Total Synthesis of Glycosylated Human Interferon-γ

Article abstract of DOI:10.1021/acs.orglett.0c02401

Interferon-γ(IFN-γ) is a glycoprotein that is responsible for orchestrating numerous critical immune induction and modulation processes and is used clinically for the treatment of a number of diseases. Herein, we describe the total chemical synthesis of homogeneously glycosylated variants of human IFN-γusing a tandem diselenide-selenoester ligation-deselenization strategy in the C- to N-terminal direction. The synthetic glycoproteins were successfully folded, and the structures and antiviral functions were assessed.

Full text of DOI:10.1021/acs.orglett.0c02401

pubs.acs.org/OrgLett
Letter
Total Synthesis of Glycosylated Human Interferon‑γ
Xiaoyi Wang, Anneliese S. Ashhurst, Luke J. Dowman, Emma E. Watson, Henry Y. Li,
Antony J. Fairbanks, Mark Larance, Ann Kwan, and Richard J. Payne*
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ABSTRACT: Interferon-γ (IFN-γ) is a glycoprotein that is responsible for orchestrating numerous critical immune induction and
modulation processes and is used clinically for the treatment of a number of diseases. Herein, we describe the total chemical
synthesis of homogeneously glycosylated variants of human IFN-γ using a tandem diselenide-selenoester ligation-deselenization
strategy in the C- to N-terminal direction. The synthetic glycoproteins were successfully folded, and the structures and antiviral
functions were assessed.
derived from recombinant expression in Escherichia coli has
nterferons (IFNs) make up a family of vertebrate cytokines,
1
Iproduced as a host response to pathogenic infection. On been approved by the U.S. Food and Drug Administration
(FDA) under the trade name ACTIMMUNE for the treatment
of malignant osteopetrosis and infections associated with
chronic granulomatous disease.9 However, pleiotropic hIFN-γ
has also exhibited efficacy for a range of other indications in
clinical and preclinical investigations, including atopic
dermatitis, Friedreich’s ataxia, hepatitis, tuberculosis, ovarian
and bladder cancer, scleroderma, idiopathic pulmonary fibrosis,
and fungal infections.10−14 The market for rhIFN is estimated
to be ∼4 billion USD per year, driven in large part by the
increased incidence of hepatitis C globally.9
the basis of sequence homology and target receptors, human
interferons can be categorized into three distinct classes,
namely, type I IFNs (IFN-α and IFN-β), type II IFN (IFN-γ),
and type III IFN (IFN-λ).2 As the sole member of the type II
IFN family, IFN-γ (also termed immune IFN) is secreted by an
array of immune cells, including natural killer (NK) cells,
natural killer T (NKT) cells, CD4+ T helper type 1 (Th1)
lymphocytes, CD8+ cytotoxic lymphocytes, professional
antigen-presenting cells (APCs), and B cells.3 Structurally,
human interferon-γ (hIFN-γ) is a symmetrical homodimeric
glycoprotein consisting of two identical peptide chains
intertwined in an antiparallel manner.4 Mature hIFN-γ is
natively N-glycosylated at two asparagine (Asn, N) residues,
N25 and N97, with two complex type N-linked oligosacchar-
ides, as well as containing an N-terminal pyroglutamate
residue. IFN-γ binds to a distinct heterodimeric interferon-γ
receptor (IFNGR) formed by IFNGR-α and IFNGR-β
subunits. This interaction facilitates immune modulation and
activation5 and regulates a diverse array of biological processes,
including cellular motility, differentiation, proliferation and
apoptosis, antigen processing and presentation, macrophage
activation, leukocyte trafficking, pathogen recognition, and
tumor surveillance.3,6
Compared to natively glycosylated hIFN-γ, unmodified
rhIFN-γ is more susceptible to proteolytic degradation leading
to shorter persistence in vivo.15 To address this issue, several
recombinant expression systems have been explored to
produce glycosylated hIFN-γ, such as baculovirus-infected
insect cells (BIICs), Chinese hamster ovary cells (CHOs),
methylotrophic yeast, and the mammary gland of transgenic
mice (TM), with the CHO-derived form affording glyco-
sylation patterns closest to native hIFN-γ.16−18 Of the two
Received: July 19, 2020
Therapeutic applications of hIFN-γ have been widely
explored, because of its potent and diverse biological activity.7,8
Specifically, an unglycosylated variant of hIFN-γ (rhIFN-γ)
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glycosylation sites, N25 was suggested to be essential for
protease resistance.18 Despite the extensive studies of ex-
pressed hIFN-γ, the effect of glycosylation on the structure and
function of the protein is still not well understood. This is due,
in large part, to the heterogeneity of the glycan modifications
on IFN-γ within the expression milieu (so-called glycoforms)
that are difficult to separate.
