Cell-Free translation of Balbiani ring RNA (75S) ofChironomus tentans salivary glands into high molecular weight products

Summary Cell-free translation of salivary gland RNA or of purified Balbiani ring RNA (75S) in a reticulocyte lysate system gives rise to high molecular weight translational products (HMTP). In addition to a common size (approx. 1×10⁶ daltons) HMTP share imunogenic determinants with the giant secretory proteins of salivary glands. This suggests that HMTP correspond to in vivo secreted proteins and thus, corroborates the notion that 75S-RNA is the messenger for these proteins. The time course of HMTP synthesis and the lack of appearance of lower molecular weight components as translational products of 75S-RNA indicate that the synthesis of HMTP (and of secretory proteins) occurs in one piece by an uninterrupted process. HMTP are regarded the largest polypeptides so far synthesized in a cell-free system.     

In vitro biosynthesis and secretion of rabbit epididymal secretory proteins: regulation by androgens

The biosynthesis and secretion of epididymal proteins were studied in an in vitro system using explants from rabbit epididymis cultured in a defined medium. Epididymal explants actively incorporated [35S]methionine into cellular proteins, about 7% of them being secreted into the medium. SDS-PAGE of the labeled proteins secreted to the medium showed regional differences in their synthesis and secretion along the epididymal tract. Castration resulted in the inhibition of the synthesis and secretion of at least two polypeptides of Mr 150,000 and 21,000, but at the same time induced the appearance of other polypeptides. Immunoprecipitations with a specific antibody indicated that the variations in the amounts of the secreted 21 kDa component were associated with differences in its rate of synthesis. Epididymis from immature rabbits synthesized some polypeptides that are repressed in the adult state. The results suggest a dual effect of testosterone on rabbit epididymal secretory proteins.     

Isoproterenol increases sorting of parotid gland cargo proteins to the basolateral pathway

Exocrine cells have an essential function of sorting secreted proteins into the correct secretory pathway. A clear understanding of sorting in salivary glands would contribute to the correct targeting of therapeutic transgenes. The present work investigated whether there is a change in the relative proportions of basic proline-rich protein (PRP) and acidic PRPs in secretory granules in response to chronic isoproterenol treatment, and whether this alters the sorting of endogenous cargo proteins. Immunoblot analysis of secretory granules from rat parotids found a large increase of basic PRP over acidic PRPs in response to chronic isoproterenol treatment. Pulse chase experiments demonstrated that isoproterenol also decreased regulated secretion of newly synthesized secretory proteins, including PRPs, amylase and parotid secretory protein. This decreased efficiency of the apical regulated pathway may be mediated by alkalization of the secretory granules since it was reversed by treatment with mild acid. We also investigated changes in secretion through the basolateral (endocrine) pathways. A significant increase in parotid secretory protein and salivary amylase was detected in sera of isoproterenol-treated animals, suggesting increased routing of the regulated secretory proteins to the basolateral pathway. These studies demonstrate that shifts of endogenous proteins can modulate regulated secretion and sorting of cargo proteins. amylase; parotid secretory protein; polarized secretion     

Polarized secretion of an ectopic protein in Drosophila salivary glands in vivo.

Epithelial cells can secrete specific proteins in a polarized manner, either from the apical or basolateral surface. Intracellular protein sorting which results in polarized secretion has previously been studied using epithelial tissue culture cells. We describe here the use of Drosophila larval salivary glands for the study of polarized secretion by epithelia in vivo, and address whether an ectopically synthesized secretory protein can be sorted and targeted to the correct cell surface for secretion. Larval cuticle proteins (LCPs) and salivary gland secretion (Sgs) proteins of Drosophila melanogaster are apically secreted proteins that are produced respectively by the epidermis and salivary glands. We have transformed Drosophila with a hybrid gene consisting of the sgs-4 promoter sequence and the coding sequence for a variant (LCP-f2) of LCP-2. We have found that transgenic late third instar larvae produce LCP-f2 only in the salivary glands and that LCP-f2 is properly secreted in vivo in a polarized manner from only the apical surface of the cells into the gland lumen. The results indicate that apical secretion does not depend on a tissue-specific targeting signal contained within the protein.     

