Skip to main content

Advertisement

SpringerLink
Log in

Biosynthesis, processing, trafficking, and enzymatic activity of mouse neprilysin 2

  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

NEP:

Neprilysin

NEP2:

Neprilysin 2

HPLC:

High-performance liquid chromatography

HEK293 cells:

Human embryonic kidney 293 cells

MAPK:

Mitogen-activated protein kinase

References

  1. Ikeda K, Emoto N, Raharjo SB et al (1999) Molecular identification and characterization of novel membrane-bound metalloprotease, the soluble secreted form of which hydrolyzes a variety of vasoactive peptides. J Biol Chem 274:32469–32477

    Article  PubMed  CAS  Google Scholar 

  2. Ghaddar G, Ruchon AF, Carpentier M et al (2000) Molecular cloning and biochemical characterization of a new mouse testis soluble-zinc-metallopeptidase of the neprilysin family. Biochem J 347:419–429

    Article  PubMed  CAS  Google Scholar 

  3. Malfroy B, Schofield PR, Kuang WJ et al (1987) Molecular cloning and amino acid sequence of rat enkephalinase. Biochem Biophys Res Commun 144:59–66

    Article  PubMed  CAS  Google Scholar 

  4. Shimada K, Takahashi M, Tanzawa K (1994) Cloning and functional expression of endothelin-converting enzyme from rat endothelial cells. J Biol Chem 269:18275–18278

    PubMed  CAS  Google Scholar 

  5. Emoto N, Yanagisawa M (1995) Endothelin-converting enzyme-2 is a membrane-bound, phosphoramidon-sensitive metalloprotease with acidic pH optimum. J Biol Chem 270:15262–15268

    Article  PubMed  CAS  Google Scholar 

  6. Lee S, Zambas ED, Marsh WL, Redman CM (1991) Molecular cloning and primary structure of Kell blood group protein. Proc Natl Acad Sci USA 88:6353–6357

    Article  PubMed  CAS  Google Scholar 

  7. Du L, Desbarats M, Viel J et al (1996) cDNA cloning of the murine Pex gene implicated in X-linked hypophosphatemia and evidence for expression in bone. Genomics 36:22–28

    Article  PubMed  CAS  Google Scholar 

  8. Kiryu-Seo S, Sasaki M, Yokohama H et al (2000) Damage-induced neuronal endopeptidase (DINE) is a unique metallopeptidase expressed in response to neuronal damage and activates superoxide scavengers. Proc Natl Acad Sci USA 97:4345–4350

    Article  PubMed  CAS  Google Scholar 

  9. Iwata N, Tsubuki S, Takaki Y et al (2000) Identification of the major Aβ1-42-degrading catabolic pathway in brain parenchyma: suppression leads to biochemical and pathological deposition. Nat Med 6:143–150

    Article  PubMed  CAS  Google Scholar 

  10. Turner AJ, Isaac RE, Coates D (2001) The neprilysin (NEP) family of zinc metalloendopeptidases: genomics and function. Bioessays 23:261–269

    Article  PubMed  CAS  Google Scholar 

  11. Rose C, Voisin S, Gros C et al (2002) Cell-specific activity of neprilysin 2 isoforms and enzymic specificity compared with neprilysin. Biochem J 363:697–705

    Article  PubMed  CAS  Google Scholar 

  12. Ouimet T, Facchinetti P, Rose C et al (2000) Neprilysin II: A putative novel metalloprotease and its isoforms in CNS and testis. Biochem Biophys Res Commun 271:565–570

    Article  PubMed  CAS  Google Scholar 

  13. Oh-hashi K, Nagai T, Tanaka T et al (2005) Determination of hypoxic effect on neprilysin activity in human neuroblastoma SH-SY5Y cells using a novel HPLC method. Biochem Biophys Res Commun 334:380–385

    Article  PubMed  CAS  Google Scholar 

  14. Florentin D, Sassi A, Roques BP (1984) A highly sensitive fluorometric assay for “enkephalinase,” a neutral metalloendopeptidase that releases tyrosine-glycine-glycine from enkephalins. Anal Biochem 141:62–69

    Article  PubMed  CAS  Google Scholar 

  15. Mumford RA, Strauss AW, Powers JC et al (1980) A zinc metalloendopeptidase associated with dog pancreatic membranes. J Biol Chem 255:2227–2230

    PubMed  CAS  Google Scholar 

  16. Hara H, Oh-hashi K, Yoneda S et al (2006) Elevated neprilysin activity in vitreous of patients with proliferative diabetic retinopathy. Mol Vis 12:977–982

    PubMed  CAS  Google Scholar 

  17. da Costa SR, Yarber FA, Zhang L et al (1998) Microtubules facilitate the stimulated secretion of β-hexosaminidase in lacrimal acinar cells. J Cell Sci 111:1267–1276

