Degradation of oxidative stress-induced denatured albumin in rat liver endothelial cells.
We previously identified conformationally denatured albumin (D2 and D3 albumin) in rats with endotoxicosis (Bito R, Shikano T, and Kawabata H. Biochim Biophys Acta 1646: 100-111, 2003). In the present study, we attempted first to confirm whether the denatured albumins generally increase in conditions of oxidative stress and second to characterize the degradative process of the denatured albumin using primary cultured rat liver endothelial cells. We used five models of oxidative stress, including endotoxicosis, ischemic heart disease, diabetes, acute inflammation, and aging, and found that serum concentrations of D3 albumin correlate with the serum levels of thiobarbituric acid-reactive substance (R = 0.87), whereas the concentrations of D2 albumin are 0.52. Ligand blot analysis showed that the D3 albumin binds to gp18 and gp30, which are known endothelial scavenger receptors for chemically denatured albumin. Primary cultured rat liver endothelial cells degraded the FITC-D3 albumin, and the degradation rate decreased to approximately 60% of control levels in response to anti-gp18 and anti-gp30 antibodies, respectively. An equimolar mixture of these antibodies produced an additive inhibitory effect on both uptake and degradation, resulting in levels approximately 20% those of the control. Furthermore, filipin and digitonin, inhibitors of the caveolae-related endocytic pathway, reduced the FITC-D3 albumin uptake and degradation to <20%. Laser-scanning confocal microscopic observation supported these data regarding the uptake and degradation of D3 albumin. These results indicate that conformationally denatured D3 albumin occurs generally under oxidative stress and is degraded primarily via gp18- and gp30-mediated and caveolae-related endocytosis in liver endothelial cells.
Bito
Ryuji
R
Laboratory for Nutritional Biochemistry, School of Agriculture, Meiji University, Kawasaki City, Kanagawa 214-8571, Japan.
Hino
Sayaka
S
Baba
Atsushi
A
Tanaka
Miharu
M
Watabe
Haruka
H
Kawabata
Hiroaki
H
D000818:Animals; D000906:Antibodies / Q000276:immunology; D021941:Caveolae / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D018384:Oxidative Stress / Q000502:physiology*; D011485:Protein Binding / Q000502:physiology; D011489:Protein Denaturation / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D012709:Serum Albumin / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D012709:Serum Albumin; C078405:albumin-binding protein gp18; C078404:albumin-binding protein gp30