| Abstract No.: | B-A2022 |
| Country: | Canada |
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| Title: | NEUROTROPHIC ACTIVITY OF RECOMBINANT, CLEAVAGE-RESISTANT PRONGF IS UNAFFECTED BY STRUCTURAL CHANGES, PURIFICATION AND ASSAY METHODS, OR EXPRESSION SYSTEM |
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| Authors/Affiliations: | 1 Raheleh Masoudi*; 1 Maria Ioannou; 1 Michael Coughlin; 2 Shelley Allen; 2 David Dawbarn; 1 Margaret Fahnestock;
1 McMaster University, Hamilton, ON, Canada; 2 University of Bristol, Bristol, United Kingdom
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| Content: | Objective: Nerve growth factor (NGF) is produced as a precursor called proNGF which is secreted by many tissues and is the predominant form of NGF in the central nervous system (CNS). In Alzheimer’s diseased brain, cholinergic neurons degenerate and can no longer transport NGF as efficiently, leading to an increase in untransported NGF in the target tissue. The NGF that accumulates in cortex and hippocampus is proNGF, with no mature NGF detectable. The role of proNGF is controversial. It has been shown that a recombinant, cleavage-resistant histidine-tagged proNGF (proNGFhis) exhibits apoptotic activity. This proNGF is expressed in mammalian cells and purified over a nickel column. In contrast, our lab produced a cleavage-resistant proNGF expressed in a baculovirus/insect cell system which is neurotrophic. Differences in the protein structures, protein expression systems, methods used for protein purification, and methods used for bioassay may affect the activity of these two proteins in such a way that one is apoptotic and the other is neurotrophic. The purpose of this study was to determine whether any of these differences could be responsible for the reported opposing activity of those recombinant proNGFs.
Material and Methods: ProNGFhis was expressed in a baculovirus/insect cell system and in mammalian cells (HEK-293). Protein purification by nickel column chromatography was followed by neurite outgrowth assays, NGF withdrawal bioassay, apoptosis assay, and by TrkA and MAP kinase phosphorylation assays. ProNGF from our lab was tested in parallel. A recombinant proNGF expressed in E. coli was also tested for TrkA and MAP kinase activation.
Results: Bioassays show that proNGFhis expressed in baculovirus/insect cells is neurotrophic, as is our proNGF, no matter which purification method is applied or which bioassay is used to test the activity of proNGF. These results are in contrast with its reported activity when expressed in mammalian cells. Furthermore, recombinant, cleavage-resistant proNGF without a histidine tag expressed in E. coli is also neurotrophic. Interestingly, proNGFhis expressed in HEK-293 cells is also neurotrophic as judged by its ability to activate MAP kinase phosphorylation.
Conclusion: None of the structural or procedural differences between the two proNGFs, including purification over a nickel column, differences in protein structure (amino acid substitutions and histidine tag), or different bioassays alter the activity of proNGFhis. Moreover, different expression systems (baculovirus/insect cell, bacterial and mammalian expression systems) all produce neurotrophic proNGF. These results support our hypothesis that cholinergic basal forebrain neurons, which lose TrkA and exhibit decreased retrograde transport in Alzheimer’s diseased brain, may be compromised because of lack of proNGF neurotrophic support.
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