Incorporation of 'click' chemistry glycomimetics dramatically alters triple-helix stability in an adiponectin model peptide

Article abstract of DOI:10.1039/c7ob01388d

Adiponectin (Adpn) has been shown to be a possible therapeutic for Type II diabetes, however the production of a therapeutic version of Adpn has proved to be challenging. Biological studies have highlighted the importance of the glycosylated lysine residues for the formation of bioactive high molecular weight oligomers of Adpn. Through the use of 'click' glycopeptide mimetics, we investigated the role of glycosylated lysine and serine residues for the formation of triple helical structures of the collagenous domain of Adpn, in the context of a collagen model peptide scaffold. The physical properties of the unglycosylated lysine and serine peptides are compared with their glycosylated analogues. Our results highlight the crucial role of lysine residues for formation of the triple helical structure of Adpn, possibly due to the extension of both intra- and interstrand hydrogen bonding networks. Strikingly, we observed a significant decrease in thermal stability upon incorporation of triazole-linked analogues of glycosylated lysine residues into the adiponectin collageneous domain, indicating possible uses of 'click' glycomimetics for bioengineering applications.

Full text of DOI:10.1039/c7ob01388d

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Incorporation of ‘click’ chemistry glycomimetics dramatically
alters triple-helix stability in an adiponectin model peptide
,
Received 00th January 20xx,
a,b
a,b
a,b
a,b
a,b
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Katherine R. Lutteroth, Paul W. R. Harris, Tom H. Wright, Harveen Kaur, Kevin Sparrow,
a,b
b,c
a,b
Sung-Hyun Yang, Garth J. S. Cooper, and Margaret A. Brimble*
Adiponectin (Adpn) has been shown to be a possible therapeutic for Type II diabetes, however the production of a
therapeutic version of Adpn has proved to be challenging. Biological studies have highlighted the importance of the
glycosylated lysine residues for the formation of bioactive high molecular weight oligomers of Adpn. Through the use of
‘
click’ glycopeptide mimetics, we investigated the role of glycosylated lysine and serine residues for the formation of triple
helical structures of the collagenous domain of Adpn, in the context of a collagen model peptide scaffold. The physical
properties of the unglycosylated lysine and serine peptides are compared with their glycosylated analogues. Our results
highlight the crucial role of lysine residues for formation of the triple helical structure of Adpn, possibly due to the
extension of both intra- and interstrand hydrogen bonding networks. Strikingly, we observed a significant decrease in
thermal stability upon incorporation of triazole-linked analogues of glycosylated lysine residues into the adiponectin
collageneous domain, indicating possible uses of ‘click’ glycomimetics for bioengineering applications.
1
08-244) (Fig. 1). Adiponectin oligomerises in vitro to form
Introduction
higher-order structures, namely low molecular weight (LMW,
ca. 180 kDa) and high molecular weight (HMW, ca. 400 kDa)
Adiponectin (Adpn) has been identified as playing a key role in
the obesity-related diseases collectively known as ‘metabolic
9
oligomers . The HMW oligomers of Adpn have been shown
-11
1
2
1
syndrome’ , including Type II diabetes mellitus (T2D),
2-4 to be the most bioactive form of the protein . Triple helix
formation mediated by the C-terminal C1q-like globular
domain, as well as the collagen-like domain, has been
5
, 6
7
cardiovascular disease and most recently melanoma; these
diseased states have all been shown to be associated with low
1
3
proposed as the first step in Adpn oligomerisation in vivo .
levels
of
plasma
adiponectin
known
as
3
,8
hypoadiponectinemia. Expression of a therapeutic form of
Adpn through recombinant techniques has proved challenging
due to difficulties with purification of the Adpn oligomers
produced in mammalian cell lines. Consequently, there has
been significant interest in developing an understanding of the
oligomerisation mechanism, in order to guide the chemical
synthesis of a bioactive form of Adpn.
Notably, the collagenous domain of native Adpn lacks the
characteristic ‘GPO’ Gly-Xaa-Yaa repeats that typically induce
triple helix formation in collagen-type proteins. Indeed, we
14
have previously demonstrated that polypeptide constructs
incorporating 30 residues of the N-terminal portion of the
collagenous domain, in addition to the variable domain, are
incapable of triple-helix formation in vitro.
