Molecular and biochemical characterization of serine protease SmSP1 in \kur{Schistosoma mansoni}

SmSP1 is a chimerical serine protease consisted of three domains (cub, LDLa and trypsin-like) and found in Schistosoma mansoni. Its characterization was performed by molecular techniques such as PCR screen, qRT-PCR and RNA interference (RNAi) to gain information about expression profile, level expression and susceptibility to RNAi. Further, protein expression was carried out to gain an antigen for immunization and recombinant for biochemical studies. Results of PCR screen and qRT-PCR suggested possible function of SmSP1 in egg and adult stages but SmSP1 gene was not found susceptible to RNAi in NTS. Recombinant from E. coli was successfully used for immunization. Active recombinant was likely expressed in Pichia pastoris but expression conditions are unstable and expression optimization is necessary

The study of \kur{dco} gene in imaginal discs of \kur{Drosophila melanogaster}

The aim of this bachelor{\crq}s thesis was to make a Drosophila line with a gene set that induces a creation of mitotic clones in wing discs and eye discs. Seven different Drosophila stocks from various sources were used in genetic crossing to obtain such line. The mitotic-clone area was composed of either cells that were homozygous for GFP or cells that were homozygous for dco3 mutation. The dco3 is a recessive lethal mutation leading to death in pupae. By induction of a small number of cells homozygous for dco3 it was possible to observed the changes in adult tissues with mutant clones

Trypsin- and Chymotrypsin-Like Serine Proteases in <i>Schistosoma mansoni</i> – ‘The Undiscovered Country’

<div><p>Background</p><p>Blood flukes (<i>Schistosoma</i> spp.) are parasites that can survive for years or decades in the vasculature of permissive mammalian hosts, including humans. Proteolytic enzymes (proteases) are crucial for successful parasitism, including aspects of invasion, maturation and reproduction. Most attention has focused on the ‘cercarial elastase’ serine proteases that facilitate skin invasion by infective schistosome larvae, and the cysteine and aspartic proteases that worms use to digest the blood meal. Apart from the cercarial elastases, information regarding other <i>S. mansoni</i> serine proteases (SmSPs) is limited. To address this, we investigated SmSPs using genomic, transcriptomic, phylogenetic and functional proteomic approaches.</p><p>Methodology/Principal Findings</p><p>Genes encoding five distinct SmSPs, termed SmSP1 - SmSP5, some of which comprise disparate protein domains, were retrieved from the <i>S. mansoni</i> genome database and annotated. Reverse transcription quantitative PCR (RT- qPCR) in various schistosome developmental stages indicated complex expression patterns for SmSPs, including their constituent protein domains. SmSP2 stood apart as being massively expressed in schistosomula and adult stages. Phylogenetic analysis segregated SmSPs into diverse clusters of family S1 proteases. SmSP1 to SmSP4 are trypsin-like proteases, whereas SmSP5 is chymotrypsin-like. In agreement, trypsin-like activities were shown to predominate in eggs, schistosomula and adults using peptidyl fluorogenic substrates. SmSP5 is particularly novel in the phylogenetics of family S1 schistosome proteases, as it is part of a cluster of sequences that fill a gap between the highly divergent cercarial elastases and other family S1 proteases.</p><p>Conclusions/Significance</p><p>Our series of post-genomics analyses clarifies the complexity of schistosome family S1 serine proteases and highlights their interrelationships, including the cercarial elastases and, not least, the identification of a ‘missing-link’ protease cluster, represented by SmSP5. A framework is now in place to guide the characterization of individual proteases, their stage-specific expression and their contributions to parasitism, in particular, their possible modulation of host physiology.</p></div

Predicted domain organization and open reading frames of SmSP proteases.

<p>CUB domains are depicted in blue, an LDLa domain in yellow and protease domains from the S1 family in red. In SmSP2 and SmSP4, N-terminal signal peptides are separated by red bars from the rest of N-terminal extensions with putative pro-peptides (protease activation peptides). Numbering indicates amino acid positions. Exon structure of the genes encoding SmSPs are shown as numbered boxes below each SmSP protein.</p

Primary sequence alignment of SmSP1 through SmSP5 with <i>S. mansoni</i> cercarial elastase 2a (SmCE2.a), bovine trypsin and bovine chymotrypsin.

