Secretion of the type 2 peritrophic matrix protein peritrophin-15 from the cardia.код для вставкиСкачать
76 Eisemann et al. Archives of Insect Biochemistry and Physiology 47:76–85 (2001) Secretion of the Type 2 Peritrophic Matrix Protein, Peritrophin-15, From the Cardia Craig Eisemann, Gene Wijffels, and Ross L. Tellam* Molecular Animal Genetics Centre, CSIRO Livestock Industries, Gehrmann Laboratories, The University of Queensland, St Lucia, Queensland, Australia The midgut of most insects is lined with a peritrophic matrix, which is thought to facilitate digestion and protect the midgut digestive epithelial cells from abrasive damage and invasion by ingested micro-organisms. The type 2 peritrophic matrix is synthesised by a complex and highly specialised organ called the cardia typically located at the junction of the cuticle-lined foregut and midgut. Although the complex anatomy of this small organ has been described, virtually nothing is known of the molecular processes that lead to the assembly of the type 2 peritrophic matrix in the cardia. As a step towards understanding the synthesis of the peritrophic matrix, the synthesis and secretion of the intrinsic peritrophic matrix protein, peritrophin15 has been followed in the cardia of Lucilia cuprina larvae using immuno-gold localisations. The protein is synthesised by cardia epithelial cells, which have abundant rough endoplasmic reticulum, Golgi, and vesicles indicative of a general secretory function. Peritrophin-15 is packaged into secretory vesicles probably produced from Golgi and transported to the cytoplasmic face of the apical plasma membrane. The vesicles fuse with the plasma membrane at the base of the microvilli and release peritrophin-15 into the inter-microvilli spaces. The protein then becomes associated with the nascent peritrophic matrix, which lies along the tips of the epithelial cell microvilli. It is proposed that peritrophin-15 binds to the ends of chitin fibrils present in the nascent peritrophic matrix, thereby protecting the fibril from the action of exochitinases. Arch. Insect Biochem. Physiol. 47:76– 85, 2001. © 2001 Wiley-Liss, Inc. Key words: peritrophic membrane; peritrophic matrix; Lucilia cuprina, peritrophin-15; cardia INTRODUCTION The peritrophic matrix (PM or peritrophic membrane) is a semi-permeable extracellular matrix that often lines the midgut of insects separating the contents of the gut lumen from the digestive epithelial cells lining the midgut. The PM is thought to have assorted functions including the facilitation of the digestive process in the in© 2001 Wiley-Liss, Inc. Contract grant sponsor: L.W. Bett Trust; Contract grant sponsor: International Wool Secretariat; Contract grant sponsor: Australian Centre for International Agricultural Research. *Correspondence to: Ross L. Tellam, Molecular Animal Genetics Centre, CSIRO Livestock Industries, Gehrmann Laboratories, Research Rd, The University of Queensland, St Lucia 4067. QLD, Australia. E-mail: Ross.Tellam@tag.csiro.au Received 18 December 2000; Accepted in revised form 8 February 2001 Secretion of Peritrophin-15 sect gut and protection of midgut epithelia from ingested abrasive particles and potentially invasive micro-organisms (Peters, 1992; JacobsLorena and Oo, 1996; Tellam, 1996; Terra, 1996; Lehane, 1997). The importance of the PM in the growth and development of insects is demonstrated by experiments showing that agents binding to the PM, such as specific antibodies or lectins, cause marked inhibition of insect growth (East et al., 1993; Eisemann et al., 1994; Casu et al., 1997; Fitches and Gatehouse, 1998; Tellam and Eisemann, 1998; Zhu-Salzman et al., 1998). It was postulated that these agents decrease the permeability of the PM, thereby leading to starvation of the insect (Eisemann et al., 1994; Casu et al., 1997). There are two distinct forms of PM that have been defined based on their sites of synthesis (Peters, 1992). Type 1 PM is synthesized by all midgut epithelial cells and forms a bag-like structure containing the ingested meal. Typically, type 1 PM is produced in direct response to the ingestion of a meal (e.g., blood-fed adult mosquitoes and black flies) but can also be constitutively produced. Type 2 PM is constitutively produced from a small highly specialized organ, the cardia, typically located in the anterior midgut region at the junction of the foregut and midgut. This PM is an open-ended sleeve-like structure and often more highly structured than type 1 PM (Peters, 1992; Tellam, 1996; Lehane, 1997). The type 2 PM is characterized by a distinctive lamellar appearance upon examination by electron microscopy. The rapid rate of growth of many insect developmental stages is accompanied