Cloning, characterisation and heterologous expression of an astacin metalloprotease, Sc-AST, from the entomoparasitic nematode Steinernema carpocapsae.
Steinernema carpocapsae is a parasitic nematode that is high virulent to insects. The parasitic juvenile reaches the insect haemocoelium by passing through mid-gut barriers and develops there. During invasion, the nematode was predicted to express a large set of proteases, including metalloproteases, one of which was sequenced and expressed in this work. A 1583-nucleotide cDNA encoding a putative metalloprotease containing a 28-aa signal peptide, a 79-aa propeptide and a 311-aa mature protease with a predicted molecular mass of 35.2 kDa and a theoretical pI of 5.9 was cloned from the parasitic stage of the nematode. Sequence analyses predicted signature sequences of the astacin metalloprotease family, an astacin domain, a zinc-binding motif and a methionine turn motif; therefore, this protein was identified as an astacin and designated as Sc-AST. The astacin domain of Sc-AST has an amino acid sequence homology of 46% to prototypical astacin from Astacus astacus and 82% to Caenorhabditis elegans NAS-8. Like NAS-8 of C. elegans, Sc-AST has a C-terminal ShK toxin domain. Recombinant Sc-AST was produced in an Escherichia coli system and was purified by affinity chromatography. Maldi-MS/MS analysis of purified recombinant protein matched the Sc-AST sequence with a significance score of 499. Sc-AST was produced in the correct folding conformation, showed activities against gelatin and azocasein substrates and was inhibited by divalent metal-chelating agents. Sc-AST presented an optimum pH of 7.5 and temperature of 37°C and K(m), V(max) and k(cat) values of 1.86 mM, 0.281 μM/min and 27.9 s(-1), respectively. Expression analyses indicated that Sc-AST is up-regulated in the parasitic stage and is strongly induced in vitro by insect tissues, thus suggesting that it plays a role in the parasitic process.
Jing
Yingjun
Y
CIRN and Department of Biology, University of Azores, Ponta Delgada, Azores, Portugal. yingjun7401@hotmail.com
Toubarro
Duarte
D
Hao
Youjin
Y
Simões
Nelson
N
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D015971:Gene Expression Regulation, Enzymologic; D015801:Helminth Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009036:Moths / Q000469:parasitology*; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017168:Rhabditida / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D016415:Sequence Alignment
D018076:DNA, Complementary; D015801:Helminth Proteins; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; C071288:astacin