Microarray-Based\nMethodology for Lipid Profiling,\nEnzymatic Activity, And Binding Assays in Printed Lipid Raft Membranes\nfrom Astrocytes and Neurons

Lipid rafts are liquid-ordered domains in which specific\nenzymes\nand receptors are located. These membrane platforms play crucial roles\nin a variety of signaling pathways. Alterations in the lipid environment,\nsuch as those elicited by oxidative stress, can lead to important\nfunctional disruptions in membrane proteins. Cell membrane microarrays\nhave emerged in the past decade as a powerful methodology for the\nstudy of both lipids and membrane proteins at large scales. Based\non that technology and the importance of liquid-ordered subdomains,\nwe have developed a new printed lipid raft technology with a preserved\nnative protein structure and lipid environment. To validate this technology\nand evaluate its potential for different aims, raft membrane microarrays\n(RMMAs) containing two different cell types (astrocytes and neurons)\nand three different conditions (astrocytes in control situation, metabolic\nstress, and oxidative stress) were developed. To study differences\nin lipid profiles between raft domains, the MALDI-MS assay was performed\non RMMAs. To evaluate the preservation of native protein activities\n(enzymatic activity and ligand binding) in the printed raft domains,\ndifferences in NADH oxidoreductase, GAPDH, cholinesterase activities,\nand sigma-1 and sigma-2 binding assays were performed. We demonstrate\nthe performance of this new microarray technology, adapted to membrane\nsubdomains, as valid to explore changes in lipid composition and protein\nactivities in raft domains from brain cell lines under different stress\nconditions relevant for neuropathology.