MINIREVIEW ISJ 9: 207-211, 2012 ISSN 1824-307X MINIREVIEW The immuneregulator role of neprilysin (NEP) in invertebrates E Ottaviani1, D Malagoli1, A Grimaldi2, M de Eguileor2 1Department of Life Sciences, University of Modena and Reggio Emilia, Via Campi 213/D,41125 Modena, Italy 2Department of Biotechnology and Life Science, University of Insubria, Via J. H. Dunant 3, 21100 Varese, Italy Accepted November 17, 2012 Abstract Neprilysin (NEP) represents an important enzyme in both vertebrates and invertebrates. In the present report we have focused our attention to invertebrates. In particular, a structure related to CD10/NEP as well as its activity in different tissues, such as immunocytes, nervous tissue and muscle of various species were detected. Moreover, the role played by the enzyme in the interactions between host and parasite has also been reported. The findings indicate that NEP immunoregulation is a well- balanced process that, with appropriate physiological and homeostatic responses to challenges, allows the survival and well-being of the species. Key Words: Neprilysin (NEP); invertebrates; immunoregulation   Introduction Neutral endopeptidase, NEP (EC 3.4.24.11) now referred to as neprilysin is a type II integral membrane protein with a MW of 93 Kda, consists of a short NH2-terminal cytoplasmic domain of 27 amino acids, a trasmembrane region of 22 hydrophobic residues, and a large extracellular domain of about 700 residues that contains zinc in the active center (Fig. 1). It cleaves substrates on the amino side of hydrophobic amino acids (Kerr et al., 1974a, b; Gafford et al., 1983; Turner and Tanzawa, 1997; Turner et al., 2001). The NEP gene exists in a single copy, extents more than 80 kb, is composed of 24 exons and is highly conserved in mammalian species (D'Adamio et al., 1989). Furthermore, NEP was shown to be identical to CD10, a tumor-associated cluster differentiation antigen expressed on the surfce of neutrophils and some lymphoid progenitors, and also known as the common acute lymphoblastic leukemia antigen (CALLA) (Letarte et al., 1988). Neprilysin (NEP) The NEP has been reported in vertebrates and invertebrates. With regards vertebrates, the majority of the data refer mammals. In particular, from the ___________________________________________________________________________ Corresponding author: Enzo Ottaviani Department of Biotechnology and Life Sciences University of Modena and Reggio Emilia Via Campi 213/D, 41125 Modena, Italy E-mail: enzo.ottaviani@unimore.it literature it emerges that this enzyme is mainly localized in the human, rat, rabbit and pig kidney (Kerr and Kenny, 1974a, b; Mumford et al., 1981; Gafford et al., 1983; Edwards et al., 1999), but also in human fibroblasts (Lorkowski et al., 1987; Kletsas et al., 1998), human genital tract (Erdös and Skidgel, 1989), rat brain (Back and Gorenstein, 1989), human blood cells (Connelly et al.,1993), rat nerve ending membranes (Vandenbulcke et al., 1994), mammalian membrane (Turner and Tanzawa, 1997), mouse mesangial cells (Ebihara et al., 2003), and so on. As far as invertebrates are concerned, a structure related to CD10/NEP as well as its activity were detected in the molluscan immunocytes of Mytilus edulis (Shipp et al., 1990), Planorbarius corneus, Viviparus ater (Ottaviani and Caselgrandi, 1997) and Mytilus galloprovincialis (Caselgrandi et al., 2000), and in the central nervous system of Aplysia californica (Zappulla et al., 1999). With refer other groups, data were reported on the hemocytes of the insect Heliothis virescens (Grimaldi et al., 2012), neural membranes from the locust Schistocerca gregaria (Isaac, 1988), from the nematode Ascaris suum muscle (Sajid and Isaac, 1995) and from the head parts of the leech Theromyzon tessulatum (Laurent and Salzet, 1995). Genomic studies revealed that Caenorhabditis elegans and Drosophila melanogaster contain 22 and 24 NEP-like genes (Turner et al., 2001). Moreover, further investigations in D. melanogaster have reported that the Nep2 gene codes for a secreted endopeptidase with a highly restricted 207   mailto:enzo.ottaviani@unimore.it pattern of expression, and this protein for its localization seems involved in renal function and in spermatogenesis (Thomas et al., 2005). We detected the presence of NEP and its activity by using different approaches. In the first