PON-1 carbamylation is enhanced in HDL of uremia patients

Abstract

High-density lipoprotein (HDL) carbamylation has been known in uremia patients. Paraoxonase-1 (PON-1) is an important HDL protein responsible for HDL anti-oxidant, arylesterase and lactonase activities. PON-1 carbamylation in uremic HDL has never been explored. We isolated HDL from uremia patients and control healthy subjects for study. Sandwich ELISA was used to estimate carbamylated PON-1 protein expression in HDL, and nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) was applied to identify the amino acid in PON-1 carbamylated. PON-1 enzyme activities were estimated by substrates conversion method. HDL anti-oxidant activity was gauged by fluorescence changes of indicator dye in the presence of H 2 O 2 . Our study results proved that the degree of PON-1 carbamylation was higher in uremic HDL than in control HDL. Sandwich ELISA study showed that carbamylated PON-1 concentration in uremic HDL was 1.49 ± 0.08 fold higher than that in HDL from controls (p < 0.05). The nanoLC-MS/MS showed that the carbamylation of lysine 290 (K290) of PON-1, a residue adjacent to PON-1 activity determining site, was detected in uremic HDL but not detected in control HDL. K290 carbamylation leads to local conformation changes that reduce accessible solvent accessibility. The HDL paraoxonase, arylesterase, and lactonase activities were all significantly lower in uremia patients than in control subjects. Additionally, HDL anti-antioxidant ability was also lower in uremia patients. Carbamylation of PON-1 in uremia patients could be one of the factors in impairing PON-1 enzyme activities and HDL anti-oxidation function.

Paraoxonase 1 and atherosclerosis‐related diseases

“…Given that free SH‐groups have the potential to neutralize oxidized phospholipids and cholesterol esters, decreased PON1 in serum, as well as in isolated HDL lipoprotein fractions may lead to oxidative damage of other lipoproteins, including HDL particles . In addition, increased uremic toxins could be an additional reason for decreased PON1 concentration in renal patients due to interference with the synthesis of PON1 itself . Furthermore, our recent study revealed significantly reduced PON3 in patients with chronic kidney disease, as well as in patients on hemodialysis .…”

“…Previous studies have demonstrated that certain uremic toxins together with increased oxidative stress and inflammation can alter HDL structure, and consequently reduce PON1 activity in renal patients . Namely, modification of PON1 by oxidation, glycation, or carbamylation significantly attenuates its activity and can lead to its dissociation from HDL . Since the kidneys play a crucial role in metabolism and excretion of uremic toxins, impaired renal function and accumulation of these molecules could significantly influence PON1's activity .…”

“…82 Namely, modification of PON1 by oxidation, glycation, or carbamylation significantly attenuates its activity and can lead to its dissociation from HDL. 78,83 Since the kidneys play a crucial role in metabolism and excretion of uremic toxins, impaired renal function and accumulation of these molecules could significantly influence PON1's activity. 84 This seemed to be the case as increased POase of PON1 after hemodialysis was found.…”

Analytically validated protein biomarkers of chronic pancreatitis and pancreatic cancer for potential clinical diagnosis with mass spectrometry

“…Recently, MALDI‐TOF MS has been approved as an in vitro diagnostic (IVD) device for bacterial identification in hospitals . Furthermore, MS can detect protein modifications as useful biosignatures or detect a panel of biomarkers to improve their diagnosis accuracy. The multiplexing capabilities of MRM by nanoLC/MS/MS can simultaneously quantify multiple protein biomarkers during a single MS analysis to improve the diagnosis accuracy and to save time, cost, as well as sample amount.…”

“…76 Recently, MALDI-TOF MS has been approved as an in vitro diagnostic (IVD) device for bacterial identification in hospitals. 77 Furthermore, MS can detect protein modifications as useful biosignatures 78,79…”