Human adiponectin is a 30 kDa peptide comprising of four
segments, the N-terminal signal peptide (residues 1-21), the
variable domain (residues 22-45), the collagenous domain
a)
2
H N
SP
Variable
Collagenous
Globular
2
CO H
1
21 22
45 46
107108
244
(residues 46-107) and a C-terminal globular domain (residues
b)
Monomer
Trimer
LMW
HMW
Fig. 1 a) The four segments of monomeric Adpn, and b) oligomers of Adpn.
1
We and Richards et al. have previously demonstrated
5
16
the crucial role of post-translationally modified lysine
(PTM-lysine) residues for formation of bioactive HMW
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0
oligomers of Adpn in vivo. For example, recombinant Adpn and protecting group choice . Importantly, such
produced in E. coli cell lines are unable to form HMW neoglycopeptides have been shown to be more resistant to
oligomers, due to the lack of PTMs in bacterially produced digestive enzymes, thus making the triazole moiety an
1
7
18
31, 32
proteins . In 1967, Spiro et al. elucidated the chemical attractive linker for potential biopharmaceuticals
.
structure of the PTM-lysine residues found in Adpn to be the
disaccharide unit 2-O-α-
D
-glucopyranosyl-
D
-galactose bound
We therefore decided to investigate whether ‘click’
to (2S,5R)-hydroxy-lysine by a β-glycosidic bond, namely analogues of PTM-lysine could modulate the triple helical
Glu-Gal-Hyl
1
(Fig. 2).
propensity in a peptide construct derived from the Adpn
collagenous domain. As a proof-of-principle, we selected
monosaccharide analogues of native PTM-lysine
1, ‘click’
galactolysine 2 and ‘click’ galactoserine 3 (Fig. 3) in order to
identify the minimal structure required to induce triple helical
propensity in Adpn glycopeptides. We rationalised this
structural simplification as, while native Adpn contains the
unusual PTM Glu-Gal-Hyl
1, structurally simpler glycosylated
residues have also been demonstrated to impart triple helical
3-36
3
propensity to collagen-type polypeptides
. Notably, β-
galactosylated threonine is found in the place of
hydroxyproline in collagens from the deep-sea hydrothermal
vent worm Riftia pachyptila, where the glycosylated residue is
Fig. 2 The chemical structure of PTM-lysine residues found in Adpn namely Glu-
Gal-Hyl
1.
3
3, 34
essential for triple helix formation
20, 37
. Further, studies from
have demonstrated the tolerance
1
9
In 2016 Ohkubo et al. demonstrated that synthetic Adpn
peptides derived from the collagenous domain containing
these PTM-lysine residues are able to form triple helical
secondary structures in solution. In addition, adoption of the
triple helical structures of Adpn peptides was shown to be the
crucial first step for oligomerisation of the peptides, with
spontaneous self-assembly into an octodecamer occurring at
the Wennemers group
of triple-helical structures for triazole-linked monosaccharides
in the Yaa position of CMP Gly-Xaa-Yaa repeats.
We chose to incorporate ‘click’ glycosides into an 18-amino
acid segment of the collagenous domain of Adpn (residues
6
6-83), as three of the four PTM-lysine residues occur within
physiological pH at 20 °C. This highlights the importance of the
PTM-lysine residues for the formation of bioactive HMW
15
this segment in native Adpn . Serine ‘click’ glycosides were
synthesised alongside the lysine glycosides to probe the
importance of the interspace distance between the glycan and
oligomers of Adpn.
1
9
the peptide backbone. In a similar manner to Ohkubo et al.,
the structural triple helical properties of our resultant 36-mer
At the outset of this study, we sought a minimalist
(
glyco)peptide model system that would recapitulate the triple
CMP-Adpn peptides
dichroism.
4-7 were then investigated using circular
helix formation observed in native adiponectin, with the aim of
establishing a facile model system for the investigation of
chemical modulation of this structure by post-translational
modification. Collagen model peptides (CMPs) have been
20,14, 21
shown to be a good mimetic for collagenous peptides
.
1
4
Our group has previously demonstrated that incorporation of
a ‘guest’ peptide sequence derived from Adpn(residues 18-74)
into a classical GPO collagen repeat ‘host’ sequence ‘rescues’
the disfavoured triple-helix formation in such constructs. Thus,
3
in the present study a CMP ‘host’ (GPO) construct was
appended on either side of the 18-mer ‘guest’ Adpn domain as
a mimic for the remainder of the Adpn peptide (Fig. 3).
