emerging new roles in health and disease

Abstract

The asialoglycoprotein receptor 1 (ASGR1) has an established role as a hepatic receptor that clears desialylated glycoproteins from the bloodstream. Recently, its role in health and disease has expanded beyond this from cardiovascular to cancer and viral infection. Circulating ASGR1 is associated with elevated risk of coronary artery disease. Preclinical studies have reinforced this, showing ASGR1 inhibition reduces atherosclerotic cardiovascular disease. ASGR1 controls platelet lifespan and function, thereby regulating coagulation and thrombosis. It also facilitates entry of bacteria and viruses into host cells, causing immunomodulatory effects, and plays dual roles in cancer - promoting and suppressing tumorigenesis. This review provides an in-depth overview of the latest mechanistic insights underlying the roles of ASGR1 in health and disease and elaborates on the progress of ASGR1-targeted therapies.

Introduction

The asialoglycoprotein receptor (ASGPR) is a c-type lectin receptor predominantly expressed in the liver. It has a well-characterized role in the circulatory clearance of glycoproteins that have had their end terminal sialic acid groups removed [1]. The process that removes the sialic acid groups is called desialylation and is strongly related to human health and illness [2]. Sialylation, by contrast, involves the addition of terminal sialic acid residues on glycoproteins and glycolipids using glycosidic linkages and is essential for many physiological functions, including cell adhesion, signalling and immunomodulation [3]. ASGPR was discovered by Gilbert Ashwell and Anatol Morell in the 1960s and hence, named the Ashwell-Morell receptor. ASGPR comprises of two subunit proteins, asialoglycoprotein receptor 1 (ASGR1) and asialoglycoprotein receptor 2 (ASGR2), which are the major and minor subunits of ASGPR, respectively [1, 4]. In humans, the genes encoding ASGR1 and ASGR2 are found on the short arm of chromosome 17. The ASGR1 gene is approximately 6 kb long and contains 8 exons, whereas the ASGR2 gene comprises 9 exons and is 13.5 kb long. The expression of ASGR1 and ASGR2 are interdependent and ASGR1 is essential for the stabilization of ASGR2 [5].

ASGR1 and ASGR2 are type II-single pass transmembrane receptors encompassing an extracellular carbohydrate recognition domain, a transmembrane domain, and a cytoplasmic domain (Fig. 1). The carbohydrate recognition domains (CRDs) have three Ca²⁺ ion binding sites [6]. The CRD with two carbonyl-containing amino acids separated by a cis-proline, coordinates Ca²⁺ and forms hydrogen bonds with sugars to determine specificity. For example: a ‘QPD’ (Gln-Pro-Asp) motif is specific for galactose (Gal) or its derivative, N-acetylgalactosamine (GalNAc), whereas a ‘EPN’ (Glu-Pro-Asn) motif confers specificity for mannose-based ligands [7]. Many proteins with a C-type CRD fold lack the conserved Ca²⁺-binding site and therefore do not bind sugars. This distinguishes true sugar-binding CRDs from the broader family of C-type lectin-like domains [8].

Fig. 1.

ASGPR function in healthy cells and its interaction with asialoglycoproteins. (A) Structure and physiological function of ASGPR in healthy hepatocytes [9]. (a) The asialoglycoprotein receptor (ASGPR) is composed of ASGR1 and ASGR2 hetero-oligomer subunits [1, 9]. The carbohydrate recognition domain, stalk, transmembrane and cytosolic domains are indicated by black arrows. Phosphorylation of ASGPR cytoplasmic domain causes the binding of specific molecular chaperones and regulates receptor trafficking [10]. (b) Glycoprotein loaded ASGPR dissociates from its ligand in the endosome [11], (c) and recycles back to the cell membrane rapidly [9, 11]. (d) The glycoprotein moves to lysosomes via vacuoles for enzymatic degradation. This applies to all cell types that ASGR1 is expressed in. (B) Interaction between the ASGPR and circulating desialylated glycoproteins with terminal Gal and GalNAc containing residues [12]

Among the Ca²⁺-dependent lectin receptors, two are especially noteworthy; ASGR1 (also known as CLEC4H1) and C-type lectin domain family 10 member A (CLEC10A), also known as the macrophage galactose-type lectin (MGL) receptor. Both receptors possess homologous CRDs that preferentially bind GalNAc over Gal [11]. ASGR1 is expressed predominantly in hepatic parenchymal cells [1], although recent studies showed its expression in immune cells such as monocytes and macrophages [13], whereas CLEC10A is expressed at multiple stages of human development, with the highest expression observed in oocytes [11]. ASGR1 primarily mediates clearance of circulating desialylated glycoproteins [9, 14], whereas CLEC10A is also expressed on immature DCs, monocyte-derived DCs as well as alternatively activated macrophages and mediate diverse immune responses such as the induction of TH1, TH2, and TH17 immune responses and promotes tolerance in naïve T cells [11].

ASGR1 and ASGR2 preferentially bind to desialylated glycoproteins containing GalNAc over Gal, with approximately 60-fold and 2-fold selectivity, respectively [11]. This, interaction between the CRD and its ligands, the desialylated glycoproteins with GalNAc and/or Gal terminal residues is calcium dependent [7, 8]. Calcium is not just supportive, it is part of the binding mechanism [9]. ASGR1 requires three bound Ca²⁺ ions for ligand binding and for the release of ligands from ASGR1 intracellularly in endosomes it depends on Ca²⁺ dissociation from ASGR1 [11]. Ca²⁺ is loaded during ER folding and recharged during receptor recycling at the cell surface [11]. In endosomes, acidification lowers Ca²⁺ levels, and once it drops below 330 µM, the ligand dissociates within minutes, likely coupled to Ca²⁺ transport into the cytosol [11]. In normal serum glycoproteins, terminal sialic acid often masks these sugars. However, when sialic acid is lost, the underlying galactose or GalNAc becomes accessible to ASGR1. This is why ASGR1 is described as a clearance receptor for asialoglycoproteins [9, 11]. Depending on glycan branching, glycoproteins display multiple terminal Gal/GalNAc residues per molecule (multivalency), enabling simultaneous binding to multiple CRDs and greatly increases overall avidity [11]. Ligand multivalency is a key feature in protein-glycan interactions, and enhances not only binding affinity but also selectivity [9]. For example, super-selective binding occurs when target ligand density surpasses a critical threshold, enabling cell discrimination based on differential glycan expression and underpinning the high liver-targeting specificity of GalNAc-conjugated therapeutics [9].

ASGR1 has a well-established role as a clearance pathway for unwanted circulating desialylated glycoproteins [9, 14]. Its role in downstream signalling pathways and alternate mechanisms of disease remains underexplored. Under physiological conditions, ASGR1 specifically binds to those glycoproteins with terminal galactose (Gal) or N-acetylgalactosamine (GalNAc) residues on proteins, lipids and nucleic acids [14, 15]. These are then internalized through clathrin-mediated endocytosis, ASGR1 dissociates from the bound ligand in the endosome and rapidly recycles back to the cell membrane [11, 16].

Beyond its long-established physiological properties, the past decade has seen the emergence of new evidence linking ASGR1 to different disease states including cardiovascular disease, cancer, infectious diseases and autoimmune conditions. This review provides an in-depth overview of the latest insights on the increasing number of mechanisms underlying the functions of ASGR1 in health and disease.

ASGR1 and atherosclerotic cardiovascular disease

In 2016, a large-scale genome-wide association study (GWAS) found two infrequent loss-of-function mutations in ASGR1, explicitly a 12-base-pair deletion in intron 4 (del12) and a 4-base-pair insertion mutation (p.W158X), associated with a 34% decreased risk of coronary artery disease in humans [17]. In this study, both del12 and p.W158X were corelated with a reduction in non-high-density lipoprotein-cholesterol (non-HDL-C) and triglycerides. Interestingly, elevations in serum alkaline phosphatase and vitamin B12 were reported, both of which are desialylated glycoproteins and ligands of ASGR1. This provided evidence that functional changes in ASGR1 were present in carriers of these mutations [17]. In support of this study, monocyte ASGR1 mRNA levels were found to positively correlate with low-density lipoprotein cholesterol (LDL-C) and total cholesterol, and were lower in patients taking cholesterol-lowering therapy compared to those who were not [18].

