Membrane Permeability of Acylated Cystatin Depends on the Fatty Acyl Chain Length

Abstract

Hydrophobization of proteins, such as chemical acylation, has been recognized as an efficient method for improving their membrane permeability. In this research, chicken cystatin, a model protein inhibitor of cysteine proteinases, was acylated with fatty acyl residues of 6-18 carbon atoms. The chemical modification was performed using fatty acyl chloride dispersion in aqueous acetonitrile solution. The reaction products were analyzed by capillary electrophoresis, SDS-PAGE and isoelectric focusing. In vitro inhibitory activity was determined by N-benzoyl-D,L-arginine-beta-naphthylamide assay and membrane permeability properties of non-acylated and acylated cystatin by measuring its efficiency to inhibit intracellular cathepsin B in MCF-10A neo T cells. The experiments showed that acylated cystatin quickly internalized into the cells and effectively inhibited cathepsin B. In contrast, non-acylated cystatin did not cause inhibition as it was unable to enter the cell. The permeability enhancement effect was shown to depend on the length of the attached fatty acyl chain as the strongest inhibition was caused by cystatin acylated with stearoyl chloride. In addition, chemical modification did not influence the protein's immunogenicity.