fragments IFN-γ(52−108) possessing an N-terminal (β-Se)-
Phe residue23,24 and functionalized at the C-terminus as a
phenylselenoester, with either no modification (6) or a β-
GlcNAc moiety (7) at N97, and (3) N-terminal fragment IFN-
γ(1−51) with an N-terminal pyroglutamate, phenylselenoester
moieties on their C-termini, and either unmodified (8) or β-
GlcNAc-derivatized (9) at N25. It was envisioned that these
fragments could then be assembled through sequential DSL
reactions at Sec and (β-Se)-Phe, followed by deselenization to
afford the four hIFN-γ (glyco)proteins 1−4 following folding
and purification (Scheme 1).
Peptide fragments 5−9 were first assembled by modified
Fmoc-based solid-phase peptide synthesis (Fmoc-SPPS)
methods (see the Supporting Information). Specifically,
fragment 5 was functionalized on the N-terminus with Boc-
selenazolidine (10),25 while bifunctional fragments 6 and 7
were synthesized with a p-methoxybenzyl (Pmb)-protected β-
selenophenylalanine residue on the N-terminus by incorpo-
ration of building block 1123,24 (see the Supporting
Information for synthetic details). Fragments 6−9 each
possessed a C-terminal phenylselenoester moiety that was
installed in solution following release of side chain-protected
(glyco)peptides from a solid support20 (see the Supporting
Information for details). The β-GlcNAc moiety was installed at
N97 of 7 using a preformed Fmoc-Asn[β-GlcNAc(OAc)3-OH]
building block, whereas β-GlcNAcylation at N25 in 9 was
achieved through late-stage on-resin aspartylation chemistry26
(see the Supporting Information for synthetic details).
We envisaged that access to homogeneously glycosylated
variants of hIFN-γ could be provided through total chemical
synthesis and that the availability of synthetic homogeneous
“glycoforms” could be used to probe the role of N-linked
glycosylation on the activity of the cytokine. Importantly, while
unglycosylated IFN-γ has recently been synthesized,19 to date
there have been no reports on the synthesis and biological
investigation of homogeneous hIFN-γ glycoproteins. In this
work, we targeted four different forms of the 138-amino acid
glycoprotein: unglycosylated hIFN-γ (1), variants bearing β-N-
acetylglucosamine (β-GlcNAc) at N25 (2) or N97 (3), and a
construct containing β-GlcNAc at both N25 and N97 (4). We
envisaged that each of these (glyco)proteins could be
constructed in the C- to N-terminal direction, with the
synthetic strategy hinging on the use of iterative one-pot
diselenide-selenoester ligation (DSL)-deselenization reac-
tions20−22 (Scheme 1). Specifically, we proposed disconnec-
tion of the hIFN-γ sequence between S51 and F52, and K108
and A109, which provided three different classes of fragments
for synthesis: (1) a C-terminal fragment, IFN-γ(109−138) 5,
functionalized at the N-terminus with a selenocystine residue
(the oxidized form of selenocysteine), (2) bifunctional
With the requisite fragments in hand, attention turned to the
total synthesis of the differentially glycosylated full length
hIFN-γ variants 1−4 through the proposed C-to-N iterative
ligation strategy. Toward this end, the first DSL reaction was
performed by reacting diselenide dimer IFN-γ(109−138) 5
(1.0 equiv on the basis of a monomeric selenopeptide) bearing
an N-terminal selenocystine with bifunctional middle fragment
6 or glycosylated IFN-γ(52−108) 7 (1.1 equiv) in ligation
buffer (pH 5.5−6.0 without adjustment, 7−8 mM with respect
to a monomeric selenopeptide) under additive-free conditions
(Scheme 2). Pleasingly, the ligation proceeded to completion
