Interaction of mammalian neprilysin with binding protein and calnexin in Schizosaccharomyces pombe

Neutral endopeptidase (neprilysin or NEP, EC 3.4.24.11) is a zinc metallo-e ndopeptidase expressed in many eukaryotic cell types and displaying several important physiological roles. In the brain land central nervous system), this enzyme is involved in the molecular mechanism of pain by its action in the degradation of enkephalin molecules. In the kidney, NEP is implicated in the degradation of regulatory factors involved in the control of arteria l pressure, including atrial natriuretic peptide and bradykinin. In this st udy we assessed the potential of the fission yeast Schizosaccharomyces pomb e to overproduce rabbit NEP and secreted NEP (sNEP, a soluble derivative of this integral membrane protein). Both recombinant NEP and sNEP were produc ed at high levels (5 mg/l) in this system. Enzymic studies revealed that th ese recombinant proteins were fully active and exhibit kinetic parameters s imilar to those of the bona fide enzyme. Immunofluorescence microscopy and enzymic assays demonstrated that recombinant NEP is correctly targeted to t he cell membrane. Furthermore, co-immunoprecipitation studies showed that f olding intermediates of NEP and sNEP, produced in S. pombe, interact in the endoplasmic reticulum (ER) with binding protein (BiP) and calnexin (Cnxlp) . The amount of sNEP coprecipitated with both BiP and Cnxlp augmented when cells were subjected to various stresses causing the accumulation of unfold ed proteins in the ER. The interactions of NEP with BiP and Cnxlp were, how ever, more refractive to the same stresses.