Investigation of the effect of modification of carbonic anhydrase II with homocysteine and methylglyoxal on enzyme activity and antigenic properties

Carbonic anhydrases (CAs) are metalloenzymes that reversibly catalyse the hydration of carbon dioxide. Studies on posttranslational modifications (PTMs) of the CA enzyme family have been evaluated mainly based on databases. Although there is a clear correlation between PTMs and the function of CAs, many modification events have not been adequately studied experimentally. In this study, we aimed to investigate the effects of in vitro modifications of human carbonic anhydrase II (hCAII) isoenzyme with homocysteine (HCY) and methylglyoxal (MGO) on the activity and antigenic properties of the enzyme. For this purpose, pure hCAII isoenzyme was purified from human erythrocytes by affinity chromatography. The obtained pure hCAII isoenzyme was modified in vitro with HCY and MGO. The presence of modifications was confirmed by Western blotting. Hydratase and esterase activities of the modified hCAII isoenzymes were measured. To determine the antigenic properties of the modified hCAII isoenzymes, Balb-c mice were immunised with these enzymes and antibody titres in mouse sera were measured by ELISA. When the enzyme activities were measured, modifications of hCAII enzyme with HCY and MGO caused dose-related inhibition of the enzyme activity due to N-homocysteinylation, while the glycation-dependent effect caused significant activation at low modification dose and inhibition at high modification dose. It was observed that modification with MGO induced a high antigenic response in Balb-c mice, while HCY did not alter antigenicity. In conclusion, modification with MGO affected both the activity and antigenic properties of hCAII enzyme, while modification with HCY had a significant effect on the activity of the enzyme, but no antigenic effect was determined.