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Expression of thioredoxin in Fasciola hepatica
Fasciola hepatica, known as a common liver fluke is a digenetic trematode which infects a wide variety of mammals including humans and commercially important livestock. The parasite possesses a complex tegument which is composed of proteins produced by developmentally regulated cytons lying beneath a superficial muscle layer. Three types of cytons, designated T0, T1, and T2 are sequentially activated to manufacture proteins throughout the fluke's migration through the mammalian host. These proteins are "shuttled" through cytoplasmic extensions for ultimate incorporation into the tegument and outer glycocalyx. A series of tegument-specific clones designated FH2020.A, FH2020.SL, and FH2020.C were isolated from a $ lambda$gt11 adult F. hepatica cDNA library. Immunohistochemical staining with anti-FH2020 serum confirmed localization of the FH2020 protein to the surface of the tegument covering the spines. Sequence analysis revealed that each of these clones contained a $ sim$500bp region with extensive homology to thioredoxin. Thioredoxin is a ubiquitous 12kD re-dox protein which is highly conserved from prokaryotes to mammals. Western blot data shows the FH2020 clones to encode a 12 kD protein, while northern analysis of total F. hepatica RNA revealed two bands at 530 bp and 1.52 kb which correspond to processed and unprocessed transcripts, respectively. A genomic southern blot indicates that the FH2020 sequence is a single copy gene. Although each of the clones contains coding sequence for thioredoxin, the initial 30bp of the 5$ sp prime$ end of the sequence shows considerable divergence. Ribonuclease protection revealed the presence of a fully protected FH2020.C thioredoxin message and a 1.52 kb transcript distinguished FH2020.C as a pre-processed thioredoxin message. This conclusion is supported by sequence data which indicates a lack of an initiator methionine in frame with the thioredoxin coding sequence as well as the lack of a poly A tail. Genomic PCR construct sequence determined that the FH2020.A clone represents a thioredoxin message with a 28 bp cis-spliced 5$ sp prime$ sequence while RT PCR with a primer coding for a unique F. hepatica trans-spliced leader sequence indicates FH2020.SL contains a thioredoxin transcript with a trans-spliced 5$ sp prime$ leader. Thus, we have isolated a F. hepatica thioredoxin localized at least to the tegument for which unprocessed as well as alternatively 5$ sp prime$ spliced transcripts can be detected in steady state RNA.