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Protective effects of recombinant kunitz-domain 1 of human tissue factor pathway inhibitor-2 against 2-chloroethyl ethyl sulfide toxicity in vitro.

Choi MS, Parikh K, Saxena A, Chilukuri N - J Burns Wounds (2007)

Bottom Line: The purified proteins were refolded, and their effects were tested in an in vitro human epidermal keratinocyte cell wounding assay.Wild-type Kunitz-domain 1 significantly improved wound healing of unexposed and 2-chloroethyl ethyl sulfide-exposed cells without influencing cell proliferation.These data suggest that wild-type Kunitz-domain 1 of human tissue factor pathway inhibitor-2 can be developed as a medical countermeasure against sulfur mustard cutaneous injury.

View Article: PubMed Central - PubMed

Affiliation: Division of Biochemistry, Department of Molecular Pharmacology, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA.

ABSTRACT

Objective: Sulfur mustard is a well-known blistering chemical warfare agent that has been investigated for its toxicological mechanisms and an efficacious antidote. Since sulfur mustard injury involves dermal:epidermal separation, proteolytic enzymes were suspected to be involved for this separation and eventual blister development. Therefore, protease inhibitors could be of therapeutic utility against sulfur mustard injury. In this study, the effects of Kunitz-domain 1 of human tissue factor pathway inhibitor-2 were evaluated against the toxic effects of 2-chloroethyl ethyl sulfide, a surrogate agent of sulfur mustard. Tissue factor pathway inhibitor-2 is a 32-kDa serine protease inhibitor produced by a variety of cell types including human epidermal keratinocytes, fibroblasts, and endothelial cells. It consists of 3 Kunitz-domains and the first Kunitz-domain contains the putative P(1) residue (arginine at position 24) responsible for protease inhibitory activity.

Methods: Recombinant wild-type and R24Q mutant Kunitz-domain 1s were expressed in Escherichia coli and purified. The purified proteins were refolded, and their effects were tested in an in vitro human epidermal keratinocyte cell wounding assay.

Results: Wild-type but not R24Q Kunitz-domain 1 inhibited the amidolytic activity of trypsin and plasmin. Wild-type Kunitz-domain1 was stable for 4 weeks at 42 degrees C and for more than 8 weeks at room temperature. Wild-type Kunitz-domain 1 significantly improved wound healing of unexposed and 2-chloroethyl ethyl sulfide-exposed cells without influencing cell proliferation. Although R24Q Kunitz-domain 1 lacked trypsin and plasmin inhibitory activity, it promoted wound closure of untreated and 2-chloroethyl ethyl sulfide-treated cells but to a much lesser degree.

Conclusion: These data suggest that wild-type Kunitz-domain 1 of human tissue factor pathway inhibitor-2 can be developed as a medical countermeasure against sulfur mustard cutaneous injury.

No MeSH data available.


