title: An apoptosis-inducing serine protease secreted by the entomopathogenic nematode Steinernema carpocapsae.
abstract: Steinernema carpocapsae is an insect parasitic nematode able to parasitise and kill the host within 48 h. Secreted products (ESP) of the parasitic stage of a virulent strain contain higher amounts of proteolytic activity than a low virulence strain, suggesting proteases are involved in virulence. From the ESP we purified a protein (Sc-SP-3) with a M(r) of 30 kDa and a pI of 7 that cleaved the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-pNA and was inhibited by phenylmethanesulfonyl fluoride, benzamidine and chymostatin, thus indicating that it belongs to the chymotrypsin-like serine protease family. Sc-SP-3 has a V(max) of 0.3 mM min(-1)ml(-1) and K(m) of 6.6 x 10(-4)M, with maximum activity at pH 8 and 40 degrees C. The full-length cDNA was obtained using degenerate oligonucleotides for serine proteases. This open reading frame encodes a preproprotein containing a putative signal peptide composed of 16 amino acid residues, a prodomain of 40 residues and a mature protease domain of 261 residues, including the catalytic triad His/Asp/Ser characteristic of trypsin-like serine proteases. The N-terminal sequence and the peptide masses fingerprint obtained by MALDI-TOF-MS for the purified protein matched the cDNA. Gene expression analysis by quantitative real-time-PCR showed that this gene is expressed only during the parasitic stage and that pre-invasive nematodes inside the mid-gut expressed higher amounts of Sc-SP-3 than those that already enter the haemocoel. Sc-SP-3 caused histolysis in the insect mid-gut. In vitro assays demonstrated that Sc-SP-3 digested extracellular proteins and induced apoptosis in Sf9 insect cells, thus suggesting Sc-SP-3 is a multifunctional chymotrypsin-like protease involved in pathogenesis.
authors.affiliation: CIRN, Departamento de Biologia, Universidade dos Açores, Apartado 1422, 9501-801 Ponta Delgada, Portugal.
authors.forename: Duarte
authors.initials: D
authors.lastname: Toubarro
authors.forename: Miguel
authors.initials: M
authors.lastname: Lucena-Robles
authors.forename: Gisela
authors.initials: G
authors.lastname: Nascimento
authors.forename: Gonçalo
authors.initials: G
authors.lastname: Costa
authors.forename: Rafael
authors.initials: R
authors.lastname: Montiel
authors.forename: Ana V
authors.initials: AV
authors.lastname: Coelho
authors.forename: Nelson
authors.initials: N
authors.lastname: Simões
mesh_terms: D000818:Animals; D017209:Apoptosis; D053818:Chymases / Q000235:genetics / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics* / Q000378:metabolism; D015801:Helminth Proteins / Q000737:chemistry / Q000378:metabolism*; D007313:Insecta / Q000469:parasitology; D016133:Polymerase Chain Reaction; D017168:Rhabditida / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D020539:Sequence Analysis, Protein; D057057:Serine Proteases / Q000235:genetics / Q000378:metabolism*
chemical_list: D018076:DNA, Complementary; D015801:Helminth Proteins; D057057:Serine Proteases; D053818:Chymases