Apyrase and α-glucosidase in the salivary glands of Aedes albopictus

An apyrase and an α-glucosidase were detected in the salivary glands extracts of adult Aedes albopictus. The apyrase is a 61,000 Da secreted protein that hydrolyses ATP and ADP. This protein is synthesized in adults and is preferentially accumulated in the distal lateral lobes of the female salivary glands. The α-glucosidase is a secreted 67,000 Da protein. This enzyme is synthesized during adult life and accumulated in the proximal-lateral lobes of both males and females. The results are discussed and compared with data previously obtained with Aedes aegypti salivary glands.     

Proteomics analysis of the excretory/secretory component of the blood-feeding stage of the hookworm, Ancylostoma caninum

Hookworms are blood-feeding intestinal parasites of mammalian hosts and are one of the major human ailments affecting approximately 600 million people worldwide. These parasites form an intimate association with the host and are able to avoid vigorous immune responses in many ways including skewing of the response phenotype to promote parasite survival and longevity. The primary interface between the parasite and the host is the excretory/secretory component, a complex mixture of proteins, carbohydrates, and lipids secreted from the surface or oral openings of the parasite. The composition of this complex mixture is for the most part unknown but is likely to contain proteins important for the parasitic lifestyle and hence suitable as drug or vaccine targets. Using a strategy combining the traditional technology of one-dimensional SDS-PAGE and the newer fractionation technology of OFFGEL electrophoresis we identified 105 proteins from the excretory/secretory products of the blood-feeding stage of the dog hookworm, Ancylostoma caninum. Highly represented among the identified proteins were lectins, including three C-type lectins and three beta-galactoside-specific S-type galectins, as well as a number of proteases belonging to the three major classes found in nematodes, aspartic, cysteine, and metalloproteases. Interestingly 28% of the identified proteins were homologous to activation-associated secreted proteins, a family of cysteine-rich secreted proteins belonging to the sterol carrier protein/Tpx-1/Ag5/PR-1/Sc-7 (TAPS) superfamily. Thirty-four of these proteins were identified suggesting an important role in host-parasite interactions. Other protein families identified included hyaluronidases, lysozyme-like proteins, and transthyretin-like proteins. This work identified a suite of proteins important for the parasitic lifestyle and provides new insight into the biology of hookworm infection.     

Balbiani ring 3 in Chironomus tentans encodes a 185-kDa secretory protein which is synthesized throughout the fourth larval instar

We have continued to map and identify genes encoding a family of secretory proteins. These proteins are synthesized in larval salivary glands of the midge, Chironomus tentans, and assemble in vivo into insoluble silk-like threads. The genes for several secretory proteins exist in Balbiani rings (BRs) on salivary-gland polytene chromosomes. A randomly primed cDNA clone, designated pCt185, hybridized in situ to BR3 and was shown on Northern blots to originate from a salivary gland-specific 6-kb poly(A) + RNA. The partial cDNA sequence contained 483 nucleotides including one open reading frame (ORF) encoding 160 amino acids (aa). A striking feature of the ORF was the periodic distribution of cysteine residues (Cys-X-Cys-X-Cys-X6-Cys) which occurred approximately every 22 aa. A cDNA-encoded 18-aa sequence was selected for chemical peptide synthesis. When affinity-purified antipeptide antibodies were incubated with a Western blot containing salivary-gland proteins they reacted specifically with a 185-kDa secretory protein (sp185). Developmental studies showed that sp185 and its mRNA were present in salivary glands throughout the fourth larval instar. Thus sp185 and a family of 1000-kDa secretory proteins are encoded by a class of genes that are expressed throughout the fourth instar. This contrasts with the developmentally regulated expression of the sp140 and sp195 genes whose expression is maximal during the prepupal stages of larval development.     