    PubMed  CAS  Google Scholar 

  18. Santell L, Marotti K, Bartfeld NS et al (1992) Disruption of microtubules inhibits the stimulation of tissue plasminogen activator expression and promotes plasminogen activator inhibitor type 1 expression in human endothelial cells. Exp Cell Res 201:358–365

    Article  PubMed  CAS  Google Scholar 

  19. Hay JC (2007) Calcium: a fundamental regulator of intracellular membrane fusion? EMBO Rep 8:236–240

    Article  PubMed  CAS  Google Scholar 

  20. Carreno FR, Goni CN, Castro LM, Ferro ES (2005) 14-3-3 epsilon modulates the stimulated secretion of endopeptidase 24.15. J Neurochem 93:10–25

    Article  PubMed  CAS  Google Scholar 

  21. Lenz W, Herten M, Gerzer R, Drummer C (1999) Regulation of natriuretic peptide (urodilatin) release in a human kidney cell line. Kidney Int 55:91–99

    Article  PubMed  CAS  Google Scholar 

  22. Kim MS, Lim WK, Park RK et al (2005) Involvement of mitogen-activated protein kinase and NF-kB activation in Ca2+-induced IL-8 production in human mast cells. Cytokine 32:226–233

    Article  PubMed  CAS  Google Scholar 

  23. Menconi MJ, Wei W, Yang H et al (2004) Treatment of cultured myotubes with the calcium ionophore A23187 increases proteasome activity via a CaMK II-caspase-calpain-dependent mechanism. Surgery 136:135–142

    Article  PubMed  Google Scholar 

  24. Sharma AK, Rohrer B (2004) Calcium-induced calpain mediates apoptosis via caspase-3 in a mouse photoreceptor cell line. J Biol Chem 279:35564–35572

    Article  PubMed  CAS  Google Scholar 

  25. Yatsu T, Kurosawa H, Hayashi M, Satoh S (2003) The role of Ca2+ in the control of renin release from dog renal cortical slices. Eur J Pharmacol 458:191–196

    Article  PubMed  CAS  Google Scholar 

  26. Ribeiro CM, McKay RR, Hosoki E et al (2000) Effects of elevated cytoplasmic calcium and protein kinase C on endoplasmic reticulum structure and function in HEK293 cells. Cell Calcium 27:175–185

    Article  PubMed  CAS  Google Scholar 

  27. Kang T, Nagase H, Pei D (2002) Activation of membrane-type matrix metalloproteinase 3 zymogen by the proprotein convertase furin in the trans-Golgi network. Cancer Res 62:675–681

    PubMed  CAS  Google Scholar 

  28. Vey M, Schäfer W, Berghöfer S et al (1994) Maturation of the trans-Golgi network protease furin: compartmentalization of propeptide removal, substrate cleavage, and COOH-terminal truncation. J Cell Biol 127:1829–1842

    Article  PubMed  CAS  Google Scholar 

  29. Oyadomari S, Mori M (2004) Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ 11:381–389

    Article  PubMed  CAS  Google Scholar 

  30. Okada T, Yoshida H, Akazawa R et al (2002) Distinct roles of activating transcription factor 6 (ATF6) and double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (PERK) in transcription during the mammalian unfolded protein response. Biochem J 366:585–594

    Article  PubMed  CAS  Google Scholar 

  31. Shirotani K, Tsubuki S, Iwata N et al (2001) Neprilysin degrades both amyloid beta peptides 1–40 and 1–42 most rapidly and efficiently among thiorphan- and phosphoramidon-sensitive endopeptidases. J Biol Chem 276:21895–21901

    Article  PubMed  CAS  Google Scholar 

  32. Roques BP, Fournie-Zaluski MC, Soroca E et al (1980) The enkephalinase inhibitor thiorphan shows antinociceptive activity in mice. Nature 288:286–288

    Article  PubMed  CAS  Google Scholar 

  33. Baumer P, Danquechin Dorval E et al (1992) Effects of acetorphan, an enkephalinase inhibitor, on experimental and acute diarrhoea. Gut 33:753–758

    Article  PubMed  CAS  Google Scholar 

  34. Bralet J, Schwartz JC (2001) Vasopeptidase inhibitors: an emerging class of cardiovascular drugs. Trends Pharmacol Sci 22:106–109

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan

    Kentaro Oh-hashi, Kazumi Ohkubo, Kaoru Shizu, Hibiki Fukuda, Yoko Hirata & Kazutoshi Kiuchi

Authors
  1. Kentaro Oh-hashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kazumi Ohkubo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kaoru Shizu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hibiki Fukuda
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yoko Hirata
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kazutoshi Kiuchi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kazutoshi Kiuchi.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Oh-hashi, K., Ohkubo, K., Shizu, K. et al. Biosynthesis, processing, trafficking, and enzymatic activity of mouse neprilysin 2. Mol Cell Biochem 313, 103–111 (2008). https://doi.org/10.1007/s11010-008-9747-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11010-008-9747-z

Keywords

Search

Navigation