Molecular Cloning of Neuropathy Target Esterase

Abstract

Organophosphate Induced Delayed Polyneuropathy (OPIDP) is a condition, characterised by a paralysing neuropathy with axonal degeneration, developing after a latent period of approximately one to three weeks. The primary target of these neuropathic OPs has been shown to be a 155 kDa neural protein with serine esterase activity designated Neuropathy Target Esterase (NTE). The OPs toxic effects are apparently due to the covalent inhibition and subsequent secondary modification of this protein. Recently, we have purified NTE to apparent homogeneity (Glynn et al. (1994) Biochem J 301: 551–556) using a novel biotinylated OP, designated S9B (1-[Saligenin-cyclic-phosphoryl]-9-biotinyl-diaminoane), and have produced sufficient pure protein for Edman degradation sequencing. Seven pig NTE sequences from V8 protease digest fragments have been produced. Of these, two have been positioned relative to the active site serine and a further three have been putatively positioned on a serine esterase multiple alignment. The two catalytic serine related peptides have been successfully used to design a pair of degenerate PCR primers which generate a short (170 bp) product designated ORF1 from pig brain cDNA. ORF1 contains an open reading frame with significant homology to the same region of other serine esterases. ORF1 is currently being used in hybridisation screening of a cDNA library, 3’ RACE and PCR with degenerate primers from the other peptides to produce further nucleotide sequences.