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-endopeptidase expressed in many eukaryotic cell types and displaying several important physiological roles. In the brain (and 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 arterial pressure, including atrial natriuretic peptide and bradykinin. In this study we assessed the potential of the fission yeast Schizosaccharomyces pombe to overproduce rabbit NEP and secreted NEP (sNEP, a soluble derivative of this integral membrane protein). Both recombinant NEP and sNEP were produced at high levels (5 mg/l) in this system. Enzymic studies revealed that these recombinant proteins were fully active and exhibit kinetic parameters similar to those of the bona fide enzyme. Immunofluorescence microscopy and enzymic assays demonstrated that recombinant NEP is correctly targeted to the cell membrane. Furthermore, co-immunoprecipitation studies showed that folding intermediates of NEP and sNEP, produced in S. pombe, interact in the endoplasmic reticulum (ER) with binding protein (BiP) and calnexin (Cnx1p). The amount of sNEP coprecipitated with both BiP and Cnx1p augmented when cells were subjected to various stresses causing the accumulation of unfolded proteins in the ER. The interactions of NEP with BiP and Cnx1p were, however, more refractive to the same stresses.
Beaulieu
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H
Département de biochimie, Faculté de médecine, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, Québec, H3C 3J7 Canada.
Elagöz
A
A
Crine
P
P
Rokeach
L A
LA
D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D037281:Calnexin; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D006031:Glycosylation; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D007158:Immunologic Techniques; D020128:Inhibitory Concentration 50; D008970:Molecular Weight; D015260:Neprilysin / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism*; D012995:Solubility; D015244:Thiorphan / Q000494:pharmacology
D002135:Calcium-Binding Proteins; D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins; C060153:KAR2 protein, yeast; D011994:Recombinant Proteins; D037281:Calnexin; D015244:Thiorphan; D015260:Neprilysin