case, the cytofluorimetric analysis of M. galloprovincialis immunocytes revealed that the cells were positive to CD10 in a range from 8 to 10 % (Fig. 2) (Caselgrandi et al., 2000), while in granulocytes of the insect H. virescens the presence of the enzyme was detected by immunocytochemical (Fig. 3) and Western Blot analysis (Grimaldi et al., 2012). For the determination of the NEP activity, we used a spectrofluorimetric procedure (Ottaviani and Caselgrandi, 1997) and the Shape Factor (SF) protocol (Ottaviani et al., 1997; Sassi et al., 1998; Caselgrandi et al., 2000). This last procedure is based on the capacity of NEP to cleave biological peptides (Duvaux-Miret et al., 1992; Otttaviani and Ceselgrandi, 1997) and cytokines (Pierart et al., 1988; Casey et al., 1993; Caselgrandi et al., 2000) molecules that, in turn, activate the cell motility of immunocytes (Scharrer and Stefano, 1994; Sassi et al., 1998; Caselgrandi et al., 2000; Ottaviani et al., 2004). The enzymatic activity of NEP was confirmed by phosphoramidon, a potent inhibitor of NEP (Fulcher et al., 1982). The induced changes in cell shape, from a round form (inactive) to an ameboid form (active), were recorded by measuring the cellular area and the perimeter allowing the evaluataion of the SF (Fig. 4). The SF formula of the American Innovision Analysis System software package (San Diego, CA) was used to express changes mathematically, as described in detail elsewhere (Schön et al., 1991). The evaluation of the SF has been described previously (Sassi et al., 1998). Briefly, this method is based on the use of a computer-assisted microscopic image analysis. A vaseline ring on a microscopy slide delimited a chamber within which 100 μl of hemolymph were placed (Fig. 5), as described in detail in a previous paper (Ottaviani et al., 1997). Functions of Neprilysin (NEP) This endopeptidase plays a regulatory role on the peptides that are involved in the physiological mechanisms of mammalian nervous, cardiovascular and immune systems (Turner et al., 2001). In the present paper we will focus our attention on its role in the invertebrate immune system. At a glance observation of literature it emerges that the enzymatic degradation of NEP induces a downregulation according to the following scheme: a first stimulus (for instance biopeptides, cytokines) activating an immunocyte upregulates NEP. Consequently immunocyte response to a second stimulus, that serves as NEP substrate, is downregulated (Fricchione and Stefano, 1994). The activation of invertebrate immunocytes was found to be suppressed by adrenocorticotropin hormone (ACTH) and alpha-melanocyte-stimulating hormone (α-MSH). The effect of ACTH is largely due to its conversion to α-MSH by immunocyte- Fig. 1 Schematic representation of NEP. associated NEP. This is the topic point, since α- MSH inhibits adherence and locomotory activity of polymorphonuclear leukocytes (PMN), monocytes and invertebrate immunocytes. It should be underlined that while α-MSH acts rapidly (min) on the cells, ACTH requires much more time (h) in order to act, and this is due its processing into α- MSH (Smith et al., 1992). Fig. 2 Cytofluorimetric analysis of M. galloprovincialis immunocytes. (A) control, (B) staning with anti-CD10 mAb. FL1 = fluorescence 1 channel; FL2 = fluorescence 2 channel. From Caselgrandi et al., 2000 (reprinted with permission). 208   Fig. 3 Immunocytochemical evidence of NEP in the in larva hemocytes of the insect H. virescens. Bar = 15 μm. 209   Fig. 4 Phase-contrast photographs of M. galloprovincialis immunocytes. (A) control, (B) activated immunocytes 5 min after the addition of 10-8 M ACTH (1-24). Bar = 10 μm. SF = 0.87 SF = 0.58 Parasites use a similar mechanism for immunosuppresion involving the proopiomelanocortin-derived peptides released from the parasite Schistosoma mansoni (Duvaux-Miret et al., 1992). S. mansoni may escape immune reactions from its vertebrate (man) by using signal molecules common to both host and parasite. In the experiments of coincubation of adult worms with human PMN or snail immunocytes has been detected the presence of α-MSH in the medium suggesting that α-MSH results from the conversion of the parasite ACTH by NEP. NEP has also been detected in the T. nigriceps/H. virescens parasitic model. During the parasitization of the insect H. virescens larva by another insect