Physicochemical characterization and in vitro interaction with brain capillary endothelial cells of artificially monoacylated ribonucleases A

Abstract

SUMMARYAcylated proteins play a crucial role in cell physiology because of their increased interaction with membranes. Their isolation is difficult as a consequence of their low cellular concentration and their chemical preparation is problematic due to solubility problems. Through the use of reversed micelles, we produced tens of milligrams of acylated ribonucleases A, chosen as a model, purified them by semi-preparative high performance liquid chromatography (HPLC) and characterized them by analytical HPLC, capillary electrophoresis, mass spectrometry, peptide mapping, Edman degradation and enzyme activity. We next scrutinized the interaction with an in vitro blood-brain barrier model and demonstrated that palmitoylated and stearoylated ribonucleases A are transported from one compartment to the other across the cellular monolayer, in contrast to the native enzyme.

Improved Acylation Method Enables Efficient Delivery of Functional Palmitoylated Cystatin into Epithelial Cells

“…The observed inhibitory activity of palmitoylated protein in the cell, which is in contrast to its inactivity in in vitro BANA assay, might be explained by its activation after an enzymatic hydrolysis of palmitoyl groups or deaggregation of possible aggregates. According to the literature data, acylated proteins can cross the cell membrane (2,4,6), but endocytosis is another possible way for cell internalization of proteins. Cystatin loaded nanoparticles were shown to be internalized in this manner (25).…”

“…The modified proteins exhibit improved transport properties and at the same time retain their biological activity (2–5). A single fatty acid chain has been reported to be sufficient for improving transmembrane crossing significantly (2,6).…”