Biochemical and molecular characterization of serine proteases from larvaeof Chrysomya bezziana, the Old World Screwworm fly

The diversity of serine proteases secreted from Chrysomya bezziana larvae w as investigated biochemically and by PCR and sequence analysis. Cation-exch ange chromatography of purified larval serine proteases resolved four tryps in-like activities and three chymotrypsin-like activities as discerned by k inetic studies with benzoyl-Arg-p-nitroanilide and succinyl-Ala-Ala-Pro-Phe -p-nitroanilide. Amino-terminal sequencing of the three most abundant fract ions gave two sequences, which were homologous to other Dipteran trypsins a nd chymotrypsins. Analysis of products generated by PCR of cDNA from whole larvae using specific primers based on the amino-terminal sequences and gen eric serine protease primers identified 22 different sequences, while phylo genetic analysis of the deduced amino acid sequences differentiated two try psin-like and four chymotrypsin-like families. Phylogenetic comparisons wit h Dipteran and mammalian serine protease sequences showed that all the Chry somya bezziana sequences clustered with Dipteran sequences. The Chrysomya b ezziana chymotrypsin-like sequences segregated within a Dipteran cluster of chymotrypsin sequences, but were well dispersed amongst these sequences. T he largest Chrysomya bezziana serine protease family, the trypB family, clu stered tightly as a group, and was closely related to a Lucilia cuprina try psin but distinct from Drosophila melanogaster alpha and beta trypsins. The trypB family contains ten highly homologous sequences and probably represe nts an example of concerted evolution of a trypsin gene in Chrysomya bezzia na. Crown Copyright (C) 2001 Published by Elsevier Science Ltd. All rights reserved.