Total chemical synthesis of homogeneous folded and misfolded N-glycosylated IL-8 for investigation of UGGT recognition
The N-glycosylation is known to plays a pivotal role in glycoprotein quality control system, especially by recognition with UDP-glucose glycoprotein glucosyltransferase (UGGT) enzyme as a folding sensor. UGGT can recognize misfolded glycoprotein with high-mannose glycan and then add a glucose as tag of misfolded glycoprotein in the endoplasmic reticulum (ER). However, how UGGT recognize and interact with misfolded glycoprotein was poorly understood due to lack of homogeneous misfolded glycoprotein for investigation. In 2012, the Kajihara group chemically synthesized homogeneous misfolded glycoprotein interleukin-8 (IL-8) 97 for the elucidation of recognition pattern by UGGT.[32] In this research, the author chose IL-8 97 as a synthetic target due to its well-studied tertiary structure, potential for disulfide shuffling and established synthetic strategy (Fig.11). The Asn36, which is not N-glycosylated in nature, was used for incorporation of mannose-9 (M9)-high mannose type N-glycan. The chemical synthesis of glycosylated IL-8 96 was achieved by NCL between peptide 94 (1-33) and glycopeptide 95 (34-72), which were prepared by SPPS.
In order to obtain the intentionally misfolded glycoprotein IL-8 for UGGT recognition investigation, the full-length IL-8 96 was treated with oxidative folding condition in the absence of redox reagents (cysteine and cystine). Monitored by LC/MS, four products were obtained which were assigned to be native structure 97, misfolded structure 98 and 99, and a misfolded dimer 100. (Fig.11) With correctly folded and misfolded glycoproteins in hand, finally, the binding assays with UGGT were examined and monitored with LC/MS by addition of one glucose residue. The results indicated that misfolded dimer 100 showed highest glucosyl transfer activity with UGGT, the misfolded glycoprotein 98, 99 showed moderate glucosyl transfer activity than folded glycoprotein 97 as we expected. This result was also in accordance with the protein surface hydrophobicity order (dimer＞misfolded＞folded). In this research, the authors successfully investigated the recognition pattern of UGGT by using chemically synthesized homogeneous intentionally misfolded glycoprotein.