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Multivalent display and receptor-mediated endocytosis of transferrin on virus-like particles.

ABSTRACT: The structurally regular and stable self-assembled capsids derived from viruses can be used as scaffolds for the display of multiple copies of cell- and tissue-targeting molecules and therapeutic agents in a convenient and well-defined manner. The human iron-transfer protein transferrin, a high affinity ligand for receptors upregulated in a variety of cancers, has been arrayed on the exterior surface of the protein capsid of bacteriophage Qbeta. Selective oxidation of the sialic acid residues on the glycan chains of transferrin was followed by introduction of a terminal alkyne functionality through an oxime linkage. Attachment of the protein to azide-functionalized Qbeta capsid particles in an orientation allowing access to the receptor binding site was accomplished by the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction. Transferrin conjugation to Qbeta particles allowed specific recognition by transferrin receptors and cellular internalization through clathrin-mediated endocytosis, as determined by fluorescence microscopy on cells expressing GFP-labeled clathrin light chains. By testing Qbeta particles bearing different numbers of transferrin molecules, it was demonstrated that cellular uptake was proportional to ligand density, but that internalization was inhibited by equivalent concentrations of free transferrin. These results suggest that cell targeting with transferrin can be improved by local concentration (avidity) effects.

SUBMITTER: Banerjee D

PROVIDER: S-EPMC4180096 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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Multivalent display and receptor-mediated endocytosis of transferrin on virus-like particles.

Banerjee Deboshri D Liu Allen P AP Voss Neil R NR Schmid Sandra L SL Finn M G MG

Chembiochem : a European journal of chemical biology 20100601 9

The structurally regular and stable self-assembled capsids derived from viruses can be used as scaffolds for the display of multiple copies of cell- and tissue-targeting molecules and therapeutic agents in a convenient and well-defined manner. The human iron-transfer protein transferrin, a high affinity ligand for receptors upregulated in a variety of cancers, has been arrayed on the exterior surface of the protein capsid of bacteriophage Qbeta. Selective oxidation of the sialic acid residues on ...[more]

PMID: 20455239

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Project description:The bloodstream form of Trypanosoma brucei expresses large poly-N-acetyllactosamine (pNAL) chains on complex N-glycans of a subset of glycoproteins. It has been hypothesised that pNAL may be required for receptor-mediated endocytosis. African trypanosomes contain a unique family of glycosyltransferases, the GT67 family. Two of these, TbGT10 and TbGT8, have been shown to be involved in pNAL biosynthesis in bloodstream form Trypanosoma brucei, raising the possibility that deleting both enzymes simultaneously might abolish pNAL biosynthesis and provide clues to pNAL function and/or essentiality. In this paper, we describe the creation of a TbGT10 null mutant containing a single TbGT8 allele that can be excised upon the addition of rapamycin and, from that, a TbGT10 and TbGT8 double null mutant. These mutants were analysed by lectin blotting, glycopeptide methylation linkage analysis and flow cytometry. The data show that the mutants are defective, but not abrogated, in pNAL synthesis, suggesting that other GT67 family members can compensate to some degree for loss of TbGT10 and TbGT8. Despite there being residual pNAL synthesis in these mutants, certain glycoproteins appear to be particularly affected. These include the lysosomal CBP1B serine carboxypeptidase, cell surface ESAG2 and the ESAG6 subunit of the essential parasite transferrin receptor (TfR). The pNAL deficient TfR in the mutants continued to function normally with respect to protein stability, transferrin binding, receptor mediated endocytosis of transferrin and subcellular localisation. Further the pNAL deficient mutants were as viable as wild type parasites in vitro and in in vivo mouse infection experiments. Although we were able to reproduce the inhibition of transferrin uptake with high concentrations of pNAL structural analogues (N-acetylchito-oligosaccharides), this effect disappeared at lower concentrations that still inhibited tomato lectin uptake, i.e., at concentrations able to outcompete lectin-pNAL binding. Based on these findings, we recommend revision of the pNAL-dependent receptor mediated endocytosis hypothesis.

Dataset Information

Multivalent display and receptor-mediated endocytosis of transferrin on virus-like particles.

Publications

Multivalent display and receptor-mediated endocytosis of transferrin on virus-like particles.

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