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Functional expression of a novel Kunitz type protease inhibitor from the human blood fluke Schistosoma mansoni.

Ranasinghe SL, Fischer K, Gobert GN, McManus DP - Parasit Vectors (2015)

Bottom Line: SmKI-1 (4 μM) delayed blood clot formation, reflected in an approximately three fold increase in activated partial thromboplastin time and prothrombin time.We 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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Parasitology Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia. shiwanthi.ranasinghe@qimrberghofer.edu.au.

ABSTRACT

Background: Schistosomes 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.

Methods: We 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.

Results: SmKI-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.

Conclusions: We 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.

No MeSH data available.


Related in: MedlinePlus

Phylogenetic analysis of SmKI-1 and functionally characterised Kunitz proteins from other taxa: Simukunin (ACH56928.1) from the blackfly Simulium vittatum, EgKU8 (ACM79010.1) from Echinococcus granulosus, Fh-KTM (AAB46830.1) from Fasciola hepatica, Ixolaris (AAK83022.1) from the hard-tick Ixodes scapularis, BPTI (1510193A) and human TFPI-2 (AAA20094)
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Fig2: Phylogenetic analysis of SmKI-1 and functionally characterised Kunitz proteins from other taxa: Simukunin (ACH56928.1) from the blackfly Simulium vittatum, EgKU8 (ACM79010.1) from Echinococcus granulosus, Fh-KTM (AAB46830.1) from Fasciola hepatica, Ixolaris (AAK83022.1) from the hard-tick Ixodes scapularis, BPTI (1510193A) and human TFPI-2 (AAA20094)

Mentions: SmKI-1 is comprised of 146 amino acids and has a signal peptide of 20 residues. The mature protein has a putative molecular mass of 15.108 kDa and an isoelectric point of 8.22. One putative N-glycosylation site is predicted for the mature SmKI-1 protein. Phylogenetic analysis (Fig. 2) with SmKI-1 and other functionally characterised Kunitz proteins revealed a high relatedness of SmKI-1 with Simukunin, a Kunitz type plasma coagulation inhibitor from the salivary glands of the black fly Simulium vittatum [37]. BLASTP analysis revealed that SmKI-1 shared greatest sequence identity (63 % identity and 34 % query cover, E value 2x10−21) with Simukunin. SmKI-1 had the second highest sequence identity (57 % identity and 39 % query cover, E value 5x10−18) with human tissue factor pathway inhibitor-2 (TFPI-2) (GenBank No AAA20094.1), which is a three tandem domain Kunitz protein.Fig. 2


Functional expression of a novel Kunitz type protease inhibitor from the human blood fluke Schistosoma mansoni.

Ranasinghe SL, Fischer K, Gobert GN, McManus DP - Parasit Vectors (2015)

Phylogenetic analysis of SmKI-1 and functionally characterised Kunitz proteins from other taxa: Simukunin (ACH56928.1) from the blackfly Simulium vittatum, EgKU8 (ACM79010.1) from Echinococcus granulosus, Fh-KTM (AAB46830.1) from Fasciola hepatica, Ixolaris (AAK83022.1) from the hard-tick Ixodes scapularis, BPTI (1510193A) and human TFPI-2 (AAA20094)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4524284&req=5

Fig2: Phylogenetic analysis of SmKI-1 and functionally characterised Kunitz proteins from other taxa: Simukunin (ACH56928.1) from the blackfly Simulium vittatum, EgKU8 (ACM79010.1) from Echinococcus granulosus, Fh-KTM (AAB46830.1) from Fasciola hepatica, Ixolaris (AAK83022.1) from the hard-tick Ixodes scapularis, BPTI (1510193A) and human TFPI-2 (AAA20094)
Mentions: SmKI-1 is comprised of 146 amino acids and has a signal peptide of 20 residues. The mature protein has a putative molecular mass of 15.108 kDa and an isoelectric point of 8.22. One putative N-glycosylation site is predicted for the mature SmKI-1 protein. Phylogenetic analysis (Fig. 2) with SmKI-1 and other functionally characterised Kunitz proteins revealed a high relatedness of SmKI-1 with Simukunin, a Kunitz type plasma coagulation inhibitor from the salivary glands of the black fly Simulium vittatum [37]. BLASTP analysis revealed that SmKI-1 shared greatest sequence identity (63 % identity and 34 % query cover, E value 2x10−21) with Simukunin. SmKI-1 had the second highest sequence identity (57 % identity and 39 % query cover, E value 5x10−18) with human tissue factor pathway inhibitor-2 (TFPI-2) (GenBank No AAA20094.1), which is a three tandem domain Kunitz protein.Fig. 2

Bottom Line: SmKI-1 (4 μM) delayed blood clot formation, reflected in an approximately three fold increase in activated partial thromboplastin time and prothrombin time.We 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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Parasitology Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia. shiwanthi.ranasinghe@qimrberghofer.edu.au.

ABSTRACT

Background: Schistosomes 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.

Methods: We 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.

Results: SmKI-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.

Conclusions: We 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.

No MeSH data available.


Related in: MedlinePlus