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Title: Uptake of the Necrotic Serpin in Drosophila melanogaster via the Lipophorin Receptor-1}
Article:
Abstract:
The humoral response to fungal and Gram-positive infections is regulated by the serpin-family inhibitor, Necrotic. Following immune-challenge, a proteolytic cascade is activated which signals through the Toll receptor. Toll activation results in a range of antibiotic peptides being synthesised in the fat-body and exported to the haemolymph. As with mammalian serpins, Necrotic turnover in Drosophila is rapid. This serpin is synthesised in the fat-body, but its site of degradation has been unclear. By “freezing” endocytosis with a temperature sensitive Dynamin mutation, we demonstrate that Necrotic is removed from the haemolymph in two groups of giant cells: the garland and pericardial athrocytes. Necrotic uptake responds rapidly to infection, being visibly increased after 30 mins and peaking at 6–8 hours. Co-localisation of anti-Nec with anti-AP50, Rab5, and Rab7 antibodies establishes that the serpin is processed through multi-vesicular bodies and delivered to the lysosome, where it co-localises with the ubiquitin-binding protein, HRS. Nec does not co-localise with Rab11, indicating that the serpin is not re-exported from athrocytes. Instead, mutations which block late endosome/lysosome fusion( dor, hk, and car) cause accumulation of Necrotic-positive endosomes, even in the absence of infection. Knockdown of the 6 Drosophila orthologues of the mammalian LDL receptor family with dsRNA identifies LpR1 as an enhancer of the immune response. Uptake of Necrotic from the haemolymph is blocked by a chromosomal deletion of LpR1. In conclusion, we identify the cells and the receptor molecule responsible for the uptake and degradation of the Necrotic serpin in Drosophila melanogaster. The scavenging of serpin/proteinase complexes may be a critical step in the regulation of proteolytic cascades.
Introduction:
The immune response to pathogen challenge in humans consists of an immediate “innate” response( mediated via cellular responses and the action of antimicrobial peptides), followed by a delayed “acquired” response( mediated by antibodies). In insects, the antibody response is absent, but these organisms synthesise antibiotic peptides, activate macrophage-like cells and mount a melanization response[1]. The necrotic( nec) gene encodes a proteinase inhibitor of the serpin family( serine proteinase inhibitor). Nec controls a proteolytic cascade which activates the innate immune response to fungal and Gram+ bacterial infections[2]. The Nec serpin carries an N-terminal extension which modifies its substrate specificity and is cleaved following immune-challenge[3]. In nec null mutants, the Toll-mediated immune response is constitutively activated, even in the absence of infection, implying that Nec continually restrains this immune response. The serpins have been extensively studied in mammals, where they regulate many extracellular proteolytic cascades. The coagulation, inflammatory and complement pathways are controlled by α1-Antithrombin, α1-Antitrypsin and C1-Inhibitor, respectively[4]–[6]; while Plasminogen Activator Inhibitor-1 modulates angiogenesis, affecting both wound-healing and tumour growth[7]. Disorders in serpin metabolism underlie a major group of human genetic diseases, the serpinopathies, which are associated with failure to clear inert serpin polymers[5],[8],[9] and homologous mutations in Necrotic similarly form inactive polymers[10],[11]. Serpins interact with their target proteinase via a “suicide-inhibition” mechanism, which destroys both serpin and proteinase and generates a covalently-linked complex[12]. Inert serpin/proteinase complexes are removed from circulation by endocytosis in the liver[13], via receptors of the low-density lipoprotein( LDLR) family[14],[15]. The LDLR family consists of a diverse group of cell surface receptors[16] that is evolutionarily conserved[17]. LDLR/ligand binding is pH-dependent, so that the complex dissociates