Background A salivary proteome-transcriptome project on the hard tick revealed that

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Background A salivary proteome-transcriptome project on the hard tick revealed that Kunitz peptides are the most abundant salivary proteins. of factor Xa but has no effect on factor VIIa kallikrein trypsin chymotrypsin thrombin urokinase plasmin tissue plasminogen activator and elastase [4]. Another example is the tick-derived protease inhibitor (TdPI) from the hard tick that is a potent β-tryptase inhibitor but not for urokinase thrombin factor Xa factor XIIa elastases kallikreins cathepsin G granzyme B chymase and chymotrypsins [5]. Hard tick feeding lasts up to a week as opposed to their distant relative the soft ticks whose feeding cycle is much faster [6]. Because of the extended hard tick ON-01910 feeding cycle a complex of host defense responses takes ON-01910 place at the injury site that is counteracted by the pharmacological properties of tick saliva [6] [7] [8]. Tick salivary protease inhibitors play a role in regulating host proteolytic events [9] and the transmission of tick-borne diseases such as Lyme disease [10] while other tick salivary proteins facilitate the transmission of rickettsioses [11] and tick-borne encephalitis [12]. Because of the known pharmacological properties of tick saliva (and the ability to facilitate tick-borne pathogen transmission) two salivary gland transcriptome and proteome ON-01910 projects – also called sialome projects – revealed secreted salivary proteins expressed from the hard tick Mouse monoclonal antibody to Pyruvate Dehydrogenase. The pyruvate dehydrogenase (PDH) complex is a nuclear-encoded mitochondrial multienzymecomplex that catalyzes the overall conversion of pyruvate to acetyl-CoA and CO(2), andprovides the primary link between glycolysis and the tricarboxylic acid (TCA) cycle. The PDHcomplex is composed of multiple copies of three enzymatic components: pyruvatedehydrogenase (E1), dihydrolipoamide acetyltransferase (E2) and lipoamide dehydrogenase(E3). The E1 enzyme is a heterotetramer of two alpha and two beta subunits. This gene encodesthe E1 alpha 1 subunit containing the E1 active site, and plays a key role in the function of thePDH complex. Mutations in this gene are associated with pyruvate dehydrogenase E1-alphadeficiency and X-linked Leigh syndrome. Alternatively spliced transcript variants encodingdifferent isoforms have been found for this gene. are defined as bilaris (two Kunitz heads) and penthalaris (five Kunitz heads). In our study we focused on the most abundant Kunitz group from the sialome project by Ribeiro et al. [14]: the monolaris group. We identified a Kunitz sequence that displays an unusal Cys ON-01910 motif when compared with the other monolaris and to previously reported Kunitz peptides. Since tick Kunitz peptides are known to inhibit serine proteases we performed an inhibitory screening demonstrating that this Kunitz inhibits several proteases as well as being a potent inhibitor of human skin β-tryptase (HSTβ). Furthermore a phylogenetic analysis using several functionally described Kunitz protease inhibitors from hematophagous arthropods nematodes and platyhelminthes reveals that this Kunitz is closely related to TdPI. We will hereafter refer to this Kunitz as tryptogalinin due to its high affinity for HSTβ. Since the crystal structure of TdPI and its complex with trypsin has been solved we used methods to elucidate the biophysical principles that determine tryptogalinin’s protein fold to predict its global tertiary structure and to hypothesize about its physicochemical interactions with serine proteases that account for its biochemical specificity – when compared with TdPI. Materials and Methods General Experimental Procedures Unless otherwise indicated standard procedures were followed according to Sambrook et al. [15]. Experiments were performed at room temperature (25±1°C). All water used was of 18-MΩ quality produced by a MilliQ apparatus (Millipore). If not otherwise stated all reagents were purchased from Sigma-Aldrich. Peptide Expression The experimental procedures for tryptogalinin (GenBank: “type”:”entrez-nucleotide” attrs :”text”:”DN971582″ term_id :”63517144″ term_text :”DN971582″DN971582) overexpression and purification were previously described in Chmelar et al. [7] with the exception that tryptogalinin overexpression was done in BL21(DE3)pLysE bacterial cells (Invitrogen). Serine Protease Inhibition Assays All assays were performed at 30°C with a total of 340 nM of tryptogalinin that was pre-incubated with each enzyme for 10 min before adding the respective fluorescent substrate of the enzyme. A (Monolaris Multiple Sequences Alignment The monolaris nucleotide sequences found in sialome [14] were submitted to the NCBI Open Reading ON-01910 Frame Finder (ORF) online server (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) to verify and edit the sequences to an appropriate start-stop codon. Accordingly we only used sequences containing a start and stop codon and a signal peptide. The translated amino acid sequences that were provided by the ORF Finder were subsequently submitted to the SignalP 4.0 server [19] and the signal peptide.