Functional analysis and localisation of a delta-class glutathione S-transferase from Sarcoptes scabiei.
The mite Sarcoptes scabiei causes sarcoptic mange, or scabies, a disease that affects both animals and humans worldwide. Our interest in S. scabiei led us to further characterise a glutathione S-transferase. This multifunctional enzyme is a target for vaccine and drug development in several parasitic diseases. The S. scabiei glutathione S-transferase open reading frame reported here is 684 nucleotides long and yields a protein with a predicted molecular mass of 26 kDa. Through phylogenetic analysis the enzyme was classified as a delta-class glutathione S-transferase, and our paper is the first to report that delta-class glutathione S-transferases occur in organisms other than insects. The recombinant S. scabiei glutathione S-transferase was expressed in Escherichia coli via three different constructs and purified for biochemical analysis. The S. scabiei glutathione S-transferase was active towards the substrate 1-chloro-2,4-dinitrobenzene, though the positioning of fusion partners influenced the kinetic activity of the enzyme. Polyclonal antibodies raised against S. scabiei glutathione S-transferase specifically localised the enzyme to the integument of the epidermis and cavities surrounding internal organs in adult parasites. However, some minor staining of parasite intestines was observed. No staining was seen in host tissues, nor could we detect any antibody response against S. scabiei glutathione S-transferase in sera from naturally S. scabiei infected dogs or pigs. Additionally, the polyclonal sera raised against recombinant S. scabiei glutathione S-transferase readily detected a protein from mites, corresponding to the predicted size of native glutathione S-transferase.
Pettersson
Eva U
EU
Department of Parasitology (SWEPAR), National Veterinary Institute and Swedish University of Agricultural Sciences, SE-751 89 Uppsala, Sweden.
Ljunggren
Erland L
EL
Morrison
David A
DA
Mattsson
Jens G
JG
D000818:Animals; D000906:Antibodies / Q000097:blood; D015153:Blotting, Western; D004283:Dog Diseases / Q000201:enzymology / Q000276:immunology / Q000469:parasitology; D004285:Dogs; D004817:Epidermis / Q000201:enzymology; D005982:Glutathione Transferase / Q000032:analysis* / Q000235:genetics / Q000502:physiology; D007422:Intestines / Q000201:enzymology; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010802:Phylogeny; D011994:Recombinant Proteins / Q000378:metabolism; D012520:Sarcoptes scabiei / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D012532:Scabies / Q000201:enzymology / Q000276:immunology / Q000662:veterinary; D017422:Sequence Analysis, DNA / Q000379:methods; D013552:Swine; D013553:Swine Diseases / Q000201:enzymology / Q000276:immunology / Q000469:parasitology
D000906:Antibodies; D011994:Recombinant Proteins; D005982:Glutathione Transferase