To circumvent the synthetic difficulties associated with the
preparation of native glycopeptide constructs, various
2
2
approaches informed by the ‘click chemistry’ concept have
been reported to provide expedient access to non-natural, but
biologically relevant glycoconjugates (termed by us
2
3-29
neoglycosides/ neoglycopeptides)
. Such glycoconjugates Fig. 3 Family of CMP-Adpn peptides 4-7 prepared in this study. The Adpn ‘guest’
sequence is highlighted in bold
.
are particularly straightforward to prepare using the
Cu(I)-catalysed azide-alkyne cycloaddition ‘click’ (CuAAC)
reaction. The CuAAC reaction is neither air nor moisture
sensitive, and is high yielding and flexible in terms of solvent
We envisaged that the CMP-Adpn peptides
prepared by Fmoc-SPPS, and the neoglycosides in
glycopeptides and could be incorporated as the building
4-7 could be
4
6
2
| J. Name., 2012, 00, 1-3
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blocks
Fmoc-Lys(neoGal(OAc))-OH
8
and
Fmoc- Subsequent removal of the acetate protecting groups on the
Ser(neoGal(OAc))-OH
9, respectively (Scheme 1). Acetate carbohydrate residues, followed by purification using reverse
protecting groups were chosen for the glycan hydroxyl groups phase high performance liquid chromatography (RP-HPLC)
3
8
as these could be easily removed using the Zemplén
procedure following peptide synthesis.
afforded the CMP-Adpn peptides
Supporting Information).
4-7 in good purity (see
Scheme 1 Retrosynthetic strategy for neoglycopeptides 4 and 6.
Results and discussion
Synthesis
Scheme 2 Synthesis of neogalactolysine
CuAAC ‘click’ reaction. Reagents and conditions: Sugar donor 11 or 13 (1.1 eq),
CuSO ·5H O (0.3 eq), sodium ascorbate (0.6 eq), degassed EtOH, µw, 80 °C, 1 h.
8 and neogalactoserine 9 using the
For the preparation of Fmoc-Lys(neoGal(OAc))-OH
Ser(neoGal(OAc))-OH , the required pre-‘click’ building blocks
namely Fmoc-lysine-azide 10 , 1-O-propargyl-2,3,4,6-tetra-O-
8 and Fmoc-
9
4
2
3
9
4
0
acetyl-β-
D
-galactopyranose 11
and 2,3,4,6-tetra-O-acetyl-β-
were first synthesised following literature methods. The We next investigated the triple helical properties of CMP-Adpn
using circular dichroism (CD), where a triple helix
then subjected to the CuAAC ‘click’ reaction using copper(II) is signified by a maximum at 225 nm and a minimum at 198
,
Fmoc-O-propargyl-serine
4
0
Circular dichroism studies
12
,
D-galactopyranosyl azide
4
2
13
pre-‘click’ coupling partners 10 and 11, and 12 and 13, were peptides
4-7
4
1
42
sulfate and sodium ascorbate with heating at 80 °C under nm . Peptides
microwave irradiation for 1 h (Scheme 2). The resulting Fmoc- buffer solution at pH 7.4 and the CD spectra obtained at 6
protected triazole-linked glycoside products and were (Fig. 4). The CD profiles revealed that the lysine-containing
isolated in 92% and quantitative yield respectively, ready for Adpn peptides
and displayed signature triple helical
incorporation into Adpn peptides and . Both and were features and were thus able to form a triple helix. In contrast,
found to be stable at room temperature and soluble in a range the serine-containing Adpn peptides
and were unable to
4-7 were dissolved in a potassium phosphate
°C
8
9
4
5
4
6
8
9
6
7
of organic and protic solvents.
adopt this secondary structure, instead adopting a random-coil
conformation. In addition, the similarity of the CD profiles for
All four CMP-Adpn peptides 4-7 were subsequently lysine-containing peptides
prepared by Fmoc-SPPS on an aminomethyl polystyrene resin peptides
and indicated that the type of residue (lysine or
functionalised with a Rink-amide linker 14 (Scheme 3, see serine) had a larger effect on triple helical conformation than
4 and 5, and serine-containing
6
7
Supporting Information for details).
the ‘clicked’ glycan moieties.
Briefly, the Fmoc-protecting groups were removed with a
For lysine-containing Adpn peptides 4 and 5, thermal melt
solution of piperidine in DMF and condensation of each studies were conducted at 215 nm to investigate the thermal
subsequent amino acid residue was performed with either stabilities of these peptides. The triazole-linked ‘click’
HATU or HCTU, either at room temperature or with microwave glycosylated peptide
4 was found to have a significantly lower
irradiation. Upon completion of the peptide sequence, the N- melting point (T of 20.4 °C) compared to the non-glycosylated
m
terminal residue of the resin bound peptides was N-acetylated peptide
with acetic anhydride and the acetylated peptides released
5
(T of 40.9 °C) (Fig. 5). This indicated that ‘click’
m
2
from the resin using a cleavage cocktail of TFA/TIPS/H O.