Molecular crowding as a mechanism for tick secretory granule biogenesis

During feeding ticks secrete bioactive components into the host to counter-act its immune and hemostatic defense systems. These bioactive components are stored in secretory granules that are secreted during feeding in an exocrine stimulus-response type of mechanism. All proteins destined for secretion are packaged into these granules during granule biogenesis. Up to date no mechanism for granule biogenesis has been proposed, except to note that biogenesis occurs under conditions of high protein and calcium concentrations in an acidic environment. Previously, the most abundant proteins (TSGPs) found in the salivary glands of the soft tick, Ornithodoros savignyi, were suggested to play a part in granule biogenesis, based on their high abundance. The TSGPs are part of the lipocalin family, of which numerous members have been identified in ticks. We consider here the high concentrations of the TSGPs in salivary glands and what effect this will have on the crowded environment inside the secretory granules. It is shown that the TSGPs occur at concentrations that will lead to molecular crowding of which one result is the non-specific aggregation of components to reduce crowding effects. Aggregation of proteins as a mechanism of granule biogenesis has been proposed before, but not in terms of molecular crowding. We thus propose molecular crowding as the general mechanism of granule biogenesis, in tick secretory granules, but can also be extended to other forms of secretory granules in general.     

Heat-stable proteins and abscisic Acid action in barley aleurone cells

[³⁵S]Methionine labeling experiments showed that abscisic acid (ABA) induced the synthesis of at least 25 polypeptides in mature barley (Hordeum vulgare) aleurone cells. The polypeptides were not secreted. Whereas most of the proteins extracted from aleurone cells were coagulated by heating to 100°C for 10 minutes, most of the ABA-induced polypeptides remained in solution (heat-stable). ABA had little effect on the spectrum of polypeptides that were synthesized and secreted by aleurone cells, and most of these secreted polypeptides were also heatstable. Coomassie blue staining of sodium dodecyl sulfate polyacrylamide gels indicated that ABA-induced polypeptides already occurred in high amounts in mature aleurone layers having accumulated during grain development. About 60% of the total protein extracted from mature aleurone was heat stable. Amino acid analyses of total preparations of heat-stable and heat-labile proteins showed that, compared to heat-labile proteins, heat-stable intracellular proteins were characterized by higher glutamic acid/glutamine (Glx) and glycine levels and lower levels of neutral amino acids. Secreted heat-stable proteins were rich in Glx and proline. The possibilities that the accumulation of the heat-stable polypeptides during grain development is controlled by ABA and that the function of these polypeptides is related to their abundance and extraordinary heat stability are considered.     

Identification, synthesis, and characterization of the yolk polypeptides of Plodia interpunctella

The mature eggs of Plodia interpunctella were found to contain four major polypeptides. These yolk polypeptides (YPs) were found to have approximate molecular weights of 153,000 daltons (YP1), 69,000 daltons (YP2), 43,000 daltons (YP3), and 33,000 daltons (YP4) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In addition, we found YP1 was resolved by a 5% polyacrylamide gel into two separate polypeptides of 153,000 and 147,000 daltons. All of the YPs could be labeled in vivo or in vitro with [35S]-methionine. Yolk peptide 1 and YP3 were synthesized by fat body of pharate adult and adult females and secreted into the hemolymph. Yolk peptide 2 and YP4 were synthesized and secreted into incubation medium by ovaries that contained vitellogenic oocytes, but these polypeptides were not found in the hemolymph. Fat bodies of males synthesized and secreted an immunoprecipitable polypeptide similar to YP3 as well as immunoprecipitable polypeptides larger than 200,000 daltons that had no counterparts in the oocytes. Peptide mapping by protease digestion showed each YP to be cleaved into unique fragments, suggesting that no precursor-product relationship exists between the YPs. Ion exchange chromatography and gel permeation chromatography separated that yolk proteins into two groups with approximate molecular weights of 462,000 and 264,000 daltons. By resolving these peaks on SDS-PAGE, it was found that YP1 and YP3 formed the 462,000-dalton yolk protein and YP2 and YP4 formed the 264,000-dalton yolk protein.     

