In vitro biological characterization of IFN- -1a major glycoforms

Mastrangeli, Renato; Rossi, Mara; Mascia, Michele; Palinsky, Wolf; Datola, Antonio; Terlizzese, Mariagrazia; Bierau, Horst

doi:10.1093/glycob/cwu082

Cited by 13 publications

(8 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, R27T also exhibited considerable variability in its glycoprofile, with tri- and tetra-antennary glycans, as well as bi-antennary glycan forms being detected. Surprisingly, rhIFN-β containing a larger proportion of higher antennary glycoforms showed more sustained bioactivity over time . Indeed, in our previous study, R27T exhibited more prolonged signaling than the mono-glycosylated rhIFN-β, with altered receptor-binding kinetics .…”

Section: Results and Discussionmentioning

confidence: 73%

“…Surprisingly, rhIFN-β containing a larger proportion of higher antennary glycoforms showed more sustained bioactivity over time. 42 Indeed, in our previous study, R27T exhibited more prolonged signaling than the mono-glycosylated rhIFN-β, with altered receptor-binding kinetics. 15 A larger portion of higher antennary components in R27T may therefore influence the cellular signaling effects.…”

Section: ■ Introductionmentioning

confidence: 88%

See 1 more Smart Citation

Glycosylation Heterogeneity of Hyperglycosylated Recombinant Human Interferon-β (rhIFN-β)

Song

Moon²,

Kim³

et al. 2020

ACS Omega

View full text Add to dashboard Cite

We previously developed a biobetter version of rhIFN-β (R27T) that possesses an additional glycosylation site compared with rhIFN-β 1a. Herein, we characterized N-glycosylation heterogeneity of R27T, which includes both N-glycan site occupancy heterogeneity (macro-heterogeneity) and complexity of carbohydrate moieties (micro-heterogeneity). N-glycan site occupancy manifested as distinct differences in size and isoelectric point. The analysis of complex carbohydrate moieties of R27T involved the common biopharmaceutical glycosylation critical quality attributes such as core fucosylation, antennary composition, sialylation, N-acetyllactosamine extensions, linkages, and overall glycan profiles using weak anion-exchange and hydrophilic interaction high-performance liquid chromatography with 2-aminobenzoic acid-labeled N-glycans. The double-glycosylated form accounted for approx. 94% R27T, while the single-glycosylated form accounted for 6% R27T. N-glycans consisted of a mixture of bi-, tri-, and tetra-antennary glycans, some with N-acetyllactosamine extensions, but neither outer arm fucose nor α-galactose was detected. Sialic acid major variants, N-acetyl- and N-glycolyl-neuraminic acid, were more abundant in R27T than in Rebif. The major N-glycan, accounting for ∼42% of total N-glycans, had a di-sialylated, core-fucosylated bi-antennary structure.

show abstract

Section: Results and Discussionmentioning

confidence: 73%

Section: ■ Introductionmentioning

confidence: 88%

Glycosylation Heterogeneity of Hyperglycosylated Recombinant Human Interferon-β (rhIFN-β)

Song

Moon²,

Kim³

et al. 2020

ACS Omega

View full text Add to dashboard Cite

show abstract

“…The results further confirmed the identity of the 19 and 27 kDa bands as unglycosylated and glycoylated HuIFNβs, respectively. IFNβ is a 166‐amino acid glycoprotein with a single N‐linked carbohydrate chain on Asn80Residue . Heterogeneous mannan residues are added at Asn‐linked glycosylation sites for glycoproteins secreted by K. lactis .…”

Section: Resultsmentioning

confidence: 99%

Secreted expression of an active human interferon‐beta (HuIFNβ) in Kluyveromyces lactis

