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. 2012;7(5):e37098.
doi: 10.1371/journal.pone.0037098. Epub 2012 May 16.

Examining the interactome of huperzine A by magnetic biopanning

Wei Guo  1 , Shupeng Liu, Jinliang Peng, Xiaohui Wei, Ye Sun, Yangsheng Qiu, Guangwei Gao, Peng Wang, Yuhong Xu
Affiliations

Examining the interactome of huperzine A by magnetic biopanning

Wei Guo et al. PLoS One. 2012.

Abstract

Huperzine A is a bioactive compound derived from traditional Chinese medicine plant Qian Ceng Ta (Huperzia serrata), and was found to have multiple neuroprotective effects. In addition to being a potent acetylcholinesterase inhibitor, it was thought to act through other mechanisms such as antioxidation, antiapoptosis, etc. However, the molecular targets involved with these mechanisms were not identified. In this study, we attempted to exam the interactome of Huperzine A using a cDNA phage display library and also mammalian brain tissue extracts. The drugs were chemically linked on the surface of magnetic particles and the interactive phages or proteins were collected and analyzed. Among the various cDNA expressing phages selected, one was identified to encode the mitochondria NADH dehydrogenase subunit 1. Specific bindings between the drug and the target phages and target proteins were confirmed. Another enriched phage clone was identified as mitochondria ATP synthase, which was also panned out from the proteome of mouse brain tissue lysate. These data indicated the possible involvement of mitochondrial respiratory chain matrix enzymes in Huperzine A's pharmacological effects. Such involvement had been suggested by previous studies based on enzyme activity changes. Our data supported the new mechanism. Overall we demonstrated the feasibility of using magnetic biopanning as a simple and viable method for investigating the complex molecular mechanisms of bioactive molecules.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The preparation of Hup-MPs.
(a)The reaction scheme of Hup-MPs. (b)FTIR scanning map of MP linked with Hup A(black line: MP linked with more drug; red line: MP linked with a little drug; blue line: MP linked with no drug).
Figure 2
Figure 2. The schematic presentation of MP based strategy for identification of Hup A-target interactions.
(a)The attachment of Hup A on the surface of magnetic particles. (b)The strategy of Hup A interacted phages screen from cDNA phage display library. From 1 to 5 was one total round of screening, several rounds of such screen were shown, and the final phages were eluted by Hup A and analyzed. (c)The strategy of Hup A target proteins screen from mice brain tissue lysate. After gel running, the specific protein bands were cut down and identified by MS.
Figure 3
Figure 3. The capillary electrophoresis confirmation on the binding between Hup A and the specific phages.
(a)The capillary electrophoresis map of the binding analysis between Hup A and the phage displaying mitochondria gene, drug concentration in the electrophoresis buffer was 0.0008 mg/ml. Hup A concentration injected from top to bottom was 0.0001, 0.0002, 0.0004, 0.001 and 0.002 mg/ml. The corresponding drug/phage ratio was indicated by the number on the right side.(•) Hup A peak; (○) phage peak, including the dissociative phage and drug-bound phage. (b)The curve and trendline between Hup A trough area and concentration using zero interpolation of internal calibration method. (c)The binding curve between Hup A and the specific phage displaying MT-ND1 gene. (d)The binding curve between Hup A and the phage displaying X-chromosome gene.
Figure 4
Figure 4. Kinetic analysis of SPR between Hup A and MT-ND1.
(a)4–12% SAS-PAGE electrophoresis map of proteins expressed by BL21(DE3) before and after inducement. 1–4 shows the different bands before and after inducement. 1,before inducement; 2,3,4,after inducement. (b)Western blot result of the expressed protein.1–3 shows the bands of loaded proteins with increased volume. C shows the binding curve got from SPR analysis between MT-ND1 and Hup A.
Figure 5
Figure 5. The possible protein-binding partners of Hup A selected from tissue lysate.
(a)SDS-PAGE silver staining of the screen results of the possible protein-binding partners of Hup A. 1, Hup A elution solution of positive beads; 2, Hup A elution solution of control beads; 3, positive beads themselves; 4, control beads themselves; 5, Marker. (b)Western blot analysis of the drug-mitochondria ATP synthase possible interaction. 1,mitochondria lysate after interaction with positive beads; 2, 2nd time washing solution of positive beads; 3,3rd time washing solution of positive beads;4, positive beads themselves; 5, mitochondria lysate after interaction with negative beads; 6, 2nd time washing solution of negative beads; 7,3rd time washing solution of negative beads;8, negative beads themselves.
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