in the low pH environment of the endosomal compartment, allowing LDLR to be recycled to the cell surface[18]–[20]. During endocytosis, the internalization of receptor-bound proteins requires Dynamin function for the pinching-off of clathrin-coated vesicles[21]. Endocytosed proteins are transported to various intracellular compartments, with the Rab family of Ras-related GTPases being critical for coordinating vesicle formation, transport and fusion with the target membrane[22]. In particular, maturation of the early endosome coincides with the replacement of Rab5 by Rab7 and the accumulation of lumenal vesicles to form multivesicular bodies( MVB)[23]. MVB correspond to a class of late endosome, which requires Hook and Fab1 for maturation[24]. Following LDLR/ligand dissociation, transport of the free LDLR back to the plasma membrane is mediated via Rab11-positive, recycling endosomes. The contents of MVBs are delivered by direct fusion, either to lysosomes, or to the plasma membrane[25]. A key component in protein sorting from late endosomes to lysosomes is ubiquitination. In this process, HRS( hepatocyte growth factor-regulated kinase substrate) binds ubiquitin and interacts with ubiquitinated cargos in the early endosome[26], while Fab1 encodes a phosphatidylinositol( 3)-phosphate 5-kinase which acts downstream of HRS[27],[28]. In Drosophila, MVB fusion with the lysosome requires the function of the deep orange( dor) and carnation( car) subunits of the HOPS( homeotypic vacuole fusion and protein sorting) trafficking complex[29]. The garland cells represent an attractive model system to study endocytotic processes, since they are giant cells that are highly active in clathrin-mediated endocytosis. They form two rows of loosely-connected cells surrounding the oesophagus at the junction with the proventriculus, but little is known about their biological function[30]–[33]. Garland cells are bi-nucleated with a highly vacuolated structure featuring deep invaginations of the plasma membrane[34]. The pericardial cells, on the other hand, are arranged in two rows of approximately 20 cells, on either side of the heart. Both cell types have a similar morphology and express the WD40 domain protein, Rudhira[34],[35] and the dot-Gal4 enhancer trap[36], while their positioning around the proventriculus and dorsal vessel ensures good contact with flowing haemolymph. In contrast to the garland cells, pericardial cells are mono-nucleate; although both cell types are bi-nucleate in Hyalophora cecropia and Calliphora erythrocephala[37],[38]. These two cell types are homologous to athrocytes in other insects[39] and share a nephrin/podocin based filtration mechanism with the vertebrate kidney glomerulus[40]. Instead of being passed to a nephric tubule, however, the ultrafiltrate is endocytosed from lacunae in the garland and pericardial cells[34],[41],[42]. There is no connection between these athrocytes and the Malpighian tubules, which regulate the concentration of plasma ions and metabolites in Insects. In this respect, the garland and pericardial cell clusters resemble more the vertebrate reticulo-endothelial cells[41],[43] than the kidney glomerulus. The reticulo-endothelial system consists of groups of phagocytic cells that take up denatured proteins, bacterial and viral components[44]–[46]. In this study, we investigate the role of LDL-family receptors in the Toll-mediated immune response of Drosophila by dsRNA-knockout. We have tested the role of the Lipophorin Receptors, LpR1 and LpR2, in Nec processing. We show that Nec is taken up in the garland and pericardial cells by LpR1, probably as a serpin/proteinase complex. Antibody staining against different proteins involved in clathrin-mediated endocytosis and intracellular trafficking establishes that Nec is sorted and degraded in the garland and pericardial athrocytes, but not recycled to the haemolymph. In general, the paucity of Rab11-positive vesicles indicates that these cells do not recycle endocytotic components, but are the site of protein and peptide degradation.