by the corresponding rapid production of type 2 PMs. Indeed, type 2 PM growth rates as fast as 7.2 mm/h have been measured in vitro (Peters, 1992). Although the cardia is a relatively small organ typically consisting of only a few hundred PM synthesising cells, it must be able to synthesise relatively large quantities of the components of the PM to accommodate this rapid growth. Therefore, the cardia cells actively synthesising PM must be heavily committed to the synthesis of the PM components and their assembly into a nascent PM. These components include chitin, intrinsic structural proteins or peritrophins, and proteoglycans (Peters, 1992; Tellam, 1996; Tellam et al., 1999). There is 77 still considerable debate regarding the relative amounts of these components. Chitin has been thought to be a significant constituent of type 2 PMs; however, this view has recently been challenged (Tellam and Eisemann, 2000). The PM varies enormously in its synthesis, structure, and organization in different species of insects (Peters, 1992). Further, different life stages of the same insect often produce different types of PM, e.g.. larval mosquito (type 2 PM) and adult mosquito (type 1 PM). This suggests that the PM has been readily adapted to the specialized diets and developmental programs of each insect species. In keeping with this view of evolutionary plasticity of the PM, the amino acid sequences of peritrophins are generally poorly conserved even between relatively closely related insects such as the higher Dipteran flies Lucilia cuprina, Chrysomya bezziana, and Drosophila melanogaster (Tellam et al., 1999). For example, the percent identity of deduced amino acid sequences of the intrinsic PM protein peritrophin48 from any pair of these species only ranges between 32–42% (Schorderet et al., 1998; Vuocolo et al., 2000). In contrast, proteins unrelated to the PM but from the same species generally have a much higher percent identity, typically greater than 70% (e.g., chymotrypsins, actin, NOTCH, chitin synthase; unpublished results). Despite this amino acid sequence variability, the major architectural feature of the peritrophin-48 proteins, which is dictated by a multiple domain structure characterised by extensive intradomain disulphide bonding, is highly conserved (Tellam et al., 1999). Indeed, one of the characteristic features of all peritrophins is the presence of multiple domains with extensive intradomain disulphide bonding (Tellam et al., 1999). The PM is thought to consist of a chitin fibril meshwork embedded with glycoproteins and proteoglycans (Peters, 1992). However, very little is known of the constituents of the PM and how they interact to form the highly ordered structure of this semi-permeable matrix. Virtually nothing is known of the molecular mechanisms underlying the synthesis of PM, i.e., where the components are synthesised, the spatial and temporal order of their secretion, and the assembly of these components into a highly structured PM. 78 Eisemann et al. To partially address some of these issues, we have followed the synthesis and secretion of the type 2 PM protein, peritrophin-15, in the cardia of larvae of the higher Dipteran fly, L. cuprina. Peritrophin-15 has a molecular mass of 8 kDa and can only be solubilized from PM using strong denaturants such as 6 M guanidine HCl or 5% SDS (Wijffels et al., 2001). The protein is a major component of the core complex of the PM and is only synthesised by the larval cardia. The amino acid sequences of orthologs of peritrophin15 from L. cuprina, C. bezziana, and D. melanogaster have been determined (Fig. 1; Wijffels et al., 2001). In contrast to all other peritrophins characterised to date, the deduced amino acid sequences of peritrophin-15 orthologs are relatively conserved ranging between 50 and 80% identity for any pair of sequences. It is likely that the relatively greater conservation of the sequence of this peritrophin reflects its fundamental role in the core assembly of the PMs from these species. Also, unlike other PM proteins, peritrophin-15 is not glycosylated (as determined by biochemical and bioinformatics analyses) and consists of a single domain structure. The characteristic feature of the amino acid sequence is the absolute conservation of 6 cysteines whose register defines the peritrophin-C type domain (Tellam et al., 1999). The cysteines probably form 3 intramolecular disulphide bonds. In addition, there is strong conservation of aromatic amino acids located between cysteines 2 and 3, 4 and 5, and 5 and 6. A recombinant form of the protein has been expressed as a soluble protein in bacteria, purified and shown to bind chitin in vitro (Wijffels et al., 2001). The presence of