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Scheme 3 Synthesis of (a)
Reagents and conditions: (a)
acid (5 eq), HCTU (4.6 eq), DIPEA (10 eq), NMP/DMF (1:4), 45 min, r.t. or Fmoc-amino acid (5 eq), HCTU (4.5 eq), DIPEA (10 eq), NMP/DMF (1:5), μW, 75 °C, 25 W, 5
min ; Coupling of glyco-AA (5 eq), HATU (4.6 eq), DIPEA (10 eq), DMF, μW, 75 °C, 25 W, 5 min; N-terminal acetylation: Ac O (40 eq), DIPEA (10 eq), NMP, 2 x 20
min, r.t.; Resin cleavage: TFA/TIPS/H O (93:3.5:3.5), μW, 35 °C, 10 W, 20 min; Glycan deacetylation: NaOMe/MeOH (1 M), 2 h, r.t.
4
, (b)
5, (c) 6 and (d) 7. Adpn-derived sequences are highlighted in bold. See Supplementary Information for full experimental details.
4
: Fmoc removal: 20% piperidine-DMF, 10 min, r.t. or 40% piperidine-DMF, μW, 75 °C, 25 W, 6 min; Fmoc-AA-OH coupling: Fmoc-amino
8
:
8
2
2
(
b)
5
: Fmoc removal: 20% piperidine-DMF, 10 min, r.t.; Fmoc-AA-OH coupling: Fmoc-amino acid (5 eq), HCTU (4.6 eq), DIPEA (10 eq), NMP/DMF (1:4), 45 min, r.t.; N-
O (40 eq), DIPEA (10 eq), NMP, 2 x 20 min, r.t.; Resin cleavage: TFA/TIPS/H O (90:5:5), 90 min, r.t.
terminal acetylation: Ac
2
2
( 6: Fmoc removal: 20% piperidine-DMF, 10 min, r.t. or 20% piperidine-DMF, μW, 75 °C, 25 W, 6 min; Fmoc-AA-OH coupling: Fmoc-amino acid (5 eq), HCTU (4.6 eq),
c)
DIPEA (10 eq), NMP/DMF (1:4), 45 min, r.t. or Fmoc-amino acid (5 eq), HATU (4.6 eq), DIPEA (10 eq), DMF, μW, 75 °C, 25 W, 5 min or Fmoc-amino acid (10 eq), HCTU
7 eq), NMM (10 eq), NMP/DMF (1:4), 20 min, r.t. N-terminal acetylation: Ac O (40 eq), DIPEA (10 eq), NMP, 2 x 20 min, r.t.; Resin cleavage: TFA/TIPS/H O (90:5:5), 90
min, r.t.; Glycan deacetylation: NaOMe/MeOH (1 M), 2 h, r.t.
(
2
2
(
d)
7
: Fmoc removal: 20% piperidine-DMF, 10 min, r.t.; Fmoc-AA-OH coupling: Fmoc-amino acid (5 eq), HCTU (4.6 eq), DIPEA (10 eq), NMP/DMF (1:4), 45 min, r.t.; N-
O (40 eq), DIPEA (10 eq), NMP, 2 x 20 min, r.t.; Resin cleavage: TFA/TIPS/H O (90:5:5), 90 min, r.t.
terminal acetylation: Ac
2
2
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1
1
5
.50E-03
.00E-03
.00E-04
a.
0
0
0
.00010
.00005
.00000
0
.00E+00
1
80
200
220
240
4
-5.00E-04
-1.00E-03
-1.50E-03
-2.00E-03
-2.50E-03
-3.00E-03
2
0
40
60
80
-
0.00005
Temperature
5
-0.00010
-0.00015
6
7
λ (nm)
b.
0.00015
0
0
0
.00010
.00005
.00000
Fig. 4 CD spectra of the CMP-Adpn peptides 4-7
.
glycosylation of the lysine residue had
destabilising effect on the triple helical structure.
a
significant
20
40
60
80
-0.00005
-0.00010
-0.00015
Temperature
Discussion
The ability of collagen-like peptides to form a triple helix is
dependent upon favourable combination of steric
constraints, interstrand hydrogen bonding networks and unglycosylated-Adpn peptide
a
Fig. 5 Thermal melt curves for a) ‘click’ neoglycosylated Adpn peptide
4 and b)
5
.
