Identification, purification and partial characterisation of an oligonucleotide receptor in membranes of HepG2 cells |
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Authors: | de Diesbach P Berens C N'Kuli F Monsigny M Sonveaux E Wattiez R Courtoy P J |
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Institution: | Philippe de Diesbach, Catherine Berens, Francisca N’Kuli, Michel Monsigny, Etienne Sonveaux, Ruddy Wattiez, and Pierre J. Courtoy |
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Abstract: | The low and unpredictable uptake and cytosolic transfer of oligonucleotides (ODN) is a major reason for their limited benefit. Improving the ODN potential for therapy and research requires a better understanding of their receptor-mediated endocytosis. We have undertaken to identify a membrane ODN receptor on HepG2 cells by ligand blotting of cell extracts with [125I]ODN and by photolabelling of living cells with a [125I]ODN-benzophenone conjugate. A major band at 66 kDa was identified by the two methods. Its labelling was saturable and competed for by unlabelled ODN of various sequences and irrespective of the presence of a phosphodiester or phosphorothioate backbone. This protein remained sedimentable after carbonate extraction, indicating strong membrane association. About half of the total cell amount resisted extensive surface proteolysis, suggesting a dual localisation at the plasma membrane and cytoplasmic vesicles. The protein was purified using a biotinylated ODN-benzophenone conjugate by photocrosslinking followed by streptavidin affinity purification. A sequence obtained by Edman degradation showed no homology with known proteins. Using anti-peptide antisera, labelling by western blotting revealed at 66 kDa a band with comparable properties as found by ligand blotting. Thus, a new membrane protein acting as an ODN receptor has been demonstrated. |
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