Discussion:
The Necrotic serpin controls activation of the Toll-mediated immune-response in Drosophila, which represents the best-studied example of serpin-regulated proteolytic cascade in insects[2],[3],[52]. The Drosophila melanogaster genome encodes 15 putative inhibitory serpin transcripts that carry secretion-signal peptides[53]. In addition to nec, the Spn27A serpin controls Toll-mediated morphogenesis in the embryo and the phenol-oxidase cascade in adults[54],[55]. Spn28D( CG7219) also regulates the phenol-oxidase cascade[56], while Spn77Ba regulates tracheal melanization, which also can trigger systemic expression of Drosomycin via the Toll pathway[57]. In addition, the Spn42Da transcript inhibits furin, which is involved in the maturation of secreted proteins[58]. In mammals, serpins are removed from circulation by endocytosis and degraded in the liver as inactive serpin/proteinase complexes. This aspect of serpin metabolism, however, has not been studied previously in Drosophila. In this study, we identify the mechanism of serpin clearance by endocytosis in the garland and pericardial cells. These cells are known to take up ferritin[59] and GFP[60] and have been suggested to be homologous to mammalian reticulo-endothelial cells[41],[43] or nephrocytes[34],[40],[61]. As in mammals, serpin turnover in Drosophila is extremely rapid, so that we were unable to detect the immune-response serpins Nec and Spn27A under normal conditions. However, freezing the pinching-off of endocytotic vesicles, using the shits1 mutation, allows us to detect serpin uptake. Endocytosed Nec is sorted, first to Rab5-positive early-endosomes and then to Rab7-positive late-endosomes. Disrupting these steps by expression of dominant negative UAS-Rab5S43 or UAS-Rab7Q67L, leads to accumulation of Nec-positive vesicles. Similarly, co-localisation of anti-Nec and anti-HRS antibody staining indicates that the serpin is present in early endosomes in the ubiquitin-dependent sorting pathway; while anti-Nec and anti-Fab1 confirms that the serpin is destined for lysosomal degradation. Blocking late-endosome/lysosome fusion using HOPS-complex mutants causes accumulation of Nec-positive endosomes/MVB, indicating that Nec sorting to MVB is required for lysosomal delivery. Co-localization of anti-Nec and anti-Lamp1 antibody staining confirms that Nec is delivered to lysosomes for degradation, while the absence of Nec staining in Rab11-positive vesicles indicates that none of the serpin is recycled from MVB to the haemolymph. In summary, Nec is cleared from the haemolymph and sorted through MVB, via the ubiquitin-dependent pathway, to lysosomes for degradation. In vitro studies in mammals have shown that different members of LDLR family have different binding specificities to different native serpins and serpin/proteinase complexes[15],[62]. In this study, we have shown that LpR1 is the Nec trafficking receptor in vivo, but that neither LpR1 nor LpR2 traffics Spn27A. By analogy to mammalian systems, Nec is probably taken up by LpR1 as a complex with its target proteinase. In addition, pre-digestion of Nec with PPE increases Nec uptake in garland cells that are not deficient for the LpR1 receptor. Our results establish that active trafficking of Nec from the haemolymph can modulate the immune response. Nec clearance is extremely rapid, but deletion of the LpR1 gene sensitises the immune response: nec transcript levels decrease and Drs transcript levels increase. These results imply a regulatory feedback loop at the transcriptional level. In this context, it is significant that LpR1 appears to bind the non-inhibitory serpin/proteinase complex, in preference to the native Nec serpin. Clearance of the serpin/protease complex through the athrocytes appears to compete with a regulatory feedback loop affecting nec transcription. In summary, we establish that the Nec serpin is taken-up via LpR1 from the haemolymph and degraded in the garland and pericardial athrocytes.
Serpin inhibitors control a wide range of rapid physiological responses that are activated by proteolytic cascades , such as blood coagulation , inflammation , the complement pathway , and angiogenesis . They interact with their target proteinases by a “suicide inhibition” mechanism , which generates an inert , denatured , serpin/proteinase complex . In mammals , humoral serpins are secreted from the liver into the blood plasma . The denatured complex is later endocytosed back into the liver and degraded . In Drosophila , the Necrotic serpin is secreted from the fat-body into the haemolymph , where it controls the humoral immune response . We show here , however , that Necrotic is not endocytosed in the fat-body , but in the garland and pericardial athrocytes . These cells clear serpins from the haemolymph extremely rapidly . The Necrotic-binding receptor for this process is LpR1 , a member of the LDLR family . The endocytosed serpin is targeted for lysosomal degradation , with none being recycled to the haemolymph . More importantly , we show that mutations in LpR1 cause a profound effect on the immune response . Thus , our results indicate that the scavenging of serpin/proteinase complexes might be a critical step in the regulation of proteolytic cascades .