Synthesis of the glycosylated polypeptide chain of an inducible costimulator on T-cells

Abstract

The glycoprotein AILIM/ICOS (Activation inducible lymphocyte immunomediately molecule/Inducible co-stimulator) on T-cells was identified in 1998 as a member of the CD28/CTLA4 family. The three-dimensional structure of the AILIM/ICOS extracellular domain has not been solved, and therefore we have examined the preparation of homogeneous glycosylated polypeptide chains of this domain having two homogeneous N-linked complex type oligosaccharides for use in folding experiments. To synthesize the glycosylated whole polypeptide chain of the AILIM/ICOS extracellular domain, the target polypeptide chain was divided into four segments, each containing a cysteine residue. Those peptide segments were synthesized by conventional SPPS, followed by thioesterification of the C-terminus. The oligosaccharide moiety, a biantennary complex type disialyloligosaccharide, was attached to the cysteine thiol in the peptide backbone using the haloacetamide method. These peptides, as well as a glycosylated peptide, were sequentially coupled by use of native chemical ligation. This process successfully afforded the desired polypeptide chain having homogeneous oligosaccharides.