Glycoproteins, Sialylation and the Ashwell Receptor The glycan branches of glycoproteins often have...

Glycoproteins, Sialylation and the Ashwell Receptor

The glycan branches of glycoproteins often have sialic acid (N-acetylneuraminic acid) added to their terminiThe process of adding sialic acid to these glycoproteins is called “SIALYLATION” and this process is critical for the function and clearance of glycoproteins from the body Glycoproteins without sialic acid residues (Asialoglycoproteins) can be rapidly cleared by the hepatic asialoglycoprotein receptor, also known as the Ashwell receptor

THE ASHWELL RECEPTOR• 1965 Gilbert Ashwell was on sabbatical at Columbia • He was having dinner with Anatol Morell from AECOM who

mentioned that he was having difficulty determining the half life of the glycoprotein that he was working on, ceruloplasmin

• Since Ashwell was working in carbohydrates, he suggested that they label a terminal galactose with a long-lasting tritium isotope to determine the half life of ceruloplasmin

• So they injected this material into a rabbit which rapidly disappeared from the circulation within 5-10 minutes and was recovered completely intact in the liver

• In 1974 they isolated and characterized this hepatic receptor as the first mammalian lectin, a receptor which recognizes particular glycoproteins, the asialoglycoproteins (glycoproteins with glycan chains lacking sialic acid)

• Being modest gentlemen they felt the appropriate name for this receptor should be the “Ashwell-Morell receptor’

THE ASHWELL RECEPTORAshwell Receptor is one of many lectins that bind asialoglycoproteins (recent data suggests that this receptor may bind sialylated glycans as well)Asialoglycoprotein receptors (ASGPR’s) are involved in finding and endocytosing various glycoproteinsAshwell Receptor is on the hepatocyte cell surface where it can remove and degrade potentially harmful circulating glycoproteinsYet the story of the Ashwell Receptor has remained mysterious since endogenous ligands have never been discovered Hopefully our discussion today will begin to shed light on the role of the Ashwell Receptor in regulating sepsis Dr. Ashwell commented that he was dumbfounded by the results of this paper since he had been working on this protein for over 30 years trying to determine its real biologic function (the knockout mice seemed to function normally) With the discovery of a ligand for the Ashwell Receptor, we have the first real evidence for the receptor’s physiologic function which may explain why it has been conserved throughout vertebrate evolution

THE ASHWELL RECEPTORAshwell Receptor is composed of 2 glycoprotein receptor subunits: Asialoglycoprotein receptor-1 (Asgr-1) Asialoglycoprotein receptor- 2 (Asgr-2)Expressed primarily in the liverOften used as markers of hepatocytesMice deficient in either glycoprotein receptor have diminished clearance of exogenous desialylated glycoproteins but endogenous asialoglycoproteins do not accumulate and the animals do not demonstrate any abnormalities

SIALIC ACID TRANFERASES (ST3Gal sialyltransferases) There are a group of enzymes that add sialic acid to the galactose residues at the end of the glycan chains; thus, they can mask endogenous ASGPR ligandsResearch on VW Disease demonstrated that an ST3Gal-4 sialyltransferase deficiency can produce prolonged bleedingThis is attributable to ASGPR-mediated clearance of certain clotting factors from the circulation (vWF and platelets)

Sialyltransferase ST3Gal-4 operates as a dominant modifier of hemostasis by concealing asialoglycoprotein receptor ligandsEllies et al. Proc Natl. Acad. Sci. 99,10042-47, 2002

A number of genetic modifiers contribute to the extensive variability of von Willebrand diseaseThis paper reported a genetic lesion that inactivated the murine ST3Gal-4 sialyltransferase; these mice were unable to sialylate their glycoproteins due to this deficiency in ST3Gal4 This produced a bleeding disorder from reduced levels of VWF and thrombocytopenia due to enhanced clearanceFirst demonstration that desialylated platelets and vWF can be removed from the circulation by Asialoglycoprotein receptorsThis study intrigued the authors of the present paper today and led them to further investigate this concept of clearance of clotting factors and its potential relationship to intravascular coagulation and sepsis

