Towards glycoprotein synthesis via chemical ligation

Abstract

Glycosylation, the most ubiquitous post-translational modification, exists in more than half of mammalian cell proteins. The diverse carbohydrate decorations in glycoproteins provide a variety of bio-functions, and are believed to play pivotal roles in many complex biological processes, including fertilization, immune surveillance, and inflammatory response. Hence, elucidation of the glycan function on proteins is crucial for understanding these biological processes. However, glycoproteins obtained from natural and recombinant approaches are normally inseparable mixtures of glycoforms. In general, heterogeneity issues associated with proteins bearing post-translational modifications make it difficult to correlate structure to function at a molecular level. Protein chemical synthesis provides a means to generate homogeneous proteins with site-specific and structure-defined natural or unnatural modifications, which is the way to solve the heterogeneity problem in the biochemical studies. Adiponectin is a circulating glycoprotein mainly produced by adipocytes. It exhibits insulin-sensitizing, anti-inflammatory, anti-atherogenic, proapoptotic, and antiproliferative effects. The metabolic regulation effects of adiponectin, that is, the governance of glucose and lipid metabolism by decreasing the hepatic glucose output and increasing fatty acid oxidation, and the enhancement of insulin sensitivity in liver and skeletal muscle, are quite notable. Although the outstanding properties of human adiponectin render it as an attractive and prospective therapeutic tool for the treatment of malignancies and metabolic syndromes, the extremely difficult accessibility of adiponectin has strongly limited its application. To overcome this obstacle, we undertook a serine/threonine chemical ligation approach to synthesize the homogenously glycosylated adiponectin collagenous domains with site-specifically installed glycans. Herein, we report a feasible and scalable strategy to chemically synthesize glycosylated adiponectin collagenous domains with all possible 15 glycoform combinations. This achievement has allowed us to define the biological role of the glycosylated adiponectin collagenous domains and further correlate the glycosylation pattern to function. In addition, we employed the Serine/Threonine/Cysteine (STC) ligation intermediates to tackle the self-assembling problem in “difficult peptide” synthesis, because the STC ligation intermediate kink structure is similar to the pseduproline. This STC ligation masking strategy resulted in the first successful chemical synthesis of programmed cell death protein 1 extracellular domain (PD-1). This homogeneous chemically synthesized PD-1 would provide an opportunity to further disclose the unsolved low responding yield problem in clinical PD-1 targeting antibody treatment. A key feature of this strategy is to introduce a temporal protecting group by simply applying STC ligation. Notably, the introduced cysteine ligation intermediate can survive in further transformations, including native chemical ligation, thiazoline deprotection and desulfurization. Therefore, the combination of this new strategy and chemical ligation approaches could pave the way to the synthesis of challenging glycoproteins We believed that this work will lay a foundation for the future synthesis of various immune cell receptors with self-assembling feature and other difficult peptides or proteins. In summary, we report the first chemical synthesis of glycosylated adiponectin collagenous domains with all possible 15 glycoform combinations and PD-1 extracellular domain via chemical ligation approaches. This work provides opportunity to realize the glycans function in glycoproteins: Adipoenctin and PD-1.