Cross-talk between Cys34 and lysine residues in human serum albumin revealed by N-homocysteinylation.
Protein N-homocysteinylation involves a post-translational modification by homocysteine (Hcy)-thiolactone. In humans, about 70% of circulating Hcy is N-linked to blood proteins, mostly to hemoglobin and albumin. It was unclear what protein site(s) were prone to Hcy attachment and how N-linked Hcy affected protein function. Here we show that Lys(525) is a predominant site of N-homocysteinylation in human serum albumin in vitro and in vivo. We also show that the reactivity of albumin lysine residues, including Lys(525), is affected by the status of Cys(34). The disulfide forms of circulating albumin, albumin-Cys(34)-S-S-Cys and albumin-Cys(34)-S-S-Hcy, are N-homocysteinylated faster than albumin-Cys(34)-SH. Although N-homocysteinylations of albumin-Cys(34)-SH and albumin-Cys(34)-S-S-Cys yield different primary products, subsequent thiol-disulfide exchange reactions result in the formation of a single product, N-(Hcy-S-S-Cys)-albumin-Cys(34)-SH. We also show that N-homocysteinylation affects the susceptibility of albumin to oxidation and proteolysis. The data suggest that a disulfide at Cys(34) of albumin promotes conversion of N-(Hcy-SH)-albumin-Cys(34)-SH to a proteolytically sensitive form N-(Hcy-S-S-Cys)-albumin-Cys(34)-SH, which would facilitate clearance of the N-homocysteinylated form of mercaptoalbumin.
Glowacki
Rafal
R
Department of Microbiology and Molecular Genetics, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, International Center for Public Health, Newark, New Jersey 07103, USA.
Jakubowski
Hieronim
H
D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003545:Cysteine / Q000378:metabolism*; D006710:Homocysteine / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008239:Lysine / Q000378:metabolism*; D010084:Oxidation-Reduction; D012709:Serum Albumin / Q000737:chemistry / Q000378:metabolism*
D012709:Serum Albumin; D006710:Homocysteine; D008239:Lysine; D003545:Cysteine