Identification of N-homocysteinylated apolipoprotein AI in normal human serum

摘要

Background: In human serum, a portion of homocysteine (Hcy) exists as an N-linked form to the 1-amino group of proteinnlysine residues. N-homocysteinylated proteins differ structurally and functionally from native proteins. The present studynstrives to develop detection and potential semi-quantification methods for N-homocysteinylated apolipoprotein AI (N-Hcy-napoAI) in human serum.nMethods: Serum treated with or without cysteamine was supplied to isoelectric focusing (IEF) followed by an immunoblotnusing an anti-apoAI antibody. Cysteamine treatment increased the isoelectric point for N-Hcy-apoAI, but not for unmodifiednapoAI, due to the presence of -SH group(s) derived from Hcy and the absence of a cysteine residue in the apoAI molecule.nN-Hcy-apoAI was semi-quantified from the scanned immunoblot pattern via a computer.nResults: After cysteamine treatment, N-Hcy-apoAI in the serum was identified by IEF at the position with a higher pI valuencompared with intact apoAI. The reproducibility (between assays) of the semi-quantification method was 19.1% CVn(coefficientofvariation) foranaverage ratio5.9%ofN-Hcy-apoAI tothewholeapoAI inthe serum.Approximately1.0–7.4%of apoAInwas N-homocysteinylated in the serum obtained from27 healthy subjects. Neither the ratio of N-Hcy-apoAI nor its concentration,ncalculated by total apoAI concentration, indicated correlation with the so-called total (free and S-linked) Hcy concentration.nConclusions:We directly found that a portion of apoAI in the serum undergoes homocysteinylation in an N-linkage manner, andnused this to develop a potential semi-quantification method for N-Hcy-apoAI.