Asialoglycoprotein receptor (ASGPR) is a C-type lectin that is highly expressed on the sinusoidal surface of the hepatocyte. ASGPR was discovered as early as 1965 by researchers in the USA. These investigators isolated ASGPR from rabbit liver by affinity chromatography using asialo-orosomucoid Sepharose. ASGPR is composed of a major 48 kDa (ASGPR-1) and a minor 40 kDa subunits (ASGPR-2). The primary role of ASGPR is binding, internalization, and subsequent clearance from the circulation of glycoproteins that contain terminal galactose or N-acetylgalactosamine residues (asialoglycoproteins). The binding of ligands to ASGPR is Ca2+ dependent and is also dependent on the position of the terminal galactose residues and has a pH optimum above 6.5. Mice lacking ASGPR are characterized by an impaired clearance of injected asialoglycoproteins but do not accumulate glycoproteins in the serum suggesting that ASGPR is not the only regulator of glycoprotein levels in the blood. Studies using knock-out mice have provided evidence for direct involvement of the ASGPR in removal of abnormally sialylated plasma glycoproteins. Mice lacking the sialyltransferase ST3Gal4 showed prolonged bleeding which was attributed to ASGPR-mediated clearance of at least one plasma hemostatic component, von Willebrand factor (VWF), that showed decreased sialylation. Studies revealed that ASGPR-deficient mice could moderate septic disseminated intravascular coagulation through hemostatic adaptation after removing desialylated hemostatic components such as VWF and platelets from bacterial neuraminidase activity. The ASGPR seems prepared to quickly eliminate plasma glycoproteins that exhibit reduced or faulty sialylation for any cause.
Mannose and galactose-binding lectins form a class of carbohydrate-binding proteins that recognize specific glycan structures on glycoproteins and glycolipids. These lectins serve multiple biological functions including immune system recognition processes together with cell adhesion mechanisms and pathogen attachment activities. The mannose receptor (MR) is a C-type lectin expressed on macrophages, dendritic cells and other antigen-presenting cells. MR has been shown to bind high-mannose glycans of a wide range of pathogens, targeting them for receptor-mediated uptake and degradation. Dendritic cell surface glycoprotein DC-SIGN attaches itself to both galactose and fucose residues found in many pathogens and self-glycoproteins. Specific ligands for the lectins can conjugate with drugs to enable targeting. Targeting macrophages and dendritic cells has been performed using mannosylated liposomes. Since these lectins have such fine specificity for particular carbohydrate ligands, off-target effects may be low.
Fig. 1 The multimeric structure of mannose-binding lectin (MBL).1,2
Siglecs are a family of structurally related animal cell surface glycan binding proteins. Humans express 14 Siglecs, 13 of which are expressed on overlapping cell types of the immune system and one (Siglec-4) on myelinating cells in the nervous system. Siglecs of the immune system are immune regulatory molecules. 9 of 13 immune system Siglecs have immunoreceptor tyrosine-based inhibitory (ITIM) motifs that generally inhibit immune responses and 3 have a positively charged transmembrane domain that mediates association with DAP12 to activate immune responses. The outermost (N-terminal) V-set Ig domain of each Siglec has a sialoglycan binding site centered by a conserved arginine residue that ligates the sialic acid carboxylate. Each Siglec binds to different sialoglycan ligands to elicit molecular and cellular responses that are important to the function of the cell(s) on which they are expressed. The CD33-related Siglecs are expressed mostly in the innate immune system and appear to be important for regulating cellular expansion and activation. CD22 is a well-characterised regulator of B-cell signalling, homeostasis and survival.
The chain type of the sugars in glycopeptides also directly affects binding affinity and specificity to a given receptor. Glycans can differ in numerous ways. They can differ in what kind of sugar residues they are built of, what type of linkage connects two adjacent residues and how branched they are. These changes are key in determining which GBPs (lectins) and receptors they will or will not bind to and with what affinity, affecting subsequent processes. For example, ASGPR mainly recognizes Gal and GalNAc residues when they are exposed as a result of terminal sialic acid removal. Tri- and tetra-antennary glycans that are more branched are higher affinity ligands for ASGPR, setting up a hierarchy of clearance. Glycans that terminate in GalNAc are also good ligands for the receptor, indicating that it might also be involved in clearing glycoproteins with a second type of glycans. Mannose receptor is another C-type lectin, that like ASGPR, binds high-mannose glycans and is involved in glycoprotein clearance from circulation. Mannose receptor's glycan recognition also plays an important role in immune recognition and binding of pathogens.