Toxoneuron nigriceps, the host activates a series of humoral and cellular defenses in which plasmatocytes and granulocytes are the circulating immunocytes (Grimaldi et al., 2012). The granulocytes activated by parasitization produce Fig. 5 Microscopy slide for cell activity assay. S = substance to test. large amount of amyloid fibrils to package melanin. The stimulation in response to parasitic attack involves a cross-talk between the immune and neuroendocrine systems with the activation of stress-sensoring circuits to produce and release molecules, such as ACTH (responsible of the autocrine/paracrine activation of cells), α-MSH (resulting in activation of melanin production), NEP and the overproduction of reactive oxygen species. In this contex NEP present on the cell surface plays an important role in controlling the ACTH/α-MSH loop modulation. The same enzyme after exocitosis of amyloid fibrils massively hydrolyzes amyloid fibrils poured in circulating fluid and this cleavage prevents the unnecessary accumulation in hemolymph of amyloid resistant material. Conclusive remaks The majority of the findings reported in the present paper on NEP have been observed in parallel also in man (Duvaux-Miret et al., 1992; Smith et al.,1992; Scharrer and Stefano, 1994) suggesting that: 1) NEP activity is present on blood cells, immunocytes, peripheral fluids and hemolymph; 2) NEP is a highly important factor in controlling the response of immunocytes in invertebrates and blood cells in man to the influence of biologically active substances; 3) the presence and the activity of NEP in invertebrates and man substantiate the importance of this intercellular regulatory mechanism of communication. In summary the NEP immunoregulation is a well-balanced process that, with appropriate physiological and homeostatic responses to challenges, allows the survival and well-being of the species. References Back SA, Gorenstein C. Histochemical visualization of neutral endopeptidase-24.11 (enkephalinase) activity in rat brain: cellular localization and codistribution with enkephalins in the globus pallidus. J. Neurosci. 9: 4439-4455,1989. Caselgrandi E, Kletsas D, Ottaviani E. Neutral endopeptidase-24.11 (NEP) deactivates PDGF- and TGF-beta-induced cell shape changes in invertebrate immunocytes. Cell Biol. Int. 24: 85- 90, 2000. Casey ML, Smith JW, Nagai K, MacDonald PC. Transforming growth factor-beta 1 inhibits enkephalinase (EC 3.4.24.11) gene expression in human endometrial stromal cells and sex skin fibroblasts in culture. J. Clin. Endocrinol. Metab. 77: 144-150, 1993. Connelly JC, Chambless R, Holiday D, Chittenden K, Johnson AR. Up-regulation of neutral endopeptidase (CALLA) in human neutrophils by granulocyte-macrophage colony-stimulating factor. J. Leukoc. Biol. 53: 685-690, 1993. D'Adamio L, Shipp MA, Masteller EL, Reinherz EL. Organization of the gene encoding common acute lymphoblastic leukemia antigen (neutral endopeptidase 24.11): multiple miniexons and separate 5' untranslated regions. Proc. Natl. Acad. Sci. USA 86: 7103-7107, 1989. Duvaux-Miret O, Stefano GB, Smith EM, Dissous C, Capron A. Immunosuppression in the definitive and intermediate hosts of the human parasite Schistosoma mansoni by release of immunoactive neuropeptides. Proc. Natl. Acad. Sci. USA 89: 778-781, 1992. Ebihara F, Di Marco GS, Juliano MA, Casarini DE. Neutral endopeptidase expression in mesangial cells. J. Renin Angiotensin Aldosterone Syst. 4: 228-233, 2003. 210   211   Edwards RM, Pullen M, Nambi P. Distribution of neutral endopeptidase activity along the rat and rabbit nephron. Pharmacology 59: 45-50, 1999. Erdös EG, Skidgel RA. Neutral endopeptidase 24.11 (enkephalinase) and related regulators of peptide hormones. FASEB J. 3: 145-151, 1989. Fricchione GL, Stefano GB. The stress response and autoimmunoregulation. Adv. Neuroimmunol. 