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Prolyl oligopeptidase is inhibited in relapsing-remitting multiple sclerosis.

Tenorio-Laranga J, Coret-Ferrer F, Casanova-Estruch B, Burgal M, García-Horsman JA - J Neuroinflammation (2010)

Bottom Line: We observed a significant decrease in POP activity in plasma of relapsing remitting MS patients relative to healthy controls, coupled with an increase of POP endogenous inhibitor.The lowered POP activity from plasma of MS patients could be rescued by reductants The decrease in circulating POP activity measured in MS is reverted by reductants.This suggests that POP inactivation in MS might be a result of the oxidative conditions prevailing in the plasma of the diseased patients.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurobiology, Centro de Investigación Príncipe Felipe, Valencia, Spain.

ABSTRACT

Background: Multiple sclerosis (MS) is a complex, inflammatory and neurodegenerative disease of the central nervous system leading to long-term disability. Recent studies indicate a close association between inflammation and neurodegeneration in all lesions and disease stages of MS. Prolyl oligopeptidase (POP) is a proline-specific serine protease that cleaves several neuroactive peptides. This peptidase has been implicated in neurodegeneration, as well as in the modulation of the inflammatory response.

Methods: We examined plasma POP and the levels of an endogenous POP inhibitor from relapsing remitting MS patients and compared these with healthy controls, by monitoring the fluorescent changes due to standard fluorescently labelled substrate cleavage. We analysed the data in relationship to patient age and disease disability status.

Results: We observed a significant decrease in POP activity in plasma of relapsing remitting MS patients relative to healthy controls, coupled with an increase of POP endogenous inhibitor. The POP activity was also correlated with patient age and disability status. The lowered POP activity from plasma of MS patients could be rescued by reductants

Conclusions: The decrease in circulating POP activity measured in MS is reverted by reductants. This suggests that POP inactivation in MS might be a result of the oxidative conditions prevailing in the plasma of the diseased patients. Plasma levels of POP activity as well as those of their endogenous inhibitor are suggested as biomarkers of inflammation and oxidative stress in MS.

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Prolyl oligopeptidase activity is sensitive to chelating agents but the alternative ZIP activity is not. Prolyl endopeptidase activity in plasma from healthy controls collected in the presence of citrate (gray bars) or EDTA (white bars). A. Z-Pro-Prolinal insensitive prolyl endopeptidase (ZIP). B. POP activity, *, p-value = 0.037. n = 4. C. Effect of increasing concentrations of citrate (-▲-) or EDTA (-■-) on human recombinant purified POP activity. Concentrations of 1.8 mg/ml EDTA or 3.2 mg/ml citrate (arrows) were used in the experiments shown in A. and B.
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Figure 1: Prolyl oligopeptidase activity is sensitive to chelating agents but the alternative ZIP activity is not. Prolyl endopeptidase activity in plasma from healthy controls collected in the presence of citrate (gray bars) or EDTA (white bars). A. Z-Pro-Prolinal insensitive prolyl endopeptidase (ZIP). B. POP activity, *, p-value = 0.037. n = 4. C. Effect of increasing concentrations of citrate (-▲-) or EDTA (-■-) on human recombinant purified POP activity. Concentrations of 1.8 mg/ml EDTA or 3.2 mg/ml citrate (arrows) were used in the experiments shown in A. and B.

Mentions: As repeated cycles of freeze-thawing inactivates POP [30], all the samples were analysed after only one freezing cycle. Plasma aliquots (25 μl) were added to each well of a 96-well plate which contained 220 μl 100 mM of sodium phosphate pH 7.0 and pre-incubated 30 min at 30°C. Then, 5 μl of the substrate Z-Gly-Pro-amino methyl coumarin (Z-GP-AMC, 10 mM, Bachem, Wheil am Rhein, Germany) were added and the activity was assayed by measuring the increase of fluorescence released from amino methyl coumarin (AMC). The measures were taken every minute over a time course of 90 min at 30°C. Two enzymes account for the total prolyl endopeptidase detected in plasma [31]. One is resistant to specific POP inhibitors and is referred to as Z-Pro-Prolinal (ZPP)-insensitive prolyl endopeptidase, or ZIP activity. A second, POP activity, corresponds to the fraction sensitive to the inhibitor. Accordingly, we report POP activity as the difference between the total activity and the fraction resistant to 50 nM of ZPP (Fig. 1).


