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

de Diesbach P, Berens C, N'Kuli F, Monsigny M, Sonveaux E, Wattiez R, Courtoy PJ

Cell Biology Unit, Christian de Duve Institute of Cellular Pathology and Universite catholique de Louvain, UCL 7541, 75 Avenue Hippocrate, B-1200 Brussels, 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 [(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.
 

PMID: 10648777, UI: 20115912