4: 13-27, 1994. Fulcher IS, Matsas R, Turner AJ, Kenny AJ. Kidney neutral endopeptidase and the hydrolysis of enkephalin by synaptic membranes show similar sensitivity to inhibitors. Biochem. J. 203: 519-522, 1982. Gafford JT, Skidgel RA, Erdös EG, Hersh LB. Human kidney "enkephalinase", a neutral metalloendopeptidase that cleaves active peptides. Biochemistry 22: 3265-3271, 1983. Grimaldi A, Tettamanti G, Congiu T, Girardello R, Malagoli D, Patrizia P, et al. The main actors involved in parasitization of Heliothis virescens larva. Cell Tissue Res. 2012 [in press]. Isaac RE. Neuropeptide-degrading endopeptidase activity of locust (Schistocerca gregaria) synaptic membranes. Biochem. J. 255: 843- 847, 1988. Kerr MA, Kenny AJ. The molecular weight and properties of a neutral metallo-endopeptidase from rabbit kidney brush border. Biochem. J. 137: 489-495, 1974a. Kerr MA, Kenny AJ. The purification and specificity of a neutral endopeptidase from rabbit kidney brush border. Biochem. J. 137: 477-488, 1974b. Kletsas D, Caselgrandi E, Barbieri D, Stathakos D, Franceschi C, Ottaviani E. Neutral endopeptidase-24.11 (NEP) activity in human fibroblasts during development and ageing. Mech. Ageing Dev. 102: 15-23, 1998. Laurent V, Salzet M. Isolation of a neuropeptide- degrading endopeptidase from the leech Theromyzon tessulatum. Eur. J. Biochem. 233: 186-191, 1995. Letarte M, Vera S, Tran R, Addis JB, Onizuka RJ, Quackenbush EJ, et al. Common acute lymphocytic leukemia antigen is identical to neutral endopeptidase. J. Exp. Med. 168: 1247- 1253, 1988. Lorkowski G, Zijderhand-Bleekemolen JE, Erdös EG, von Figura K, Hasilik A.Neutral endopeptidase-24.11 (enkephalinase). Biosynthesis and localization in human fibroblasts. Biochem. J. 248: 345-350, 1987. Mumford RA, Pierzchala PA, Strauss AW, Zimmerman M. Purification of a membrane- bound metalloendopeptidase from porcine kidney that degrades peptide hormones. Proc. Natl. Acad. Sci. USA 78: 6623-6627, 1981. Ottaviani E, Caselgrandi E. Neutral endopeptidase- 24.11 (NEP)-like activity in molluscan hemocytes. Peptides 18:1107-1110, 1997. Ottaviani E, Malagoli D, Franchini A. Invertebrate humoral factors: cytokines as mediators of cell survival. Prog. Mol. Subcell. Biol. 34: 1-25, 2004. Ottaviani E, Sassi D, Kletsas D. PDGF- and TGF-b- induced changes in cell shape of invertebrate immunocytes: effect of calcium entry blockers. Eur. J. Cell Biol. 74: 336-341, 1997. Pierart ME, Najdovski T, Appelboom TE, Deschodt- Lanckman MM. Effect of human endopeptidase 24.11 ("enkephalinase") on IL-1-induced thymocyte proliferation activity. J. Immunol. 140: 3808-3811, 1988. Sajid M, Isaac RE. Identification and properties of a neuropeptide-degrading endopeptidase (neprilysin) of Ascaris suum muscle. Parasitology 111 ( Pt 5): 599-608, 1995. Sassi D, Kletsas D, Ottaviani E. Interactions of signaling pathways in ACTH (1-24)-induced cell shape changes in invertebrate immunocytes. Peptides 19: 1105-110, 1998. Scharrer B, Stefano GB. Neuropeptides and autoregulatory immune processes. In: Scharrer B, Smith EM, Stefano GB (eds), Neuropeptides and immunoregulation, Springer-Verlag, Berlin, Germany, pp 1-13, 1994. Schön JC, Torre-Bueno J, Stefano GB. Microscopic computer-assisted analysis of conformational state: reference to neuroimmunology. Adv. Neuroimmunol. 1: 252-259, 1991. Shipp MA, Stefano GB, D'Adamio L, Switzer SN, Howard FD, Sinisterra J, Scharrer B, Reinherz EL. Downregulation of enkephalin-mediated inflammatory responses by CD10/neutral endopeptidase 24.11. Nature 347: 394- 396,1990. Smith EM, Hughes TK Jr, Hashemi F, Stefano GB. Immunosuppressive effects of corticotropin and melanotropin and their possible significance in human immunodeficiency virus infection. Proc. Natl. Acad. Sci. USA 89: 782-786, 1992. Thomas JE, Rylett CM, Carhan A, Bland ND, Bingham RJ, Shirras AD, et al. Drosophila melanogaster NEP2 is a new soluble member of the neprilysin family of endopeptidases with implications for reproduction and renal function. Biochem. J. 386 (Pt 2): 357-366, 2005. Turner AJ, Isaac RE, Coates D. The neprilysin (NEP) family of zinc metalloendopeptidases: genomics and function. BioEssays 23: 261-269, 2001. Turner AJ, Tanzawa K. Mammalian membrane metallopeptidases: NEP, ECE, KELL, and PEX. FASEB J. 11: 355-364, 1997. Vandenbulcke F, Ciofi P, Beauvillain JC. Detection of neutral endopeptidase (NEP, enkephalinase, E.C.3.4.24.11) in relation to dopaminergic and gonadoliberinergic nerve endings in the median eminence of the male rat: a double labeling ultrastructural study. J. Neuroendocrinol. 6: 655-664, 1994. Zappulla JP, Wickham L, Bawab W, Yang XF, Storozhuk MV, Castellucci VF, et al. Cloning and characterization of Aplysia neutral endopeptidase, a metallo-endopeptidase involved in the extracellular metabolism of neuropeptides in Aplysia californica. J. Neurosci. 19: 4280-4292, 1999.