schenckii sspla 2 gene. Figure 4A shows the sequencing strategy used for sequencing the sspla 2 gene. The size and location in the gene of the various fragments obtained from PCR and RACE are shown. Figure 4B shows the genomic and derived amino acid sequence of the sspla 2 gene. Non-coding regions are given in lower case letters, coding regions and amino acids are given in upper Blebbistatin cell line case letters. The invariant

amino acids required for phospholipase activity are shown in red. The potential EF hands are shaded in yellow and the putative calmodulin binding domain is shaded in gray. The cPLA2 signature motif is shaded in green and the serine proteases, subtilase family, aspartic acid active site motif is shaded in blue green. Bioinformatic

characterization of SSPLA2 The PANTHER Classification System identified this protein as a member of the cytosolic phospholipase A2 family (PTHR10728) (residues 132–827) with an extremely significant E value of 6.4 e-97 [40]. BLAST analysis of the derived amino acid sequence of the S. schenckii SSPLA2, showed a phospholipase domain extending from amino acids 177 to 750 [39]. Pfam analysis shows similar results, and in this domain the PLA2 signature GXSG [G, S] (Pfam: Family PLA2_B PF 01735) is present as GVSGS in the active site (highlighted green in Figure 4B) [41, 42]. The Batimastat clinical trial amino acids needed for catalytic activity R235, S263 and D553 are given in red in this same figure [43]. S263 is essential for the formation of arachidonyl Aspartate serine needed for the transfer of the arachidonyl group to glycerol or to water. The amino acids D511 to L523, D583 to G595 and D738 to A750 (highlighted in yellow) comprise putative EF hand

domains of the protein (76% identity, probability, 3.33e-06). In Figure 4B a putative calmodulin binding domain was identified from amino acids Q806 to L823 using the Calmodulin Target Database [44] and highlighted in gray. A serine protease, subtilase family, aspartic acid active site motif was identified using Scan Prosite with an E value of 5.283e-07 from amino acids 549 to 559 and is shaded in blue green in Figure 4B[45]. This motif is characteristic of both yeast and fungal cPLA2 homologues [43]. Figure 5 shows the multiple sequence CRM1 inhibitor alignment of the derived amino acid sequence of S. schenckii PLA2 homologue to that of other PLA homologues or hypothetical proteins from N. crassa, A. nidulans, M. grisea, Chaetomium globosum, Podospora anserina and Gibberella zeae. This figure shows that the important domains are very similar, although variations occur in the N terminal and C terminal regions. The alignment shown includes only the catalytic domain, the complete alignment is given as additional material (Additional file 1). Figure 5 Amino acid sequence alignments of SSPLA 2 with other PLA 2 homologues. The S. schenckii SSPLA2 was aligned to other PLA2 fungal homologues as described in Methods. The fungal PLA2 used for the alignment were: E.