Preclinical studies support these epidemiological findings. ASGR1 inhibition with a monoclonal antibody enhanced cholesterol excretion, reduced blood cholesterol and decreased atherosclerotic plaque size in atherosclerotic apolipoprotein E^−/− (Apoe^−/−) mice [19]. Heterozygous Asgr1^+/− and homozygous Asgr1^−/− genetically modified mice also had lower plasma and hepatic levels of total cholesterol and triglycerides, relative to their wild-type counterparts [19]. Delineation of the mechanisms revealed Asgr1 deletion activates AMP-activated protein kinase (AMPK) [19, 20], which increases liver X receptor alpha (LXRα) and liver X receptor beta (LXRβ) and in turn their target genes encoding ATP binding cassette transporter A1 (ABCA1) and ATP binding cassette subfamily G members 5 and 8 (ABCG5, ABCG8) [19]. ABCA1, ABCG5 and ABCG8 all play an important role in mediating the efflux of cholesterol from cells. ABCG5 and ABCG8 form a heterodimer that specifically facilitates biliary secretion of cholesterol. Interestingly, deletion of Asgr1 increased biliary cholesterol levels. ASGR1 inhibition also suppressed mammalian target of rapamycin complex 1 (mTORC1), which downregulates sterol regulatory element binding protein 1 (SREBP1), an important transcription factor for cholesterol handling mechanisms. Therefore, via synergistic activation of AMPK activation and suppression of mTORC1, ASGR1 inhibition or deletion reduces cholesterol biosynthesis and increases hepatobiliary cholesterol secretion, both of which provide an explanation for the reduction in plasma cholesterol levels [19, 21].

CRISPR-generated Asgr1^−/+ and Asgr1^−/− deficient pigs were found to have lower triglycerides, non-HDL-c and smaller atherosclerotic lesions compared to wildtype controls. Asgr1^−/+ pigs were found to have reduced hepatic 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), the critical rate limiting enzyme in cholesterol synthesis [22, 23]. Liver transcriptome profiling identified that 12 genes involved in cholesterol metabolism pathways were downregulated in Asgr1^−/− pig livers [22]. However, they also noted that Asgr1 deficiency was accompanied by increased levels of liver enzymes, particularly alkaline phosphatase (ALP), suggesting an increase in liver inflammation [22]. In summary, ASGR1 deficiency appears to engage a broadly conserved metabolic axis in which reduced ASGR1 signalling lowers circulating cholesterol via AMPK activation and mTORC1 inhibition in mice [19, 21], with similar reductions in triglycerides, non-HDL-c and atherosclerotic lesions observed in ASGR1 deficient large‑animal models such as pigs [22]. Interestingly, previous studies showed that mTORC1 is a conserved serine/threonine kinase that integrates nutrient and energy cues to regulate cellular metabolism, including lipid metabolism [24]. AMPK is also known as an evolutionarily conserved energy sensor that inhibits lipid metabolism by phosphorylating and suppressing acetyl-CoA carboxylase (ACC) and HMGCR [25]. This indicates that ASGR1 deficiency-associated modulation of AMPK-mTORC1 signalling in atherosclerosis may be evolutionarily conserved across mammals and supports ASGR1 as a translational therapeutic target.

Apoe^−/− x Asgr1^−/− mice, placed on a high-fat atherogenic diet for 12 weeks, were found to develop smaller atherosclerotic plaques. Very-low-density lipoprotein (VLDL) and LDL-c levels were also lower and there was a decrease in VLDL generation and increased fecal cholesterol relative to Apoe^−/− control mice [26]. The reported mechanism of action was focussed on the reduction in VLDL production, mediated by reduced expression of microsomal triglyceride transfer protein (MTTP), important for VLDL and chylomicron assembly [26]. There was also a reduction in angiopoietin-like protein 3/ angiopoietin-like protein 8 (ANGPTL3/8) and an increase in lipoprotein lipase (LPL) activity, both part of VLDL metabolism [26]. As has been reported by other studies, ASGR1 deletion led to increased hepatic LDL receptor (LDLR) levels, suggesting enhanced LDL-c clearance [22, 26]. Increased biliary total cholesterol was also observed, supported by increases in LXRα, ABCA1 and ABCG5 [26]. This study also found Asgr1^−/x Apoe^−/− mice had elevated plasma ALT and AST levels [26].

In vitro studies have helped to elucidate the mechanisms of action of ASGR1 in relation to lipid metabolism. Proprotein convertase subtilisin/kexin type 9 (PCSK9), a SREBP1-targeted gene that mediates the ubiquitination and degradation of the LDLR, is found to be lower in ASGR1 deficient hepatocytes [27]. ASGR1 deficiency enhances retention of SREBPs in the endoplasmic reticulum by upregulating insulin-induced gene 1 (INSIG1/2). This hinders SREBP activation by anchoring SREBPs in the endoplasmic reticulum with SREBP-cleavage-activating protein (SCAP) and insulin-induced gene 1/2 (INSIG1/2)[27]. It was hypothesized that in the presence of ASGR1, the SCAP/SREBP complex dissociates from INSIG1/2 and is packaged into vesicles to travel into the Golgi apparatus. In the Golgi, SREBP is proteolytically cleaved to form active nuclear SREBPs that enter the nucleus and bind to the sterol response element to enhance lipogenesis. Consistent with this, ASGR1 inhibition increased LDL-c uptake from the circulation and clearance by hepatocytes [27].

Interestingly, these LDLR-related changes by ASGR1 deletion/inhibition appear to occur independently of PCSK9. Whilst overexpression of PCSK9 causes expected decreases in the LDLR, this does not impact ASGR1, yet ASGR1 overexpression still increases LDLR. ASGR1 is thought therefore to mediate its effects on the LDLR via its carbohydrate recognition domain in a carbohydrate dependent and/or independent manner [28]. Furthermore, heterobifunctional molecules that bind simultaneously to ASGR1 and PCSK9, increase PCSK9 clearance from the circulation, and in the absence of these heterobifunctional molecules ASGR1 had no effect on PCSK9 clearance [29]. Overall, this suggests ASGR1 deficiency regulates lipid homeostasis via LDLR upregulation, independent of changes in PCSK9 (Fig. 2).

Fig. 2.

Pathways influenced by ASGR1 with relevance to atherosclerosis. ASGR1 deletion activates AMPK, a cellular energy sensor that plays a key role in regulating metabolism [19, 20]. Activated AMPK triggers the activation of lipid metabolism regulating genes specifically LXRα and LXRβ followed by their target gene ABCG5/8 and ABCA1 upregulation [19]. ABCG5/8 promotes cholesterol excretion into the bile whereas ABCA1 enhances cholesterol efflux [21]. ASGR1 inhibition activates INSIG1/2 and downregulates mTORC1, both synergize in suppressing lipogenesis by downregulating SREBP [19]. Once the SREBPs anchored to the ER with SCAP and INSIG1/2, the complex prevents SREBP movement into Gologi and its’ proteolytic cleavage and activation [30]. ASGR1 deficiency suppresses cholesterol synthesis regulating enzyme, HMGCR, which in turn upregulates LDLR and LDL uptake, and hence, decreases circulatory LDL [19]. A deficit in ASGR1 inhibited MTTP followed by a decreased VLDL production and chylomicron assembly leading to a decreased bloodstream LDL and TG [26, 27]. ASGR1 deficiency increases VLDL and TG clearance by downregulating ANGPTL3/8 and upregulating LPL activity [26]. At the same time, ANGPTL3/8 suppression enhances HDL functionality [31]. ASGR1 deficiency supresses inflammation through downregulating Notch1 activation [32]. Notch1 suppression downregulates JACK2/STAT3 phosphorylation leading to reduced ROS production, and hence, impedes inflammation [33]. Likewise, inhibited Notch1 signalling downregulates ICAM-1 [34], important adhesion molecule for monocyte [35] and neutrophil [36] extravasation, ultimately impairing their recruitment and inflammation [35, 36]. Suppressed Notch1 signalling also favours T regs and other anti-athrogenic immune cells development while suppressing pro-athrogenic inflammatory macrophages, all of which are anti-inflammatory [37]. A heterobifunctional antibody that binds simultaneously to ASGR1 and PCSK9 enhanced PCSK9 degradation, which in turn increased LDL clearance via the LDLR pathway [29]. Abbreviations: AMPK, AMP-activated protein kinase; LXRα, Liver X receptor alpha; LXRβ, Liver X receptor beta, ABCG5/8, ATP-binding cassette transporters G5 and G8; ABCA1, ATP-binding cassette transporter A1; INSIG1/2, Insulin-induced genes 1 and 2; mTORC1, Mechanistic target of rapamycin complex 1; SREBP, Sterol regulatory element-binding protein; ER, Endoplasmic reticulum; SCAP, SREBP cleavage-activating protein; HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase; LDLR, Low-density lipoprotein receptor; LDL, Low-density lipoprotein; MTTP, Microsomal triglyceride transfer protein; VLDL, Very low-density lipoprotein; TG, Triglycerides; ANGPTL3/8, Angiopoietin-like proteins 3 and 8; LPL, Lipoprotein lipase; PCSK9, Proprotein convertase subtilisin/kexin type 9; JACK2/STAT3, Janus kinase 2/signal transducer and activator of transcription 3; ICAM-1, Intercellular adhesion molecule 1

Monocytes and macrophages are key cellular players in atherosclerosis. An in vitro study found inflammatory stimulation with lipopolysaccharide (LPS) increased ASGR1 protein expression in a human monocytic cell line (THP-1) [13]. Furthermore, ASGR1 mediated the differentiation of monocytes into macrophages through upregulation of NF-κB signalling and activation of transcription factor 5 (ATF5) in wildtype mice. Conversely, ASGR1 depleted mice had reduced monocyte to macrophage differentiation and fewer Ly6C⁺ inflammatory macrophages in the liver [13]. Suppression of macrophage differentiation represents an atheroprotective mechanism of ASGR1 deletion.