conserved aromatic amino acids in peritrophin-15 is consistent with this function (Elgavish and Shaanan, 1997). In the current study, specific serum raised to the recombinant Fig. 1. Alignment of the amino acid sequences of peritrophin-15 from three higher Dipterans. Only the mature polypeptide sequences are shown (Wijffels et al., 2001). The absolutely conserved cysteine residues are boxed. DmPM15a, D. melanogaster peritrophin-15a; LcPM15, L. cuprina peritrophin-15; CbPM15, C. bezziana peritrophin-15. D. protein has been used for immuno-gold localizations to follow the synthesis and secretion of peritrophin-15 in the cardia epithelial cells from L. cuprina larvae. MATERIALS AND METHODS Culture of L. cuprina Larvae Laboratory populations of L. cuprina, which had originated from fly-struck sheep, were maintained on an artificial medium for up to 10 generations. Eggs were collected by placing small trays of minced liver covered with fine nylon gauze, inside cages of adult L. cuprina for 4–5 h. The eggs were then incubated overnight at 16°C and 100% relative humidity before being transferred to 34°C until hatching. Larvae were reared to third instar on a diet containing 10% w/v skim milk powder and 2% w/v brewer’s yeast in 1% w/ v agar gel. Production and Purification of Recombinant Hexahis-Peritrophin-15 The recombinant protein, hexahis-peritrophin-15 derived from the C. bezziana peritrophin15 sequence (Wijffels et al., 2001), was expressed using the pQE9 vector (Qiagen) essentially according to the manufacturer’s instructions. Only the DNA encoding the mature form of the protein was used. The soluble protein was purified by Ni-NTA affinity chromatography (Qiagen). Production of Serum to Hexahis-Peritrophin-15 Recombinant hexahis-peritrophin-15 (70 µg) was homogenized in Montanide ISA70 (Seppic, Paris, France) and subcutaneously injected into a rabbit. The rabbit received two further injections 4 and 6 weeks after the primary immunization. Serum was collected 2 weeks after the final im- melanogaster contains two peritrophin-15-like sequences. For simplicity, only one of these is shown. The asterisks show positions where an amino acid is absolutely conserved in the three orthologs. Dashes have been introduced in the sequences to optimise the alignment. Secretion of Peritrophin-15 munization. The “Australian code of practice for the care and use of animals for scientific purposes” was followed for all procedures involving the rabbit. Immuno-blots and ELISAs confirmed the specificity of the antibody. Although the serum was raised to C. bezziana hexahis-peritrophin-15, it reacted strongly with L. cuprina peritrophin-15 in immuno-blots and ELISAs as would be expected for two proteins with 82% amino acid sequence identity (results not shown). Immuno-Gold Localization Cardiae were dissected from third instar larvae in PBS and fixed in 4% paraformaldehyde and 0.3% glutaraldehyde in PBS for 1 h at room temperature. The dissected cardiae were washed in PBS, dehydrated through an ethanol series (30, 50, 70, 90%) and embedded in medium grade LR White embedding resin (London Resin Co., Reading, Berkshire, UK), which was then polymerised in air-tight gelatin capsules at 50°C overnight. Ultra-thin sections were cut longitudinally through individual cardia on an LKB Ultrotome Nova ultramicrotome and the sections taken up on Butvarcoated copper grids. Sections on grids were processed by transferring to drops of buffer A (PBS with 0.5% ovalbumin [Sigma] and 0.1% Tween 20) containing: (1) 10% normal goat serum (1.5 h); (2) pre- or post-vaccination rabbit serum to recombinant hexahis-peritrophin-15 (diluted 1/500) for 1 h; (3) goat anti-rabbit Ig antibody conjugated to 10-nm-diameter colloidal gold particles diluted 1/ 100 (British Biocell International) for 1.5 h all at room temperature. The sections were washed (3× 15 min) in drops of buffer A after steps (2) and (3) and finally in distilled water. Each section was then stained (5 min each) in 2% aqueous uranyl acetate and 0.1 M lead citrate and examined in a JEOL 1010 transmission electron microscope. 