Evolutionary analysis of the cystatin family in three Schistosoma species

Abstract

The cystatin family comprises cysteine protease inhibitors distributed in 3 subfamilies (I25A-C). Family members lacking cystatin activity are currently unclassified. Little is known about the evolution of Schistosoma cystatins, their physiological roles, and expression patterns in the parasite life cycle. The present study aimed to identify cystatin homologs in the predicted proteome of three Schistosoma species and other Platyhelminthes. We analyzed the amino acid sequence diversity focused in the identification of protein signatures and to establish evolutionary relationships among Schistosoma and experimentally validated human cystatins. Gene expression patterns were obtained from different developmental stages in Schistosoma mansoni using microarray data. In Schistosoma, only I25A and I25B proteins were identified, reflecting little functional diversification. I25C and unclassified subfamily members were not identified in platyhelminth species here analyzed. The resulting phylogeny placed cystatins in different clades, reflecting their molecular diversity. Our findings suggest that Schistosoma cystatins are very divergent from their human homologs, especially regarding the I25B subfamily. Schistosoma cystatins also differ significantly from other platyhelminth homologs. Finally, transcriptome data publicly available indicated that I25A and I25B genes are constitutively expressed thus could be essential for schistosome life cycle progression. In summary, this study provides insights into the evolution, classification, and functional diversification of cystatins in Schistosoma and other Platyhelminthes, improving our understanding of parasite biology and opening new frontiers in the identification of novel therapeutic targets against helminthiases.

Keywords: bayesian inference; function prediction; phylogenomics; proteinase inhibitor; schistosomiasis.

Figure 1

Cystatin classification. The I25 family is classified in three subfamilies (I25A–C) according to MEROPS. Several family members remain unclassified. UniProt accession numbers of human homologs are listed.

Figure 2

Cystatin architecture in three Platyhelminthes. Accession numbers correspond to those assigned in each genome project. Domain limits (above) and sequence length (below) are provided for each protein. A signal peptide (SP) and the conserved domain (PF00031) with significant (blue) or insignificant (?) matches are indicated.

Figure 3

Alignment of Schistosoma and human homologs. Multiple sequence alignment of the conserved domain (PF00031) of I25A and I25B proteins of S. haematobium (Sha), S. japonicum (Sjp), S. mansoni (Smp), and Homo sapiens (UniProt accession numbers). Amino acid sequences were aligned using MAFFT with iterative refinement by the G-INS-i strategy. Conserved motifs (Q-x-V-x-G and PW) and disulfide bridges (S-S) are indicated.

Figure 4

Evolutionary relationships of I25A and I25B cysteine protease inhibitors. A total of 22 amino acid sequences and 96 sites comprising the conserved domain (PF00031) of homologs encoded by S. haematobium (brown), S. japonicum (green), S. mansoni (blue), and Homo sapiens (black) were analyzed. The phylogeny was reconstructed by two methods using WAG was as the best fit model. In the Bayesian inference, support values for each node were estimated as posterior probability (above). In the maximum likelihood analysis, they were estimated using the Akaike Likelihood Ratio Test (aLRT) (below). Only support values higher than 70% are shown.

Figure 5

Cystatins mRNA expression patterns in the S. mansoni life cycle. Public microarray data available at ArrayExpress (E-MEXP-2094) was downloaded to a local server in order to identify cystatins expression patterns of the I25A subfamily member Smp_006390 (A) and the I25B subfamily member Smp_034420 (B) in S. mansoni. Bars correspond to the mean normalized values for each oligonucleotide probe named Smp_006390 and Smp_034420 in 13 different life stages.