Another pathway of ASGR1 regulation was revealed in which Asgr1^−/− mice had increased visceral and subcutaneous adipose tissue relative to wildtype controls. This suggested lipids from the plasma were being redirected towards adipose tissues, with no change in glucose homeostasis [38]. Interestingly, Asgr1^−/− mice had mitochondrial dysfunction in their visceral adipocytes which was linked to downregulation of oxidative phosphorylation [38]. Impaired mitochondrial oxidative phosphorylation is known to decrease coronary atherosclerosis severity by reducing adiponectin in adipose tissue [39], This suggests ASGR1 deficiency may be atheroprotective by inhibiting adiponectin through downregulation of oxidative phosphorylation. Moreover, liver transcriptomic analysis in Asgr1^−/− mice showed that 181 genes were dysregulated, 77 upregulated and 104 downregulated. These genes were enriched in metabolic pathways and 12 were linked to lipid metabolism. Apolipoprotein B (ApoB) was also lower in the serum and livers of the Asgr1^−/− mice [27].

In another study, proteomic profiling of visceral adipose tissue showed that Asgr1 deficiency was accompanied by downregulation of proteins associated with lipogenesis and upregulation of those involved in lipoprotein uptake [38]. Of note, CD36 scavenger receptor, fatty acid synthase, and malate dehydrogenase were downregulated, while cytochrome P450 oxidases like CYP1A2, CYP2C8, and CYP2E1 were upregulated. Visceral adipose tissue analyses also found fatty acid synthase, acetyl-CoA carboxylase, stearoyl-Coenzyme A desaturase 1, lysosomal associated membrane protein, and sterol carrier protein 2 were downregulated [38], all changes associated with atheroprotection [40–42].

In vitro studies in mouse primary cells demonstrated that inhibition of hepatic ASGR1, using an anti-ASGR1 antibody or the ASGPR inhibitor Asialofetuin, which acts as a competitive ligand or agonist that blocks the binding of endogenous desialylated glycoproteins, decreased activation of Notch1 signalling and hepatic thrombopoietin production, regulators of thrombopoiesis [32]. Conversely, treatment of hepatocytes with desialylated platelets stimulated Notch1 signalling and thrombopoietin production. However, ASGR1 and ASGR2 protein levels were unchanged in Notch1 deficient hepatocytes [32], suggesting Notch1 deletion does not alter hepatocyte ASGR1 and ASGR2. In vivo macrophage specific Notch1 deletion (Notch1^MAC−KO) supressed atherosclerosis by reducing inflammatory macrophages and increasing the activation and polarization of anti-atherogenic macrophage phenotypes in Apoe^−/−Notch1^MAC−KOmice, when compared to Apoe^−/− control mice [43]. Suppression of Notch1 also reduces low shear stress-induced formation of atherosclerotic plaques [34] and the expression of intercellular adhesion molecule 1 (ICAM-1), important for monocyte and neutrophil recruitment and atherosclerotic plaque development [34, 35]. Another consequence of ASGR1 deletion-induced Notch inhibition includes an increase in endothelial cell proliferation [44], an important function for re-endothelialisation. Taken together, ASGR1 inhibition may have atheroprotective and endothelial protective effects via suppression of Notch signalling.

Other studies have found ASGR1 inhibition suppresses signal transducer and activator of transcription 3 (STAT3) phosphorylation through Nemo-like kinase (NLK) which interacts with the Src Homology 2 domain of STAT3 [45]. STAT3 is associated with increased inflammation (IL-6, IL-1β, TNF-α, IκBα) and enhanced oxidized LDL (ox-LDL) uptake into macrophages [46]. Thus, ASGR1 inhibition, through its suppression of STAT3 phosphorylation may inhibit inflammation and ox-LDL uptake, both atheroprotective mechanisms.

In vitro treatment of Lewis lung carcinoma (3LL) cells with mouse recombinant ASGR1 was found to trigger the activation of the epidermal growth factor receptor - extracellular signal regulated kinase (EGFR-ERK) signalling pathway and the upregulation of matrix metalloproteinase-9 (MMP-9) transcription [47]. This is a mechanism that increases atherosclerotic plaque instability [48]. Consistent with this, elevated Egfr mRNA levels have been reported in atherosclerotic lesions and correlate significantly with plaque size [49]. EGFR inhibition reduces plaque macrophages, oxidative stress, pro-inflammatory cytokine production and SMC proliferation in high fat diet fed Apoe^−/− mice [50]. Moreover, activation of the EGFR-ERK pathway upregulates Forkhead box M1 (FOXM1), that supresses reactive oxygen species (ROS) [51], nitric oxide synthase (NOS) [52], IL-6 [53], the C-C motif chemokine receptor type 2 (CCR2) [54] and C-X3-C motif chemokine receptor 1 (CX3CR1) [55], all of which are involved in promoting atherosclerosis [56]. Collectively, there is interplay between the inhibition of ASGR1, reduced levels of oxidative stress and inflammation as well as an association with improved plaque stability.

Demina et al. found neuraminidases (NA) NA1 and NA3, which remove sialic acid from glycolipids and LDL-associated glycoproteins to enable interaction with ASGR1, promote atherosclerosis development [57]. Another study found higher Na1, Na3 and Na4 mRNA levels in atherosclerotic lesions of thoracic aorta relative to healthy tissue [58]. LDL desialylation also increases LDL accumulation in the intima by first enhancing LDL attachment to arterial proteoglycans [59] and improving LDL uptake by plaque macrophages via ASGR1 in Apoe^−/− mice [57]. Overall, these studies show ASGR1 inhibition may reduce atherosclerosis via suppression of LDL uptake into macrophages.

In addition to its emerging atheroprotective effects, ASGR1 deficiency has also been linked with protection against type 2 diabetes mellitus, a common co-morbidity in cardiovascular disease patients. In Asgr1^−/− mice fed high fat diet, there was increased hepatic insulin-triggered phosphorylation of insulin receptor β and protein kinase B, suggesting a role for ASGR1 in glucose homeostasis through insulin signalling [60]. The same study demonstrated decreased serum insulin levels in Asgr1^−/− and Asgr1^+/− mice relative to wildtype mice [60], indicating enhanced insulin sensitivity in the presence of high fat diet-induced metabolic stress [61]. Hepatic insulin resistance is the major driver of systemic insulin resistance [62].

In contrast to the growing body of evidence that ASGR1 deficiency is associated with a reduction in atherosclerotic cardiovascular disease [17, 19, 22, 23, 27, 63], an epidemiological study found a 2.4 fold reduction of ASGR1 mRNA expression in isolated peripheral blood mononuclear cells (PBMCs) from Iranian patients with coronary artery disease, when compared to healthy controls. The same study also found that whilst PBMC ASGR1 mRNA expression was inversely associated with the incidence of coronary artery disease, it was not associated with its severity [64]. The findings of this study are contrary to another similarly designed study which found a positive correlation between soluble serum ASGR1 and LDL-C levels [65].

In summary, ASGR1 plays a role in lipid metabolism and atherosclerotic cardiovascular disease. Loss-of-function variants and experimental ASGR1 inhibition reduce plasma cholesterol, triglycerides, VLDL production, and atherosclerotic plaque size, partly through enhanced LDL receptor activity, AMPK activation, and mTORC1 suppression. ASGR1 deficiency also modulates inflammation by reducing monocyte-to-macrophage differentiation, STAT3 and Notch1 signalling, and LDL uptake into plaque macrophages, all atheroprotective mechanisms.

