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Pigment epithelium-derived factor: neurotrophic activity and identification as a member of the serine protease inhibitor gene family.

ABSTRACT: Cultured pigment epithelial cells of the fetal human retina secrete a protein, pigment epithelium-derived factor (PEDF), that induces a neuronal phenotype in cultured human retinoblastoma cells. Morphological changes include the induction of an extensive neurite meshwork and the establishment of corona-like cellular aggregates surrounding a central lumen. The differentiated cells also show increases in the expression of neuron-specific enolase and the 200-kDa neurofilament subunit. Amino acid and DNA sequence data demonstrate that PEDF belongs to the serine protease inhibitor (serpin) family. The PEDF gene contains a typical signal-peptide sequence, initiator methionine codon, and polyadenylylation signal and matches the size of other members of the serpin superfamily (e.g., alpha 1-antitrypsin). It lacks homology, however, at the putative serpin reactive center. Thus, PEDF could exert a paracrine effect in the embryonic retina, influencing neuronal differentiation by a mechanism that does not involve classic inhibition of serine protease activity.

SUBMITTER: Steele FR

PROVIDER: S-EPMC45907 | biostudies-other | 1993 Feb

REPOSITORIES: biostudies-other

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Pigment epithelium-derived factor: neurotrophic activity and identification as a member of the serine protease inhibitor gene family.

Steele F R FR Chader G J GJ Johnson L V LV Tombran-Tink J J

Proceedings of the National Academy of Sciences of the United States of America 19930201 4

Cultured pigment epithelial cells of the fetal human retina secrete a protein, pigment epithelium-derived factor (PEDF), that induces a neuronal phenotype in cultured human retinoblastoma cells. Morphological changes include the induction of an extensive neurite meshwork and the establishment of corona-like cellular aggregates surrounding a central lumen. The differentiated cells also show increases in the expression of neuron-specific enolase and the 200-kDa neurofilament subunit. Amino acid an ...[more]

PMID: 8434014

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Project description:BackgroundPigment epithelium derived factor (PEDF), a member of the serpin family, regulates cell proliferation, promotes survival of neurons, and blocks growth of new blood vessels in mammals. Defining the molecular phylogeny of PEDF by bioinformatic analysis is one approach to understanding the link between its gene structure and its function in these biological processes.ResultsFrom a comprehensive search of available DNA databases we identified a single PEDF gene in all vertebrate species examined. These included four mammalian and six non-mammalian vertebrate species in which PEDF had not previously been described. A five gene cluster around PEDF was found in an approximate 100 kb region in mammals, birds, and amphibians. In ray-finned fish these genes are scattered over three chromosomes although only one PEDF gene was consistently found. The PEDF gene is absent in invertebrates including Drosophila melanogaster (D. melanogaster), Caenorhabditis elegans (C. elegans), and sea squirt (C. intestinalis). The PEDF gene is transcribed in all vertebrate phyla, suggesting it is biologically active throughout vertebrate evolution. The multiple actions of PEDF are likely conserved in evolution since it has the same gene structure across phyla, although the size of the gene ranges from 48.3 kb in X. tropicalis to 2.9 kb in fugu, with human PEDF at a size of 15.6 kb. A strong similarity in the proximal 200 bp of the PEDF promoter in mammals suggests the existence of a possible regulatory region across phyla. Using a non-synonymous/synonymous substitution rate ratio we show that mammalian and fish PEDFs have similar ratios of <0.13, reflecting a strong purifying selection of PEDF gene. A large number of repetitive transposable elements of the SINE and LINE class were found with random distribution in both the promoter and introns of mammalian PEDF.ConclusionThe PEDF gene first appears in vertebrates and our studies suggest that the regulation and biological actions of this gene are preserved across vertebrates. This comprehensive analysis of the PEDF gene across phyla provides new information that will aid further characterization of common functional motifs of this serpin in biological processes.

Project description:The predatory natural enemy Eocanthecona furcellata plays a crucial role in agricultural ecosystems due to its effective pest control measures and defensive venom. Predator venom contains serine protease inhibitors (SPIs), which are the primary regulators of serine protease activity and play key roles in digestion, development, innate immunity, and other physiological regulatory processes. However, the regulation mechanism of SPIs in the salivary glands of predatory natural enemies is still unknown. In this study, we sequenced the transcriptome of E. furcellata salivary gland and identified 38 SPIs genes named EfSPI1∼EfSPI38. Through gene structure, multiple sequence alignment and phylogenetic tree analysis, real-time quantitative PCR (RT-PCR) expression profiles of different developmental stages and different tissues were analyzed. RNAi technology was used to explore the gene function of EFSPI20. The results showed that these 38 EfSPIs genes contained 8 SPI domains, which were serpin, TIL, Kunitz, Kazal, Antistasin, Pacifastin, WAP and A2M. The expression profile results showed that the expression of different types of EfSPIs genes was different at different developmental stages and different tissues. Most of the EfSPIs genes were highly expressed in the egg stage. The EfSPI20, EfSPI21, EfSPI22, and EfSPI24 genes of the Pacifastin subfamily and the EfSPI35 gene of the A2M subfamily were highly expressed in the nymphal and adult stages, which was consistent with the RT-qPCR verification results. These five genes are positively correlated with each other and have a synergistic effect on E. furcellata, and they were highly expressed in salivary glands. After interfering with the expression of the EfSPI20 gene, the survival rate and predatory amount of male and female adults were significantly decreased. Taken together, we speculated some EfSPIs may inhibit trypsin, chymotrypsin, and elastase, and some EfSPIs may be involved in autoimmune responses. EfSPI20 was essential for the predation and digestion of E. furcellata, and the functions of other EfSPIs were discussed. Our findings provide valuable insights into the diversity of EfSPIs in E. furcellata and the potential functions of regulating their predation, digestion and innate immunity, which may be of great significance for developing new pest control strategies.

