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Dr Peter M Smooker
Department of Biotechnology and Environmental Biology
RMIT University
Building 223, Level 1, Room 35, Plenty Road, Bundoora 3083
Email: peter.smooker@rmit.edu.au
Tel: +61-3-9925 7129
Fax: +61-3-9925 7110
Homepage: http://www.rmit.edu.au/biotechnology/
| Research |
Current projects include the structure/function/evolutionary relationships between secreted liver fluke proteases, and an evaluation of the secreted cathepsin B- and L-like proteases as potential vaccination/drug targets to protect against Fasciolosis. Liver fluke secrete multiple cathepsin L proteases, both from newly-excysted juvenile and adult parasites. There is temporal expression however- cathepsin B is secreted from the newly-excysted stage until approximately 5 weeks, while cathepsin L is secreted throughout the mammalian infection stage. Over thirty sequences of cathepsin L proteases have been reported. This relatively large family of variant proteases gives an opportunity for evolutionary studies, and the correlation between primary sequence and enzymatic activity. The liver fluke proteases form a monophyletic clade within the papain-like cysteine protease family. By molecular modelling and recombinant protein expression we have identified an amino acid lining the S2 subsite that has a large influence of the substrate specificity of the enzymes. Cathepsin L variants with the same amino acid at the crucial position lie in the same clade of a neighbour-joining tree.
Both cathepsin B and L have been expressed as recombinant proteins in yeast and we have also delivered these to rodents as experimental DNA vaccines. Both proteases classes are immunogenic in sheep and mice after either experimental infection or vaccination, and show promise as vaccines.
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| Collaborations |
Dr Robert Pike, Monash University
Professor Terry Spithill, McGill University
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| Publications |
Smooker PM, Whisstock JC, Siyaguna S, Irving JA, Spithill TW & Pike RN (2000). A single point mutation in a cysteine protease from Fasciola hepatica confers an enhanced ability to accommodate proline residues in substrates. Protein Science 9, 2567-2572
Smooker PM, Kennedy NJ, Steeper KR, Christoupoulos H & Spithill TW (2001). Vaccination of mice with Fasciola antigens: humoral responses generated to cathepsin L and fatty-acid binding protein delivered as DNA vaccines. Experimental Parasitology 97, 154-160
Irving JA, Spithill TW, Pike RN, Whisstock JC, Smooker PM (2003). The evolution of enzyme specificity in Fasciola spp. Journal of Molecular Evolution 57, 1-15
Law RH*, Smooker PM*, Irving JA, Piedrafita D, Ponting R, Kennedy NJ, Whisstock JC, Pike RN & Spithill TW (2003). Cloning and expression of the major secreted cathepsin B-like protein from juvenile Fasciola hepatica and analysis of immunogenicity following liver fluke infection. Infection and Immunity 71, 6921-6932. *Joint first authors
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