ASGR1 and coagulopathy

Platelets control the coagulation pathway. Their activation markers include the sialylated glycoproteins: glycoprotein-Ib-alpha (GPIbα) subunit and P-selectin (CD62P) [66]. Desialylation of platelets triggers their hepatic clearance through an ASGR1-mediated pathway which, in turn, activates thrombopoietin production and new platelet synthesis [67]. Aging and some microbial infections can trigger desialylation of platelet glycoproteins and interrupt platelet synthesis and their removal cycle, which affects the coagulation pathway [66, 68]. Plasma von Willebrand factor (VWF) is a glycoprotein and the presence of a terminal sialic acid group is crucial for preventing VWF degradation and its plasma levels [69]. Loss of the terminal sialic acid group by genetic inactivation of ST3 beta-galactoside alpha-2,3-sialyltransferase 4 (ST3GAL4), a sialyltransferase, was associated with hyposialylation and increased VWF clearance through ASGR1 [70]. Consistent with this, VWF clearance was severely attenuated in ASGR1 deficient mice [71]. ASGR1 deficient mice also showed slower bleeding times compared to wildtype littermates [70, 72], whereas ASGR2 deficient mice exhibited no differences in bleeding times, demonstrating ASGPR subunit specificity [70]. ASGR1 and macrophage galactose-type lectin (MGL) are receptors with special binding preference to N-acetylgalactosamine (GalNAc), with both receptors being expressed on antigen presenting cells including macrophages and dendritic cells [9, 73]. In vivo macrophage depletion showed decreased clearance of hyposialylated VWF [71, 74]. VWF maintains hemostasis by controlling platelet adhesion to subendothelial collagen and also the intrinsic coagulation pathway through stabilizing factor VIII (FVIII) [75]. Overall, sialylation-controlled VWF clearance explains the mechanism by which ASGR1 regulates the coagulation and thrombosis pathway.

ASGR1 and liver injury

Some studies find ASGR1 deficient mice exhibit no apparent phenotypic changes, with normal body weight, life span [5, 60], liver mass and liver function, indicated by alanine aminotransferase (ALT) and aspartate aminotransferase (AST) [60]. An in vitro study using the human embryonic stem cell line WA01 found a normal karyotype and morphology, pluripotency and differentiation ability following ASGR1 knockdown [76].

In contrast, other studies have found increases in liver injury, ALT and AST with ASGR1 deficiency [22, 77–79]. Mice lacking ASGR1 exhibit more severe liver damage following carbon tetrachloride induced toxic liver injury [77]. The mechanism for this was purported to be via galactose, which exclusively binds to hepatic ASGR1 and upregulates anti-apoptotic hepatic genes (e.g. TNF alpha induced protein 3 and baculoviral IAP repeat containing 2), through NF-κB signalling. In the absence of ASGR1, this galactose-mediated anti-apoptotic pathway is therefore suppressed. Interaction between galactose and ASGR1 also downregulates caspase-3-mediated hepatocyte apoptosis in wildtype mice [80]. Likewise, a recent study revealed ASGR1 deficiency exacerbates hepatic injury in mice exposed to the liver toxin carbon tetrachloride, compared to wildtype controls. The same study found ASGR1 binds with golgi protein 73 (GP73), a mediator of endoplasmic reticulum stress, which enhances GP73 lysosomal degradation to prevent hepatic injury [78]. A deficiency in ASGR1 would therefore remove this protective mechanism and increase endoplasmic reticulum stress, a key driver of hepatic injury [81, 82].

Consistent with this, a study in pigs found ASGR1 deficiency was associated with increased hepatic endoplasmic reticulum stress [22]. Increased liver damage was also found in mice with ASGR1 deficiency, accompanied by an increased hepatic inflammatory cell infiltrate and higher mRNA levels of proinflammatory cytokines Tnfa, Il6, Ccl2, and Il1b [78]. There were increased plasma levels of biomarkers of liver dysfunction, ALT, AST, alkaline phosphatase, and gamma-glutamyl transferase [78]. Furthermore, a clinical study found hepatic ASGR1 protein and mRNA levels were lower in cirrhosis patients [78].

In non-alcoholic steatohepatitis, ASGR1 and cytochrome P450 family 2 subfamily E member 1 (CYP2E1) protein levels were upregulated in mice fed chow and high cholesterol diet [83]. Interestingly, CYP2E1 is activated in carbon tetrachloride-induced liver damage [84]. However, a recent study demonstrated that the level of CYP2E1 was unaffected by ASGR1 deficiency following tetrachloride and acetaminophen treatment [78]. This suggests that enhanced liver injury in ASGR1 deficient mice occurs independently of the CYP2E1 pathway. The phenotypic effects of ASGR1 knockout in animal models of atherosclerotic cardiovascular disease are summarized in Table 1.

Table 1.

Phenotypic effects of ASGR1 knockout across various animal models of atherosclerotic cardiovascular diseases

Animal model	Lipid profile	Liver function test	Metabolic effects	Disease outcomes	References
Mouse Asgr1−/− on HF/HC/BS	↓ Plasma and liver TC and TGs.	↑ Serum ALT and AST (mild increase)	↑ AMPK activation, LXRα protein and cholesterol efflux. No change in blood glucose.	Reduced arterial lipid deposition and plaque formation and is cardioprotective.	Wang et al. [19].
Apoe−/− x Asgr1−/−on WD	↓ Plasma VLDL and LDL-C.	↑ Serum ALT and AST (mild increase)	↑ LDLR and LPL. Reduced VLDL production and increased faecal cholesterol.	Decreased arterial lipid deposition and plaque formation and is cardioprotective.	Zhang et al. [26].
Asgr1−/+ on HF/HC/BS diet	↓ Plasma and liver TC and TGs.	No change in serum ALT and AST	No change in blood glucose.	Cardioprotective via reduced arterial lipids and plaque formation.	Wang et al. [19].
Pig ASGR1−/−	↓ Plasma TC, non-HDL-C, TGs and LDL-C at 17 months of age.	↑ Plasma ALP, ALT and AST	↑ Plasma vitamin B12	↓ Atherosclerotic lesions. Cardioprotective effect	Xie et al. [22].
PigASGR1−/+	↓ Plasma TC, LDL-C, TGs and HMGCR.	↓ Plasma ALT and no change in AST	↓ Cholesterol synthesis and uptake genes mRNA expression	Cardioprotective effect and no liver injury.	Yin et al. [85].
Human ASGR1 LoF variants	↓ Plasma non-HDL-C and TGs.	↑ Serum ALP	↑ Serum vitamin B12	↓ Coronary artery disease risk. Cardioprotective effect.	Nioi et al. [17].

ALP, Alkaline Phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; AMPK, AMP-activated protein kinase; Apoe^−/−, apolipoprotein E^−/−; ASGR1, Asialoglycoprotein receptor 1; non-HDL-C, non- high-density lipoprotein cholesterol; HF/HC/BS, High-fat/ high-cholesterol/bile salt diet; HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase; LDL-C, Low-density lipoprotein cholesterol; LDLR, Low-density lipoprotein receptor; LoF, Loss-of-Function; LPL, Lipoprotein lipase; LXRα, Liver X receptor alpha; mRNA, messenger Ribonucleic Acid; TC, Total cholesterol; TGs, Triglycerides; VLDL, Very-low-density lipoprotein; WD, Western diet

In summary, there is evidence that ASGR1 deficiency enhances liver injury and inflammation through suppression of key anti-apoptotic and anti-inflammatory pathways.

Balancing cardiovascular benefit and liver injury risk in ASGR1 deficiency

ASGR1 deficiency consistently improves cardiovascular outcomes in animal models primarily by lowering circulating lipids and attenuating atherosclerosis progression [19]. In Apoe^−/− Western diet fed mice, ASGR1 deficiency reduces plasma cholesterol and triglycerides, including VLDL and LDL cholesterol as well as atherosclerotic plaque area compared to ASGR1 wild type littermates [26]. In humans, genetic studies show that ASGR1 loss-of-function variants are associated with lower non-HDL/LDL cholesterol and reduced risk of coronary artery disease [17], reinforcing the protective role of ASGR1 deficiency in atherosclerotic cardiovascular diseases.

Conversely, ASGR1 deficiency exacerbates liver injury as marked by increased plasma ALT and AST [22] and elevated hepatic ER stress [78], highlighting a key trade-off. ASGR1 deficient mice showed increased circulating GP73 and stronger GP73‑ immunoglobulin heavy-chain-binding protein (BIP) interaction, promoting ER stress driven hepatic injury [78]. In Apoe^−/− ASGR1 deficient mice fed western diet for 16 weeks, decreased lipid profile, atherosclerosis plaque area and necrotic core are paralleled by increased plasma AST/ALT ratio [86]. These findings underscore that therapeutic ASGR1 targeting in animals must balance reduced cardiovascular risk against heightened hepatic vulnerability and ER stress. Targeting ASGR1 inhibition strategies to specific sites, such as atherosclerotic plaques, may help to bypass potential side effects elicited by pan-ASGR1 inhibition.

ASGR1 and the immune system

Plasma proteomic analyses in Asgr1^−/− mice revealed there were 291 upregulated proteins related to coagulation, proteolysis and the immune system [87]. As a regulator of glycoprotein biosynthesis [88], ASGR1 has the ability to control the immune system through changes in glycosylation [89, 90]. For example, ASGR1 facilitates antigen uptake by dendritic cells, which in turn promotes the expansion of IL-10-producing CD4⁺ T cells. These T cells adopt an immunosuppressive/regulatory phenotype that contributes to immune tolerance [89, 91].

