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Characterisation of quiescin-sulfhydryl oxidase and nematode astacin mutants using functional studies in caenorhabditis elegans.

Birnie, Andrew J. (2008) Characterisation of quiescin-sulfhydryl oxidase and nematode astacin mutants using functional studies in caenorhabditis elegans. PhD thesis, University of Glasgow.

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Abstract

Nematodes, both free-living and parasitic, are dependant upon their Extra Cellular Matrix (ECM) for multiple aspects of functionality. Two distinct ECMs are present in Caenorhabditis elegans, the basement membrane and the cuticle. The cuticle of C. elegans, like other nematodes is composed largely of collagen-like proteins, with the trimeric collagenous proteins forming approximately 80% of the cuticle. Cuticle collagens are believed to be highly processed in a manner similar to vertebrate collagen maturation, with collagens being; co-translationanly modified, folded into triple helices and proteolytically cleaved at the C- and N- termini. Cross-linking of mature triple helical collagens into higher order structures leads to the generation of a flexible yet robust cuticle. Disulphide bonding is crucial in the formation of the cuticle, with cysteine cross-linking mutants having been shown to produce severely disrupted cuticles and associated lethal phenotypes. During the life cycle, C. elegans progresses through four moults during which a new cuticle is synthesised and the old cuticle is shed. Moulting occurs by proteolytic digestion and shedding of an anterior cuticular cap which provides an opening for the nematode to escape the previous stage cuticle. Both free-living and parasitic nematodes shed and exsheath their cuticles in this manner. Two distinct phases of cuticle processing become apparent: cuticle synthesis and cuticle degradation. Of the enzymes involved with processing of cuticular collagens, the quiescin sulfhydryl oxidases (QSOX), and the nematode astacins (NAS) are of particular interest with regard to cuticle synthesis and proteolytic cleavage of cuticular collagens respectively. QSOX have been shown to be linked directly to the generation of disulphide bonds, and have also been shown to associate with other essential proteins of cuticle formation, namely the protein disulphide isomerases. There are three distinct QSOX family members found within the C. elegans genome, which have been shown to temporally coincide with lethargus (cuticle synthesis) and have been proven to spatially localise to the C. elegans hypodermis, the tissue responsible for cuticle secretion. Characterisation of qsox mutants reveals weak cuticular phenotypes when disrupted singly; but, in combination, silencing of qsox-1 and qsox-2 resulted in blistered cuticles and lethality, by RNA mediated interference and double knockouts respectively. This demonstrates the essential nature of the cuticle associated QSOX enzymes, and to my knowledge represents the first loss-of-function mutant in a QSOX enzyme. xv Investigation of the NAS enzymes focused on the group V astacins, members of which exhibit the only notable defects associated with disruption of C. elegans nas genes, namely: dumpy body shape, nas-35/dpy-31; hatching, nas-34/hch-1; and moult defects, nas-36 and 37. With regard to proteolytic degradation of cuticular components, NAS-36 and NAS-37 were of specific interest as mutants resulted in moult defective nematodes unable to digest and fully escape their previous stage cuticles; in addition, spatial expression illustrated an association of these gene products with regions of cuticle attachment and degradation. C. elegans NAS-36 and NAS-37 were also shown to digest isolated L3(2M) trichostrongylid cuticles of parasites of veterinary importance, suggesting that the metalloprotease and cuticle substrates involved in exsheathment is conserved between trichostrongylid and free-living nematodes. Conservation is poor between ecdysozoan and non-moulting organisms, meaning that proteins such as NAS-36 and 37 could become specific novel targets for anti-nematode drug development.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: quiescin sulfhydryl oxidases, neamatode astacins, C. elegans, moulting, disulphide bond formation, cuticle collagens
Subjects: Q Science > Q Science (General)
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Veterinary Medicine
Supervisor's Name: Page, Prof. Antony P.
Date of Award: 2008
Depositing User: Mr Andrew J Birnie
Unique ID: glathesis:2008-209
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 25 Jun 2008
Last Modified: 10 Dec 2012 13:16
URI: http://theses.gla.ac.uk/id/eprint/209

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