Identification, purification and partial characterisation of an oligonucleotide receptor in membranes of HepG2 cells.

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 [(125)I]ODN and by photolabelling of living cells with a [(125)I]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 phosphoro-thioate 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.