Biosynthesis, processing, trafficking, and enzymatic activity of mouse neprilysin 2.
Neprilysin 2 (NEP2) has been recently identified as a new member of the M13 subfamily of zinc-dependent metalloproteases and shares a highly homologous amino acid sequence with neprilysin (EC 3.4.24.11, NEP). NEP2 has been reported to exist as membrane-bound and soluble secreted variants. To investigate mechanisms of regulating NEP2 activity, we developed a simple and sensitive method for measuring NEP2 activity using synthetic substrates with a fluorescent probe. NEP2 only cleaved Suc-Ala-Ala-Phe-AMC, while NEP cleaved both Dansyl-D-Ala-Gly-p-nitro-Phe-Gly and Suc-Ala-Ala-Phe-AMC. Using HEK293 cells stably expressing mouse NEP2, we evaluated the effects of various reagents affecting post-translational modification and protein trafficking on extracellular NEP2 activity secreted into the culture medium. Inhibition of N-glycosylation by tunicamycin reduced both the enzymatic activity of extracellular NEP2 and the molecular size of intracellular NEP2. Disruption of the Golgi apparatus with brefeldin A markedly reduced extracellular NEP2 activity in parallel with intracellular NEP2 protein level in HEK293 cells. In contrast, the cytoskeleton disrupting reagents, nocodazole and cytochalasin B barely affected NEP2 activity. Two distinct calcium-perturbing reagents, a calcium ionophore A23187 and thapsigargin, reduced extracellular NEP2 activity. However, A23187-mediated down-regulation was not rescued by co-treatment with inhibitors of MAPK, calmodulin, or the proteasome/calpains. In conclusion, we established a simple and sensitive protocol which was able to discriminate NEP2 and NEP activity, and showed that intracellular transport and secretion of NEP2 is regulated by processes such as glycosylation, ER-Golgi transport, and intracellular calcium levels.
Oh-hashi
Kentaro
K
Department of Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
Ohkubo
Kazumi
K
Shizu
Kaoru
K
Fukuda
Hibiki
H
Hirata
Yoko
Y
Kiuchi
Kazutoshi
K
D000818:Animals; D001921:Brain / Q000201:enzymology; D020126:Brefeldin A / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D017077:Culture Media, Conditioned; D003513:Cycloheximide / Q000494:pharmacology; D000091342:Endoplasmic Reticulum Chaperone BiP; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005110:Extracellular Space / Q000187:drug effects / Q000201:enzymology; D005786:Gene Expression Regulation / Q000187:drug effects; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000201:enzymology; D051379:Mice; D008810:Mice, Inbred C57BL; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D015260:Neprilysin / Q000096:biosynthesis* / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012995:Solubility / Q000187:drug effects; D013379:Substrate Specificity / Q000187:drug effects; D051742:Transcription Factor CHOP / Q000235:genetics / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology
D017077:Culture Media, Conditioned; C497250:Ddit3 protein, mouse; D000091342:Endoplasmic Reticulum Chaperone BiP; D004791:Enzyme Inhibitors; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D012333:RNA, Messenger; D014415:Tunicamycin; D051742:Transcription Factor CHOP; D020126:Brefeldin A; D003513:Cycloheximide; D015260:Neprilysin; C530673:neprilysin 2, mouse; D002118:Calcium