within 5 min, as determined by UPLC-MS analysis (and the
precipitation of Ph2Se2), giving the desired product as a
mixture of product selenoester 12I (or 13I for ligation with 7)
and asymmetric diselenide dimer 12II (or 13II from ligation
with 6). The ligation mixture was first extracted with hexane to
remove precipitated Ph2Se2 and then diluted with an equal
volume of ligation buffer containing TCEP (50 equiv) and
DTT (50 equiv). The resulting mixture was then thoroughly
sparged with argon to remove any dissolved oxygen prior to
incubation at 37 °C. After 16 h, UPLC-MS analysis confirmed
smooth deselenization of Sec to Ala and subsequent
purification by HPLC afforded PMB-protected IFN-γ(52−
138) 14 (from ligation with 6) and 15 (from ligation with 7)
in good yields over the one-pot ligation deselenization (62%
and 57%, respectively). The PMB protecting groups on the Sec
residues of 14 and 15 were subsequently removed under
oxidative deprotection conditions [1:1:3 (v/v/v) DMSO/
buffer comprising 6 M Gdn·HCl and 0.1 M Na2HPO4 (pH
7.2)/TFA, 15 mg/mL] to generate IFN-γ(52−138) disele-
nides 16 (90%) and 17 (81%), respectively. It is important to
note that these deprotection reactions proceeded with
concomitant oxidation of methionine residues 77, 117, and
134 to the corresponding sulfoxides. Having successfully
prepared C-terminal fragments 16 and 17, we initiated the
Scheme 1. Retrosynthesis of Differentially Glycosylated
IFN-γ Variants 1−4 (Q1 is pyrrolidone carboxylic acid)
a
a
The native protein exists as a homodimer.
B
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hIFN-γ(1−51) selenoester 8 or 9 (1.5 equiv), in ligation buffer
[6 M Gdn·HCl and 0.1 M Na2HPO4 (pH 7.2)]. In this case,
the reaction was conducted at a higher dilution (1−2 mM with
respect to a monomeric selenopeptide) due to the poorer
solubility of the N-terminal selenoester fragments 8 and 9 in
ligation buffer. The final pH of the ligation was adjusted to
5.6−6.0 to slow the hydrolysis of selenoester 8 or 9 (40%
hydrolysis occurred after 1 h at pH >6.1). The ligation was
incubated at 30 °C, and after 30 min, UPLC-MS analysis
indicated complete consumption of IFN-γ(52−138) diselenide
16 or 17, affording full length hIFN-γ as the phenylselenyl
asymmetric diselenide as the sole ligation product (see the
Supporting Information for data). Without purification, the
reaction mixture was subjected to modified radical deseleniza-
tion conditions necessary to prevent oxidation of the benzylic
position of the Phe residue at the ligation junction. These
conditions involved treatment with TCEP (50 equiv) and
DTT (200 equiv) as described above but included the water-
soluble radical initiator VA-044 (8 equiv) and the benzylic
radical competitor 18. Gratifyingly, under these conditions, the
reactions proceeded to completion in 5 min (cf. 16 h under
VA-044-free conditions) and no β-hydroxylated byproduct was
observed. Each of the four hIFN-γ(1−138) variants could be
isolated in excellent yields over the two steps following reverse-
phase HPLC purification (54−59%). All that remained for the
synthesis of the native hIFN-γ (glyco)protein sequences was
the reduction of the three methionine sulfoxide moieties. This
was achieved by incubation of the DSL-deselenization
products with a reductive cocktail comprising TFA, thio-
anisole, H2O, Me2S, iPr3SiH, phenol, and NH4I.27 After 2 h,
clean conversion to the target hIFN-γ (glyco)proteins 1−4 was
observed by UPLC-MS; purification by reverse-phase HPLC
led to excellent isolated yields (71% for 1, 81% for 2, 73% for
3, and 78% for 4).
