Surface display of LRP1-IV on E. coli and ApoE3 binding: the key to open the blood-brain barrier

Gisbert Fenoy C, Raudszus B, Nienberg C, Langer K, Jose J

Abstract

The blood-brain barrier (BBB) surrounding the central nervous system (CNS) protects it from widely different potential hazards. Due to its complex structure the pass of compounds across the BBB is highlyrestricted. This is an important drawback for the therapy of diseases like brain tumors or Alzheimer's disease. The high molecular weight drugs used are normally not able to reach its target specifically and inlarge amounts, without the integrity of the BBB has been previously disrupted [1]. Many specific transport systems, like LRP1 (Low-density lipoprotein receptor-related protein-1), contribute to the flow ofsubstances between bloodstream and the CNS. LRP1 has diverse biological functions, including Apolipoprotein E (ApoE) binding and endocytosis. Nanoparticles modified with ApoE, loaded with a drug ofinterest, could bind to LRP1. The whole system will be internalized and the drug could pass though the BBB, thereby reaching its target [2]. Previous studies showed the high binding affinity of ApoE to thefourth ligand-binding domain of LRP1 (LRP1-IV) [3]. In this work, LRP1-IV was displayed on the surface of E. coli using Autodisplay technology. For this purpose an artificial gene encoding the autotransporterfusion protein was constructed. The fusion protein consisted of an N-terminal signal peptide, directing the protein across the inner membrane of E. coli, the LRP1-IV domain as passenger, and the C-terminalautotransporter unit, facilitating the transport of LRP1-IV to the surface of the cell. Once LRP1-IV is displayed on the surface, whole cells can be used for binding studies with ApoE3, avoiding cumbersomeLRP1-IV purification [4]. Expression of LRP1-IV was verified by western blot of outer membrane preparations and flow cytometry of whole cells with a specific LRP1-IV domain antibody. Cells displayingLRP1-IV domain bind purified ApoE3 and fluorescently-labeled ApoE3, certifying the functionality of the receptor domain on the surface of E. coli. The next step will be to test the binding of ApoE3 modifiednanoparticles to surface displayed LRP1-IV. These nanoparticles could be used as carriers across the BBB for drugs which normally are not able to cross this barrier [5, 6]. The assay established in this studyallows further investigation relevant for the development of nanoparticles, since binding affinities between ApoE3 variants and LRP1-IV or variants thereof could be easily analyzed.