Molecular cloning of neuropathy target esterase

A single ingestion of certain organophosphorus esters (OPs) can cause a syndrome known as Organophosphate Induced Delayed Polyneuropathy (OPIDP), a paralysing neuropathy with degeneration of long axons, developing after a latent period of approximately one to three weeks. The primary target of these OPs has been shown to be a 155kDa neural protein designated Neuropathy Target Esterase (NTE), and the toxic effects apparently due to the covalent inhibition and subsequent secondary modification of this protein. Recently NTE has been purified to apparent homogeneity using a novel biotinylated OP and sufficient pure protein was produced for limited problem sequencing.;The aim of this project was to clone NTE cDNA using peptide sequence data. Initially, these sequences were used to design degenerate oligonucleotide primers for amplifying sections of brain cDNA by polymerase chain reaction (PCR). These approaches were unsuccessful. Subsequently, a database searching with the peptide sequences identified a number of Expressed Sequence Tags (EST)s; these could be aligned to form an initial contig of 2.2kbp which encoded the 3' end of NTE cDNA. The 5' end of NTE cDNA, comprising a further 2.2kbp, was obtained by PCR-based technique. The final 4.4kbp contig encoded a 1327 residue polypeptide predicted to have a molecular mass of 146kDa and at least one transmembrane domain. A novel serine esterase domain of approximately 200 residues was present near the C-terminus. NTE is unrelated to any known serine hydrolases but homologous proteins are predicted to be present in diverse prokaryotic and eukaryotic organisms. The homologue in Drosophila is associated with the swisscheese phenotype, an age-dependent neurodegeneration of the brain. NTE was also mapped to chromosome 19p 13.3 between markers D19216 and the D19S413 (using the UniGene database) and an OMIM search reveals that this is near the locus of cerebellar ataxia (Cayman type).