To maintain immune cell homeostasis, hepatocytes eliminate activated lymphocytes through perforin-dependent mechanisms and CD95 ligand-CD95 interactions [92]. This occurs via endocytosis and a process termed suicidal emperipolesis, in which T cells are engulfed and subsequently killed by hepatocytes [93, 94]. Importantly, hepatocyte cytotoxic effector function is modulated by surface glycoprotein desialylation of the target cells, the level of ASGR1 expression on hepatocytes, and the surrounding cytokine milieu [92] (Fig. 3). Consistent with this, neuraminidase treatment has been shown to induce desialylation of CD95-bearing P815 cells (a murine mastocytoma-derived mast cell line), as well as CD95-deficient K562 cells (a human erythroleukemia cell line), in vitro.

Fig. 3.

The role of ASGR1 in immune responses. (a) Under physiological conditions, tightly regulated cell-surface sialylation contributes to normal cell signalling and helps maintain the balance between apoptosis (programmed cell death) and cell proliferation, thereby supporting tissue homeostasis [95]. (b) However, alterations in sialylation dynamics are observed upon immune activation, with much heterogeneity. For instance, activated T cells expressing B220 epitope, a sialic acid depleted glycoform of CD45, exhibit markedly increased surface desialylation [92]. This activates ASGR1 to take place its effector function [92] and will target desialated cells including desialated B cells and T cells for endocytosis [96, 97], and hence, contributes to immunological hemostasis through contracting the immune system and maintaining a steady immunological environment [92]. Moreover, desialylation also enhances hepatocytes to kill activated cells through perforin-dependent and CD95 ligand-CD95 pathways [92]

In vitro, cultured WCM-260 hepatocytes and human HepG2 cells killed both CD95-bearing P815 and CD95-deficient K562 target cells in an ASGR1-mediated manner. This was further supported by reduced target cell killing observed in primary mouse hepatocytes following siRNA-mediated ASGR1 knockdown [92]. In the absence of CD95, activated CD4⁺ T lymphocytes expressing B220, a CD45 form with depleted sialic acid, were also cleared by hepatocytes via ASGR1 [92, 98]. Conversely, hepatocytes with ASGR1 knockdown displayed significantly diminished target cell killing [92], underscoring a critical role for ASGR1 in clearing desialylated lymphocytes, independent of CD95.

Sialic acids on B and T cell membranes are essential for their maturation, differentiation, migration and survival by counteracting apoptosis and other different death-inducing pathways [96, 97]. Mice deficient in B cell-specific cytidine monophosphate sialic acid synthase (CMAS), the enzyme that mediates sialoglycan synthesis, exhibited severe B-cell deficiency [96]. Likewise, mice deficient in T cell-specific CMAS displayed peripheral T cell deficiency accompanied by increased activated caspase-3, indicating an ongoing apoptosis and impaired T cell survival [99]. These findings highlight the critical role of sialylation in lymphocyte homeostasis and reinforce the function of ASGR1 in selectively clearing desialylated B [96] and T cells [99], thereby contributing to the regulation of adaptive immune cell populations.

ASGR1 is recognized as the major antigenic component of liver-specific proteins and has identified as a liver specific autoantigen in autoimmune hepatitis [100, 101]. Löhr et al. reported that 50% of patients with autoimmune hepatitis and 15% of those with primary biliary cirrhosis process autoantibodies against human hepatic ASGPR [102]. Beyond its role as an autoantigen, ASGR1 has been implicated in the clearance of apoptotic cells [15], removal of cellular fibronectin [103] and IgA clearance from the circulation [104, 105].

Deficiency in ASGR1 supresses endocytosis and lysosomal breakdown of glycoproteins [19]. Transient inhibition of endocytosis activates immune responses through antibody-dependent cellular cytotoxicity, by increasing target antigen availability to effector cells and thereby enhancing natural killer cell function [106]. In line with this, Asgr1^−/− mice displayed increased numbers of innate immune cells, including monocytes, natural killer cells, and dendritic cells, in visceral adipose tissue, whereas neutrophils, macrophages, CD4⁺ T cells, CD8⁺ T cells, and B cells were unchanged. In contrast, liver immune profiling in Asgr1^−/− mice revealed reduced natural killer cells and dendritic cells, while other immune cell subsets remained unaffected [38]. The expression of ASGR1 (also known as CLEC4H1) was negatively correlated with neutrophils, dendritic cells, macrophages, CD8⁺ T cells, CD4⁺ T cells, and B cells in hepatocellular carcinoma [107].

In transplantation immunology, in vitro hepatic xenograft transplantation models using pig donor and human recipient tissues demonstrated ASGR1 deletion is associated with reduced human platelet destruction compared with wildtype porcine livers during perfusion [108]. This shows a promising role for ASGR1 in hepatic transplantation tolerance induction. However, thrombocytopenia is a clinical barrier for liver xenograft transplantation [109]. Furthermore, ASGR1 inhibition can impede the elimination of outdated platelets and the production of fresh ones, impairing their physiological function [23].

In summary, ASGR1 has roles in immune regulation - clearing desialylated lymphocytes, facilitating IL-10–producing regulatory T cell induction, and modulating innate immune cell populations. Additionally, ASGR1 functions as a liver-specific autoantigen, contributes to apoptotic cell and IgA clearance, and influences platelet survival in transplantation settings. Collectively, these roles highlight ASGR1 as a central regulator of immune tolerance and systemic homeostasis.

ASGR1 and cancer

Increased ASGR1 mRNA levels have been identified in hepatocellular carcinoma cell lines including HepG2 [107, 110], SMMC-7721 [74], C3A [110], and Hep3B [110] cells. The high abundance of the ASGR1 transcript in HepG2 cells was also identified by RNA-seq analysis, while Western blotting verified its high protein abundance [107]. Likewise, increased ASGR1 mRNA expression has been found in colon, stomach and rectum adenocarcinoma, and head and neck squamous cell carcinoma [111]. This was further corroborated by increased ASGR1 protein in hepatocellular carcinoma and cholangiocarcinoma patients [112]. Overall, this presents ASGR1 as a novel tissue biomarker that associates with the presence and extent of tumor development in liver cancer patients [112]. Another study also showed higher ASGR1 mRNA levels in hepatocellular carcinoma, relative to normal tissue, but ASGR1 decreased with cancer progression in immune cells including neutrophils, dendritic cells, macrophages, CD8⁺ T cells, CD4⁺ T cells, and B cells [107]. An inverse correlation was also found between ASGR1 mRNA levels and the same immune cells in cholangiocarcinoma. Conversely, a positive association was found between ASGR1 and the same immune cells in kidney renal papillary cell carcinoma and neck squamous cell carcinoma patients [111].

Asgr1^−/− mice were found to have lower colon cancer metastasis relative to wildtype littermates [113]. This is consistent with the ability of ASGR1 to increase the adhesion and proliferation of colon cancer cell lines via cell membrane glycoconjugate interactions [113]. Gene regulatory network analysis identified ASGR1 and other genes including Notch1 and erythroblast transformation-specific related gene (ERG) as a hub gene involved in prostate cancer pathophysiology in humans [114]. ASGR1 was also found to be a hub gene in pancreatic-ductal adenocarcinoma, including its metastasis to the liver and negative effects on overall patient survival [115]. Interestingly, coculture of hepatocytes from wildtype mice with Lewis lung carcinoma 3LL cells induces MMP-9 protein release from 3LL cells, whereas this effect was absent when hepatocytes from Asgr1^−/− mice were used. Moreover, treatment of 3LL cells with mouse recombinant ASGR1 activates the EGFR-ERK pathway, leading to increased MMP-9 transcription and its proteinase activity [47], all of which promote cancer development. ASGR1-induced MMP-9 production in 3LL cells was inhibited by small molecules such as PD98059 and AG1478, which inhibit ERK and EGFR phosphorylation, respectively [47]. Consistent with this, wildtype and Asgr1^−/− mice intra-hepatically injected with 3LL cells containing the luciferase reporter gene (3LL-Luc2 cells) showed Asgr1^−/− mice had decreased lung metastasis than wildtype littermates. Overall, ASGR1 promotes cancer propagation by increasing MMP-9 via the EGFR–ERK pathway. This is ameliorated by ASGR1 inhibition [47].

