Immunohistochemical detection of N-homocysteinylated proteins in humans and mice.
N-homocysteinylation of epsilon-amino group of protein lysine residues by homocysteine (Hcy) thiolactone has been implicated in vascular disease in humans. We have previously generated polyclonal rabbit anti-N-Hcy-protein IgG antibodies that specifically recognize the Nepsilon-Hcy-Lys epitope on N-homocysteinylated proteins. The present work was undertaken to examine the utility of these antibodies for the immunohistochemical detection of N-homocysteinylated proteins in biological samples. We found that the rabbit antibody specifically detected N-Hcy-protein in a dot-blot assay, that the signal resulting from the reaction of the antibody with N-Hcy-protein depended on the amount of the antigen, and that the sensitivity of the assay was protein-dependent. The rabbit anti-N-Hcy-protein IgG also specifically detected Nepsilon-Hcy-Lys epitopes in human tissues, as shown by positive immunohistochemical staining of myocardium and aorta samples from cardiac surgery patients, and a lack of staining when the antibody was pre-adsorbed with N-Hcy-albumin. We also observed increased immunohistochemical staining for N-Hcy-proteins in aortic lesions from ApoE-/- mice with hyperhomocysteinemia induced by a high methionine diet, relative to ApoE-/- mice fed a control chow diet. In conclusion, polyclonal rabbit anti-N-Hcy-protein antibody can detect and monitor N-homocysteinylated proteins in human and mouse tissues with good sensitivity and specificity.
Perła-Kaján
Joanna
J
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland.
Stanger
Olaf
O
Luczak
Michał
M
Ziółkowska
Agnieszka
A
Malendowicz
Ludwik K
LK
Twardowski
Tomasz
T
Lhotak
Sárka
S
Austin
Richard C
RC
Jakubowski
Hieronim
H
D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D001057:Apolipoproteins E / Q000235:genetics; D045744:Cell Line, Tumor; D004032:Diet; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006433:Hemocyanins / Q000737:chemistry; D006710:Homocysteine / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology / Q000378:metabolism; D007150:Immunohistochemistry; D007202:Indicators and Reagents; D008297:Male; D008715:Methionine / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008875:Middle Aged; D009206:Myocardium / Q000201:enzymology / Q000378:metabolism; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism
D001057:Apolipoproteins E; D007074:Immunoglobulin G; D007202:Indicators and Reagents; D011506:Proteins; D006710:Homocysteine; D006433:Hemocyanins; D008715:Methionine; C032808:keyhole-limpet hemocyanin