Neurotrophin binding to the Trk receptors transduces positive signals like growth, survival, or differentiation, whereas binding to the p75 NTR/Trk heterodimer can transduce positive or negative signals ( 12– 16). The two receptors, Trk and p75 NTR, are structurally unrelated with neurotrophins interacting primarily with the Ig-like C2 (IgGC2) domain of the Trk receptors but with the cysteine-rich domains of the p75 NTR receptor ( 9– 11). In addition to a selective Trk receptor for each neurotrophin, there is a common neurotrophin receptor (NTR), p75 NTR ( 7, 8). NGF binds to TrkA, BDNF and NT-4/5 selectively bind to TrkB, and NT-3 binds to TrkC ( 1, 6). Each neurotrophin binds to a 140-kDa tyrosine kinase receptor, Trk receptor. Neurotrophins play a crucial role in neuronal survival, differentiation, growth, and apoptosis ( 5). Other members of the family include brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), NT-4/5, and NT-6 ( 1– 4). Nerve growth factor (NGF), the first member of the family, was discovered by Levi-Montalcini and coworkers ( 2) over 50 years ago. The neurotrophin family includes structurally related proteins that promote the survival, growth, and maintenance of neurons in the central and peripheral nervous systems ( 1). This stable proNGF molecule demonstrated proapoptotic activity on rat pheocytochroma PC12 cells, PC12nnr cells, C6 glioblastoma cells, and RN22 schwannoma cells. The construct with all three sites mutated (termed proNGF123) gave all proNGF with no mature NGF and was not cleaved by three proconvertases (furin, PACE-4, and PC-2) known to proteolyze proneurotrophins in vivo. Purification involved strong cation-exchange chromatography and N60 immunoaffinity column chromatography.
Expression was performed in stably transfected Sf21 insect cells. The two consecutive basic residues at each of the three sites were mutated to neutral alanine residues. Here we describe the construction, expression, and purification of this more stable proNGF molecule.
Prepros no me funciona pro#
On introducing additional mutations in the pro domain at the other two dibasic sites, we found the stability of proNGF to increase significantly. We found that mutating this major processing site (site 3) resulted in a form of proNGF that was only partially stable. Of these processing sites, site 3, adjacent to the N terminus of mature NGF, was thought to be the major site responsible for processing of proNGF to mature NGF. The proNGF molecule is processed by proteases at three dibasic sites found in the pro domain to form mature NGF (termed herein as sites 1, 2, and 3 from the proNGF N terminus).
Precursor of nerve growth factor (proNGF) has been found to be proapoptotic in several cell types and mediates its effects by binding to p75 neurotrophin receptor (p75 NTR) and sortilin.