Scheme 2. Chemical Synthesis and Refolding of Synthetic
IFN-γ Glycoforms 1−4
a
After the successful preparation of the full length hIFN-γ
glycoforms, the next critical step was to refold the (glyco)-
proteins. We initially followed a dialysis-based protocol that
had been successfully employed on recombinant hIFN-γ
(rhIFN-γ) following E. coli expression.28 This involved
solubilizing the lyophilized (glyco)proteins in 4.0 M Gdn·
HCl followed by dialysis against 10 mM NH4OAc at pH 7.0
for 24 h at 4 °C. Unfortunately, this approach gave rise only to
partially folded hIFN-γ, which was confirmed by far-ultraviolet
1
circular dichroism (CD) and H NMR spectroscopy (see the
a
(a) DSL: 6 M guanidine-HCl (Gdn·HCl), 0.1 M Na2HPO4, pH
Supporting Information). We next attempted a rapid dilution
folding approach by first solubilizing the lyophilized (glyco)-
proteins in 7 M urea and 0.1 M NH4OAc at pH 7.5 at a
concentration of 1 mg/mL. The (glyco)protein solutions were
then rapidly diluted to 0.1 mg/mL using a 0.01 M NH4OAc
solution at pH 7.5 and 4 °C for 4 days.29 Purification by cation
exchange chromatography eluting with 20 mM Tris-HCl and
0.5 NaCl (pH 8.0) provided folded hIFN-γ protein 1 and
hIFN-γ glycoproteins 2−4. The folded (glyco)proteins were
characterized by one-dimensional NMR spectroscopy and CD
spectroscopy. The CD spectra showed diagnostic minima at
208−209 and 220−221 nm, characteristic of α-helical
structural elements, which was consistent with the refolded
recombinant hIFN-γ (rhIFN-γ) from E. coli (Figure 1).
5.5−6.0, 25 °C, 5 min. (b) Deselenization: 50 equiv of tris-
(carboxyethyl)phosphine (TCEP), 50 equiv of 1,4-dithiothreitol
(DTT), pH 5.1−5.5, 25 °C, 16 h. (c) PMB deprotection: 3:1:1 (v/
v/v) TFA/DMSO/ligation buffer (6 M Gdn·HCl, 0.1 M Na2HPO4,
pH 7.2), 25 °C, 30 min. 16 and 17 were produced with oxidation at
Met 77, 117, and 134. (d) DSL: 6 M Gdn·HCl, 0.1 M Na2HPO4, pH
5.6−6.0, 30 °C, 30 min. (e) Deselenization: 50 equiv of TCEP, 200
equiv of DTT, 8 equiv of 2,2′-azobis[2-(2-imidazolin-2-yl)propane]-
dihydrochloride (VA044), 6 equiv of 18, pH 5.5−6.8, 37 °C, 5 min.
(f) Sulfoxide reduction: 62.5:5:5:5:10:7.5:5 (v/v/v/v/v/w/w) TFA/
thioanisole/H2O/Me2S/iPr3SiH/phenol/NH4I, 25 °C, 2 h. (g)
Refolding: dropwise dilution from 7 M urea, 0.1 M NH4OAc, pH
7.5, into 0.01 M NH4OAc, pH 7.5, folding reaction incubated at 4 °C
for 4 days (final buffer of 0.7 M urea, 0.01 M NH4OAc, pH 7.5),
purification by cation exchange chromatography.
At this point, we considered the possibility of increasing the
diversity of the hIFN-γ glycoprotein library via late-stage
glycan elaboration with endo-β-N-acetylglucosaminidase (EN-
Gase) enzymes,30,31 specifically, wild type (WT) Endo A and
the commercially available glycosynthase mutant Endo M
second DSL reaction at the N-terminal (β-Se)-Phe by
dissolving hIFN-γ(52−138) diselenide dimers 16 and 17
(1.0 equiv on the basis of a monomeric selenopeptide) and
C
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Figure 1. Exemplar characterization data for doubly glycosylated IFN-γ 4. (a) Reverse-phase UPLC profile of 4 (0−70% MeCN in H2O over 5
min, 0.1% TFA, λ = 214 nm, ACQUITY UPLC Peptide BEH C18 300 Å, 1.7 μm, 2.1 mm × 50 mm column). (b) ESI mass spectrum of 4. (c)
MALDI-TOF mass spectrum (linear positive mode) of 4. (d) Circular dichroism (CD) spectra of rIFN-γ from E. coli and synthetic IFN-γ 1−4 [in
