title: The anti-apoptotic activity of albumin for endothelium is inhibited by advanced glycation end products restricting intramolecular movement.
abstract: Human serum albumin (HSA) inhibits endothelial apoptosis in a highly specific manner. CNBr fragmentation greatly increases the effectiveness of this activity, suggesting that this type of protection is mediated by a partially cryptic albumin domain which is transiently exposed by intramolecular movement. Advanced glycation end-product (AGE) formation in HSA greatly reduces its intra-molecular movement. This study aimed to determine if this inhibits the anti-apoptotic activity of HSA, and if such inactivation could be reversed by CNBr fragmentation. HSA-AGE was prepared by incubating HSA with glucose, and assessed using the fructosamine assay, mass spectrometry, SDS-PAGE and fluorometry. Low levels of AGE in the HSA had little effect upon its anti-apoptotic activity, but when the levels of AGE were high and the intra-molecular movement was reduced, endothelial cell survival was also found to be reduced to levels equivalent to those in cultures without HSA or serum (p > 0.001). Survival was restored by the inclusion of native HSA, despite the presence of HSA with high levels of AGE. Also, CNBr fragmentation of otherwise inactive HSA-AGE restored the anti-apoptotic activity for endothelium. Apoptosis was confirmed by DNA gel electrophoresis, transmission electron microscopy and fluorescence-activated cell sorting analysis, and there was no evidence for direct toxicity in the HSA-AGE preparations. The results are consistent with the proposed role of intra-molecular movement in exposing the anti-apoptotic domain in HSA for endothelium. The levels of AGE formation required to inhibit the anti-apoptotic activity of HSA exceeded those reported for diabetes. Nonetheless, the data from this study seems to be the first example of reduced protein function due to AGE-restricted intra-molecular movement.
authors.affiliation: Cellular and Molecular Pathology Research Unit, Department of Oral Pathology and Oral Medicine, Westmead Centre for Oral Health, The University of Sydney, Westmead, NSW, 2145, Australia. hzoellne@mail.usyd.edu.au
authors.forename: Hans
authors.initials: H
authors.lastname: Zoellner
authors.forename: Salman
authors.initials: S
authors.lastname: Siddiqui
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Kelly
authors.forename: Heather
authors.initials: H
authors.lastname: Medbury
mesh_terms: D000418:Albumins; D017209:Apoptosis; D002478:Cells, Cultured; D003488:Cyanogen Bromide / Q000737:chemistry; D019610:Cytoprotection; D003920:Diabetes Mellitus / Q000378:metabolism; D004727:Endothelium / Q000166:cytology / Q000378:metabolism; D017127:Glycation End Products, Advanced / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D046529:Microscopy, Electron, Transmission; D011485:Protein Binding; D012709:Serum Albumin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000418:Albumins; D017127:Glycation End Products, Advanced; D012709:Serum Albumin; D003488:Cyanogen Bromide