Functional studies on nerve growth factor and its precursor from naja sputatrix

Abstract

The nerve growth factor from Naja sputatrix has been purified by gel filtration followed by RP-HPLC. The protein showed a very high ability to induce neurite formation in PC12 cells relative to the mouse nerve growth factor. Two cDNAs encoding isoforms of NGF have been cloned and an active recombinant nerve growth factor, sputa NGF has been produced in E. coli as a his-tagged fusion protein. The effects of sputa NGF was accessed at both gene (real-time PCR and microarray) and protein (western blot and SELDI-TOF) levels. These studies showed that sputa NGF upregulated the expression of the endogenous NGF in PC12 cells. In addition, the function of the precursor NGF was investigated, using the Tet on/off system in CHO cells. Functional studies (gene and protein) showed that it behaved in a similar manner as the mature NGF. In addition, the pro-domain was found to be apoptotic, while both precursor and mature NGF were neuroprotective for both normal and ischemic PC12 cells. Another component from Naja sputatrix, neutral phospholipase A2 (nPLA2), was found to act via glutamate metabotrophic receptors (mGluR1).