Variable ASGR1 mRNA expression levels have been reported among hepatocellular carcinoma patients. ASGR1 expression decreases with increasing carcinoma grade and is higher in cirrhotic specimens [116]. Lower ASGR1 mRNA levels were identified in cholangiocarcinoma [111], hepatocellular carcinoma [117] and in proliferative or metastatic tumours [116, 118]. Overexpression of ASGR1 inhibits the invasiveness and cell migration of hepatocellular carcinoma HCCLM3 cells in vitro. In vivo, male BALB/c nude mice, injected systemically with ASGR1-overexpressing HCCLM3 cells developed fewer metastatic lung nodules compared to mice receiving HCCLM3 cells with shRNA-mediated ASGR1 knockdown. The same study also found hepatocellular carcinoma patients with low ASGR1 protein levels have shorter survival, higher recurrence risk and advanced tumour-node-metastasis staging [117]. Another study found patients with higher circulating epithelial cells, that lack ASGR1 protein, associates with a higher incidence of hepatocellular carcinoma. This demonstrates that increases in ASGR1 may be a prognostic biomarker for reduced cancer risk and metastasis [118]. Mechanistically, ASGR1 exerts antitumorigenic effects in hepatocellular carcinoma by suppressing ATP6L subunit of vacuolar H+-ATPase (V-ATPase) through direct interaction with and activation of longevity assurance homolog 2 (LASS2) [117] (Table 2). V-ATPase promotes cancer metastasis by enhancing the acidity of the microenvironment [119], which promotes drug resistant, metastatic and invasive cancer phenotypes [117]. In mice, LASS2 deletion is associated with liver tumorigenesis [120] and hence ASGR1-LASS2-induced suppression of V-ATPase is a mechanism to inhibit tumour metastasis [117].

Table 2.

The dual role of ASGR1 in cancer and its association with the tumour microenvironment and molecular switches

Tumor type	ASGR1 status	Functional role	Pathways involved	Experimental evidence	Effect on tumor microenvironment	Proposed molecular switch	References
HCC	WT/high expression	ASGR1 supress tumour	LASS2 / V-ATPase inhibition	ASGR1 reduced cancer invasion in HCCLM3 cells in vitro and tumour metastasis in BALB/c nude mice in vivo. Also, decreased ASGR1 in HCC patients was related to higher recurrence and poor survival rate	ASGR1 suppress acidic TME by suppressing ATP6L subunit of V-ATPase	ASGR1 reduced TME acidity by interacting with LASS2 and regulates lysosomal acidification and intracellular pH.	Gu et al. [117].
Lewis lung carcinoma	WT/KO	ASGR1 promotes tumour development and metastasis	EGFR-ERK pathway	MMP-9 was elevated in ASGR1 WT 3LL cells via EGFR-ERK activation, but not in Asgr1−/− mice hepatocytes, indicating a pro-tumorigenic role for ASGR1.	ASGR1 activates the EGFR-ERK pathway	ASGR1 promotes pro-metastatic & growth factor rich TME. Inhibiting ERK-EGFR by AG1478 decreased ASGR1-induced MMP-9 production in 3LL cells.	Ueno et al. [47].
Liver cancer	WT/KO	ASGR1 supress tumour	JAK1/STAT3 through GP130/JAK1-mediated STAT3 activation	ASGR1 interacts with NLK and inhibits STAT3 activation. Over expression of ASGR1 increased liver cancer cell apoptosis. ASGR1 knockdown HCCLM3 cells showed increased STAT3 expression. ASGR1 expression correlates with tumour size.	ASGR1 overexpression inhibited IL6 induced STAT3 phosphorylation. ASGR1 CpG island hypermethylation silences ASGR1-NLK pathway.	ASGR1 inhibits STAT3 phosphorylation by promoting the binding of NLK to STAT3.	Zhu et al. [45].

ASGR1, Asialoglycoprotein receptor 1; Asgr1^−/−, Asgr1 knockout; CpG, cytosine-phosphate-guanine; EGFR, Epidermal growth factor receptor; ERK, Extracellular signal regulated kinase; GP130, Glycoprotein 130; HCC, Hepatocellular carcinoma; IL6, Interleukin 6; JAK1, Janus kinase 1; KO, knockout; LASS2, longevity assurance homolog 2; 3LL, Lewis lung carcinoma; MMP-9, Matrix metalloproteinase-9; NLK, Nemo-like kinase; pH, potential of hydrogen; STAT3, Signal transducer and activator of transcription 3; TME, tumour microenvironment; V-ATPase, vacuolar H+-ATPase; WT, Wild type

ASGR1 can also supress cancer progression by upregulating Nemo-like kinase (NLK). NLK competes with glycoprotein, GP130, by binding to the Src homology 2 (SH2) domain of STAT3, thereby inhibiting GP130/JAK1-mediated STAT3 phosphorylation [45]. Conversely, hypermethylation of the CpG island of ASGR1, a region of short DNA sequences rich in cytosine-guanine dinucleotides in the promoter, silences the ASGR1–NLK pathway, causing STAT3 activation and liver tumour propagation [45]. Consistent with this, a strong association has been found between ASGR1 with DNA methyltransferases [111], reinforcing ASGR1 as a tumour suppressor gene that is regulated by DNA methylation [45].

Elevated sialyltransferase activity increases sialylation within the tumor microenvironment, which in turn suppresses sialidase expression [121, 122]. This alters the balance of sialic acid production and degradation which, in turn, affects ASGR1 function [121]. Siglecs are sialic acid-binding immunoglobulin-type lectins and immune checkpoint inhibitor proteins [122]. Siglec-sialic acid interactions augment tumour sialylation which in turn downregulates immune cell responses and assist cancer cell evasion of immunological surveillance mechanisms [122]. Sialyltransferase overexpression not only contributes to cancer development but also promotes atherosclerosis by increasing inflammatory cell recruitment [123] and suppresses anti-atherogenic B1-cell function [124].

In summary, ASGR1 plays dual roles in cancer, promoting tumor adhesion and metastasis in some contexts while exerting tumor-suppressive effects through LASS2–V-ATPase inhibition and NLK-mediated blockade of STAT3 signalling. Its regulation by DNA methylation and tumor-associated sialyation highlights the importance of epigenetic and microenvironmental factors in its function. ASGR1 presents as both a prognostic biomarker and a potential therapeutic target for cancer (Fig. 4).

Fig. 4.

Dual roles of ASGR1 in cancer biology. (a) HCCLM3 cancer cells with ASGR1 overexpression demonstrated less HCC invasiveness and migration in vitro [117]. (b) Six-week-old male BALB/c nude mice tail vein injected with HCCLM3-ASGR1, ASGR1 protein overexpressing cells has fewer metastatic lung nodules [117]. (c) Six-week-old male BALB/c nude mice tail vein injected with HCCLM3-ASGR1, ASGR1 knockdown cells has advanced metastatic lung nodules [117]. (d) HCC clinical patients with low ASGR1 protein have higher risk of HCC recurrence, advanced tumour node metastasis and decreased overall survival rate [117]. (e) Delineated the mechanism by which ASGR1 supress HCC. Hypomethylation of the CpG island of ASGR1 promoter enhances cell cycle and high ASGR1 protein production [45]. ASGR1 protein directly interacts and activates LASS2, which in turn supresses the activity of V-ATPase followed by decreased acidic TME [117]. Suppressed acidic TME decreases HCC progression and metastasis [125]. On the other hand, ASGR1 enhances the binding of NLK to SH2 domain of STAT3 and prevents GP130/JAK1 mediated phosphorylation of STAT3 [45]. Inhibited STAT3 phosphorylation ultimately suppresses HCC progression and metastasis [45]. (f) The 3LL cells cocultured with hepatocytes isolated form Asgr1^+/+ mice showed increased MMP-9 production in the cell culture supernatant [47]. (g) Treatment of 3LL cells with mouse rASGR1 increased the ERK and EGFR phosphorylation that ultimately activates MMP-9 transcription. As a result, increased MMP-9 protein and its activity favours cancer progression [47]. (h) The PD98059 and AG1478, small molecules which inhibits the ERK and EGFR phosphorylation, respectively suppressed rASGR1 induced MMP-9 production in 3LL cells [47]. (i) Intrahepatic injection of 3LL-Luc2 cells to Asgr1^−/− mice showed decreased lung metastasis relative to Asgr1^+/+ littermates [47]. Abbreviations: HCC, Hepatocellular carcinoma; LASS2, Longevity assurance homolog 2, V-ATPase, Vacuolar H⁺-ATPase; NLK, Nemo-like kinase; SH2, Src homology 2; STAT3, Signal transducer and activator of transcription 3, GP130/JAK1, Glycoprotein 130/Janus kinase 1; MMP-9, Matrix metalloproteinase-9; ERK, Extracellular signal-regulated kinase; EGFR, Epidermal growth factor receptor; Luc, Luciferase

ASGR1 and infectious diseases

Bloodstream infection caused by sialidase (neuraminidase) expressing microbial agents induces rapid desialylation in pathological conditions and alters the balance of endogenous circulatory glycoproteins [70]. For example, during Streptococcus pneumoniae-induced sepsis, thrombocytopenia happens because of hepatic ASGR1 mediated platelet destruction in response to platelet desialylation caused by the bacterial ‘NanA’ sialidase enzyme [70].