Salivary gland-specific gene expression in the malaria vector Anopheles gambiae

Molecular studies on the tissue-specific gene expression in the salivary glands of Anopheles gambiae may provide useful tools for the development of new strategies for the control of the most efficient malaria vector in the sub-Saharan Africa. We summarize here the results of a recent investigation focused on the isolation of secreted factors and putative receptors from the salivary glands of An. gambiae. Using the Signal Sequence Trap technique we have identified the first cDNAs specifically expressed in the An. gambiae salivary glands. Among these, four are exclusively expressed in female glands and encode factors presumably involved in blood-feeding, whereas two other cDNAs seem to be expressed both in male and in female glands and are likely implicated in sugar-feeding. Homologues of genes previously identified in the yellow fever mosquito Aedes aegypti, like the apyrase and D7, as well as novel salivary gland-specific cDNAs, were identified. The isolation and characterization of promoter sequences from the corresponding genes may prove useful for the expression of anti parasitic agents in the salivary glands of transgenic mosquitoes.     

Synthesis of the major adult cuticle proteins of Drosophila melanogaster during hypoderm differentiation

Differentiating imaginal hypodermal cells of Drosophila melanogaster form adult cuticle during the second half of the pupal stage (about 40 to 93 hr postpupariation). A group of proteins with molecular weights of 23,000, 20,000, and 14,000 is identified as putative major wing cuticle proteins with the following biological properties: These proteins are abundant components of cuticle and are major synthetic products of cuticle-secreting hypodermal cells. They are leucine-rich and methionine-free and are the most prominent proteins of this type synthesized by wing hypoderm at 65 hr, during the period of procuticle formation. Electron microscopic autoradiography shows that leucine-rich, methionine-free proteins specifically localize to the apical cell surface and newly secreted cuticle of 65-hr wing cells. This strongly suggests the export of these proteins to the cuticle. Lastly, these proteins undergo a reduction in extractability just after eclosion, during the period of cuticle protein crosslinking (sclerotization). The synthesis of these major hypoderm proteins is temporally regulated in development. In wing cells, the 14-kDa proteins are synthesized first, from 53 to 78 hr, and the 20- and 23-kDa proteins are synthesized from 63 to 93 hr. The pattern of synthesis for these proteins is similar in abdominal cells but delayed by 6 to 10 hr. Two-dimensional gel electrophoresis shows that each of the 23-, 20-, and 14-kDa size classes contains at least two component polypeptides. Patterns of protein synthesis in cells of the imaginal hypodermis are regulated in a precise temporal sequence during the production of adult cuticle. Their study yields a useful system for the analysis of molecular events in gene control and cell differentiation.     

Lipid-independent secretion of a Drosophila Wnt protein

Wnt proteins comprise a large class of secreted signaling molecules with key roles during embryonic development and throughout adult life. Recently, much effort has been focused on understanding the factors that regulate Wnt signal production. For example, Porcupine and Wntless/Evi/Sprinter have been identified as being required in Wnt-producing cells for the processing and secretion of many Wnt proteins. Interestingly, in this study we find that WntD, a recently characterized Drosophila Wnt family member, does not require Porcupine or Wntless/Evi/Sprinter for its secretion or signaling activity. Because Porcupine is involved in post-translational lipid modification of Wnt proteins, we used a novel labeling method and mass spectrometry to ask whether WntD undergoes lipid modification and found that it does not. Although lipid modification is also hypothesized to be required for Wnt secretion, we find that WntD is secreted very efficiently. WntD secretion does, however, maintain a requirement for the secretory pathway component Rab1. Our results show that not all Wnt family members require lipid modification, Porcupine, or Wntless/Evi/Sprinter for secretion and suggest that different modes of secretion may exist for different Wnt proteins.     