Related in: MedlinePlus

Prokaryotic expression and purification of wt-KD1: Panel A: SDS-PAGE of His-affinity column purified wt-KD1. Lanes are (1) molecular weight markers; (2) column purified wt-KD1 and (3) inclusion body extract. The arrow points to the location of wt-KD1. Panel B: SDS-PAGE of FPLC fractions. FPLC fractions were processed by SDS-PAGE, using 15% polyacrylamide gel, and proteins were identified by silver staining. Lanes are (1) molecular size markers; (2) wt-KD1 purified with His-affinity column; and (3–10) FPLC fractions from A6–A13. Fractions A8–A12 (lanes 5–9) contained the pure wt-KD1 fusion protein. Panel C: SDS-PAGE of wt-KD1 without His-tag. Lanes are (1) molecular size marker; (2) wt-KD1 with His-tag (solid arrow); and (3) wt-KD1 without His-tag (dashed arrow). Panel D: Molecular composition of refolded wt-KD1. Proteins were fractionated using 15% polyacrylamide gels and identified by Western blotting using anti-TFPI-2 antibody. Lanes are (1) wt-KD1 with His-tag and (2) wt-KD1 without His-tag. Wt-KD1 with His-tag contains both monomers (dashed arrow) and multimers (solid arrows) where as wt-KD1 without His-tag contains monomers only.
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Figure 1: Prokaryotic expression and purification of wt-KD1: Panel A: SDS-PAGE of His-affinity column purified wt-KD1. Lanes are (1) molecular weight markers; (2) column purified wt-KD1 and (3) inclusion body extract. The arrow points to the location of wt-KD1. Panel B: SDS-PAGE of FPLC fractions. FPLC fractions were processed by SDS-PAGE, using 15% polyacrylamide gel, and proteins were identified by silver staining. Lanes are (1) molecular size markers; (2) wt-KD1 purified with His-affinity column; and (3–10) FPLC fractions from A6–A13. Fractions A8–A12 (lanes 5–9) contained the pure wt-KD1 fusion protein. Panel C: SDS-PAGE of wt-KD1 without His-tag. Lanes are (1) molecular size marker; (2) wt-KD1 with His-tag (solid arrow); and (3) wt-KD1 without His-tag (dashed arrow). Panel D: Molecular composition of refolded wt-KD1. Proteins were fractionated using 15% polyacrylamide gels and identified by Western blotting using anti-TFPI-2 antibody. Lanes are (1) wt-KD1 with His-tag and (2) wt-KD1 without His-tag. Wt-KD1 with His-tag contains both monomers (dashed arrow) and multimers (solid arrows) where as wt-KD1 without His-tag contains monomers only.

Mentions: Wt-KD1 protein was expressed in E coli grown in LB containing 100 μg/mL of ampicillin. Induction at 37°C with 1 mM IPTG at mid-log phase for 2 hours showed the highest amount of wt-KD1 expression (Figure 1A, lane 3). These conditions were adopted for the large-scale expression of both wt- and R24Q-KD1s.


Protective effects of recombinant kunitz-domain 1 of human tissue factor pathway inhibitor-2 against 2-chloroethyl ethyl sulfide toxicity in vitro.

Choi MS, Parikh K, Saxena A, Chilukuri N - J Burns Wounds (2007)

Prokaryotic expression and purification of wt-KD1: Panel A: SDS-PAGE of His-affinity column purified wt-KD1. Lanes are (1) molecular weight markers; (2) column purified wt-KD1 and (3) inclusion body extract. The arrow points to the location of wt-KD1. Panel B: SDS-PAGE of FPLC fractions. FPLC fractions were processed by SDS-PAGE, using 15% polyacrylamide gel, and proteins were identified by silver staining. Lanes are (1) molecular size markers; (2) wt-KD1 purified with His-affinity column; and (3–10) FPLC fractions from A6–A13. Fractions A8–A12 (lanes 5–9) contained the pure wt-KD1 fusion protein. Panel C: SDS-PAGE of wt-KD1 without His-tag. Lanes are (1) molecular size marker; (2) wt-KD1 with His-tag (solid arrow); and (3) wt-KD1 without His-tag (dashed arrow). Panel D: Molecular composition of refolded wt-KD1. Proteins were fractionated using 15% polyacrylamide gels and identified by Western blotting using anti-TFPI-2 antibody. Lanes are (1) wt-KD1 with His-tag and (2) wt-KD1 without His-tag. Wt-KD1 with His-tag contains both monomers (dashed arrow) and multimers (solid arrows) where as wt-KD1 without His-tag contains monomers only.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1937028&req=5

