University of Technology, Sydney. Faculty of Science.Thioredoxins are a family of small proteins conserved through evolution, essential for
cellular homeostasis. The 'classic' thioredoxin, identified in most species, is a 12 kDa
protein with a Cys-Gly-Pro-Cys (CGPC) active site. The thioredoxin system, composed
of thioredoxin, thioredoxin reductase and peroxiredoxin, is essential to protect cells
from metabolically produced reactive oxygen. This and the diversification of this
system through evolution identified it as a target for the control of many diseases,
including parasitic infections. This work characterises the thioredoxins of Haemonchus
contortus, a parasite with increasing economic impact on sheep and wool production in
Australia. Five thioredoxin proteins were identified, expressed and characterised
(HcTrx1-5).
H. contortus contained the classic thioredoxin (HcTrx1), but the major thioredoxin was
a 16 kDa protein (HcTrx3) with a Cys-Pro-Pro-Cys (CPPC) active site, which is related
to tryparedoxin, a unique protein in Trypanosomes. Both proteins were expressed
through the lifecycle and both had a similar ability to reduce the disulphide bonds of
insulin compared to the classic thioredoxins in Escherichia coli and sheep. Both
proteins were regenerated by thioredoxin reductase, but unlike the ovine thioredoxin,
both were also able to reduce oxidised glutathione, directly reduce hydrogen peroxide
and indirectly reduce hydrogen peroxide coupled with H.contortus peroxiredoxin.
Two thioredoxin-like proteins were identified with homology to thioredoxins reported
in human cells, a 31 kDa protein with a CGPC classic active site (HcTrx2) and a 28 kDa
protein with a Cys-Pro-Ala-Cys (CPAC) active site, a transmembrane domain and an
endoplasmic reticulum localisation signal (HcTFx4). These had different activities to the
classic HcTrx1 in that HcTrx2 could not directly reduce insulin, but could when
coupled to thioredoxin reductase. In contrast, HcTrx4 could directly reduce insulin, but
could not react with thioredoxin reductase. The results suggest that HcTrx4 is not a
thioredoxin, but acts as a protein-disulphide isomerase (PDI). Other characterised PDis,
contain at least two active sites, in contrast to the one active site in the H.contortus
protein.
HcTrx5 is a 20 kDa protein with a unique active site Cys-Arg-Ser-Cys (CRSC).
Although this active site has a charge change, HcTrx5 was able to reduce insulin, be
regenerated by thioredoxin reductase and react with H.contortus peroxiredoxin.
However, HcTrx5 was also regenerated by glutathione reductase coupled with
glutathione, showing it had the activity of a glutaredoxin as well as a thioredoxin, an
activity not reported for any thioredoxin.
This study characterised the thioredoxins of a parasitic nematode. The differences
identified may provide new drug targets for the control of many tropical diseases for
which drug resistance is emerging as a major problem. Preliminary investigations
showed increased H.contortus thioredoxin expression in a drug resistant strain. HcTrxl
was highly increased in ivermectin resistant parasites and may provide a marker of drug
resistance