The Ashwell receptor mitigates the lethal coagulopathy of sepsis

(Grewal, et al.Nature Medicine 14:6,2008)

Figure 1a: Liver sections of 8 week old mice(vWF localizes primarily with the Asgpr-1 chain)


(Grewal, et al.Nature Medicine 14:6,2008)

Figure 1b demonstrates less colocalization of vWF with Asgr-2


(Grewal, et al.Nature Medicine 14:6,2008)Figure 1c (null mutations in Asgpr 1 or 2 but the Asgr glycoprotein

remained expressed - vWF quantification in asgr-1 or 2 deficient hepatocytes)

The Ashwell receptor mitigates the lethal coagulopathy of sepsis(Grewal, et al.Nature Medicine 14:6.2008)

Figure 1d demonstrates a 1.5x increase in circulating plasma vWF in Asgr-1 deficient miceFigure 1e demonstrates an increased plasma factor 8 (since this procoagulant factor normally binds to and is stabilized by vWF in the circulation)Figure 1f demonstrates an increased vWF circulating ½ life in the Asgpr-1 deficient animal This data thus suggests that the Ashwell receptor is involved in vWF homeostasis


(Grewal, et al.Nature Medicine 14:6,2008)(Figures 1g, 1h – BT &/or vWF in mice with various genotypes)Do the Ashwell receptor and ST3 Gal-4 jointly control vWF homeostasis?

The Ashwell receptor mitigates the lethal coagulopathy of sepsis (Grewal, et al. Nature Medicine 14:6,2008) Does the Ashwell receptor also control platelet homeostasis?Figure 2a: ST3Gal-4 deficiency=low platelet count which normalized with either Asgr-1 or 2 deficiencyFigure 2b-d: Normalization of platelet volume, newly formed platelets (retic) and ½ life as well


(Grewal, et al.Nature Medicine 14:6,2008)Figure 2

The Ashwell receptor mitigates the lethal coagulopathy of sepsis(Grewal, et al.Nature Medicine 14:6,2008)

Figure 3: Bacterial Sialidase activity and the Ashwell receptor3a: WT mice were given lethal doses of S. pneumoniae – platelets were desialylated – 3a notes an increase in galactose exposure

The Ashwell receptor mitigates the lethal coagulopathy of sepsis (Grewal, et al Nature Medicine 14:6,2008)Figure 3b: Injection with the NanA mutant strain of S. pneumoniae did not produce any Asialo-platelets in the circulation Figure 3c: Adding NanA returned platelet desialylation to 3a levelFigure 3d: S.pna produced thrombocytopenia dependent upon presence of absence of asgr-1 receptor, S pna NanA mutant+/- addition of NanA gene


(Grewal, et al.Nature Medicine 14:6,2008)Figure 3e:platelet ½ life diminished with S. pna in WTFigure 3f: increased desialylation & clearance of asialo-vWF with sepsis

The Ashwell receptor mitigates the lethal coagulopathy of sepsis(Grewal, et al.Nature Medicine 14:6,2008)

Figures 4-6: The Ashwell receptor reduces coagulopathy and increases survival4a: Asgr-1 or 2 deficient mice died rapidly compared to WT4b: Mice infected with the S. pneumoniae NanA- mutant strain (lacking the sialidase) also died sooner than the mice infected with the WT Strep. But when complemented with NanA, the survival curve returned to former WT baseline

The Ashwell receptor mitigates the lethal coagulopathy of sepsis(Grewal, et al. Nature Medicine 14:6,2008)

Figure 4c & d: A histopathologic study to assess whether the Ashwell receptor may modify the severity of sepsis-induced DIC4c: splenic hemorrhage severe with Asgr-1 def./absent in WT4d: renal vascular occlusion & fibrin deposition with Asgr-1 def

