Two analytical methods to study the interaction of AGEs with cell surface proteins.
Advanced glycation end products (AGEs) are sugar-modified proteins that are known to appear in vivo and are suspected to be involved in the pathogenesis of several diseases. Although different cellular responses to AGEs can be measured in cell culture studies, knowledge about the nature of AGE-binding and their cell surface receptors is poor. In the present paper a method for the purification of AGE-binding proteins from membrane fractions derived from different rat organs as well as a method for assaying the binding of fluorescein labelled AGEs to the surface of cells of different cell lines are described. The presence of more than 10 proteins interacting with AGEs could be shown in membrane fractions obtained from rat organs. Additionally, binding of AGE-modified BSA to different cells could be shown using fluorescence-labelled ligands in a flow cytometric approach. The presented methods provide an option to isolate AGE-interacting proteins which is a precondition for the identification of these proteins. Furthermore, the measurement of AGE-binding to cell surfaces bears the potential to gain a deeper understanding about the nature of AGE-binding to cell surface proteins and might be applied as a preliminary test before performing cell culture studies about AGE effects.
Schmitt
Annett
A
Interdisciplinary Centre of Clinical Research (IZKF) at the Faculty of Medicine at the University of Leipzig, Inselstr. 22, 04103 Leipzig, Germany. annett_schmitt@web.de
Meiners
Ina
I
Schmitt
Johannes
J
Nöller
Joachim
J
Ihling
Christian
C
Münch
Gerald
G
Sinz
Andrea
A
Nieber
Karen
K
D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D005434:Flow Cytometry / Q000379:methods; D019793:Fluorescein; D017127:Glycation End Products, Advanced / Q000378:metabolism*; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D017208:Rats, Wistar; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic / Q000302:isolation & purification / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
D002352:Carrier Proteins; D017127:Glycation End Products, Advanced; D008565:Membrane Proteins; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic; D012710:Serum Albumin, Bovine; D019793:Fluorescein