79 tains the oesophagus surrounded by highly modified foregut including a cuticular lining and this, in turn, is surrounded by specialized midgut cells that synthesise the PM. Only a single PM is produced by the larval cardia. In contrast, the adult cardia simultaneously produces 3 PMs (Binnington, 1988). The region of the cardia marked F in Figure 2 represents the region where sections were examined in detail for the presence of peritrophin15 using immuno-gold localizations. In total, the sections encompassed the cuticular lining contributed by the foregut, the adjacent PM, and the underlying epithelial cells characterised by extensive microvilli. The order of the following figures has been arranged to progressively show the spatial aspects of peritrophin-15 production, secretion, and addition to the nascent PM. The epithelial cells underlying the nascent PM in the cardia are characterised by extensive rough endoplasmic reticulum, Golgi, and secretory vesicles, collectively indicative of a secretory cell (Fig. 3). The vesicle labeled in Figure 3a is surrounded by membranous layers, probably RESULTS The cardia from the larvae of higher Dipterans is a small highly specialized organ located at the junction of the cuticle-lined foregut (oesophagus) and midgut, and formed by intussusception of both tissues. For orientation, Figure 2 shows a diagrammatic representation of a sagittal section through the cardia of a higher Dipteran larva (Binnington, 1988; Peters, 1992). The cardia con- Fig. 2. Schematic representation of a sagittal section through the larval cardia of a higher Dipteran. B, bacteria; Cu, cuticle; F, formation zone of the PM; FG, foregut; IC, imaginal cells; O, oesophagus; PM, peritrophic matrix. The region marked F represents the sections in the PM formation zone examined for the synthesis of peritrophin-15. Reproduced from Peters (1992) with permission of the publisher, Springer-Verlag, Berlin. 80 Eisemann et al. Fig. 3. Immuno-gold localization of peritrophin-15 in the cytoplasm of cardia epithelial cells from third instar L. cuprina larvae. The sections were from the cardia PM formation zone. a: Pre-vaccination control. b–d: Post-vaccination serum. Arrows denote examples of gold particles associated with the rough endoplasmic reticulum and vesicles. G, Golgi; M, mitochondria; N, nucleus; RER, rough endoplasmic reticulum; V, intracellular vesicle. The bars represent (a) 267 nm; (b) 267 nm; (c) 133 nm; (d) 133 nm. Golgi. This suggests that the vesicle is in intimate contact with Golgi and possibly derived from it. Close examination of the immuno-gold-labeled cells reveals the presence of gold particles associated with the rough endoplasmic reticulum, Golgi, and vesicles (Fig. 3b–d). The labeling of the rough endoplasmic reticulum is consistent with the production of a protein destined for secretion. Not all vesicles were labeled to the same density and a minority contained no gold particles (e.g., Fig. 3c,d). The vesicles, therefore, may have contained multiple cargoes and in some cases no peritroph- in-15. Figure 3a is a control using pre-vaccination serum. This section shows virtually no gold labeling, thereby demonstrating the specificity of the immuno-localization. Figure 4 shows concentrations of heavily labeled vesicles immediately underlying the base of microvilli at the apical plasma membrane of the cardia epithelial cells. The vesicles are generally larger than those present further within the cell and often contain internal “nodes.” These are particularly discernible in the pre-vaccination control shown in Figure 4a but are also apparent Secretion of Peritrophin-15 81 Fig. 4. Immuno-gold localization of peritrophin-15 in intracellular vesicles adjacent to the apical plasma membrane of cardia epithelial cells. a: Pre-vaccination control. b–d: Post-vaccination serum. Arrows denote examples of intrac- ellular vesicles labeled with gold particles. M, mitochondria; MF, microfilaments; MV, microvilli; RER, rough endoplasmic reticulum; V, intracellular vesicle. The bars represent (a) 267 nm; (b) 267 nm; (c) 133 nm; (d) 133 nm. in the labeled vesicles shown in Figure 4b–d. Again, this suggests multiple cargoes in the vesicles. Often the vesicles are associated with a microfilament extending from the base of a microvillus back into the cell (Fig. 4c). The microfilament may be a means of transporting the vesicles from their site of synthesis to the cell periphery or may be involved in organization of vesicles at the cell membrane as a prelude to fusion of the vesicle with the plasma membrane and subse- quent exocytosis. The vesicles appear to fuse with the cell membrane lying between two microvilli and, therefore, presumably disgorge their vesicle contents between the microvilli (Fig. 4c,d). There is no evidence that any of the vesicles travel within the microvillus to its tip before membrane fusion. What appear to be the remnants of the vesicles, which now are much larger and contain little or no gold labeling, are present extracellularly between the microvilli (Fig. 4a,d). It is not 82 Eisemann et al. clear whether these putative vesicle remnants are membrane bound. Further, there is no indication that the putative vesicle remnants contain peritrophin-15 or any other cargoes. The putative vesicle remnants progressively move through the inter-microvillar spaces to