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Identification, purification and partial characterisation of an oligonucleotide receptor in membranes of HepG2 cells
P de Diesbach1, C Berens, F N'Kuli
1Cell Biology Unit, Christian de Duve Institute of Cellular Pathology and Université catholique de Louvain, UCL 7541, 75 Avenue Hippocrate, B-1200 Brussels, Belgium.
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 [(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.
Insights
Researchers identified a novel 66 kDa membrane protein on HepG2 cells that acts as an oligonucleotide (ODN) receptor. This discovery could improve ODN delivery for therapeutic and research applications.
Area of Science:
- Molecular biology
- Cell biology
- Biochemistry
Background:
- Oligonucleotide (ODN) therapy is limited by poor cellular uptake and unpredictable cytosolic transfer.
- Understanding receptor-mediated endocytosis is crucial for enhancing ODN efficacy.
Purpose of the Study:
- To identify a specific membrane receptor responsible for oligonucleotide (ODN) uptake in HepG2 cells.
- To characterize the properties and localization of this potential ODN receptor.
Main Methods:
- Ligand blotting of cell extracts with radiolabeled ODN.
- Photolabeling of living cells with a radiolabeled ODN-benzophenone conjugate.
- Protein purification via photocrosslinking and streptavidin affinity chromatography.
- Edman degradation for protein sequencing and Western blotting.
Main Results:
- A 66 kDa membrane protein was identified as a potential ODN receptor using two distinct labeling methods.
- The protein exhibited saturable binding, competed with unlabeled ODN, and showed strong membrane association.
- Evidence suggested dual localization at the plasma membrane and cytoplasmic vesicles.
- Purification and sequencing revealed a novel protein with no homology to known receptors.
Conclusions:
- A new 66 kDa membrane protein functioning as an oligonucleotide (ODN) receptor has been identified in HepG2 cells.
- This discovery offers a potential target for improving ODN delivery and therapeutic strategies.