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Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death
Published on: April 24, 2011
Engineered annexin A5 variants have impaired cell entry for molecular imaging of apoptosis using pretargeting
Lisette Ungethüm1, Heidi Kenis, Gerry A Nicolaes
1Department of Biochemistry of the Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands.
Abstract:
Phosphatidylserine (PS) on apoptotic cells is a target for diagnosis and therapy using annexin A5 (anxA5). Pretargeting is a strategy developed to improve signal to background ratio for molecular imaging and to minimize undesired side effects of pharmacological and radiotherapy. Pretargeting relies on accessibility of the target finder on the surface of the target cell. anxA5 binds PS and crystallizes in a two-dimensional network covering the PS-expressing cell surface. Two-dimensional crystallization is the driving force for anxA5 internalization by PS-expressing cells. Here, we report structure/function analysis of anxA5 internalization. Guided by structural bioinformatics including protein-protein docking, we revealed that the amino acids Arg(63), Lys(70), Lys(101), Glu(138), Asp(139), and Asn(160) engage in intermolecular salt bridges within the anxA5 trimer, which is the basic building block of the two-dimensional network. Disruption of the salt bridges by site-directed mutagenesis does not affect PS binding but inhibits trimer formation and cell entry of surface-bound anxA5. The anxA5 variants with impaired internalization are superior molecular imaging agents in pretargeting strategies as compared with wild-type anxA5.
Insights
Researchers identified key amino acids in annexin A5 (anxA5) essential for its internalization by cells. Disrupting these salt bridges improves anxA5 as a molecular imaging agent for pretargeting strategies.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Phosphatidylserine (PS) on apoptotic cells is a diagnostic and therapeutic target, often utilizing annexin A5 (anxA5).
- Pretargeting strategies enhance molecular imaging and reduce side effects by improving target accessibility.
- Annexin A5 (anxA5) binds PS and forms 2D crystals, driving its internalization by PS-expressing cells.
Purpose of the Study:
- To analyze the structure and function of annexin A5 (anxA5) internalization.
- To investigate the role of specific amino acids and salt bridges in anxA5 trimer formation and cell entry.
- To evaluate the potential of modified anxA5 variants in pretargeting molecular imaging.
Main Methods:
- Structural bioinformatics and protein-protein docking were employed to guide mutagenesis.
- Site-directed mutagenesis was used to disrupt intermolecular salt bridges within the anxA5 trimer.
- PS binding affinity and anxA5 internalization were assessed for wild-type and mutant variants.
Main Results:
- Specific amino acids (Arg63, Lys70, Lys101, Glu138, Asp139, Asn160) form salt bridges crucial for anxA5 trimerization.
- Disruption of these salt bridges did not affect PS binding but inhibited trimer formation and cell internalization.
- Annexin A5 (anxA5) variants with impaired internalization demonstrated superior performance in pretargeting molecular imaging.
Conclusions:
- Intermolecular salt bridges within the annexin A5 (anxA5) trimer are critical for its internalization mechanism.
- Modulating anxA5 internalization through targeted mutagenesis enhances its utility as a molecular imaging agent.
- These findings advance the application of pretargeting strategies in molecular imaging and therapy.
