The role of interchain disulfide bond in a recombinant human interleukin-17A variant

Wu, Bingyuan; Muzammil, Salman; Jones, Brian H.; Nemeth, Jennifer F.; Janecki, Dariusz; Baker, Audrey; Elloso, M. Merle; Naso, Michael F.; Carton, Jill M.; Taudte, Susann

doi:10.1016/j.cyto.2013.11.007

Cited by 3 publications

(4 citation statements)

References 12 publications

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“…Drawing inspirations from the folding recipe for a recombinant variant of IL-17A utilized by Wu and co-workers, glycerol and concentrated arginine were applied in the buffer as additives to stabilize the folded proteins and intermediates. We were delighted to find that no precipitation of aggregated proteins was observed using a folding buffer containing cysteine/cystamine, and the formation of dimeric proteins was significantly improved, as indicated by SDS-PAGE (Figure , and see Supporting Information, Pages S46, Table S3, entries 4–5).…”

Section: Results and Discussionmentioning

confidence: 99%

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

Zhang

et al. 2021

J. Am. Chem. Soc.

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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.

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Section: Results and Discussionmentioning

confidence: 99%

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

Zhang

et al. 2021

J. Am. Chem. Soc.

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“…The full length IL-17A with signal peptide removed (residues 24–155, UniProtKB accession Q16552) was expressed in E. coli and refolded from inclusion bodies 23 32 . The refolded protein was purified by size exclusion chromatography and subjected to RP-HPLC for separation of the doubly disulfide-linked IL-17A homodimer from the dissociable non-covalent dimer 32 . The covalent linked IL-17A dimer was used in co-crystallization and compound 4 labeling studies.…”

Section: Methodsmentioning

confidence: 99%

Binding site elucidation and structure guided design of macrocyclic IL-17A antagonists

Liu

Dakin

et al. 2016

Sci Rep

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Interleukin-17A (IL-17A) is a principal driver of multiple inflammatory and immune disorders. Antibodies that neutralize IL-17A or its receptor (IL-17RAInterleukin-17 (IL-17) cytokines are homo or heterodimeric proteins formed by combinations of six distinct polypeptides designated IL17A-F 1 . They are essential to a fully functional immune system 2,3 , but dysregulated expression of IL-17A is implicated in autoimmune disorders such as psoriasis, psoriatic arthritis, rheumatoid arthritis and multiple sclerosis 4-6 . The IL-17A covalent homodimer's significance in psoriasis is evidenced by the recent success of anti-IL-17A biologics as therapeutics. Secukinumab (Costentyx TM ), a monoclonal antibody targeting IL-17A, was recently approved for the treatment of moderate to severe plaque psoriasis 7,8 and is being investigated in other IL-17A-driven immunological diseases 9 . Additionally, two other biologics, ixekizumab (anti-IL17A) 10,11 and brodalumab (an antibody to the IL-17 receptor, IL-17RA) 12,13 , have shown efficacy in psoriasis in late stage clinical trials.IL-17A signaling occurs through its membrane-bound receptors, IL-17RA and IL-17RC, and elicits multiple inflammatory and immune responses [14][15][16] . The cytokine binds to IL-17RA with low single-digit nanomolar affinity 14,15,17,18 . and the structure of their complex is known 17 . The emerging biologics block this interaction by binding to one or other of the partners, but our goal was to determine whether it could be blocked or modulated with a small molecule as this could afford orally active agents.Small-molecule inhibition of a protein-protein interaction (PPI) is invariably challenging 19 . Even the discovery of early lead matter tends to be difficult because corporate compound collections are largely designed to target the active centers of enzymes, and are deficient in compounds suitable to the longer and shallower binding sites on which PPIs tend to depend. As the industry expands the "druggable genome", continued efforts at small molecule inhibition of PPIs will be required 20 . ResultsLead small molecule IL-17A antagonists. Our effort to discover small-molecule antagonists of IL-17A was initiated from disclosed inhibitors 21,22 exemplified by compound 1 (Fig. 1), a polyamide with clear structure-activity relationships (SAR) representative of the series. For example, the amide bonds, correct chiral center and cyclopentyl group were all required for activity. Surface plasmon resonance (SPR) measurements showed

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“…While in the refolding buffer (100 mM Tris-HCl, pH 8.0, 1M L-arginine), equal or more oxidized glutathione, with GSH and GSSG at a 1:5 molar ratio (0.2 mM GSH and 1 mM GSSG), or with GSH and GSSG at 1:1 molar ratio (0.3 mM GSH and 0.3 mM GSSG), was the best ratio to produce the correctly refolded IL-17A covalent dimer (Table 1, bottom row; Figure 1, band indicated by arrow). This isoform is the most active of the four, containing two interchain disulfide bonds [11]. As the refolding duration was 40 h, the percentages of this isoform present were similar (25-30%) with both equal and more GSSG, as shown in Figure 1A (lanes 1 and 2).…”

Section: Benchmarkmentioning

confidence: 68%

“…In contrast, other groups chose E. coli expression due to its multiple advantages, such as high expression levels, lack of posttranslational modifications and cost-effectiveness [6,10]. A total of four isoforms of a recombinant human IL-17A variant post-refolding were reported, differing by their interchain disulfide structures [11].…”

Section: Introductionmentioning

confidence: 99%

Optimized Methods for IL-17A Refolding and Anti-IL17A Fab Production for Co-crystallization With Small Molecules

Meng¹,

Wei²,

Li³

et al. 2020

BioTechniques

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Refolding of human interleukin 17A (IL-17A) has been reported; however, the key refolding protocol was not robust enough to deliver consistent results and to be easily scaled up for crystallization. Here we report an optimized refolding method for IL-17A. Although co-crystal structures of IL-17A with ligands have been obtained with a high-affinity peptide and an anti-IL-17A Fab as stabilizers, neither the production yield nor the characterization of the IL-17A/Fab complex was reported. To facilitate co-crystallization of IL-17A with small-molecule compounds derived from our DNA encoded library, we also describe the method for yield enhancement of anti-IL-17A Fab production and characterize the IL-17A/Fab complex for the first time, providing an essential prerequisite for structure-based drug discovery targeting IL-17A.

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The role of interchain disulfide bond in a recombinant human interleukin-17A variant

Cited by 3 publications

References 12 publications

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

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

Binding site elucidation and structure guided design of macrocyclic IL-17A antagonists

Optimized Methods for IL-17A Refolding and Anti-IL17A Fab Production for Co-crystallization With Small Molecules

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