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.
de Diesbach
P
P
Cell Biology Unit, Christian de Duve Institute of Cellular Pathology and Université catholique de Louvain, UCL 7541, 75 Avenue Hippocrate, B-1200 Brussels, Belgium.
Berens
C
C
N'Kuli
F
F
Monsigny
M
M
Sonveaux
E
E
Wattiez
R
R
Courtoy
P J
PJ
D001483:Base Sequence; D015153:Blotting, Western; D006801:Humans; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D019920:Photoaffinity Labels; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D013347:Subcellular Fractions; D014407:Tumor Cells, Cultured
D009838:Oligodeoxyribonucleotides; D019920:Photoaffinity Labels; D011956:Receptors, Cell Surface