Researchers can change glycan-peptide interaction and glycopeptide function by designing various peptide scaffolds and spacers. The peptide component of glycopeptides may affect the conformation and accessibility of glycans that can impact their recognition by receptors. Moreover, spacer design can be utilized to enhance the stability and function of glycopeptides. Figure 13 shows an example where glycopeptide design considerations with respect to peptide scaffold and spacer were applied to the selectin family of adhesion molecules. As cell adhesion molecules selectins identify specific glycan structures on leukocytes they facilitate leukocyte adhesion and rolling throughout immune responses. The peptide scaffold and spacers direct glycan presentation into an optimal conformation for selectin binding and spacers modulate the distance between the glycan and peptide structure. Glycopeptides targeting the mannose receptor show that the design of peptide scaffold and spacer affects receptor-ligand interactions. Since mannose receptor can recognize sulfated glycans, the peptide scaffold and spacer design can potentially influence the recognition and binding of these glycans. For instance, the receptor's CRD can accommodate both sulfated and nonsulfated glycans, and the FN2 domain can recognize a range of non-mannose containing glycans such as 3-SO4-LEWIS(X).
Controlled-length glycopeptides are glycopeptides with a defined glycan chain length that is consistent and uniform. The use of controlled-length glycopeptides with defined glycan structures and peptide sequences can also be used to achieve an optimal ligand density that is tuned to the binding requirements of the target receptors. This can be a useful approach to ensure that the glycopeptide ligands are of optimal length for effective receptor binding while minimizing steric hindrance. For instance, glycopeptides for ASGPR can be synthesized with residues such as galactose or GalNAc that have high affinity for the ASGPR, and the length and branching of the glycans can be controlled to tune the binding affinity and specificity. Controlled-length glycopeptides can also be utilized to optimize ligand density on the surface of nanocarriers. Ligand density can be controlled by regulating the number of glycopeptides attached to the nanocarrier surface, avoiding overcrowding that can lead to reduced binding efficiency.
Another approach to improve the ligand density is through co-delivery strategies that would allow multivalent presentation of ligands. Multivalency can be simply defined as the presentation of multiple ligands on a single nanocarrier. The benefits of multivalent presentation of ligands on the nanocarrier are avidity effects, which can dramatically increase the binding affinity and specificity. Co-delivery strategies can be designed to simultaneously present different ligands on the surface of the same nanocarrier. For example, a nanocarrier can be decorated with glycopeptides targeting ASGPR, and at the same time decorated with antibodies for targeting other cell surface markers. In addition, multivalent presentation of ligands can also be achieved through clustering the ligands on the nanocarrier surface. This clustering effect could increase the binding affinity due to higher local concentration of ligands at the receptor binding site. For example, multiple copies of glycopeptides can be attached to the same nanocarrier using click chemistry to allow for high-density presentation of ligands.
Our customized portfolio of receptor-specific glycopeptide solutions includes a diverse range of pre-validated glycopeptide ligands, specifically designed to bind your target receptor with high affinity and specificity. Our pre-validated glycopeptides are rigorously tested to ensure optimal binding to receptors such as the ASGPR or mannose receptor which are highly overexpressed on your target cells (e.g. hepatocytes, immune cells etc.) Our pre-validated glycopeptides are synthesized with controlled-length glycans to increase receptor binding and prevent steric hindrance in order to ensure targeting and internalization are as efficient as possible. Ideal for researchers interested in capitalizing on glycopeptide targeting for their drug delivery system without extensive validation studies.
We expand our glycopeptide library with customized on-demand synthesis for specific targets. Our service enables quick production of custom glycopeptides specifically designed to interact with chosen receptors or cell types. The platform delivers rapid glycopeptide synthesis services specifically designed for novel research targets. We have expertise in the design and synthesis of glycopeptides to target novel receptors or cell types, enabling researchers to rapidly generate glycopeptides for research and development purposes. Our on-demand synthesis service can be used to target unexplored receptors or to explore new therapeutic strategies.
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