Probing N-Glycan Functions in Human Interleukin-17A Based on Chemically Synthesized Homogeneous Glycoforms

Abstract

N-Glycosylation represents an essential type of posttranslational modification for proteins. However, deciphering the functions of N-glycosylation remains a challenge due to the lack of analytical and biochemical methods to accurately differentiate the protein glycoforms with various intact glycans. Here we report our synthesis and evaluation of homogeneously glycosylated interleukin-17A (IL-17A), based on a synthetic approach combining solid-phase synthesis of (glyco)­peptides, chemoenzymatic glycan modification on segments, and chemical ligations. The obtained homogeneous glycoproteins allow for the demonstration of the stabilizing role of N-glycans during the folding step. A comparison of three IL-17A glycoforms in a normal human dermal fibroblast (NHDF) assay reveals dose-dependent interleukin-6-inducing activities in all cases, wherein the glycoform with sialyl undecasaccharides displays much weaker stimulatory effect than that of the GlcNAc- or GlcNAc­(β1→4)­GlcNAc-modified proteins. Further surface plasmon resonance (SPR) and hydrogen/deuterium exchange mass spectroscopic experiments confirm that the evaluated complex type N-glycan impedes the binding between IL-17A and its receptor IL-17RA. This structure–activity relationship study on glycoproteins highlights the viability of applying the de novo approach to probe the roles of N-glycans.

Nanobodies as solubilization chaperones for the expression and purification of inclusion-body prone proteins

“…3, the far-UV CD spectra of IL-17A–EPEA and IL-6–EPEA display standard absorbance peaks of β-sheet and α-helix, respectively. These results were highly consistent with the literature reports, 15,21 and verified the formation of native secondary structures of both EPEA-fused ILs.…”

Synthetic Thiol and Selenol Derived Amino Acids for Expanding the Scope of Chemical Protein Synthesis

“…Recently, β-thiolated Asp has also been used for the design and synthesis of a homogenously N-glycosylated interleukin-17A (IL-17A) with the aim to elucidate the impacts of N-glycan on protein (76-105) 105 was modified with a C-terminal alkyl thioester (Scheme 12B) (Fang et al, 2011;Li et al, 2021b). A β-thiolderived Asp was introduced to the N-terminus of segment (42-75) 104, which permits a NCL reaction to form the first half of IL-17A (1-75), whereas two orthogonally protected cysteines were adopted in preparing the N-terminal residues of segments (76-105) 105 and (106-132) 106.…”

“…The peptide assembly of each glycosylated form of IL-17A began with performing NCL between segments (75-105) 105 and (106-132) 106 and peptide hydrazine ligation between segments (1-41) 103 and (42-75) 104 in parallel to furnish the first and second halves of IL-17A. Unmasking of the thiazolidine-protected Cys76 enabled the final convergent assembly of the two main fragments (1-75) and (76-105) to provide the full-length IL-17A 102 (Li et al, 2021b). This convergent approach provided plasticity in the synthesis of discrete glycosylated forms of IL-17A since each segment could be modified and optimized independently to maximize the synthetic yield and PTM diversity.…”

“…Recently, β-thiolated Asp has also been used for the design and synthesis of a homogenously N-glycosylated interleukin-17A (IL-17A) with the aim to elucidate the impacts of N-glycan on protein folding, thermal stability, and inflammatory cytokine inducing ability ( Li et al, 2021b ). In particular, the ligation-desulfurization methodology provided a powerful means to access individual glycosylated forms of IL-17A that are otherwise difficult to recombinantly derive.…”

“…IL-17A ( 102 ) was disconnected into four peptide segments prepared through Fmoc-based SPPS method, and methionine (Met) residues were mutated to norleucines to prevent unwanted oxidation. Both segments (1-41) 103 and (42-75) 104 contained a C-terminal peptide hydrazine which serves as a latent thioester, whilst segment (76-105) 105 was modified with a C-terminal alkyl thioester ( Scheme 12B ) ( Fang et al, 2011 ; Li et al, 2021b ). A β-thiol-derived Asp was introduced to the N-terminus of segment (42-75) 104 , which permits a NCL reaction to form the first half of IL-17A (1–75), whereas two orthogonally protected cysteines were adopted in preparing the N-terminal residues of segments (76-105) 105 and (106–132) 106 .…”