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. 2009 May 30;14(4):575–586. doi: 10.2478/s11658-009-0021-5

The anti-apoptotic activity of albumin for endothelium is inhibited by advanced glycation end products restricting intramolecular movement

Hans Zoellner 1,✉, Salman Siddiqui 1, Elizabeth Kelly 1, Heather Medbury 2
PMCID: PMC6275583  PMID: 19484197

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.

Key words: Apoptosis, Cryptic domain, Endothelium, HSA, HSA-AGE

Full Text

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Abbreviations used

AGE

advanced glycation end product

HSA

human serum albumin

HUVEC

human umbilical vein endothelial cells

PBS

phosphate-buffered saline

SCS

supplemented calf serum

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