Kentaro Oh-hashi, Kazumi Ohkubo, Kaoru Shizu, Hibiki Fukuda, Yoko Hirata, Kazutoshi Kiuchi
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.
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Animals
Brain
Brefeldin A
Calcium
Cell Line
Culture Media, Conditioned
Cycloheximide
Endoplasmic Reticulum Chaperone BiP
Enzyme Inhibitors
Extracellular Space
Gene Expression Regulation
Heat-Shock Proteins
Humans
Intracellular Space
Mice
Mice, Inbred C57BL
Molecular Chaperones
Neprilysin
Protein Processing, Post-Translational
Protein Transport
RNA, Messenger
Solubility
Substrate Specificity
Transcription Factor CHOP
Tunicamycin
Culture Media, Conditioned
Ddit3 protein, mouse
Endoplasmic Reticulum Chaperone BiP
Enzyme Inhibitors
Heat-Shock Proteins
Molecular Chaperones
RNA, Messenger
Tunicamycin
Transcription Factor CHOP
Brefeldin A
Cycloheximide
Neprilysin
neprilysin 2, mouse
Calcium