Characterization of proteins secreted by sheep oviduct epithelial cells and their function in embryonic development

The role in early development of proteins secreted by oviduct epithelial cells has been investigated. Secreted proteins devoid of serum contamination have been produced by the surgical removal and immediate incubation of oviduct cells in [35S]methionine-containing medium. After electrophoretic separation, secreted polypeptides could be divided into those that were secreted uniformly throughout the oestrous cycle and a second class that showed a cyclical pattern of secretion. The first class of proteins represented a small proportion of total output whilst the predominant second class was composed mainly of polypeptides of Mr 92 and 46 x 10(3), respectively. Both of these polypeptide species, referred to as sheep oviduct proteins 92 and 46 (SOP 92, SOP 46), are detected only during the first 4 to 5 days after oestrus when the embryos are located in the oviduct. Oviduct cells collected at oestrus and maintained thereafter in culture secrete the same pattern of proteins and follow the same time course as their counterparts in vivo. The interaction between the oviduct proteins and the developing embryo was studied firstly by determining whether any of the secreted proteins bound to the zona pellucida. The results of iodination studies showed that two polypeptides of Mr 92 and 46 x 10(3), respectively, were bound to the zona pellucida of eggs removed from the oviduct but were absent from eggs that had not had contact with the oviduct epithelium. That these newly acquired proteins represent SOP 92 and 46 is suggested by their electrophoretic mobility and their ability to bind to the zona of follicular eggs when added in vitro and by the fact that they both disappear from the zonae of embryos after exit from the oviduct. The collection of unlabelled secreted proteins enabled us to produce a monoclonal antibody, which was used in the second series of experiments on oviduct-embryo interactions. The results confirmed that SOP 92 binds to the zona pellucida and moreover showed that this protein crosses the zona and becomes associated with the individual blastomeres of the developing embryo. These findings provide evidence that the mammalian oviduct probably plays a direct role in supporting embryonic development through specific polypeptides produced by its epithelium.     

Shared weapons of blood- and plant-feeding insects: Surprising commonalities for manipulating hosts

Insects that reprogram host plants during colonization remind us that the insect side of plant–insect story is just as interesting as the plant side. Insect effectors secreted by the salivary glands play an important role in plant reprogramming. Recent discoveries point to large numbers of salivary effectors being produced by a single herbivore species. Since genetic and functional characterization of effectors is an arduous task, narrowing the field of candidates is useful. We present ideas about types and functions of effectors from research on blood-feeding parasites and their mammalian hosts. Because of their importance for human health, blood-feeding parasites have more tools from genomics and other – omics than plant-feeding parasites. Four themes have emerged: (1) mechanical damage resulting from attack by blood-feeding parasites triggers “early danger signals” in mammalian hosts, which are mediated by eATP, calcium, and hydrogen peroxide, (2) mammalian hosts need to modulate their immune responses to the three “early danger signals” and use apyrases, calreticulins, and peroxiredoxins, respectively, to achieve this, (3) blood-feeding parasites, like their mammalian hosts, rely on some of the same “early danger signals” and modulate their immune responses using the same proteins, and (4) blood-feeding parasites deploy apyrases, calreticulins, and peroxiredoxins in their saliva to manipulate the “danger signals” of their mammalian hosts. We review emerging evidence that plant-feeding insects also interfere with “early danger signals” of their hosts by deploying apyrases, calreticulins and peroxiredoxins in saliva. Given emerging links between these molecules, and plant growth and defense, we propose that these effectors interfere with phytohormone signaling, and therefore have a special importance for gall-inducing and leaf-mining insects, which manipulate host-plants to create better food and shelter.