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Functionally Expression of Metalloproteinase in Taenia solium Metacestode and Its Evaluation for Serodiagnosis of Cysticercosis.

Zhang Y, Bae YA, Zong HY, Kong Y, Cai GB - Iran J Parasitol (2016 Jan-Mar)

Bottom Line: After refolding and purification, enzymatic properties of the recombinant metalloproteinase were observed.The recombinant TsMP protein showed proteolytic activity, which preferred host extracellular matrix proteins such as collagen and fibronectin as degradable substrates.TsMP might be involved in the processing of numerous host proteins and play an important role in the parasite life cycle.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Medical Genetics, Wuhan University School of Basic Medicial Sciences, Wuhan 430071, China.

ABSTRACT

Background: Parasite proteases have important roles in cleavage of host proteins during the invasion of host tissues and participate in the parasite's evasion from the host's immune response. The aim of the present study was to estimate a metalloproteinase properties of Taenia solium metacestode (TsMP) during host-parasite interactions, and evaluate its potential as a serodiagnostic antigen for cysticercosis.

Methods: The cDNA coding for the mature catalytic domain of TsMP was cloned into pGEX-6P-1 expression vector. A recombinant glutathione S-transferase and TsMP fusion protein was induced. After refolding and purification, enzymatic properties of the recombinant metalloproteinase were observed. Immunoblot assay was processed to evaluate its potential as a serodiagnostic antigen for cysticercosis.

Results: The recombinant TsMP protein showed proteolytic activity, which preferred host extracellular matrix proteins such as collagen and fibronectin as degradable substrates. In immunoblot assay, 87.5% of sera from patients with cysticercosis showed strong reactivity. In sera from patients with other parasitic infections and from normal controls, it showed high specificity.

Conclusions: TsMP might be involved in the processing of numerous host proteins and play an important role in the parasite life cycle. A single recombinant TsMP antigen could have a potential value for serodiagnosis of cysticercosis.

No MeSH data available.


Related in: MedlinePlus

Effects of protease inhibitor (A) and metal ion (B) on proteolytic activity of recombinant mTsMP protein by gel zymograph analysis. Lane 1, recombinant mTsMP; lane 2, recombinant mTsMP + 10 mM EDTA (metalloproteinase specific inhibitor); lane 3, recombinant mTsMP +2 µM E-64 (cysteine proteinase specific inhibitor); lane 4, recombinant mTsMP + 2 mM PMSF (serine proteinase specific inhibitor); lane 5, recombinant mTsMP + 1 µM pepstatin A (aspartic proteinase specific inhibitor); lane 6, recombinant mTsMP; lane 7, recombinant mTsMP + 2 mM Ca2+; lane 8, recombinant mTsMP + 2 mM Mg2+; lane 9, recombinant mTsMP +1 µM Zn2+; lane 10, recombinant mTsMP + 5 mM Zn2+
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Figure 3: Effects of protease inhibitor (A) and metal ion (B) on proteolytic activity of recombinant mTsMP protein by gel zymograph analysis. Lane 1, recombinant mTsMP; lane 2, recombinant mTsMP + 10 mM EDTA (metalloproteinase specific inhibitor); lane 3, recombinant mTsMP +2 µM E-64 (cysteine proteinase specific inhibitor); lane 4, recombinant mTsMP + 2 mM PMSF (serine proteinase specific inhibitor); lane 5, recombinant mTsMP + 1 µM pepstatin A (aspartic proteinase specific inhibitor); lane 6, recombinant mTsMP; lane 7, recombinant mTsMP + 2 mM Ca2+; lane 8, recombinant mTsMP + 2 mM Mg2+; lane 9, recombinant mTsMP +1 µM Zn2+; lane 10, recombinant mTsMP + 5 mM Zn2+

Mentions: Purified recombinant mTsMP protein was used to observe the proteinase properties. The effects of protease inhibitors on the recombinant proteinase were examined by gel zymograph. Zinc specific metal chelator EDTA (10 mM) strongly inhibited enzyme activity. It was uninhibited by serine proteinase inhibitor (PMSF, 2 mM), aspartic proteinase inhibitor (pepstatin A, 1 µM) and cysteine proteinase inhibitor (E-64, 10 µM) (Fig. 3 A). The addition of Mg2+ (2 mM), Ca2+ (2 mM) and Zn2+ (1 µM) could enhance but did not significantly affect enzyme activity. However, Zn2+ in a concentration greater than 1 mM inhibited enzyme activity, with 5 mM Zn2+ resulting in an almost half reduction of proteinase activity (Fig. 3 B).


