The Spn4 gene of Drosophila encodes a potent furin-directed secretory pathway serpin

Abstract

Proprotein convertases (PCs) are an important class of host-cell serine endoproteases implicated in many physiological and pathological processes. Owing to their expanding roles in the proteolytic events required for generating infectious microbial pathogens and for tumor growth and invasiveness, there is increasing interest in identifying endogenous PC inhibitors. Here we report the identification of Spn4A, a previously uncharacterized secretory pathway serine protease inhibitor (serpin) from Drosophila melanogaster that contains a consensus furin cleavage site, -Arg P4 -Arg-LysArg P12 -, in its reactive site loop (RSL). Our biochemical and kinetics analysis revealed that recombinant Spn4A inhibits human furin (K i, 13 pM; k ass , 3.2 ؋ 10 7 M ؊1 ⅐s ؊1 ) and Drosophila PC2 (K i , 3.5 nM; k ass , 9.2 ؋ 10 4 M ؊1 ⅐s ؊1 ) by a slow-binding mechanism characteristic of serpin molecules and forms a kinetically trapped SDS-stable complex with each enzyme. For both PCs, the stoichiometry of inhibition by Spn4A is nearly 1, which is characteristic of known physiological serpin-protease interactions. Mass analysis of furinSpn4A reaction products identified the actual reactive site center of Spn4A to be -Arg P4 -Arg-Lys-Arg P12 -. Moreover, we demonstrate that Spn4A's highly effective PC inhibition properties are critically dependent on the unusual length of its RSL, which is composed of 18 aa instead of the typical 17-residue RSL found in most other inhibitory serpins. The identification of Spn4A, the most potent and effective natural serpin of PCs identified to date, suggests that Spn4A could be a prototype of endogenous serpins involved in the precise regulation of PC-dependent proteolytic cleavage events in the secretory pathway of eukaryotic cells.A critical cellular event shared by many endogenous proproteins and pathogen molecules is the absolute requirement for a selective proteolytic cleavage to yield biologically active products or the metastable protein components required for microbial infections (1-3). A class of serine endoproteases in eukaryotic cells responsible for these cleavage events is the kexin͞furin family of processing proteases, or subtilisin-like proprotein convertases (PCs) (4, 5). PC-mediated proteolytic activation has been identified in plants and in other eukaryotes ranging from Hydra to higher animals (6). In humans, seven members of the PC family have been identified: PC1͞3, PC2, PC4, PC5͞6, PC7͞LPC͞8, PACE 4, and furin (4). By contrast, Drosophila melanogaster is known to have only three PC genes (6): two furin-like genes (7) and amontillado, encoding the Drososphila homolog of mammalian PC2 (8, 9).The conservation and crucial biological roles of PCs within the secretory pathway of eukaryotic cells predict the presence of endogenous inhibitors and͞or helper proteins to regulate their important endoproteolytic actions (2). However, to date, only three naturally occurring protein regulators have been identified: 7B2 (10, 11), proSAAS (11, 12), and CRES (13). Moreover, all three of these are ...

Processed short neuropeptide F peptides regulate growth through the ERK‐insulin pathway in Drosophila melanogaster

“…Flanking dibasic amino acids of these peptides in the precursor are proteolytic sites cleaved by the evolutionary conserved proprotein convertase (PC). Three PCs are found in D. melanogaster [18]. PCs sometimes cleave a monobasic amino acid, which suggests that sNPF2 may be cleaved further and produce sNPF2 [1][2][3][4][5][6][7][8][9][10] (WFGDVNQKPI) and sNPF2 [12][13][14][15][16][17][18][19] (SPSLRLRFa) found in the larval and adult tissues.…”

“…In the mammalian CHO cell based bioluminescence assay for sNPFR1, sNPF1, sNPF2 [12][13][14][15][16][17][18][19] , sNPF3, and sNPF4 peptides show high calcium responses [12]. However, in the Xenopus oocytes electrophysiological assay for sNPFR1, sNPF1, sNPF2, and sNPF2 [9][10][11][12][13][14][15][16][17][18][19] peptides show higher inward chloride current responses than sNPF3 or sNPF4 [13].…”

“…However, in the Xenopus oocytes electrophysiological assay for sNPFR1, sNPF1, sNPF2, and sNPF2 [9][10][11][12][13][14][15][16][17][18][19] peptides show higher inward chloride current responses than sNPF3 or sNPF4 [13]. In the mammalian CHO-K1 cell based radioreceptor assay, sNPF1, sNPF2, and sNPF2 [9][10][11][12][13][14][15][16][17][18][19] peptides are more potent than sNPF2 [12][13][14][15][16][17][18][19] , sNPF3 or sNPF4 [10] (Supplementary Table S1). Since the sNPF peptides show different biological activities for sNPFR1 in vitro and heterologous assays and its functions are unknown, we examined whether sNPF peptides regulate growth through the ERK-insulin pathway.…”

“…2A). However, in previous mass spectrometry studies using larval and adult tissues, AQRSPSLRLRFa (sNPF1), SPSLRLRFa (sNPF2 [12][13][14][15][16][17][18][19] ), SDPDMLNSIVE (sNPF-AP-1), WFGDVNQKPI (sNPF2 [1][2][3][4][5][6][7][8][9][10] ), SPSLRLRFa (sNPF2 [12][13][14][15][16][17][18][19] ) peptides are found [7][8][9]. PQRLRWa (sNPF3) and PMRLRWa (sNPF4) are also identified in adult body extract [10,11].…”

The serpin Spn5 is essential for wing expansion in Drosophila melanogaster

“…The third Drosophila serpin that has been studied in detail is Spn4; it is transiently expressed in the developing nervous system and appears to be the closest Drosophila homolog to neuroserpin, a vertebrate neuronal serpin (Osterwalder et al, 2004). Recombinant Spn4 binds and inhibits in vitro human furin and its Drosophila homolog Prohormone Convertase 2 (PC2) (Oley et al, 2004;Osterwalder et al, 2004;Richer et al, 2004), which belong to the subtilisin-like proprotein convertase (SPC) family known to regulate the maturation of many secreted proteins (Steiner, 1998).…”