Trivalent, Gal/GalNAc-containing ligands designed for the asialoglycoprotein receptor

Khorev, Oleg; Stokmaier, Daniela; Schwardt, Oliver; Cutting, Brian; Ernst, Beat

doi:10.1016/j.bmc.2008.03.017

Cited by 130 publications

(106 citation statements)

References 37 publications

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“…The epitope recognised by the antibody is located on the opposite side of the carbohydrate binding site comprising the sequence 239 Gln-Asn 264 , consistent with the lack of a neutralising effect of the antibody on carbohydrate binding; moreover, the epitope binding by the antibody was ascertained to be independent from the presence of calcium ions. These results confirm the feasibility of the B01 antibody as a molecular tool for the isolation and functional characterisation of carbohydrate complexes of H1CRD [5]. Furthermore, proteolytic epitope excision mass spectrometry is shown here as a highly efficient approach to reveal molecular details of antigen-antibody interactions.…”

Section: Discussionsupporting

confidence: 78%

“…Proteolytic epitope excision and extraction mass spectrometry of an antibody complex of the H1 subunit of the carbohydrate recognition domain of the asialoglycoprotein receptor H1CRD provide the identification of the epitope comprising the N-terminal domain 5 T-R 23 , with proteolytic accessibility observed at the R-16 and R-23 residues. The primary structure characterisation and identification of disulfide linkages of H1CRD was found to be an essential prerequisite for the identification of the antibody epitope.…”

Section: Discussionmentioning

confidence: 99%

“…The recombinant carbohydrate recognition domain of H1 (H1CRD) was expressed in E. coli and purified by affinity chromatography and size exclusion chromatography as previously described [5,23]. The cDNA comprised the amino acid residues 147-290 of the H1 subunit of ASGPR and an initiator methionine (see sequence numbering of the recombinant H1CRD in the Supplementary Figure 1).…”

Section: Preparation Of H1crdmentioning

confidence: 99%

“…The FTICR mass spectra of the affinity-bound epitope peptide fraction provided the identification of two tryptic peptides, 5 TCCPVNWVEHER 16 (T2) containing the vicinal Cys-6-Cys-7 disulfide linkage, and 17 SCYWFSR 23 (T3) (Figure 4a, b). These peptides identified the location of the epitope at the N-terminal domain within residues 5-23 of H1CRD.…”

Section: Epitope Structure Identification Of a Monoclonal Antibody Tomentioning

confidence: 99%

“…Both subunits are composed of a cytosolic N-terminal domain, a single transmembrane segment, a stalk domain and a C-terminal carbohydrate recognition domain (CRD) (Figure 1a). The carbohydrate recognition domain of subunit H1 (H1CRD) binds glycoproteins with terminal galactose (Gal) and N-acetylgalactosamine (GalNAc) motifs [4,5]. The receptor provides clearance of circulating ligand glycoproteins by receptormediated endocytosis followed by degradation in the lysosome, and it is then recycled to the cell surface.…”

Section: Introductionmentioning

confidence: 99%

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Epitope Structure of the Carbohydrate Recognition Domain of Asialoglycoprotein Receptor to a Monoclonal Antibody Revealed by High-Resolution Proteolytic Excision Mass Spectrometry

