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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

Clustal alignment of the Kunitz domains of Smp_147710, Smp_179120, Smp_139840, Smp_147730 (SmKI-1), the 1st Kunitz domain of Smp_052230, the 4th and 6th Kunitz domains of Smp_180810 and BPTI. The six conserved cysteine residues are highlighted in black, the P1 site is shown by the black arrow head and the Kunitz family signature is shown as the double headed arrow
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Fig1: Clustal alignment of the Kunitz domains of Smp_147710, Smp_179120, Smp_139840, Smp_147730 (SmKI-1), the 1st Kunitz domain of Smp_052230, the 4th and 6th Kunitz domains of Smp_180810 and BPTI. The six conserved cysteine residues are highlighted in black, the P1 site is shown by the black arrow head and the Kunitz family signature is shown as the double headed arrow

Mentions: Sequence searches revealed the presence of seven putative Kunitz proteins in S. mansoni (Additional file 1). One contained two (Smp_052230) Kunitz domains while the remaining Kunitz molecules were associated with other domains such as chitin binding type-2 (Smp_180810) or spondin (Smp_180240), with Smp_180240 having two distorted Kunitz domains. The Kunitz domain of Smp_147710 did not contain the characteristic inhibitory amino acid at the P1 site and probably has an alternative function to protease inhibition. Two amino acid sequences with a single Kunitz domain (Smp_179120 and Smp_139840) were each devoid of two essential cysteine residues. Smp_147730 (SmKI-1 - Gene bank accession number CCD77156) was selected for further characterisation as it had a single secretory type Kunitz protein domain containing the inhibitory amino acid at the P1 site and was of full length. Furthermore, the Smp_147730 gene had been shown previously to be highly expressed in mechanically transformed S. mansoni schistosomula [21]. Clustal alignment (Fig. 1) of the Kunitz domains of these putative S. mansoni Kunitz proteins and BPTI showed that the amino acid sequence is highly conserved within the domain. Smp_180240 was excluded due to its two highly distorted Kunitz domains.Fig. 1


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)

Clustal alignment of the Kunitz domains of Smp_147710, Smp_179120, Smp_139840, Smp_147730 (SmKI-1), the 1st Kunitz domain of Smp_052230, the 4th and 6th Kunitz domains of Smp_180810 and BPTI. The six conserved cysteine residues are highlighted in black, the P1 site is shown by the black arrow head and the Kunitz family signature is shown as the double headed arrow
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Clustal alignment of the Kunitz domains of Smp_147710, Smp_179120, Smp_139840, Smp_147730 (SmKI-1), the 1st Kunitz domain of Smp_052230, the 4th and 6th Kunitz domains of Smp_180810 and BPTI. The six conserved cysteine residues are highlighted in black, the P1 site is shown by the black arrow head and the Kunitz family signature is shown as the double headed arrow
Mentions: Sequence searches revealed the presence of seven putative Kunitz proteins in S. mansoni (Additional file 1). One contained two (Smp_052230) Kunitz domains while the remaining Kunitz molecules were associated with other domains such as chitin binding type-2 (Smp_180810) or spondin (Smp_180240), with Smp_180240 having two distorted Kunitz domains. The Kunitz domain of Smp_147710 did not contain the characteristic inhibitory amino acid at the P1 site and probably has an alternative function to protease inhibition. Two amino acid sequences with a single Kunitz domain (Smp_179120 and Smp_139840) were each devoid of two essential cysteine residues. Smp_147730 (SmKI-1 - Gene bank accession number CCD77156) was selected for further characterisation as it had a single secretory type Kunitz protein domain containing the inhibitory amino acid at the P1 site and was of full length. Furthermore, the Smp_147730 gene had been shown previously to be highly expressed in mechanically transformed S. mansoni schistosomula [21]. Clustal alignment (Fig. 1) of the Kunitz domains of these putative S. mansoni Kunitz proteins and BPTI showed that the amino acid sequence is highly conserved within the domain. Smp_180240 was excluded due to its two highly distorted Kunitz domains.Fig. 1

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