Chemical Synthesis of Intentionally Misfolded Homogeneous Glycoprotein: A Unique Approach for the Study of Glycoprotein Quality Control

Abstract

Biosynthesis of glycoproteins in the endoplasmic reticulum employs a quality control system, which discriminates and excludes misfolded malfunctional glycoproteins from a correctly folded one. As chemical tools to study the glycoprotein quality control system, we systematically synthesized misfolded homogeneous glycoproteins bearing a high-mannose type oligosaccharide via oxidative misfolding of a chemically synthesized homogeneous glycopeptide. The endoplasmic reticulum folding sensor enzyme, UDP-glucose:glycoprotein glucosyltransferase (UGGT), recognizes a specific folding intermediate, which exhibits a molten globule-like hydrophobic nature.

Studies on the Precise Chemical Synthesis of Human Glycoproteins

“…Therefore, our research group concluded that UGGT recognizes these hydrophobic surfaces formed on the misfolded glycoprotein surface. 38 Our chemical approach confirmed that UGGT indeed recognizes the characteristic nature of misfolded glycoproteins, and this finding enabled us to consider how UGGT recognizes the glycoprotein folding intermediates in which hydrophobic amino acids may be exposed on the glycoprotein. There are many folding intermediates, such as the random coil structure 46, native form folded 48, misfolded form 50, and heavily aggregated 51 in the course of the folding process ( Figure 9A), and some of these folding intermediates are in equilibrium with each other.…”

“…Segment 1 (1 33 position), the peptide-α-thioester, and segment 2 (3472 position), a glycopeptide bearing a high-mannose-type oligosaccharide at the 36 position, were successfully synthesized by Boc-SPPS and Fmoc-SPPS, respectively, and subsequent NCL yielded the target glycosylpolypeptide 42 ( Figure 8A). 38 All of the synthetic intermediate and the target glycosylpolypeptide 42 were analyzed by ESI-mass spectrometry.…”

“…For the folding experiments of glycosylpolypeptide, our research group employed Anfinzen's protocol using 1.0 M guanidineHCl and cysteinecystine redox conditions to obtain native IL8 under thermodynamically controlled folding processes (data not shown), 38 while a kinetic folding process ( Figure 8C) in the absence of the redox condition yielded the misfolded IL8 43, 44, and 45 due to the shuffling of the disulfide bond formation along with the correctly folded IL8 41.…”

Chemical Approaches to Elucidate Enzymatic Profiles of UDP-Glucose: Glycoprotein Glucosyltransferase

“…13) Recently, Kajihara's group reported the synthesis of interleukin-8 and crambin carrying M9 glycan (M9-IL8 14) and M9-crambin 15) ) by the NCL method. The ability of UGGT to discriminate glycoprotein's folding status was confirmed by using conformers of M9-IL8 that were prepared by swapping of the disulfide linkage.…”

Deciphering the Roles of Glycan Processing in Glycoprotein Quality Control through Organic Synthesis

“…More recently, Kajihara's group achieved the synthesis of an intentionally misfolded glycoprotein as a structurally defined, homogeneous substrate of UGGT. 51) They selected IL-8 as a model, modified by M9 at Asn36 (M9-IL-8). Synthesis was achieved by native chemical ligation of two segments, a 1-33 thioester and a 34-72 glycopeptide.…”