0.01 M NH4OAc (pH 7.0)].
N175Q (see the Supporting Information for details). We
initially investigated glycan transfer to the unfolded variants of
1−4; however, these species were insoluble in the aqueous
buffers that were compatible with the Endo enzymes. As such,
we performed Endo-catalyzed glycan transfer on the fully
folded proteins that possessed excellent solubility in aqueous
buffer (phosphate-buffered saline). Interestingly, while we
could observe Endo M N175Q-catalyzed transfer of a complex
biantennary sialylglycan (activated for transfer as the
corresponding oxazoline32) onto β-N-acetylglucosaminylated
hIFN-γ 2, bearing an GlcNAc at N25, we did not observe any
transfer to 3, where the GlcNAc was positioned at N97. In
contrast, WT Endo A was capable of transferring the complex
biantennary glycan to the bridgehead GlcNAc residues of both
hIFN-γ 2 and hIFN-γ 3; however, these enzymatic reactions
(including to N25 and N97 on 4) could not be pushed to
completion (see the Supporting Information). Unfortunately,
despite significant optimization, the ENGase-catalyzed transfer
of the complex biantennary glycan could not be driven to
completion, leading to the generation of inseparable glyco-
forms; as such, we did not pursue this strategy further here.
The effect of N-glycosylation at N25 and/or N97 on
antiviral activity was next interrogated by assessing the
synthetic hIFN-γ glycoproteins in viral resistance assays
(Figure 2). Specifically, human foreskin fibroblast (HFF-1)
cells (ATCC) were cultured in the presence of synthetic hIFN-
γ constructs 1−4, with E. coli- and CHO-expressed hIFN-γ as
controls, for 24 h before infection with herpes simplex virus-1
[HSV-1; multiplicity of infection (MOI) of 0.125, 1 h] that
expresses eGFP under the control of the ICP47 early gene
Figure 2. Synthetic hIFN-γ induces resistance to viral infection,
irrespective of glycosylation. HFF-1 cells were treated with various
IFN-γ constructs for 24 h and then infected with HSV-1-eGFP. After
one round of viral replication, the proportions of eGFP+ cells were
determined by flow cytometry. Data are the mean
the standard
error of the mean for three independent biological replicates.
promoter.32 After incubation to allow viral replication (14 h,
37 °C, 5% CO2), cells were detached and the proportion of
GFP+ cells was determined by flow cytometry (see the
Supporting Information). Gratifyingly, all glycosylated IFN-γ
variants were biologically active, inhibiting viral replication in a
dose-dependent manner. However, antiviral activity was not
modulated by the presence or position of N-glycosylation.
In summary, we have developed an efficient synthetic
approach to generating the 16.5 kDa hIFN-γ using two
consecutive DSL-deselenization reactions in the C- to N-
D
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terminal direction. Using this strategy, we have successfully
prepared four differentially glycosylated variants of the protein
that were successfully folded and characterized. Future work in
our laboratories will involve the generation of larger synthetic
and/or semisynthetic libraries of hIFN-γ bearing larger glycans
to assess the effect of these more complex modifications on
structure, function, and stability.
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ASSOCIATED CONTENT
* Supporting Information
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Authors
Xiaoyi Wang − School of Chemistry, The University of Sydney,
Sydney, NSW 2006, Australia
Anneliese S. Ashhurst − School of Chemistry, School of Medical
Sciences, Faculty of Medicine and Health, and Charles Perkins
Centre, The University of Sydney, Sydney, NSW 2006, Australia
Luke J. Dowman − School of Chemistry, The University of
Sydney, Sydney, NSW 2006, Australia
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The authors acknowledge grant support from an ARC
Discovery Project (DP190101526), an ARC LIEF grant
(LE180100050), and the Royal Society of New Zealand
Marsden Fund (UOC1608). The authors also thank the John
A. Lamberton Research Scholarship for Ph.D. funding (to
X.W.). The authors thank A. Prof. Barry Slobedman, Dr. Brian
McSharry, and Carolyn Samer, The University of Sydney, for
technical advice, the provision of HFF-1 stocks, and HSV-1
GFP stock (originally provided by Prof. David Tscharke,
Australian National University).
E
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