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      Prolyl 4-Hydroxlase Activity Is Essential for Development and Cuticle Formation in the Human Infective Parasitic Nematode Brugia malayi*

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          Abstract

          Background: Collagen prolyl 4-hydroxylases (C-P4H) are involved in the formation of extracellular matrices.

          Results: The full complement of C-P4H enzymes from the human infective parasite Brugia malayi have been bioinformatically, biochemically, and functionally characterized.

          Conclusion: C-P4H enzymes are essential for development in B. malayi.

          Significance: Unique features of these essential enzymes may be exploited in future control mechanisms.

          Abstract

          Collagen prolyl 4-hydroxylases (C-P4H) are required for formation of extracellular matrices in higher eukaryotes. These enzymes convert proline residues within the repeat regions of collagen polypeptides to 4-hydroxyproline, a modification essential for the stability of the final triple helix. C-P4H are most often oligomeric complexes, with enzymatic activity contributed by the α subunits, and the β subunits formed by protein disulfide isomerase (PDI). Here, we characterize this enzyme class in the important human parasitic nematode Brugia malayi. All potential C-P4H subunits were identified by detailed bioinformatic analysis of sequence databases, function was investigated both by RNAi in the parasite and heterologous expression in Caenorhabditis elegans, whereas biochemical activity and complex formation were examined via co-expression in insect cells. Simultaneous RNAi of two B. malayi C-P4H α subunit-like genes resulted in a striking, highly penetrant body morphology phenotype in parasite larvae. This was replicated by single RNAi of a B. malayi C-P4H β subunit-like PDI. Surprisingly, however, the B. malayi proteins were not capable of rescuing a C. elegans α subunit mutant, whereas the human enzymes could. In contrast, the B. malayi PDI did functionally complement the lethal phenotype of a C. elegans β subunit mutant. Comparison of recombinant and parasite derived material indicates that enzymatic activity may be dependent on a non-reducible covalent link, present only in the parasite. We therefore demonstrate that C-P4H activity is essential for development of B. malayi and uncover a novel parasite-specific feature of these collagen biosynthetic enzymes that may be exploited in future parasite control.

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          Most cited references31

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          Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans.

          Genetic interference mediated by double-stranded RNA (RNAi) has been a valuable tool in the analysis of gene function in Caenorhabditis elegans. Here we report an efficient induction of RNAi using bacteria to deliver double-stranded RNA. This method makes use of bacteria that are deficient in RNaseIII, an enzyme that normally degrades a majority of dsRNAs in the bacterial cell. Bacteria deficient for RNaseIII were engineered to produce high quantities of specific dsRNA segments. When fed to C. elegans, such engineered bacteria were found to produce populations of RNAi-affected animals with phenotypes that were comparable in expressivity to the corresponding loss-of-function mutants. We found the method to be most effective in inducing RNAi for non-neuronal tissue of late larval and adult hermaphrodites, with decreased effectiveness in the nervous system, in early larval stages, and in males. Bacteria-induced RNAi phenotypes could be maintained over the course of several generations with continuous feeding, allowing for convenient assessments of the biological consequences of specific genetic interference and of continuous exposure to dsRNAs.
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            Draft genome of the filarial nematode parasite Brugia malayi.

            Parasitic nematodes that cause elephantiasis and river blindness threaten hundreds of millions of people in the developing world. We have sequenced the approximately 90 megabase (Mb) genome of the human filarial parasite Brugia malayi and predict approximately 11,500 protein coding genes in 71 Mb of robustly assembled sequence. Comparative analysis with the free-living, model nematode Caenorhabditis elegans revealed that, despite these genes having maintained little conservation of local synteny during approximately 350 million years of evolution, they largely remain in linkage on chromosomal units. More than 100 conserved operons were identified. Analysis of the predicted proteome provides evidence for adaptations of B. malayi to niches in its human and vector hosts and insights into the molecular basis of a mutualistic relationship with its Wolbachia endosymbiont. These findings offer a foundation for rational drug design.
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              The draft genome of the parasitic nematode Trichinella spiralis

              Genome-based studies of metazoan evolution are most informative when phylogenetically diverse species are incorporated in the analysis. As such, evolutionary trends within and outside the phylum Nematoda have been less revealing by focusing only on comparisons involving Caenorhabditis elegans. Herein, we present a draft of the 64 megabase nuclear genome of Trichinella spiralis, containing 15,808 protein coding genes. This parasitic nematode is an extant member of a clade that diverged early in the evolution of the phylum enabling identification of archetypical genes and molecular signatures exclusive to nematodes. Comparative analyses support intrachromosomal rearrangements across the phylum, disproportionate numbers of protein family deaths over births in parasitic vs. a non-parasitic nematode, and a preponderance of gene loss and gain events in nematodes relative to Drosophila melanogaster. This sequence and the panphylum characteristics identified herein will advance evolutionary studies and strategies to combat global parasites of humans, food animals and crops.
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                Author and article information

                Journal
                J Biol Chem
                J. Biol. Chem
                jbc
                jbc
                JBC
                The Journal of Biological Chemistry
                American Society for Biochemistry and Molecular Biology (9650 Rockville Pike, Bethesda, MD 20814, U.S.A. )
                0021-9258
                1083-351X
                18 January 2013
                7 December 2012
                7 December 2012
                : 288
                : 3
                : 1750-1761
                Affiliations
                From the []Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Garscube Estate, Bearsden Road, Glasgow G61 1QH, Scotland, United Kingdom and
                the [§ ]Biocenter Oulu and Department of Medical Biochemistry and Molecular Biology, Oulu Center for Cell Matrix Research, University of Oulu, FIN-90014 Oulu, Finland
                Author notes
                [1 ] To whom correspondence may be addressed: Institute of Infection, Immunity, and Inflammation, College of Medical, Veterinary, and Life Sciences, University of Glasgow, Garscube Estate, Bearsden Rd., Glasgow G61 1QH, UK. Tel,: 44-141-330-5759; Fax: 44-141-330-2271; E-mail: Alan.Winter@ 123456glasgow.ac.uk .
                [2 ] To whom correspondence may be addressed: Institute of Infection, Immunity, and Inflammation, College of Medical, Veterinary, and Life Sciences, University of Glasgow, Garscube Estate, Bearsden Rd., Glasgow G61 1QH, UK. Tel.: 44-141-330-1997; Fax: 44-141-330-2271; E-mail: Tony.Page@ 123456glasgow.ac.uk .
                Article
                M112.397604
                10.1074/jbc.M112.397604
                3548485
                23223450
                ad6ae994-6386-4192-a3e8-ee4eeb6e3db9
                © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

                Author's Choice—Final version full access.

                Creative Commons Attribution Non-Commercial License applies to Author Choice Articles

                History
                : 3 July 2012
                : 16 November 2012
                Categories
                Glycobiology and Extracellular Matrices

                Biochemistry
                c. elegans,collagen,extracellular matrix,extracellular matrix proteins,hydroxylase,hydroxyproline,parasitology,post translational modification,protein synthesis

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