Total Chemical Synthesis of Correctly Folded Disulfide-Rich Proteins Using a Removable O-Linked β-N-Acetylglucosamine Strategy

Abstract

Disulfide-rich proteins are useful as drugs or tool molecules in biomedical studies, but their synthesis is complicated by the difficulties associated with their folding. Here, we describe a removable glycosylation modification (RGM) strategy that expedites the chemical synthesis of correctly folded proteins with multiple or even interchain disulfide bonds. Our strategy comprises the introduction of simple O-linked β-N-acetylglucosamine (O-GlcNAc) groups at the Ser/Thr sites that effectively improve the folding of disulfide-rich proteins by stabilization of their folding intermediates. After folding, the O-GlcNAc groups can be efficiently removed using O-GlcNAcase (OGA) to afford the correctly folded proteins. Using this strategy, we completed the synthesis of correctly folded hepcidin, an iron-regulating hormone bearing four pairs of disulfide-bonds, and the first total synthesis of correctly folded interleukin-5 (IL-5), a 26 kDa homodimer cytokine responsible for eosinophil growth and differentiation.

DNA damage-induced YTHDC1 O-GlcNAcylation promotes homologous recombination by enhancing N6-methyladenosine binding

“…When chemical synthesis does succeed, it no doubt pinpoints the exact role of O-GlcNAc on the specific protein or peptide. For instance, when a removable glycosylation modification (RGM) method was used in total chemical synthesis, O-GlcNAc was found to improve disulfide-rich protein folding (42). In the neurodegenerative disease-causing α -synuclein, O-GlcNAc inhibits amyloid aggregation (43).…”

Total synthesis of interleukin-2 via a tunable backbone modification strategy

“…[14][15][16][17][18][19][20][21][22][23][24] In particular, protein chemical synthesis provides a unique means to generate proteins with tailor-made modications, which are difficult or impossible for expression systems. [25][26][27][28][29][30][31][32][33][34][35][36] Despite these achievements in protein chemical synthesis, the synthesis of proteins or peptides with aggregation-prone properties remains a challenging task and requires case by case analysis and study. The dilemma can be classied into two types: (a) the peptides aggregate on the resin beads during solid-phase peptide synthesis (SPPS), leading to peptide elongation failure or poor product quality; 37,38 (b) the peptide products tend to form insoluble or colloidal solids aer successful synthesis and cleavage from the resin, which makes reverse-phase high-performance liquid chromatography (RP-HPLC) purication or performing peptide ligation difficult.…”

Synthesis of disulfide surrogate peptides incorporating an ethylene glycol bridge

“…Disulfide-rich peptides play important roles in metabolic regulation and therapy as bioactive molecules 1 ( e.g. , oxytocin, conotoxin and insulin).…”