Other respiratory pathogens, such as Pseudomonas aeruginosa, Haemophilus influenzae, and influenza virus express different neuraminidases that target host glycoproteins for adherence to the mucosa or for cellular penetration of the target cell to establish the disease process [126, 127]. The presence of an increased variety of sialidases including NEU1, NEU2, and NEU4 in adipocytes and CD4⁺ T cells have been reported among human immunodeficiency virus (HIV)-1 positive patients on antiretroviral therapy [128]. Another study found desialylation of PBMCs from HIV-1 positive patients promotes viral growth through increased synthesis of reverse transcriptase and p24 antigen [129]. Consistent with this, ASGR1 transcriptome expression is upregulated in HIV-1 positive long-term non-progressor patients [130]. Network analysis of protein-protein interactions showed ASGR1 interacts with HIV-1 proteins directly [130]. This suggests ASGR1 acts as a receptor that either facilitates infection or mediates CD4⁺ T cell destruction, contributing to immunosuppression in the presence of elevated sialidase activity during HIV infection. Desialylation coupled with CD4⁺ T cell killing reinforces the notion that desialylated cells, such as B-cells, are tagged for ASGR1-mediated apoptosis [96].

Elevated ASGR1 mRNA levels are reported in SARS-CoV-2 infection [131, 132] and hepatotropic viral infections such as hepatitis C virus [133]. Elevated ASGR1 protein levels have been identified in hepatitis B [134] and hepatitis E virus infections [135]. Mechanistically, the binding and viral internalization of hepatitis C virus subtype 1 by HepG2 cells and primary human hepatocytes in vitro is impaired when ASGR1 is inhibited (e.g. by asialofetuin) [133]. ASGR1 knockout PLC/PRF/5 liver cells also displayed decreased hepatitis E virus binding to HeLa hepatocytes, as does inhibition with ASGR1 ligands (e.g. fibronectin, asialofetuin, asialoganglioside) [135]. Overall, these studies directly implicate ASGR1 in the binding and entry of hepatitis viruses (C and E) into human hepatocytes.

ASGR1 binds directly with preS1, a large envelop protein on hepatitis B virus surface antigen (HBsAg), both in vitro and in vivo, mediating its entry into hepatocytes [134]. Moreover, in vitro studies found 50% of HepG2 cells secreted HBVsAg after hepatitis B virus inoculation via ASGR1-hepatitis B virus interaction. In the presence of ASGR1 competitive inhibitor asialofetuin, this was reduced to 5% [136]. Another study found viral binding and entry into hepatocytes was enhanced in the presence of neuraminidase and viral preS1-desialylation, which increases interactions with ASGR1 [137]. This did not occur in the presence of acylated preS1-derived lipopeptides [138–140] and asialofetuin [137]. Consistent with this, the anti-ASGR1 antibody, ASGR3-10 M, inhibited epidermal growth factor receptor (EGFR) and activated cell division control 42 (CDC42)-associated kinase 1 (Ack1) endocytosis, which is vital for hepatitis B viral infection [141]. ASGR1 mRNA is also expressed in Sertoli cells, Leydig cells, and seminiferous tubules stromal cells but rarely in spermatogonia cells. This suggests ASGR1 provides hepatitis B virus and other sexually transmitted microbes access to the human testis luminal compartment to invade germ cells and promote the spread of sexually transmitted infections [142].

Detailed host cell membrane protein receptome profiling uncovered the multi-organ tropism and pathogenicity of SARS‑CoV‑2. Twelve SARS-CoV-2 capsid spike protein binding membrane receptors were identified that included ASGR1 [143]. Hepatic ASGR1 mRNA levels are also higher following coronavirus disease 2019 (COVID-19) infection, indicating ASGR1 binding to the SARS‑CoV‑2 spike protein is used to enhance viral entry into hepatocytes [131, 132]. This notion is in line with another study that revealed higher ASGR1 protein in lung alveolar macrophages [144], increased ASGR1 mRNA in alveolar organoids [145], and oral cavity following SARS‑CoV‑2 infection [146].

In general, cells or tissues with a higher expression of the SARS‑CoV‑2 primary receptor, angiotensin converting enzyme 2 (ACE2), such as lung, testis, intestine, heart, liver and kidney are more severely affected by SARS‑CoV‑2 infection [131]. However, primary hepatic-parenchymal and epithelial cell lines with low ACE2 levels have efficient SARS‑CoV‑2 infection capacities, which are supressed by siRNA deletion of ASGR1 or anti-ASGR1 antibody inhibition. ASGR1 is found to bind to multiple spike proteins from different SARS-CoV-2 strains, implicating SARS-CoV-2 infection as an ASGR1-dependent process [132]. Consistent with this, ASGR1 was identified as a functional receptor for SARS-CoV-2, which can utilize a distinct combination of ACE2/ASGR1/KREMEN1 (the ASK complex) to enter host cells [143]. Neutralizing antibody cocktails targeting the ASK receptors more effectively blocked SARS-CoV-2 infection in human lung organoids compared to single-receptor antibodies [143].

In summary, ASGR1 facilitates bacterial and viral infections by recognizing desialylated glycoproteins, promoting platelet clearance, immune cell apoptosis, and viral entry. It serves as a receptor for hepatotropic viruses and SARS-CoV-2. These findings position ASGR1 as a key mediator of pathogen tropism and a potential target for therapeutic intervention (Fig. 5).

Fig. 5.

ASGR1 and infectious diseases. (a) different cell lines including PLC/PRF/5, HepG2 and primary human hepatocytes cocultured with different viruses such as HBV, HCV and HEV have decreased viral binding and entry into these individual cells in the presence of ASGR1 knockout/inhibition and/or asialofetuin [133, 135]. (b) SARS-CoV-2 infects host cells via ACE2, ASGR1 and KREMEN1 combination of receptors [143]. (c) ASGR1 inhibition showed decreased SARS-CoV-2 binding and entry into host cells [132]. (d) circulating HBV binds and enters to hepatocytes through the NTCP, EGFR [147] and ASGR1 [9, 134], left panel. ASGR1 inhibition decreased HBV binding and entry into these cells [136], right panel. ASGR1 binds with preS1 part of HBV surface protein and facilitates HBV entry into hepatocytes [134]. (e) microbial neuraminidase enhances microbial attachment to host cells and ultimately their entry [126, 127]. Thereafter, bacterial and/or viral neuraminidase induces host cell desialylation in a pathological way ultimately leading to increased ASGR1 mediated host cell destruction [70] including immune cells [96]. During bacterial infection, desialylation also enhances biofilm formation [126] and favours the development of antibiotic-resistant mutants [148]. (f) HIV infected CD4⁺T cells showed increased expression of several sialidases [128], which may promote desialylation in a pathogenic manner and ultimately encourage the virus to produce large amounts of reverse transcriptase and p24 antigen, which improves viral replication [129]. Likewise, increased desialylation promotes ASGR1 mediated CD4⁺T cells destruction, which results in cell death and immunodeficiency [130]. Abbreviations: HBV, Hepatitis B virus; HCV, Hepatitis C virus; HEV, Hepatitis E virus; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; ACE2, Angiotensin-converting enzyme 2; KREMEN1, Kringle-containing transmembrane protein 1; NTCP, sodium taurocholate cotransporting polypeptide; EGFR, epidermal growth factor receptor

ASGR1 as a target for therapy and future directions

In vitro and in vivo murine studies found ASGR1 inhibition using an anti-ASGR1 monoclonal antibody blocked hepatitis B virus infection [141]. Furthermore, the fusogenic peptide 10 M-D42AN containing an anti-ASGR1 antibody prevented hepatitis B virus infection via inhibition of hepatocyte endocytosis [141]. This study presents ASGR1 as a promising strategy to reduce viral infections.

Injection of wild type mice, on a non-atherogenic high-fat/high-carbohydrate/butylated-sulfite diet, with the anti-ASGR1 neutralizing antibody, 4B9-IgG, every other day for 14 days, lead to alteration in lipid handling mechanisms [19]. These included decreases in proteins that promote cholesterol synthesis (SREBP1c, fatty acid synthase and SCD1) and increases in proteins that promote cholesterol efflux (ABCG5, ABCG8 ABCA1) [19]. Consistent with this, 4B9-IgG decreased total cholesterol and triglyceride levels in wildtype mice, regardless of whether they were on lipid lowering statin therapy (atorvastatin) or not. Treatment with 4B9-IgG also exhibited synergistic effects when used with intestinal cholesterol absorption inhibitor, ezetimibe [19]. ASGR1 inhibition using an anti-ASGR1 neutralizing antibody shows a therapeutic promise to prevent atherosclerotic cardiovascular diseases.

The expression of ASGR1 in different tumour types including liver, ovarian, breast, and colon cancers [149], raises the possibility that ASGR1 can be targeted for drug delivery. For example, a nanodrug combining the anticancer agent ursolic acid with ASGR1 and the near-infrared probe indocyanine green demonstrates efficient uptake into hepatocellular carcinoma cells, leveraging ASGR1’s to recognize lactobionic acid on the cancer cell surface [150]. In another study, ASGR1-mediated recognition improved nanomedicine internalization by cancerous HepG2 cells, inhibiting their proliferation [151]. Thus, the incorporation of ASGR1 into nanoparticle delivery systems, is a novel approach to prevent cancer growth [150, 152].