Madhavan

Sukumaran

2016

Engineering in Life Sciences

View full text Add to dashboard Cite

Interferon‐beta (IFNβ) is a cytokine involved in the antiviral, anti‐proliferative and immunomodulatory responses of cells, and a potent drug for multiple sclerosis. Human interferon beta (HuIFNβ) gene fused with the glucoamylase signal sequence in the N‐terminus and 6 His Tag in the C‐terminus was cloned into pKlac1 vector and introduced in Kluyveromyces lactis to allow secreted expression and one‐step purification of the protein. Recombinant yeast transformant with the highest level of HuIFNβ production was identified, and this secreted up to 1 mg/L of the cytokine after 72 h of incubation. Glycosylated and non‐glycosylated forms of the cytokine were elaborated by the yeast, the latter in higher quantities. His tag of the protein allowed easy one‐step purification by nickel‐nitriloacetic acid affinity chromatography, yielding close to 100% purity. SDS‐PAGE, western blot and MALDI‐TOF‐TOF confirmed the identity of the protein. The biological activity of the recombinant HuIFNβ was confirmed by its anti cell proliferative activity on HeLa cells. Expression of HuIFNβ in K. lactis is advantageous since it is a very safe organism to produce proteins for therapeutic applications, allows glycosylation and offers a cost effective method for large scale production as it can be grown in cheap carbon sources.

show abstract

“…For example, an N-linked glycan on residue 80 of IFN-β-1a has been reported to stabilize the IFN-β-1a structure (Runkel et al, 1998a), and the R27T structure can be stabilized by the hydrogen bond between 35R and N25-linked glycan moieties (Song et al, 2014). It has been reported that the individual glycoforms of IFN-β-1a enable to affect the alternation in biological activities (Mastrangeli et al, 2014). In addition, the glycan enables to decrease the flexibility of the protein backbone and increase protein conformation stability (Wormald and Dwek, 1999).…”

Section: Discussionmentioning

confidence: 99%

A Glycoengineered Interferon-β Mutein (R27T) Generates Prolonged Signaling by an Altered Receptor-Binding Kinetics

et al. 2019

View full text Add to dashboard Cite

The glycoengineering approach is used to improve biophysical properties of protein-based drugs, but its direct impact on binding affinity and kinetic properties for the glycoengineered protein and its binding partner interaction is unclear. Type I interferon (IFN) receptors, composed of IFNAR1 and IFNAR2, have different binding strengths, and sequentially bind to IFN in the dominant direction, leading to activation of signals and induces a variety of biological effects. Here, we evaluated receptor-binding kinetics for each state of binary and ternary complex formation between recombinant human IFN-β-1a and the glycoengineered IFN-β mutein (R27T) using the heterodimeric Fc-fusion technology, and compared biological responses between them. Our results have provided evidence that the additional glycan of R27T, located at the binding interface of IFNAR2, destabilizes the interaction with IFNAR2 via steric hindrance, and simultaneously enhances the interaction with IFNAR1 by restricting the conformational freedom of R27T. Consequentially, altered receptor-binding kinetics of R27T in the ternary complex formation led to a substantial increase in strength and duration of biological responses such as prolonged signal activation and gene expression, contributing to enhanced anti-proliferative activity. In conclusion, our findings reveal N-glycan at residue 25 of R27T is a crucial regulator of receptor-binding kinetics that changes biological activities such as long-lasting activation. Thus, we believe that R27T may be clinically beneficial for patients with multiple sclerosis.

show abstract

In vitro biological characterization of IFN- -1a major glycoforms

Cited by 13 publications

References 22 publications

Glycosylation Heterogeneity of Hyperglycosylated Recombinant Human Interferon-β (rhIFN-β)

Glycosylation Heterogeneity of Hyperglycosylated Recombinant Human Interferon-β (rhIFN-β)

Secreted expression of an active human interferon‐beta (HuIFNβ) in Kluyveromyces lactis

A Glycoengineered Interferon-β Mutein (R27T) Generates Prolonged Signaling by an Altered Receptor-Binding Kinetics

Contact Info

Product

Resources

About