Glycosylation is one of the most important post-translational modifications (PTMs) of proteins affecting their physicochemical and biological properties. The biosynthesis of glycoproteins is a complex process that delivers mixture of the same protein sequence with different glycosylation patterns. To overcome the difficulty in the isolation of single glycoproteins from natural sources, synthetic and semi-synthetic strategies are emerging as a powerful alternative for the production of homogeneous glycoproteins. These strategies will help to study the role of carbohydrates in the biological function of glycoproteins. However, there are still limitations to obtain glycoproteins having multiple glycosylations.
Thy-1 (CD90) is a 12 kDa glycoprotein having a glypiation and three N-glycosylations sites. The protein is found on the outer leaflet of the membrane of T-cells and is speculated to participate in cell-cell and cell-matrix interactions, nerve regeneration, apoptosis, metastasis, inflammation, and fibrosis. Furthermore, this glycoprotein can act as a tumor suppressor and be a potential probe for cancer drug targeting. To study the biological function and the effect of glypiation and glycosylations on the folding and activity of Thy-1, we are working on a strategy for the synthesis of homogeneous glypiated and glycosylated Thy-1 variants.
Here, we report a strategy based on a combination of sequential native chemical ligation and chemoenzymatic techniques to obtain Thy-1 having three N-glycosylations. Three peptide fragments were designed, two of them containing N-glycosylated asparagine residues. The fragments were synthesized as peptide hydrazides as thioester precursors using solid phase peptide synthesis (SPPS). The glycosylations were introduced coupling a glycosylated asparagine building block in the specific positions. The challenging Thy-1 fragment 38-103, with two N-glycosylations, was obtained by condensation of the glycopeptide fragments (38-65) and (66-103). In addition to the assembly of the protein, we present the progress for the introduction of differentiated oligosaccharide structures on each glycosylation site using the mutant glycosynthase Endo-M and in the ligation reactions, to obtain a glypiated glycoprotein.