Identification and characterisation of the excreted/secreted serine proteases of larvae of the old world screwworm fly, Chrysomya bezziana.
Serine proteases are the major proteolytic activity excreted or secreted from Chrysomya bezziana larvae as demonstrated by gelatin gel analyses and the use of specific substrates, benzoyl-Arg-p-nitroanilide and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. Serine proteases were identified through their inhibition by 4-(2-aminoethyl)-benzene sulphonyl fluoride and classified as trypsin- and chymotrypsin-like on the basis of inhibition by tosyl-L-lysine chloromethyl ketone and tosyl-L-phenylalanine chloromethyl ketone, respectively. Like most insect serine proteases, the C. bezziana enzymes were active over broad pH range from mildly acidic to alkaline. The excreted or secreted serine proteases were purified by affinity chromatography using soybean trypsin inhibitor. A different subset of the serine proteases was isolated by salt elution from washed larval peritrophic matrices. Amino-terminal sequencing identified both trypsin and chymotrypsin-like sequences in the excreted or secreted pool with the latter being the dominant protease, whereas trypsin was the dominant species in the peritrophic matrix eluant. These results suggest that trypsin was possibly preferably adsorbed by the peritrophic matrix and may act as a final proteolytic processing stage as partially digested and ingested polypeptides pass through the peritrophic matrix. Immunoblot analysis on dissected gut tissues indicated that the anterior and posterior midguts were the main source of the serine proteases, although a novel species of 32 kDa was predominantly associated with the peritrophic matrix. Proteases are a target for a partially protective immune response and understanding the complexity of the secreted and digestive proteases is a necessary part of understanding the mechanism of the host's immunological defence against the parasite.
Muharsini
S
S
CSIRO Tropical Agriculture, Molecular Animal Genetics Centre, Level 3, Gehrmann Laboratories, University of Queensland, 4072, Queensland, Australia. sri.santoso@tag.csiro.au
Sukarsih
Riding
G
G
Partoutomo
S
S
Hamilton
S
S
Willadsen
P
P
Wijffels
G
G
D000595:Amino Acid Sequence; D000818:Animals; D004175:Diptera / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D007814:Larva / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012697:Serine Endopeptidases / Q000737:chemistry*
D012697:Serine Endopeptidases