the tips of the microvilli immediately adjacent to the nascent PM. Here, they are considerably smaller in size (Fig. 5a). Close examination of the inter-microvilli spaces reveals the presence of gold particles but at relatively low density (Fig. 5c). This would be expected if the contents of the intracellular vesicles were diluted into the larger extracellular spaces associated with the microvilli. Fig. 5. Immuno-gold localization of peritrophin-15 on nascent PM. a: Pre-vaccination control. b,c: Post-vaccination serum. Solid black arrows denote examples of gold particles associated with the PM, intracellular vesicles, and microvilli. Broken white arrows denote the positions of the cuticle (C), microvilli (MV), vesicles (V), and peritrophic matrix (PM) in (a) and (b). The bars represent (a) 350 nm; (b) 350 nm; (c) 133 nm. Secretion of Peritrophin-15 Figure 5a and b show sections of the PM formation zone encompassing the cuticle, nascent PM, and microvilli of the cardia epithelial cells. The microvilli that characterize these cells are relatively long (~ 2–3 µm). The sections show labeled vesicles strongly concentrated at the apical face of the plasma membrane, presumably awaiting membrane fusion and secretion of their contents. Also clearly discernible are the putative vesicle remnants between the microvilli adjacent the cell proper and smaller remnants at the distal ends of the microvilli. The microvilli extend directly to the nascent PM, which is strongly and uniformly labeled with gold (Fig. 5c). In contrast, the highly defined cuticle, which is located immediately adjacent to the nascent PM but opposite the microvilli, shows no gold labeling. This is consistent with the view that peritrophin-15 is restricted in its final location to only the larval PM. Indeed, tissue-specific immuno-blots have demonstrated the presence of peritrophin-15 in larval cardia and PM but not in any other larval tissues (Wijffels et al., 2001). The PM at the position in the cardia where the sections were taken does not show the marked lamellar appearance that characterises the fully mature PM isolated from larval midgut. Presumably, the PM visualized in these sections is immature. DISCUSSION The current study has demonstrated that peritrophin-15 is synthesised by epithelial cells in the PM formation zone of the cardia. These cells package peritrophin-15 into secretory vesicles, which originate from deep within the cell, probably derived from Golgi. The vesicles are then transported to the apical face of the cell where they concentrate at the base of microvilli. The process of vesicular movement and organization at the plasma membrane may be facilitated by microfilaments originating from the base of the microvilli. The vesicles presumably fuse with the plasma membrane and release their cargoes into the inter-microvillar space via a process of exocytosis. Peritrophin-15 is strongly associated with the nascent PM, which is situated at the tips of the microvilli of the cardia epithelial cells. What is not clear is how the protein moves from the base of the microvilli to the PM. This may be a 83 passive diffusional process that relies on the binding of peritrophin-15 to the nascent PM and the subsequent movement of the PM away from this site thereby creating a concentration gradient of peritrophin-15 in the intermicrovillar spaces. Alternatively, pulsatile contractions of the cardia may facilitate such movement (Peters, 1992). The process of intracellular vesicular transport of peritrophin-15 followed by its exocytosis at the base of microvilli also describes the production and secretion of a peritrophin (IIM) from midgut cells of a lepidopteran insect producing type 1 PM (Harper and Granados, 1999). Thus, the same mechanisms of peritrophin synthesis and secretion may underlie the formation of both type 1 and type 2 PMs. The cardia may be an evolutionary adaptation in some insects, allowing synthesis of relatively large quantities of peritrophins in a highly localized gut region that acts as an organising centre for the nascent PM. One of the significant differences between type 2 and type 1 PMs is the greater level of structural organization in the former PMs (Peters, 1992). One putative function of peritrophin-15 is to cap the ends of chitin polymers or fibrils in the PM, thereby possibly regulating the lengths of the polymer and also protecting its ends from attack by exochitinases (Wijffels et al., 2001). This model was based on the evidence that peritrophin-15 bound strongly to chitin but at low stoichiometries (~1 molecule of peritrophin-15 to 10,000 GlcNAc molecules) in vitro and that the protein was probably a monomer consisting of a single protein domain. This model is