Solid-phase synthesis of a biotin-labelled thiopalmitoylated myelin proteolipid protein epitope and application in the study of uptake of antigen by macrophages

Abstract

Proteolipid protein (PLP) is the most abundant protein of CNS myelin, and is posttranslationally acylated by covalent attachment of palmitic acid to cysteine residues viaa thioester linkage. It was previously shown that thiopalmitoylation of encephalitogenic PLP T-cell epitopes enhanced immune responses as well as the development and chronicity of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). To understand how the thiopalmitoylated peptides (S-palm peptide) can play a role in the development of autoreactivity in EAE and MS, the mechanism by which they are taken up by antigen presenting cells (APC) and presented to the immune system must be determined. This paper describes the solid-phase synthesis and purification of biotin-labelled thiopalmitoylated PLP(139–151) for use in studying the uptake of S-palm peptides by macrophages using flow cytometry analysis. Our aim was to obtain the labelled lipopeptide after on-resin biotinylation and palmitoylation, but, due to the reactivity of biotin towards acylation, we had to modify the conditions of thiopalmitoylation we had previously described, i.e. palmitic acid activated ester instead of palmitoyl chloride. Using flow cytometry analysis, we were able to show that the entry of S-palm peptide in the macrophages is very rapid compared to the non-palmitoylated peptide, and that the lipopeptide is taken up more efficiently into the macrophages.