ASGR1 recognises galactose and GalNAc moieties, a property that has been exploited to deliver drugs specifically to ASGR1-expressing tissues, particularly the liver [16, 149]. GalNAc-conjugated antisense oligonucleotides (ASOs) and siRNA have been employed in preclinical and clinical settings to enhance hepatic uptake and therapeutic efficacy. For example, in diethylnitrosamine-induced hepatocellular carcinoma models, GalNAc-ASOs improve cellular uptake and antitumor activity [153]. GalNAc-siRNA conjugates are also in clinical trials for the treatment of different diseases [154]. For example, the GalNAc-siRNA conjugate, Olpasiran, which targets lipoprotein (a) (Lp(a)), attenuated Lp(a) assembly efficiently and safely in hepatocytes, leading to a significant reduction in Lp(a) in the circulation [155]. Likewise, a clinical trial using GalNAc-siRNA conjugate, VIR-2218, targeting chronic hepatitis B virus infection, silenced hepatitis B viral transcripts and demonstrated favourable pharmacokinetics in healthy volunteers [156].

Other ASGR1-targetting GalNAc-siRNA conjugates that have been regulatory approved for clinical use include Inclisiran that targets PCSK9 and causes long-lasting reductions in total cholesterol levels. Givosiran is another that is designed to treat acute hepatic porphyria, a group of rare genetic disorders caused by deficiencies in enzymes of the heme biosynthesis pathway. Lumasiran targets primary hyperoxaluria type 1, a rare genetic disorder that causes kidney stones and kidney damage. Finally, Vutrisiran for hereditary transthyretin amyloidosis that causes polyneuropathy and cardiomyopathy [157]. The therapeutic advantages and limitations of ASGR1 targeting in cardiovascular disease, cancer, viral infections, and liver-directed RNA therapeutics are summarized in Table 3.

Table 3.

Benefits and limitations of ASGR1 as a therapeutic target in cardiovascular disease, cancer, viral infections and liver-directed RNA therapeutics

Disease conditions		Therapeutic rationale of ASGR1 targeting	Benefits (Pros)	Limitations (Cons)	Mechanisms or pathways	References
Atherosclerosis and/or atherosclerotic cardiovascular disease		ASGR1 deficiency or inhibition lowers atherogenic lipoproteins and cardiovascular risk.	Cardioprotective via reducing LDL-C, non-HDL-C, TGs, arterial lipid deposition and plaque formation.	Potential hepatic stress or injury and impaired desialylated glycoprotein clearance.	Enhanced cholesterol efflux, AMPK activation, mTORC1 inhibition, and reduced cholesterol biosynthesis	Wang et al. [19], Xiao et al. [9].
Lewis lung carcinoma	ASGR1 promotes pro-metastatic & growth factor rich TME such as MMP-9		Targeting ASGR1 suppresses MMP-9 via EGFR-ERK pathway inhibition and impedes cancer metastasis and growth.	Tumour-specific contradictory effects of ASGR1. For example, in HCC, ASGR1 overexpression reduced TME acidity via LASS2/V-ATPase inhibition [117].	The ERK-EGFR inhibition by PD98059 and AG1478 reduced ASGR1-induced MMP-9 production in 3LL cells.	Ueno et al. [47].
Viral infections involving hepatocytes	ASGR1 binds HBV preS1 on HBVsAg [134] and SARS-CoV-2 capsid spike protein [143] to promote viral entry into hepatocytes.		ASGR1 can be used to modulate viral hepatotropism and liver-specific delivery of antiviral therapeutics.	ASGR1 inhibition might alter normal hepatic immune clearance mechanisms and glycoprotein clearance.	ASGR1-mediated endocytosis of hepatotropic viruses and glycoprotein recognition	Xiao et al. [9].
Liver-directed RNA therapeutics	ASGR1 is highly expressed in hepatocytes and enables selective liver delivery through GalNAc conjugation [9].		Hepatocyte-specific efficient drug delivery with reduced systemic exposure. For example, Olpasiran, (GalNAc-siRNA conjugate) safely reduced hepatocyte Lp(a) assembly and decreased circulatory Lp(a) [155].	Therapeutic efficacy depends on intact ASGR1 expression, receptor recycling, and pH-sensitive GalNAc-ASGR1 interactions [158].	GalNAc-ASGR1 mediated endocytosis [158].	Zhang et al. [157].

AMPK, AMP-activated protein kinase; ASGR1, Asialoglycoprotein receptor 1; EGFR, epidermal growth factor receptor; ERK, extracellular signal regulated kinase; GalNAc, N-acetylgalactosamine; HBV, Hepatitis B virus; HBVsAg, Hepatitis B surface antigen; HCC, Hepatocellular carcinoma; non-HDL-C, non- high-density lipoprotein cholesterol; LASS2, longevity assurance homolog 2; LDL-C, Low-density lipoprotein cholesterol; 3LL, Lewis lung carcinoma; Lp(a), Lipoprotein(a); MMP-9, Matrix metalloproteinase-9; mTORC1, Mechanistic target of rapamycin complex 1; preS1, pre-surface 1; RNA, Ribonucleic acid; SARS-CoV-2, Severe acute respiratory syndrome coronavirus 2; siRNA, Small interfering RNA; TGs, Triglycerides; TME, tumour microenvironment; V-ATPase, vacuolar H+-ATPase

Overall, ASGR1 presents not only as a therapeutic target but is also being increasingly utilized as a facilitator of drug delivery systems.

Conclusions

ASGR1 is a hepatic receptor that clears desialylated glycoproteins from the circulation. Its roles in health and disease are now known to extend to lipid modulating activities that regulate atherosclerosis, platelet regulation, immunomodulation, the promotion of cancer metastasis yet also suppression of tumorigenesis, and finally facilitation of hepatotropic viral entry into cells. Through its many and varied roles ASGR1 presents as an exciting target for disease prevention. ASGR1’s unique ability to recognize the GalNAc epitope has revolutionized liver-targeted drug delivery, enabling the development of highly efficient therapeutic approaches that are now in clinical use.

Abbreviations

ASGPR

Asialoglycoprotein receptor

ASGR1

Asialoglycoprotein receptor 1

ASGR2

Asialoglycoprotein receptor 2

AMPK

AMP-activated protein kinase

GalNAc

N-acetylgalactosamine

GWAS

Genome-wide association study

LXRα

Liver X receptor alpha

LXRβ

Liver X receptor beta

ABCG5/8

ATP-binding cassette transporters G5 and G8

ABCA1

ATP-binding cassette transporter A1

INSIG1/2

Insulin-induced genes 1 and 2

mTORC1

Mechanistic target of rapamycin complex 1

SREBP

Sterol regulatory element-binding protein

ER

Endoplasmic reticulum

SCAP

SREBP cleavage-activating protein

HMGCR

3-hydroxy-3-methylglutaryl-CoA reductase

LDLR

Low-density lipoprotein receptor

LDL

Low-density lipoprotein

LDL-C

Low-density lipoprotein cholesterol

HDL-C

High-density lipoprotein cholesterol

VLDL

Very low-density lipoprotein

TG

Triglycerides

ApoB

Apolipoprotein B

ANGPTL3/8

Angiopoietin-like proteins 3 and 8

LPL

Lipoprotein lipase

PCSK9

Proprotein convertase subtilisin/kexin type 9

MTTP

Microsomal triglyceride transfer protein

JACK2/STAT3

Janus kinase 2/signal transducer and activator of transcription 3

ICAM-1

Intercellular adhesion molecule 1

HCC

Hepatocellular carcinoma

LASS2

Longevity assurance homolog 2

V-ATPase

Vacuolar H⁺-ATPase

NLK

Nemo-like kinase

SH2

Src homology 2

MMP-9

Matrix metalloproteinase-9

ERK

Extracellular signal-regulated kinase

EGFR

Epidermal growth factor receptor

SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2

ACE2

Angiotensin-converting enzyme 2

KREMEN1

Kringle-containing transmembrane protein 1

NTCP

Sodium taurocholate cotransporting polypeptide

ALT

Alanine aminotransferase

AST

Aspartate aminotransferase

Author contributions

D.G. and C.A.B conceived the idea for the manuscript. D.G. wrote the manuscript and C.A.B was a major contributor to editing the manuscript. J.T.M.T. and P.J.P provided editing and oversight of the manuscript. All authors read and approved the final manuscript.

Funding

The authors acknowledge funding from the National Health and Medical Research Centre to C.A.B (Ideas grant: APP2029117 and MRFF 2016053). C.A.B. is the recipient of the Lin Huddleston Heart Foundation Fellowship. D.G. received a University of Adelaide Research Training Program International Stipend. P.J.P. receives a Level 3 Future Leader Fellowship from the National Heart Foundation of Australia [FLF 106656].

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.