Biochemical and molecular characterization of serine proteases from larvae of Chrysomya bezziana, the Old World Screwworm fly.
The diversity of serine proteases secreted from Chrysomya bezziana larvae was investigated biochemically and by PCR and sequence analysis. Cation-exchange chromatography of purified larval serine proteases resolved four trypsin-like activities and three chymotrypsin-like activities as discerned by kinetic studies with benzoyl-Arg-p-nitroanilide and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. Amino-terminal sequencing of the three most abundant fractions gave two sequences, which were homologous to other Dipteran trypsins and chymotrypsins. Analysis of products generated by PCR of cDNA from whole larvae using specific primers based on the amino-terminal sequences and generic serine protease primers identified 22 different sequences, while phylogenetic analysis of the deduced amino acid sequences differentiated two trypsin-like and four chymotrypsin-like families. Phylogenetic comparisons with Dipteran and mammalian serine protease sequences showed that all the Chrysomya bezziana sequences clustered with Dipteran sequences. The Chrysomya bezziana chymotrypsin-like sequences segregated within a Dipteran cluster of chymotrypsin sequences, but were well dispersed amongst these sequences. The largest Chrysomya bezziana serine protease family, the trypB family, clustered tightly as a group, and was closely related to a Lucilia cuprina trypsin but distinct from Drosophila melanogaster alpha and beta trypsins. The trypB family contains ten highly homologous sequences and probably represents an example of concerted evolution of a trypsin gene in Chrysomya bezziana.
Muharsini
S
S
CSIRO Livestock Industries, Molecular Animal Genetics Centre, Level 3 Gehrmann Laboratories, The University of Queensland, Qld. 4072, Australia.
Dalrymple
B
B
Vuocolo
T
T
Hamilton
S
S
Willadsen
P
P
Wijffels
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002918:Chymotrypsin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018076:DNA, Complementary; D004175:Diptera / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D007700:Kinetics; D007814:Larva; D008322:Mammals; D008969:Molecular Sequence Data; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D014357:Trypsin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D018076:DNA, Complementary; D012697:Serine Endopeptidases; D002918:Chymotrypsin; D014357:Trypsin