Ştefănescu

Born

Moise

et al. 2011

J. Am. Soc. Mass Spectrom.

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Recent studies suggest that the H1 subunit of the carbohydrate recognition domain (H1CRD) of the asialoglycoprotein receptor is used as an entry site into hepatocytes by hepatitis A and B viruses and Marburg virus. Thus, molecules binding specifically to the CRD might exert inhibition towards these diseases by blocking the virus entry site. We report here the identification of the epitope structure of H1CRD to a monoclonal antibody by proteolytic epitope excision of the immune complex and highresolution MALDI-FTICR mass spectrometry. As a prerequisite of the epitope determination, the primary structure of the H1CRD antigen was characterised by ESI-FTICR-MS of the intact protein and by LC-MS/MS of tryptic digest mixtures. Molecular mass determination and proteolytic fragments provided the identification of two intramolecular disulfide bridges (seven Cys residues), and a Cys-mercaptoethanol adduct formed by treatment with β-mercaptoethanol during protein extraction. The H1CRD antigen binds to the monoclonal antibody in both native and Cys-alkylated form. For identification of the epitope, the antibody was immobilized on N-hydroxysuccinimide (NHS)-activated Sepharose. Epitope excision and epitope extraction with trypsin and FTICR-MS of affinity-bound peptides provided the identification of two specific epitope peptides (5-16) and (17-23) that showed high affinity to the antibody. Affinity studies of the synthetic epitope peptides revealed independent binding of each peptide to the antibody.

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Section: Discussionsupporting

confidence: 78%

Section: Discussionmentioning

confidence: 99%

Section: Preparation Of H1crdmentioning

confidence: 99%

Section: Epitope Structure Identification Of a Monoclonal Antibody Tomentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Epitope Structure of the Carbohydrate Recognition Domain of Asialoglycoprotein Receptor to a Monoclonal Antibody Revealed by High-Resolution Proteolytic Excision Mass Spectrometry

Ştefănescu

Born

Moise

et al. 2011

J. Am. Soc. Mass Spectrom.

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Rational Design of Copper and Iron Chelators to Treat Wilson's Disease and Hemochromatosis

Gateau¹,

Mintz²,

Delangle³

2014

Ligand Design in Medicinal Inorganic Chemistry

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Galactose Encapsulated Multifunctional Nanoparticle for HepG2 Cell Internalization

Lai

Lin

et al. 2010

Adv Funct Materials

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The fluorescent dye Cy3 and galactose derivatives are covalently assembled with different ratios on the surfaces of magnetic nanoparticles (MNPs) to produce multifunctional HepG2 cancer cell–targeting agents and the effect of ligand spatial orientation on the MNP surface is investigated on targeting specificity. By using a mixture of bis‐N‐hydroxysuccinimide ester (a bifunctional linker) and OSu‐activated Cy3 (w/w = 30:1), stable and quantifiable fluorescent MNPs (Cy3@MNPs) are synthesized that could be subsequently loaded with galactosyl ligands. A mono‐antennary and two different tri‐antennary galactosyl ligands are individually immobilized on Cy3@MNPs, and the uptake efficiencies of the resulting galactosyl Cy3@MNPs by HepG2 and HeLa cells are investigated using confocal microscopy. The confocal images show that galactosyl Cy3@MNPs are sprayed over cytoplasm of the HepG2 cells, indicating that the MNP uptake occurs via receptor‐mediated endocytosis that is followed by release from endosomes. The results also reveal that the ligand spatial orientation affects the efficiency of the receptor‐mediated endocytosis and one of the tri‐antennary galactosyl ligands shows the best uptake efficiency owing to the optimal spatial presentation of the galactosyl moieties. Overall, it is shown that the MNP is a good ligand carrier and that, when pre‐assembled, the multivalent ligand structure enhances the interactions between the surface ligands of the MNPs and receptors of HepG2 cells. Additionally, the galactosyl Cy3@MNPs are not cytotoxic, indicating that they may potentially be used for in vivo applications.

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Trivalent, Gal/GalNAc-containing ligands designed for the asialoglycoprotein receptor

Cited by 130 publications

References 37 publications

Epitope Structure of the Carbohydrate Recognition Domain of Asialoglycoprotein Receptor to a Monoclonal Antibody Revealed by High-Resolution Proteolytic Excision Mass Spectrometry

Epitope Structure of the Carbohydrate Recognition Domain of Asialoglycoprotein Receptor to a Monoclonal Antibody Revealed by High-Resolution Proteolytic Excision Mass Spectrometry

Rational Design of Copper and Iron Chelators to Treat Wilson's Disease and Hemochromatosis

Galactose Encapsulated Multifunctional Nanoparticle for HepG2 Cell Internalization

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