PON-1 carbamylation is enhanced in HDL of uremia patients

Chiz-Tzung Chang ^a,b,c,1, Yun-Ping Lim ^d,e,f,1, Chi-Wen Lee ^g,

Hsin-Yi Liao ^h, Feng-Yu Chen ^c, Chia-Ming Chang ^c, Feng-Yao Tang ^g, Chao-Yuh Yang ^c, Chao-Jung Chen ^h,i,*

^a College of Medicine, China Medical University, Taichung, Taiwan

^b Division of Nephrology, China Medical University Hospital, Taichung, Taiwan

^c Cardiovascular Research Laboratory, China Medical University Hospital, Taichung, Taiwan

^d Department of Pharmacy, College of Pharmacy, China Medical University, Taichung, Taiwan

^e Department of Internal Medicine, China Medical University Hospital, Taichung, Taiwan

^f Department of Medical Research, China Medical University Hospital, Taichung, Taiwan

^g Department of Nutrition and Institute of Nutrition, China Medical University, Taiwan

^h Proteomics Core Laboratory, Department of Medical Research, China Medical University Hospital, Taichung, Taiwan

ⁱ Graduate Institute of Integrated Medicine, China Medical University, Taichung, Taiwan

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 H₂O₂. 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.