Project description:Rhipicephalus (Boophilus) microplus evades the host's haemostatic system through a complex protein array secreted into tick saliva. Serine protease inhibitors (serpins) conform an important component of saliva which are represented by a large protease inhibitor family in Ixodidae. These secreted and non-secreted inhibitors modulate diverse and essential proteases involved in different physiological processes.The identification of R. microplus serpin sequences was performed through a web-based bioinformatics environment called Yabi. The database search was conducted on BmiGi V1, BmiGi V2.1, five SSH libraries, Australian tick transcriptome libraries and RmiTR V1 using bioinformatics methods. Semi quantitative PCR was carried out using different adult tissues and tick development stages. The cDNA of four identified R. microplus serpins were cloned and expressed in Pichia pastoris in order to determine biological targets of these serpins utilising protease inhibition assays.A total of four out of twenty-two serpins identified in our analysis are new R. microplus serpins which were named as RmS-19 to RmS-22. The analyses of DNA and predicted amino acid sequences showed high conservation of the R. microplus serpin sequences. The expression data suggested ubiquitous expression of RmS except for RmS-6 and RmS-14 that were expressed only in nymphs and adult female ovaries, respectively. RmS-19, and -20 were expressed in all tissues samples analysed showing their important role in both parasitic and non-parasitic stages of R. microplus development. RmS-21 was not detected in ovaries and RmS-22 was not identified in ovary and nymph samples but were expressed in the rest of the samples analysed. A total of four expressed recombinant serpins showed protease specific inhibition for Chymotrypsin (RmS-1 and RmS-6), Chymotrypsin / Elastase (RmS-3) and Thrombin (RmS-15).This study constitutes an important contribution and improvement to the knowledge about the physiologic role of R. microplus serpins during the host-tick interaction.

Project description:Serine protease inhibitors (serpins) regulate proteolytic events within diverse biological processes, including digestion, coagulation, inflammation and immune responses. The presence of serpins in Fasciola hepatica excretory-secretory products indicates that the parasite exploits these to regulate proteases encountered during its development within vertebrate hosts. Interrogation of the F. hepatica genome identified a multi-gene serpin family of seven members that has expanded by gene duplication and divergence to create an array of inhibitors with distinct specificities. We investigated the molecular properties and functions of two representatives, FhSrp1 and FhSrp2, highly expressed in the invasive newly excysted juvenile (NEJ). Consistent with marked differences in the reactive centre loop (RCL) that executes inhibitor-protease complexing, the two recombinant F. hepatica serpins displayed distinct inhibitory profiles against an array of mammalian serine proteases. In particular, rFhSrp1 efficiently inhibited kallikrein (Ki = 40 nM) whilst rFhSrp2 was a highly potent inhibitor of chymotrypsin (Ki = 0.07 nM). FhSrp1 and FhSrp2 are both expressed on the NEJ surface, predominantly around the oral and ventral suckers, suggesting that these inhibitors protect the parasites from the harmful proteolytic effects of host proteases, such as chymotrypsin, during invasion. Furthermore, the unusual inhibition of kallikrein suggests that rFhSrp1 modulates host responses such as inflammation and vascular permeability by interfering with the kallikrein-kinin system. A vaccine combination of rFhSrp1 and rFhSrp2 formulated in the adjuvant Montanide ISA 206VG elicited modest but non-significant protection against a challenge infection in a rat model, but did induce some protection against liver pathogenesis when compared to a control group and a group vaccinated with two well-studied vaccine candidates, F. hepatica cathepsin L2 and L3. This work highlights the importance of F. hepatica serpins to regulate host responses that enables parasite survival during infection and, coupled with the vaccine data, encourages future vaccine trials in ruminants.

Dataset Information

Pigment epithelium-derived factor: neurotrophic activity and identification as a member of the serine protease inhibitor gene family.

Publications

Pigment epithelium-derived factor: neurotrophic activity and identification as a member of the serine protease inhibitor gene family.

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