Prolyl oligopeptidase is inhibited in relapsing-remitting multiple sclerosis.

Tenorio-Laranga J, Coret-Ferrer F, Casanova-Estruch B, Burgal M, García-Horsman JA - J Neuroinflammation (2010)

Prolyl oligopeptidase activity is sensitive to chelating agents but the alternative ZIP activity is not. Prolyl endopeptidase activity in plasma from healthy controls collected in the presence of citrate (gray bars) or EDTA (white bars). A. Z-Pro-Prolinal insensitive prolyl endopeptidase (ZIP). B. POP activity, *, p-value = 0.037. n = 4. C. Effect of increasing concentrations of citrate (-▲-) or EDTA (-■-) on human recombinant purified POP activity. Concentrations of 1.8 mg/ml EDTA or 3.2 mg/ml citrate (arrows) were used in the experiments shown in A. and B.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2864237&req=5

Figure 1: Prolyl oligopeptidase activity is sensitive to chelating agents but the alternative ZIP activity is not. Prolyl endopeptidase activity in plasma from healthy controls collected in the presence of citrate (gray bars) or EDTA (white bars). A. Z-Pro-Prolinal insensitive prolyl endopeptidase (ZIP). B. POP activity, *, p-value = 0.037. n = 4. C. Effect of increasing concentrations of citrate (-▲-) or EDTA (-■-) on human recombinant purified POP activity. Concentrations of 1.8 mg/ml EDTA or 3.2 mg/ml citrate (arrows) were used in the experiments shown in A. and B.
Mentions: As repeated cycles of freeze-thawing inactivates POP [30], all the samples were analysed after only one freezing cycle. Plasma aliquots (25 μl) were added to each well of a 96-well plate which contained 220 μl 100 mM of sodium phosphate pH 7.0 and pre-incubated 30 min at 30°C. Then, 5 μl of the substrate Z-Gly-Pro-amino methyl coumarin (Z-GP-AMC, 10 mM, Bachem, Wheil am Rhein, Germany) were added and the activity was assayed by measuring the increase of fluorescence released from amino methyl coumarin (AMC). The measures were taken every minute over a time course of 90 min at 30°C. Two enzymes account for the total prolyl endopeptidase detected in plasma [31]. One is resistant to specific POP inhibitors and is referred to as Z-Pro-Prolinal (ZPP)-insensitive prolyl endopeptidase, or ZIP activity. A second, POP activity, corresponds to the fraction sensitive to the inhibitor. Accordingly, we report POP activity as the difference between the total activity and the fraction resistant to 50 nM of ZPP (Fig. 1).

Bottom Line: We observed a significant decrease in POP activity in plasma of relapsing remitting MS patients relative to healthy controls, coupled with an increase of POP endogenous inhibitor.The lowered POP activity from plasma of MS patients could be rescued by reductants The decrease in circulating POP activity measured in MS is reverted by reductants.This suggests that POP inactivation in MS might be a result of the oxidative conditions prevailing in the plasma of the diseased patients.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurobiology, Centro de Investigación Príncipe Felipe, Valencia, Spain.

ABSTRACT

Background: Multiple sclerosis (MS) is a complex, inflammatory and neurodegenerative disease of the central nervous system leading to long-term disability. Recent studies indicate a close association between inflammation and neurodegeneration in all lesions and disease stages of MS. Prolyl oligopeptidase (POP) is a proline-specific serine protease that cleaves several neuroactive peptides. This peptidase has been implicated in neurodegeneration, as well as in the modulation of the inflammatory response.

Methods: We examined plasma POP and the levels of an endogenous POP inhibitor from relapsing remitting MS patients and compared these with healthy controls, by monitoring the fluorescent changes due to standard fluorescently labelled substrate cleavage. We analysed the data in relationship to patient age and disease disability status.

Results: We observed a significant decrease in POP activity in plasma of relapsing remitting MS patients relative to healthy controls, coupled with an increase of POP endogenous inhibitor. The POP activity was also correlated with patient age and disability status. The lowered POP activity from plasma of MS patients could be rescued by reductants

Conclusions: The decrease in circulating POP activity measured in MS is reverted by reductants. This suggests that POP inactivation in MS might be a result of the oxidative conditions prevailing in the plasma of the diseased patients. Plasma levels of POP activity as well as those of their endogenous inhibitor are suggested as biomarkers of inflammation and oxidative stress in MS.

Show MeSH
Related in: MedlinePlus