Functional characterization of single-domain cystatin-like cysteine proteinase inhibitors expressed by the trematodeFasciola hepatica

Abstract

Cystatins are small, phylogenetically conserved proteins that are tight-binding inhibitors of cysteine proteinases. The liver fluke Fasciola hepatica uses a diverse set of cysteine proteinases of the papain superfamily for host invasion, immune evasion and nutrition, but little is known about the regulation of these enzymes. The aim of this work is to characterize the cystatin repertoire of F. hepatica. For this purpose, we first surveyed the available sequence databases, identifying three different F. hepatica single-domain cystatins. In agreement with the in silico predictions, at least three small proteins with cysteine proteinase binding activity were identified. Phylogenetic analyses showed that the three cystatins (named FhStf-1, -2 and -3) are members of the I25A subfamily (stefins). Whereas FhStf-1 grouped with classical stefins, FhStf-2 and 3 fell in a divergent stefin subgroup unusually featuring signal peptides. Recombinant rFhStf-1, -2 and -3 had potent inhibitory activity against F. hepatica cathepsin L cysteine proteinases but differed in their capacity to inhibit mammalian cathepsin B, L and C. FhStf-1 was localized in the F. hepatica reproductive organs (testes and ovary), and at the surface lamella of the adult gut, where it may regulate cysteine proteinases related with reproduction and digestion, respectively. FhStf-1 was also detected among F. hepatica excretion-secretion (E/S) products of adult flukes. This suggests that it is secreted by non-classical secretory pathway and that it may interact with host lysosomal cysteine proteinases.

Fasciola hepatica serine protease inhibitor family (serpins): Purposely crafted for regulating host proteases

“…These include several developmentally-regulated cathepsin L-and B-like cysteine proteases that are specially adapted to degrading host tissue proteins, such as collagen and fibronectin, and blood proteins such haemoglobin [10,11]. Additionally, several cysteine protease inhibitors, such as cystatins [12] and Kunitz-type inhibitors [13] have been shown to be co-secreted with the cysteine proteases and are proposed to be important in regulating the parasite protease/anti-protease balance.…”

Expression of cysteine proteinases and cystatins in parasites and use of cysteine proteinase inhibitors in parasitic diseases. Part I: Helminths

“…It regulated proteolytic activity of cathepsin B to protect the parasite from intra-cellular autoproteolysis, and the investigators recommended further studies to elucidate role of FgStefin-2 expressed in the reproductive organs [217] . Recently, FgStefin-3 (type II) was identified and its recombinant showed potent regulatory function of cathepsin L, similar to the other CYSs [218] . Similarly, stefin-1 was expressed from all C. sinensis developmental stages, and it was immunolocalized where CsCatF synthesis.…”

Characteristics, Structure, and Biological Role of Stefins (Type-1 Cystatins) of Human,

“…109 Three different stefins, designated rFhStf-1, rFhStf-2, and rFhStf-3, expressed by the trematode Fasciola hepatica exhibited differences in their inhibition profile against various tested enzymes. 110 Immunomodulatory properties of FhStefins could be used in order to evaluate their therapeutic potential against inflammatory diseases. The inhibitors FhStf-2 and FhStf-3 fall into an atypical subgroup of stefins due to the presence of a signal peptide, similar to the previously mentioned FgStefin-2.…”

“…105 Similarly, the oligomerization from monomers (10 kDa) to oligomers of various sizes (over 100 kDa) in F. hepatica stefin-type inhibitors (rFhStf-1, rFhStf-2, and rFhStf-3) was reported very recently. 110 An important step in elucidating the oligomerization of cystatins was determining the crystal structure of dimerized domain-swapped human cystatin C 141 and of chicken cystatin and human stefin A in solution. 142 Then, it was demonstrated that the domain-swapped dimer of chicken cystatin oligomerizes to a tetramer as a transient intermediate prior to oligomerization.…”