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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010The Characterization of Staphopains Enzyme Family fromStaphylococcus aureus Using Combinatorial Chemistry MethodsAdam Lesner 1 , Magdalena Wysocka 1 , Marcelina Jaros 1 ,Anna Łęgowska 1 , Katarzyna Guzow 1 , Grzegorz Dubin 2 ,Benedykt Władyka 2 , Wiesław Wiczk 1 , and Krzysztof Rolka 11 Gdansk University, Faculty of Chemistry, Gdansk, 80-952, Poland; 2 JagiellonianUniversity, Faculty of Biochemistry, Biophysics and Biotechnology,Cracow, 30-357, PolandIntroductionStaphylococcus aureus is a main cause of nosocomial infections of all kinds. It colonizesand infects virtually every tissue of the body. To encounter the environmental diversityfaced, the bacteria is well equipped with a broad spectrum of secreted proteins. Threedifferent catalytic classes, including metallo-, serine- and cysteine proteases are foundamong the secreted staphylococcal proteins. A variety of different functions including, butnot limited to, tissue degradation [1], defense against host immune response [2],interception of host enzymes and bacterial adhesion regulation [3] have been attributed tothese proteins. Staphopains A, B and C are the major secreted cysteine proteases ofS. aureus. The literature contains evidence both for and against their role as virulencefactors. All three proteases are members of the papain superfamily of enzymes and areencoded on the genome as preproenzymes. Staphopain A and B shared 47% of amino acidhomology. The structure of mature staphopain A is known. It shows that staphopains areremote members of the papain superfamily of enzymes with cathepsin B as their closeststructural neighbor among the eukaryotic enzymes. Proteolytical activity of those enzymesare strictly controlled by the endogenous inhibitors called staphostatines [4].The main goal of this research was to obtain detailed information about specificity ofthose three enzymes in substrate segment comprised positions P 4 – P 1 . To do so, we appliedcombinatorial chemistry methods to synthesize the library of peptides with the generalformula:ABZ-X 4 -X 3 -X 2 -X 1 -ANB-NH 2where:ABZ – 2-aminobenzoic acid (donor of fluorescence),ANB-NH 2 – amide of 5-amino-2-nitrobenzoic acid (acceptor of fluorescence),X 4 , X 3 , X 2 and X 1 = all proteinogenic amino acid residues were present.The presence of ABZ and ANB-NH 2 in peptides synthesized indicates that the library wasdesigned to contain fluorogenic substrates of the investigated proteases.Results and DiscussionThe library synthesized was screened in solution against three members of cystein proteasefamily (staphopain A (StpA), staphopain B (StpB) and staphopain C (StpC)) using iterativemethod of deconvolution. All enzymes displayed the highest activity measured as increaseof the fluorescence in time when in position X 4 (equivalent to the substrate P 4 position) ofthe library the hydrophobic residues were present. StpA prefers the aromatic Phe followedby Ile and Leu since for StpB and StpC this trend was reversed; Ile predominates over Pheand Tyr. The charged residues present in discussed position significantly reduced thehydrolysis rate of the library for all three proteinases. In position P 3 of the library, allenzymes reveal broad specificity. StpA prefers library with Gly followed by Ala and Met.Asp and Pro in this position reduced the rate of proteolysis. StpB rapidly hydrolyzedpeptide library when Ala or Gly were present in discussed position. This was not true forTrp and low fluorescence readout was observed. A quite similar picture was observed in thecase of StpC. Ala followed by Ile, Gly and Arg(!) present in the position P 3 reveal thehighest hydrolysis rate. Again Trp and Pro introduced in the discussed position yieldedinactive peptides.In position P 2 , for two enzymes (StpA and StpB) the most intense fluorescence wasdetected for Ala. All other amino acids displayed at least twice lower hydrolysis rate. In thecase of StpC, sub-libraries with Lys in position P 2 followed by Gly, Arg and Glu were the202