BMC Genomics 2007, 8:249 http://www.biomedcentral.com/1471-2164/8/249 gFMeiognleuerceula3r characterization of a new serine endopeptidase Molecular characterization of a new serine endopeptidase gene. A) Graphic representation of the genomic alignment of the new transcript (SP212) identified by ORE-5. The small arrows localize the primers used for cloning the SP212 cDNA. The asterisk indicates the primer that was based on the ORE-5 sequence. CG-33329 was elec- tronically annotated. B) Sequence of the SP212 cDNA and the amino-acid sequence encoded in the largest open reading frame (GenBank accession number: EF108315). Grey boxes mark the motifs containing the SP catalytic residues, which are underlined. identity with serine proteases (SP), thus revealing a new D. melanogaster SP protein. SP proteins are proteolytic enzymes that require serine for their catalytic activity. They are ubiquitous peptidases, which perform a wide array of important physiological functions, including digestion, blood coagulation, fibri- nolysis, cellular and humoral immunity, fertilization and embryonic development [21]. In a previous work that intended to map all SPs and SP related proteins in the genome of D. melanogaster, Ross and colleagues [22] per- formed a series of similarity searches (PSI-BLAST) and found a total of 211 GenBank entries encoding SPs and SPHs (SP homologs – proteins in which one or more res- idues of the catalytic triad are missing). Following the nomenclature criteria used by these authors, we named the additional SP gene characterized here as SP212. PROSITE analysis [23] revealed that D. melanogaster SP212 presents the conserved catalytic triad ordered His, Asp, Ser (HDS), a characteristic of SP chymotrypsin fam- ily. These residues form two diads, Ser-His and His-Asp, that operate in concert for the acyl mechanism of catalysis [24]. These catalytic residues are, as in most SPs, embed- ded into highly conserved motifs: SAAHC, DIAL and GDSGGG. SP212 also presents the signal peptide sequence in its amino terminal end with the cleavage between residues 17 and 18, an indication that this pepti- dase might be secreted (figure 3B). The SP212 gene is located in a chromosomal region (88A12-B1) that contains three predicted genes, CG9631, CG9649 and CG31326, which codify two SPs and one SPH respectively, named: SP60, SP55 and SPH144. In order to evaluate the similarity level between SP212 and related proteins, we performed a multiple alignment com- parison with a selected group of SPs and SPHs. The pro- tein sequences for this alignment were selected based on their similarity (E value < -20) with SP212 using the BLASTx tool. Multiple alignment [25] of the catalytic domain of these proteins (50 residues upstream of the conserved His and 50 residues downstream of the Ser cat- alytic site) showed that SP212 is more closely related to SP55 and SPH144, which are located in the 88A12-B1 chromossomic region. Thus, SP212, SP55 and SPH144 genes were possibly generated through gene duplication during evolution of the SP family, probably being derived from a common ancestor (figure 4). SP212 gene is upregulated in response to infection SP212 is 528 amino acid residues long and presents the potential to form three disulfide-bridges. These structural features of SP212 indicate that this enzyme is probably associated with physiological functions other than diges- tion. Digestive SPs are much smaller, contain approxi- mately 250 amino acid residues and have a relatively Page 6 of 9 (page number not for citation purposes)