An apoptosis-inducing serine protease secreted by the entomopathogenic nematode Steinernema carpocapsae.
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.
Toubarro
Duarte
D
CIRN, Departamento de Biologia, Universidade dos Açores, Apartado 1422, 9501-801 Ponta Delgada, Portugal.
Lucena-Robles
Miguel
M
Nascimento
Gisela
G
Costa
Gonçalo
G
Montiel
Rafael
R
Coelho
Ana V
AV
Simões
Nelson
N
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*
D018076:DNA, Complementary; D015801:Helminth Proteins; D057057:Serine Proteases; D053818:Chymases