<p>For SmSP1 to SmSP4, only the protease domains are shown; the upstream sequences (except a short sequence stretch) forming N-terminal extensions and non-proteolytic domains are not included in the alignment. Also, a downstream C-terminal extension of SmSP5 is not included. The catalytic residues His, Asp and Ser are highlighted in bold and black-boxed; critical Asp residues in the S1 subsite that account for trypsin-like activity are in bold red; Cys residues that are predicted to form disulfide bonds are indicated by the same color; putative unpaired Cys residues are highlighted in olive, and predicted N-glycosylation signals are in bold and underlined. Glu residues binding a Ca²⁺ ion in the trypsin molecule are blue-boxed. The upper line numbering is according to SmSP1; the predicted mature protease domain starts with 1, the suffix p indicates pro-peptide/N-terminal extension numbering. GenBank accession numbers are as follows: SmSP1 (KF535923), SmSP2 (KF510120), SmSP3 (KF510121), SmSP4 (KF510122), SmSP5 (KF939306), SmCE2a (AAM43941), bovine trypsinogen (XP_871686) and bovine chymotrypsinogen A (XP_003583409).</p

RT-qPCR to evaluate the expression of SmSP genes among <i>S. mansoni</i> developmental stages.

<p>mRNA levels are displayed as the percentage of expression compared to the constitutively expressed <i>S. mansoni</i> cytochrome oxidase I (SmCOX I). The value 0.01% was used as a significance threshold. The gene expression analysis of the protease domains of SmSPs. Each unit represents the -fold change in the transcription level using the log₂ scale.</p

List of studied serine proteases and their accession numbers.

<p>List of studied serine proteases and their accession numbers.</p

Profiling SP activities in extracts of <i>S. mansoni</i> developmental stages.

<p>The kinetic assays, performed at pH µM fluorogenic substrates (P1 and P2 positions are highlighted) that are specific for trypsin- and chymotrypsin/elastase-type proteases. SP activities (sensitive to inhibition by PMSF and Pefabloc SC) are expressed as relative fluorescence units (RFU/s) and normalized to the protein content of extracts. Data are displayed in a heat map.</p

Maximum likelihood phylogenetic tree of 101 selected members of the S1 family of serine proteases with emphasis on trematode SPs.

<p>Numbers in the collapsed branches (triangles) indicate the number of taxa included in the branch. Maximum likelihood and maximum parsimony bootstrap supports are shown at nodes, bootstrap percentages with <50% support are not shown. Branches in the trematode clade 5 SPs are shortened to one third of their original length as indicated by the two diagonal lines. For clades 1 and 4, two <i>S. japonicum</i> orthologs are missing due to their absence in the GenBank nr database. However, both sequences can be retrieved from the SchistoDB database under the identifiers Sjp_0012180 (SjSP1) and Sjp_0047680 (Sj SP4).</p

Homology model of the SmSP1 protease domain in complex with leupeptin.

<p>The model was built using the template X-ray structure of bovine trypsin in complex with substrate-like inhibitor leupeptin (N-acetyl-L-leucyl-L-leucyl-L-argininal; PDB code 1jrt). (<b>A</b>) Surface representation of the SmSP1 active site colored by electrostatic potential (at a scale from −10 kT/e (red) to +10 kT/e (blue)). Carbon atoms of leupeptin are yellow; heteroatoms have a standard color coding (O, red; N, blue). (<b>B</b>) The same detail as (A) but viewed from above (the surface display was clipped for a better view). (<b>C</b>) Schematic view of the active site residues of SmSP1 (green) forming hydrogen bonds (dashed lines) with leupeptin (yellow). Note the interactions between Asp182 (in the S1 protease subsite) and the basic P1 residue of leupeptin that mimic the S1-P1 salt bridge that is critical for trypsin-like substrate specificity. Catalytic residues (cyan) are shown, including the covalent linkage of leupeptin with the catalytic Ser188.</p