Application of serine/threonine ligation to the synthesis of the glycosylated adiponectin collagenous domain and bioactive stapled peptides

Abstract

Abstract Protein glycosylation is one of the most abundant protein post-translational modifications (PTM) that account for the complexity in higher or complicated organisms. Cell surface carbohydrates are involved in many cellular events, including viral and bacterial infections and cell-cell interactions. Protein glycosylation needs to be strictly controlled. Abnormal glycosylations are often associated with many diseases such as rheumatoid arthritis and cancer metastasis. Therefore, to understand the effect of glycosylation on the protein structures and functions is valuable to reveal the principle of life and understand the pathology.

Adiponectin, which comprises of 244 amino acid residues, has direct anti-diabetic, anti-atherogenic, anti-inflammatory properties and is associated with several malignancies. The collagenous domain of adiponectin bearing post-translational modifications, including hydroxylation of lysine and glycosylation, predominately contributes to the formation and stabilization of High Molecular Weight (HMW) adiponectin. The precise role of lysine hydroxylation and glycosylation in the biosynthesis of HMW adiponectin and the role in anti-diabetic and anti-tumor biological activities are still poorly understood at this stage. To investigate this issue, the collagenous domain of adiponecin and its analogues were chemically synthesized. Our research group has recently developed chemoselective serine/threonine ligation (STL) allowing peptides to ligate at the Ser/Thr site using the side chain unprotected fragments. We have applied the STL method successfully to the chemical synthesis of adiponectin collagenous domain and its variants with different glycosylation patterns.

In addition, we investigated if the intramolecular STL could be used for the synthesis of stapled peptides, which have been developed into promising therapeutics to block protein-protein interactions (PPIs) or increase protease resistance. Many biological processes are mediated by PPIs, but discovering small drug-like molecules to target PPIs has been challenging due to the large polar interacting surface areas involved and only very shallow ligand-binding hydrophobic clefts. In this study, a series of stapled peptides have been successfully synthesized by intramolecular STL. The synthetic stapled peptides exhibit appreciable α-helicity in water away from a helix-stabilizing protein environment.

In summary, we have successfully applied STL method developed by our research group to the chemical synthesis of adiponectin collagenous domain and its analogues. And STL provides us a new strategy to form the stapled peptides, which are potential therapeutic targets.