Chemical Synthesis of Ubiquitinated High-Mannose-Type N-Glycoprotein CCL1 in Different Folding States

Abstract

Degradation of misfolded glycoproteins by the ubiquitin-proteasome system (UPS) is a very important process for protein homeostasis. To demonstrate the accessibility toward a ubiquitinated glycoprotein probe for the study of glycoprotein degradation by UPS, we synthesized ubiquitinated glycoprotein CC motif chemokine 1 (CCL1) bearing a high-mannose type N-glycan, starting from six peptide segments. A native isopeptide linkage was constructed using δ-thiolysine (thioLys)-mediated chemical ligation. CCL1 glycopeptide with a high-mannose-type N-glycan as well as a δ-thioLys residue was synthesized chemically. The chemical ligation between δ-thioLys-containing glycopeptide and ubiquitin-α-thioester successfully yielded a ubiquitinated glycopeptide with a native isopeptide bond after desulfurization, even in the presence of a large N-glycan. In vitro folding experiments under reduced and redox conditions gave the desired two types of ubiquitinated glycosylated CCL1s, consisting of unfolded CCL1 and folded ubiquitin, and folded form of both CCL1 as well as ubiquitin. We achieved the chemical synthesis of a complex protein molecule that contains not only the two major post-translational modifications, ubiquitination and glycosylation, but also controlled folding states of ubiquitin and CCL1. These chemical probes could have useful applications in the study of complex ubiquitin biology and glycobiology.