Functionally Expression of Metalloproteinase in Taenia solium Metacestode and Its Evaluation for Serodiagnosis of Cysticercosis.

Zhang Y, Bae YA, Zong HY, Kong Y, Cai GB - Iran J Parasitol (2016 Jan-Mar)

Effects of protease inhibitor (A) and metal ion (B) on proteolytic activity of recombinant mTsMP protein by gel zymograph analysis. Lane 1, recombinant mTsMP; lane 2, recombinant mTsMP + 10 mM EDTA (metalloproteinase specific inhibitor); lane 3, recombinant mTsMP +2 µM E-64 (cysteine proteinase specific inhibitor); lane 4, recombinant mTsMP + 2 mM PMSF (serine proteinase specific inhibitor); lane 5, recombinant mTsMP + 1 µM pepstatin A (aspartic proteinase specific inhibitor); lane 6, recombinant mTsMP; lane 7, recombinant mTsMP + 2 mM Ca2+; lane 8, recombinant mTsMP + 2 mM Mg2+; lane 9, recombinant mTsMP +1 µM Zn2+; lane 10, recombinant mTsMP + 5 mM Zn2+
© Copyright Policy
Related In: Results  -  Collection

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Figure 3: Effects of protease inhibitor (A) and metal ion (B) on proteolytic activity of recombinant mTsMP protein by gel zymograph analysis. Lane 1, recombinant mTsMP; lane 2, recombinant mTsMP + 10 mM EDTA (metalloproteinase specific inhibitor); lane 3, recombinant mTsMP +2 µM E-64 (cysteine proteinase specific inhibitor); lane 4, recombinant mTsMP + 2 mM PMSF (serine proteinase specific inhibitor); lane 5, recombinant mTsMP + 1 µM pepstatin A (aspartic proteinase specific inhibitor); lane 6, recombinant mTsMP; lane 7, recombinant mTsMP + 2 mM Ca2+; lane 8, recombinant mTsMP + 2 mM Mg2+; lane 9, recombinant mTsMP +1 µM Zn2+; lane 10, recombinant mTsMP + 5 mM Zn2+
Mentions: Purified recombinant mTsMP protein was used to observe the proteinase properties. The effects of protease inhibitors on the recombinant proteinase were examined by gel zymograph. Zinc specific metal chelator EDTA (10 mM) strongly inhibited enzyme activity. It was uninhibited by serine proteinase inhibitor (PMSF, 2 mM), aspartic proteinase inhibitor (pepstatin A, 1 µM) and cysteine proteinase inhibitor (E-64, 10 µM) (Fig. 3 A). The addition of Mg2+ (2 mM), Ca2+ (2 mM) and Zn2+ (1 µM) could enhance but did not significantly affect enzyme activity. However, Zn2+ in a concentration greater than 1 mM inhibited enzyme activity, with 5 mM Zn2+ resulting in an almost half reduction of proteinase activity (Fig. 3 B).

Bottom Line: After refolding and purification, enzymatic properties of the recombinant metalloproteinase were observed.The recombinant TsMP protein showed proteolytic activity, which preferred host extracellular matrix proteins such as collagen and fibronectin as degradable substrates.TsMP might be involved in the processing of numerous host proteins and play an important role in the parasite life cycle.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Medical Genetics, Wuhan University School of Basic Medicial Sciences, Wuhan 430071, China.

ABSTRACT

Background: Parasite proteases have important roles in cleavage of host proteins during the invasion of host tissues and participate in the parasite's evasion from the host's immune response. The aim of the present study was to estimate a metalloproteinase properties of Taenia solium metacestode (TsMP) during host-parasite interactions, and evaluate its potential as a serodiagnostic antigen for cysticercosis.

Methods: The cDNA coding for the mature catalytic domain of TsMP was cloned into pGEX-6P-1 expression vector. A recombinant glutathione S-transferase and TsMP fusion protein was induced. After refolding and purification, enzymatic properties of the recombinant metalloproteinase were observed. Immunoblot assay was processed to evaluate its potential as a serodiagnostic antigen for cysticercosis.

Results: The recombinant TsMP protein showed proteolytic activity, which preferred host extracellular matrix proteins such as collagen and fibronectin as degradable substrates. In immunoblot assay, 87.5% of sera from patients with cysticercosis showed strong reactivity. In sera from patients with other parasitic infections and from normal controls, it showed high specificity.

Conclusions: TsMP might be involved in the processing of numerous host proteins and play an important role in the parasite life cycle. A single recombinant TsMP antigen could have a potential value for serodiagnosis of cysticercosis.

No MeSH data available.


Related in: MedlinePlus