Identification of N-homocysteinylated apolipoprotein AI in normal human serum.
BACKGROUND
In human serum, a portion of homocysteine (Hcy) exists as an N-linked form to the epsilon-amino group of protein lysine residues. N-homocysteinylated proteins differ structurally and functionally from native proteins. The present study strives to develop detection and potential semi-quantification methods for N-homocysteinylated apolipoprotein AI (N-Hcy-apoAI) in human serum.


METHODS
Serum treated with or without cysteamine was supplied to isoelectric focusing (IEF) followed by an immunoblot using an anti-apoAI antibody. Cysteamine treatment increased the isoelectric point for N-Hcy-apoAI, but not for unmodified apoAI, due to the presence of -SH group(s) derived from Hcy and the absence of a cysteine residue in the apoAI molecule. N-Hcy-apoAI was semi-quantified from the scanned immunoblot pattern via a computer.


RESULTS
After cysteamine treatment, N-Hcy-apoAI in the serum was identified by IEF at the position with a higher pI value compared with intact apoAI. The reproducibility (between assays) of the semi-quantification method was 19.1% CV (coefficient of variation) for an average ratio 5.9% of N-Hcy-apoAI to the whole apoAI in the serum. Approximately 1.0-7.4% of apoAI was N-homocysteinylated in the serum obtained from 27 healthy subjects. Neither the ratio of N-Hcy-apoAI nor its concentration, calculated by total apoAI concentration, indicated correlation with the so-called total (free and S-linked) Hcy concentration.


CONCLUSIONS
We directly found that a portion of apoAI in the serum undergoes homocysteinylation in an N-linkage manner, and used this to develop a potential semi-quantification method for N-Hcy-apoAI.
Ishimine
N
N
Analytical Laboratory Chemistry, Graduate School of Health Care Sciences, Tokyo Medical and Dental University, Tokyo.
Usami
Y
Y
Nogi
S
S
Sumida
T
T
Kurihara
Y
Y
Matsuda
K
K
Nakamura
K
K
Yamauchi
K
K
Okumura
N
N
Tozuka
M
M
D016632:Apolipoprotein A-I / Q000097:blood* / Q000378:metabolism*; D050197:Atherosclerosis / Q000097:blood; D015415:Biomarkers / Q000097:blood / Q000378:metabolism; D001774:Blood Chemical Analysis / Q000379:methods*; D006710:Homocysteine / Q000031:analogs & derivatives / Q000378:metabolism*; D006801:Humans; D008075:Lipoproteins, HDL / Q000378:metabolism
D016632:Apolipoprotein A-I; D015415:Biomarkers; D008075:Lipoproteins, HDL; D006710:Homocysteine; C007957:homocysteine thiolactone