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Cloning, characterization and expression analysis of a novel gene encoding Kunitz-type protease inhibitor from Dolichos biflorus

ABSTRACT: This paper reports the presence of a Kunitz-type protease inhibitor (HGPI) gene in Dolichos biflorus for the first time. A full-length protease inhibitor gene with complete open reading frame of 669 bp encoding 222 amino acids was cloned from D. biflorus using a PCR-based method. BlastN search showed that the HGPI gene shared 100% homology with Cicer arietinum trypsin inhibitor mRNA and 97% with Cajanus cajan protease inhibitor. The deduced amino acid sequence exhibited homology with Kunitz-type trypsin inhibitor from chickpea, pigeon pea and soybean. The deduced amino acid sequence contained N-terminal signal sequence of 18 amino acids and had a molecular weight of 24 kDa. The phylogenetic tree also showed close relationship with Cicer arietinum and Cajanus cajan. Southern blotting revealed the presence of only one copy of HGPI gene in the D. biflorus genome. Homology modeling was employed to predict secondary structure and 3D-structural models for the protease inhibitor. The gene was found to be constitutively expressed in different tissues as determined by semi-quantitative RT-PCR. The novel HGPI gene has been submitted to the GenBank with Accession No. FN666416. The isolated novel HGPI gene will broaden the pool of plant defense genes and might be an ideal choice for developing transgenic crops resistant to insect pests, as well as pathogens. In addition, it could be used as a probe for selection of insect- and pathogen-resistant genotypes.

SUBMITTER: Kuhar K

PROVIDER: S-EPMC3433878 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundSchistosomes are able to survive for prolonged periods in the blood system, despite continuous contact with coagulatory factors and mediators of the host immune system. Protease inhibitors likely play a critical role in host immune modulation thereby promoting parasite survival in this extremely hostile environment. Even though Kunitz type serine protease inhibitors have been shown to play important physiological functions in a range of organisms these proteins are less well characterised in parasitic helminths.MethodsWe have cloned one gene sequence from S. mansoni, Smp_147730 (SmKI-1) which is coded for single domain Kunitz type protease inhibitor, E. coli-expressed and purified. Immunolocalisation and western blotting was carried out using affinity purified polyclonal anti-SmKI-1 murine antibodies to determine SmKI-1 expression in the parasite. Protease inhibitor assays and coagulation assays were performed to evaluate the functional roles of SmKI-1.ResultsSmKI-1 is localised in the tegument of adult worms and the sub-shell region of eggs. Furthermore, this Kunitz protein is secreted into the host in the ES products of the adult worm. Recombinant SmKI-1 inhibited mammalian trypsin, chymotrypsin, neutrophil elastase, FXa and plasma kallikrein with IC50 values of 35 nM, 61 nM, 56 nM, 142 nM and 112 nM, respectively. However, no inhibition was detected for pancreatic elastase or cathepsin G. SmKI-1 (4 μM) delayed blood clot formation, reflected in an approximately three fold increase in activated partial thromboplastin time and prothrombin time.ConclusionsWe have functionally characterised the first Kunitz type protease inhibitor (SmKI-1) from S. mansoni and show that it has anti-inflammatory and anti-coagulant properties. SmKI-1 is one of a number of putative Kunitz proteins in schistosomes that have presumably evolved as an adaptation to protect these parasites from the defence mechanisms of their mammalian hosts. As such they may represent novel vaccine candidates and/or drug targets for schistosomiasis control.

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Cloning, characterization and expression analysis of a novel gene encoding Kunitz-type protease inhibitor from Dolichos biflorus

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