Figure 1: Prokaryotic expression and purification of wt-KD1: Panel A: SDS-PAGE of His-affinity column purified wt-KD1. Lanes are (1) molecular weight markers; (2) column purified wt-KD1 and (3) inclusion body extract. The arrow points to the location of wt-KD1. Panel B: SDS-PAGE of FPLC fractions. FPLC fractions were processed by SDS-PAGE, using 15% polyacrylamide gel, and proteins were identified by silver staining. Lanes are (1) molecular size markers; (2) wt-KD1 purified with His-affinity column; and (3–10) FPLC fractions from A6–A13. Fractions A8–A12 (lanes 5–9) contained the pure wt-KD1 fusion protein. Panel C: SDS-PAGE of wt-KD1 without His-tag. Lanes are (1) molecular size marker; (2) wt-KD1 with His-tag (solid arrow); and (3) wt-KD1 without His-tag (dashed arrow). Panel D: Molecular composition of refolded wt-KD1. Proteins were fractionated using 15% polyacrylamide gels and identified by Western blotting using anti-TFPI-2 antibody. Lanes are (1) wt-KD1 with His-tag and (2) wt-KD1 without His-tag. Wt-KD1 with His-tag contains both monomers (dashed arrow) and multimers (solid arrows) where as wt-KD1 without His-tag contains monomers only.
Mentions: Wt-KD1 protein was expressed in E coli grown in LB containing 100 μg/mL of ampicillin. Induction at 37°C with 1 mM IPTG at mid-log phase for 2 hours showed the highest amount of wt-KD1 expression (Figure 1A, lane 3). These conditions were adopted for the large-scale expression of both wt- and R24Q-KD1s.

Bottom Line: The purified proteins were refolded, and their effects were tested in an in vitro human epidermal keratinocyte cell wounding assay.Wild-type Kunitz-domain 1 significantly improved wound healing of unexposed and 2-chloroethyl ethyl sulfide-exposed cells without influencing cell proliferation.These data suggest that wild-type Kunitz-domain 1 of human tissue factor pathway inhibitor-2 can be developed as a medical countermeasure against sulfur mustard cutaneous injury.

View Article: PubMed Central - PubMed

Affiliation: Division of Biochemistry, Department of Molecular Pharmacology, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA.

ABSTRACT

Objective: Sulfur mustard is a well-known blistering chemical warfare agent that has been investigated for its toxicological mechanisms and an efficacious antidote. Since sulfur mustard injury involves dermal:epidermal separation, proteolytic enzymes were suspected to be involved for this separation and eventual blister development. Therefore, protease inhibitors could be of therapeutic utility against sulfur mustard injury. In this study, the effects of Kunitz-domain 1 of human tissue factor pathway inhibitor-2 were evaluated against the toxic effects of 2-chloroethyl ethyl sulfide, a surrogate agent of sulfur mustard. Tissue factor pathway inhibitor-2 is a 32-kDa serine protease inhibitor produced by a variety of cell types including human epidermal keratinocytes, fibroblasts, and endothelial cells. It consists of 3 Kunitz-domains and the first Kunitz-domain contains the putative P(1) residue (arginine at position 24) responsible for protease inhibitory activity.

Methods: Recombinant wild-type and R24Q mutant Kunitz-domain 1s were expressed in Escherichia coli and purified. The purified proteins were refolded, and their effects were tested in an in vitro human epidermal keratinocyte cell wounding assay.

Results: Wild-type but not R24Q Kunitz-domain 1 inhibited the amidolytic activity of trypsin and plasmin. Wild-type Kunitz-domain1 was stable for 4 weeks at 42 degrees C and for more than 8 weeks at room temperature. Wild-type Kunitz-domain 1 significantly improved wound healing of unexposed and 2-chloroethyl ethyl sulfide-exposed cells without influencing cell proliferation. Although R24Q Kunitz-domain 1 lacked trypsin and plasmin inhibitory activity, it promoted wound closure of untreated and 2-chloroethyl ethyl sulfide-treated cells but to a much lesser degree.

Conclusion: These data suggest that wild-type Kunitz-domain 1 of human tissue factor pathway inhibitor-2 can be developed as a medical countermeasure against sulfur mustard cutaneous injury.

No MeSH data available.


Related in: MedlinePlus