The Ashwell receptor mitigates the lethal coagulopathy of sepsis (Grewal, et al. Nature Medicine 14:6, 2008) Figure 4e: Severe DIC in the liver of Asgr-1 deficient mice with increased hepatocyte death


(Grewal, et al. Nature Medicine 14:6,2008)

Figure 5: Severe DIC develops when mice are septic with the S. Pneumoniae Nan A – mutant


(Grewal, et al. Nature Medicine 14:6,2008)Figure 5: Once again, note the severe fibrin deposition,

thrombosis and hemorrhage produced with the Nan A mutant

The Ashwell receptor mitigates the lethal coagulopathy of sepsis(Grewal, et al.Nature Medicine 14:6.2008)

Figure 6: using a lower dose of S.Pneumoniae37% survival in the WT vs 100% mortality in the 15 Asgr-1-deficient miceThus, the Ashwell receptor decreases mortality in S.PNA sepsis, inhibiting DIC

CONCLUSIONSThe Ashwell Receptor as a protective

mechanism in sepsis• The function and clearance of glycoproteins from the

circulation are dependent upon whether or not they are sialylated (the addition of sialic acid to the glycan branch)

• Desialylated glycoproteins can be rapidly removed from the circulation by ASGPR’s

• The hepatic Ashwell receptor is such an ASGPR and is composed of 2 glycoprotein subunits, Asgpr-1 and Asgpr-2

• Although the Ashwell receptor was discovered in 1974, endogenous ligands for the Ashwell receptor have only recently been found when Ellies et al (PNAS 2002) first discovered that desialylated platelets and vWF can be removed from the circulation by ASGPR’s (as opposed to exogenous ligands)


mechanism in sepsis• The present study examined mice deficient in 1 of the 2

subunits of the Ashwell receptor as well as mice deficient in ST3Gal-4, the sialyl transferase protein

• They demonstrated that a reduction in vWF in ST3Gal4 deficient mice is dependent on Asgr-1 but that depletion of platelets is dependent upon both subunits of the Ashwell receptor

• That led to the question as to whether platelets were desialylated in sepsis since pathogens may express sialidases

• A Strep pneumoniae sepsis model was used to demonstrate that the sialidase activity of pathogens can alter endogenous glycoproteins, thus producing a ligand for the hepatic Ashwell receptor

• The receptor then promotes clearance of platelets which protects the host from DIC


mechanism in sepsis• Thus, thrombocytopenia in sepsis may be due to hepatic

uptake of platelets by the Ashwell receptor , once desialylated

• The hepatocyte may play a role in the procoagulant response to sepsis via the Ashwell receptor

• Sepsis by a mutant Strep pneumoniae strain that lacked sialidase could not produce asialylated platelets and did not produce thrombocytopenia

• Asgr-1 deficient mice also did not develop thrombocytopenia

• Thrombocytopenia produced by the Ashwell receptor clearance of asgp-covered platelets protected against DIC and those animals survived longer


mechanism in sepsis• This study demonstrates the presence of endogenous ligands for the

Ashwell receptor which may play a role in mitigating the severe coagulopathy of sepsis

• This study puts forth a new concept in the pathophysiology of sepsis – the development of thrombocytopenia as a way to limit the lethal coagulation cascade complicating sepsis

• What are the important questions that need to be addressed in determining the relevance of these experiments re: sepsis, DIC and the role of asialylated glycoproteins in disease? Needless to say, there are many.

• There are a number of pathways involved in the coagulation cascade. Since DIC is a complicated disease, these other pathways also need to be explored in terms of their involvement in promoting the prothrombotic state in sepsis

• Sepsis is a very complicated pathophysiologic state and there are also many pathogens that do not express sialidases. So does the Ashwell receptor play a role in sepsis from these other pathogens?

• The Ashwell receptor is present on the hepatocytes of mammalian species and is thought to have originated from a single ancestral gene. It may have a number of important pathophysiologic roles and this study may just be touching the tip of the iceberg

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