Serine proteases from nematode and protozoan parasites: isolation of sequence homologs using generic molecular probes.

Abstract

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.Serine proteases are one of the most important families of enzymes found in nature. Members ofthis ubiquitous class of proteases hydrolyze peptide bonds and are involved in a broad range of biological processes including intra-and extracellular protein metabolism, digestion, blood coagulation, clot dissolution, immunological response, developmental regulation, and fertilization (1-4). The three-dimensional structure of serine proteases, in addition to biophysical, molecular biological, and enzymological studies, provides very useful models to understand the mechanism of enzyme action, the basis of substrate specificity, and the molecular evolution of the enzymes themselves (5-15).Aside from their role in the physiology and metabolism of organisms, serine proteases have also been implicated in the pathogenesis of a number of infectious diseases. Among the most prevalent of these are parasitic diseases, such as schistosomiasis and onchocerciasis (African river blindness), which

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“…A degenerate primer set (Sakanari et al 1989) was modified to amplify protease gene fragments from gDNA of the triactinomyxon stage of Myxobolus cerebralis by PCR. The sequence of the degenerate primers are: serine protease forward primer: 5'-TTG GTI GTI ACI GCI GCI CA(C/T) TG-3', serine protease reverse primer 5'-A(A/G)I GGI CCI CCI (A/T)(C/G)(A/G) CT(T/G) CC-3'.…”

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“…Serine proteases are members of one of the most biologically important and widely distributed families of enzymes found throughout nature. Members of this ubiquitous class of proteases are involved in a broad range of biological processes and have also been implicated in pathogenesis of a number of infectious diseases; parasite serine proteases may facilitate invasion of host tissue, metabolism of host proteins and evasion of the host immune response (46). In the case of Leishmania parasites, a serine oligopeptidase from L. amazonensis promastigotes has been described (10).…”

Kallikrein 7 enhances pancreatic cancer cell invasion by shedding E‐cadherin

“…10 A pool of 4 duct RNA samples and 4 tumor RNA samples were prepared and reverse-transcribed using MMLV-RT and oligo (dT) and random primers. The cDNAs prepared from pools of duct or tumor RNA were amplified using the degenerate oligonucleotide primers, subcloned into pGEM-T Easy (Promega, Madison, Wis), and the plasmid DNA was sequenced.…”