Prolyl oligopeptidase is inhibited in relapsing-remitting multiple sclerosis

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.

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.

Figure 2

Prolyl oligopeptidase activity in plasma and the variation with age of healthy subject and RR-MS patients. POP activity in plasmas from healthy controls (white bar, n = 17) and RR-MS patients (gray bar, n = 11) (A.) ***, p-value = 0.0076 t-student unpaired test. B. Relationship between POP activity and subject age for both healthy controls and RR-MS patients. Correlation for healthy controls, n = 17, Pearson r = -0.5153, p-value (two-tailed) = 0.03, R-square = 0.2656.

Figure 3

Prolyl oligopeptidase level and MS disability status. Correlation between plasma POP activity with the EDSS score from RR-MS patients; n = 10, Pearson r = -0.74, p-value (two-tailed) = 0.0144, R-square = 0.5476.

Figure 4

Levels of endogenous prolyl oligopeptidase inhibitor, and its relation with prolyl oligopeptidase activity, in plasma from healthy and RR-MS patients. A. Inhibitory effect of plasma (25 μl) from healthy controls (white bar, n = 17) and RR-MS patients (gray bar, n = 11) on the activity of pure POP (basal activity 100 nmol/min/mg of POP), ***, p-value = 0.0002. B. Relationship between plasma POP activity, and endogenous POP inhibitor content, expressed as % of inhibition of pure POP (data from part A.) in healthy controls (n = 17) and RR-MS patients (n = 11). No statistically significant correlation was found.

Figure 5

Effect of DTT on prolyl oligopeptidase levels in plasma from healthy and RR-MS patients. POP activity in plasma from healthy controls (white bar) and RR-MS patients (gray bar) after plasma pre-incubation in the absence, or presence (diagonal pattern), of DTT (5 mM). n = 10, p-values, **, 0.0024, ***, 0.0004.