Serine proteases from nematode and protozoan parasites: isolation of sequence homologs using generic molecular probes.
Serine proteases are one of the biologically most important and widely distributed families of enzymes. Isolation of serine protease genes from organisms of widely diverged phylogenetic groups would provide a basis for studying their biological function, the relationship between structure and function, and the molecular evolution of these enzymes. Serine proteases for which little structural information is known are those that are important in the pathogenesis of parasitic nematode and protozoan diseases. Identification and isolation of protease genes from these organisms is a critical first step in understanding their function for the parasite and possibly suggesting innovative approaches to arresting parasitic diseases. Serine protease gene fragments were isolated from genomic DNA of the parasitic nematode Anisakis simplex by using degenerate oligonucleotide primers and the polymerase chain reaction. Primers were designed based upon the consensus sequence of amino acids flanking the active site serine and histidine residues of eukaryotic serine proteases. Four serine protease gene fragments from this parasite were sequenced and one is 67% identical to the rat trypsin II gene. Alignment of these two genes revealed that the intron-exon junctions are conserved between nematode and rat suggesting that this Anisakis serine protease is structurally and functionally similar to rat trypsin. The generality of this approach to identify serine protease genes from genomic DNA of two very divergent species, a parasitic protozoan and a mammal, was also confirmed. Genes for other enzymes or any protein with conserved structural motifs can be identified and isolated using this technology. Using a similar strategy, a cathepsin B-like cysteine (thiol) protease gene fragment was isolated from Caenorhabditis elegans DNA.
Sakanari
J A
JA
Department of Pathology, University of California, San Francisco 94143.
Staunton
C E
CE
Eakin
A E
AE
Craik
C S
CS
McKerrow
J H
JH
D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D008099:Liver / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D009348:Nematoda / Q000201:enzymology* / Q000235:genetics; D051381:Rats; D012550:Schistosoma mansoni / Q000201:enzymology* / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D012697:Serine Endopeptidases / Q000235:genetics*; D013045:Species Specificity; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000235:genetics
D012697:Serine Endopeptidases