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Nucleic Acids Research, 2000, Vol. 28, No. 4 868-874
© 2000 Oxford University Press

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

Philippe de Diesbach1, Catherine Berens1,2, Francisca N’Kuli1, Michel Monsigny3, Etienne Sonveaux2, Ruddy Wattiez4 and Pierre J. Courtoy1,*

1Cell Biology Unit, Christian de Duve Institute of Cellular Pathology and Université catholique de Louvain, UCL 7541, 75 Avenue Hippocrate, B-1200 Brussels, Belgium, 2Laboratoire de Chimie Thérapeutique et de Radiopharmacie, Université catholique de Louvain, UCL 7340, 73 Avenue E. Mounier, B-1200 Brussels, Belgium, 3Glycobiologie, Centre de Biophysique Moléculaire and Université d’Orléans, CNRS UPR 4301, rue Ch. Sardon, F-45071 Orléans Cedex 02, France and 4Service de Chimie Biologique, Université de Mons-Hainaut, 6 Pentagone, B-7000 Mons, Belgium

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.

* To whom correspondence should be addressed. Tel: +32 2 764 75 69; Fax: +32 2 764 75 43; Email: courtoy@cell.ucl.ac.be


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