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.

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.
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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 Biodiversity, One Health & 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: https://theses.gla.ac.uk/id/eprint/209