4
3, 44
electrostatic interactions
. For glycosylated CMPs the
hydrogen bonding network extends to additional glycan-glycan
4
and glycan-peptide backbone interactions .
5
In the case of non-glycosylated peptide
5 it is probable
that, in the tested buffer solution at pH 7.4, the ionised Lys
The difference in triple helix formation observed for the residues may form stabilising ion-pairs with neighbouring
lysine- and serine-containing CMP-Adpn peptides highlights strand Glu residues, thus stabilising the triple helical structure
+
the critical role of lysine residues in the Yaa position for triple of the peptide. Furthermore, the charged NH
helix induction in Adpn peptides. The importance of lysine in present in
this position could be due to both hydrogen bonding and hydrogen bonding network, similar to those observed in
electrostatic interactions, dependent upon the length of the published crystal structures of CMPs such as
amino acid side-chain. We postulate that the longer lysine H-(POG) EKG(POG) -OH reported by Berman et al.
side-chain length in lysine-containing Adpn peptides and
facilitates the formation of intrastrand and interstrand
hydrogen bonding networks, thus stabilising the triple helical β-galactose into the lysine-containing Adpn peptide
structure. Conversely, the serine analogues and
may not be dramatically lowered the thermal stability of glycosylated
able to adopt the correct conformation for hydrogen bonding,
peptide . This is in stark contrast to previous studies of
thus destabilising the structure and resulting in a random coil glycosylated CMP scaffolds in which glycosylation has typically
3
residues
5
could also have contributed to a stabilising
5
0
4
5
4
5
It is interesting to note that incorporation of triazole-linked
5
6
7
4
conformation.
resulted in either enhanced triple helical propensity and
thermal stability, or only moderate reduction. Most
3
4
In addition to favourable hydrogen bonding interactions, dramatically, Bann et al. synthesised repeat sequences of the
the lysine-containing Adpn peptide
may also have additional (Gly-Pro-Thr) motif found in the cuticle collagen of the
electrostatic interactions favouring the collagen-type triple hydrothermal vent worm Riftia pachyptila and demonstrated
helical structure, which the serine analogues and
may lack. triple helix formation only if the threonine residue was β-
have reported the importance of galactosylated. The stabilising influence of galactosylation in
4
6
7
4
6-49
Several groups
electrostatic interactions between positively and negatively that system was ascribed to carbohydrate-mediated ‘shielding’
charged residues for the structural stability of collagens. of peptide amide-water hydrogen bonds, favouring the
4
Indeed, Brodsky et al. noted that ionisable residues (Lys, Arg, formation of inter-strand hydrogen bonding and thus triple
6
Glu and Asp) account for 15-20% of total residues in helix formation. Conformational restriction of the galactosyl
collagenous peptides, indicating
electrostatic interactions in the formation and/or stabilisation such a mechanism operating in the case of our glycosylated
of the triple helical structure and subsequent fibril formation peptide . Further, linker length has been shown to be a key
a
key role for such residue due to the presence of the triazole linker may prevent
4
51
of collagenous peptides.
variable in the conformational preferences of glycopeptides ,
an observation consistent with the differing triple helical
propensities in ‘click’ glycopeptides 4 and 6.
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Wennemers et al. have reported functionalization of glycosides to modify the thermal properties of collagenous
azidoproline-containing CMP constructs with galactosyl materials.
residues. In these studies, only a relatively slight decrease (5
°
C) in thermal stability was reported upon incorporation of a
triazole-linked galactosylated hydroxyproline (O*) residue into
the CMP Ac-(POG) PO*G-(POG) -NH
Acknowledgements
3
3
2
.
The authors would like to thank the Maurice Wilkins Centre for
Molecular Biodiscovery for financial support of this work.
The observed decrease in thermal stability is also in
contrast to previous literature reports of native glycosylated
3
Adpn and the recently published work by Ohkubo et al.
3
19
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1
.
I. Padmalayam and M. Suto, Current pharmaceutical
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.
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2
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2
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5
.
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5
3
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peptide 4.
6
.
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1
9
7.
Y. Sun and H. F. Lodish, PLoS One, 2010, 5, e11987.
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significant opportunity for the use of Adpn ‘guest’ sequences
in concert with ‘click’ neoglycosides in collagen-type materials
to manipulate the thermal properties of these materials.
8
9
1
.
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