consistent with the presence of an immature PM at the position in the cardia where peritrophin-15 is secreted and presumably added, as well as the uniform distribution of peritrophin-15 throughout the nascent PM. It is interesting to note the complete absence of peritrophin-15 in the cuticle layer lying adjacent to the nascent PM but opposite the microvilli of the cardia epithelia cells. The cuticle, like the PM, is also thought to contain chitin (Hackman, 1974). Substantial analyses of cuticular proteins from various sources have never revealed the presence of peritrophin-like proteins (Anderson et al., 1995). The cuticular proteins in their mature state nearly always contain no cysteine residues, a feature that easily distinguishes the two groups. Presumably, the chitin present within the 84 Eisemann et al. cuticle layer in the cardia is not available for binding peritrophin-15. The cuticle layer at this position in the PM could be relatively mature and bound cuticle proteins may block any binding sites for peritrophin-15. Alternatively, chitin present in the PM and cuticle may be in very different conformations, which could dictate the specific binding interactions with the different families of proteins associated with these structures. It will be important to follow up the current studies with multiple labeling for several PM proteins to determine whether these proteins are made simultaneously by the same cells and secreted from common or different vesicles or whether there is a spatially distinct distribution of synthesis along the cardia epithelial cells that allows the progressive and ordered addition of specific PM proteins to the nascent PM. Of particular importance will be the determination of those cells producing chitin destined for inclusion in the PM. Presumably, chitin is the principal component of the nascent PM onto which peritrophin-15 is added. Cardia epithelial cells located further toward the origins of the PM formation zone of the cardia could be producing chitin that assembles into the nascent PM, which then progressively moves as it is synthesised toward the posterior of the cardia. Another possibility is that chitin is synthesised by the same cells producing peritrophin-15. Here, the tips of the microvilli may be sites for chitin synthesis and secretion. However, these regions contain no specialized structures normally associated with chitin production at least in those cells synthesising cuticular chitin (Locke, 1996). Alternatively, the epithelia cells of the PM formation zone could package chitin in the same secretory vesicles that carry peritrophin15 and perhaps other peritrophins as well. The preassembled contents of these vesicles could be released into the inter-microvillar spaces and then finally assemble into the nascent PM at the tips of microvilli. To distinguish between these models it will be necessary to localise chitin- and peritrophinsynthesising cells. The detection of chitin in tissue sections is not easy (Tellam and Eisemann, 2000). The results obtained from the traditional method of localisation of chitin using gold-labeled wheat germ lectin are difficult to interpret as many peritrophins are glycoproteins with strong affinity for this lectin (East et al., 1993; Casu et al., 1997; Tellam et al., 1999). Perhaps the best way of characterising the cells synthesising chitin is to identify those that express chitin synthase. The recent molecular characterization of insect chitin synthase should facilitate the localization of this enzyme in the insect cardia (Tellam et al., 2000). The current study has followed the exocytosis and assembly of the integral PM protein, peritrophin-15, onto newly formed PM in the cardia of L. cuprina larvae. This is a small step in a complex assembly process involved in the production of PM from a small but highly specialized group of cells, the cardia. Many questions still remain unanswered. For example: what is the full repertoire of specific components, particularly the peritrophins, present in the PM; where is chitin produced; what is the conformation of chitin in the PM; what controls the order, if any, of the addition of specific components to the PM; what specific protein-protein, protein-chitin, and protein-oligosaccharide interactions are required for assembly of the PM; and what are the precise molecular and biological functions of the PM? The recent determination of the Drosophila genomic sequence and the molecular characterization of several insect PM proteins (Tellam et al., 1999; and our unpublished results) now enable the identification of the majority of the principal protein components of the PM from higher Dipterans. 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