Physicochemical studies on the copper(ii) binding by glycated collagen telopeptides

Article abstract of DOI:10.1039/c4ob02536a

Emerging evidence indicates that levels of advanced glycation end-products (AGEs) correlate with age- and diabetes-related organ damage and may play a causative role in such damage. Increased chelation of Cu(ii) ions appears to play an important role in this process, however, the precise relationship between formation of AGEs and accumulation of Cu(ii) is yet to be determined. The interaction between AGEs and Cu(ii) has been investigated using a collagenous peptide that has been site-specifically modified by a key AGE. Potentiometric titration showed that introduction of this AGE increased the capacity of the host-peptide to bind Cu(ii). This result was confirmed by mass spectrometric characterisation of the AGE-modified peptide-Cu(ii) system. This journal is

Full text of DOI:10.1039/c4ob02536a

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Biomolecular Chemistry
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Physicochemical studies on the copper(II) binding
by glycated collagen telopeptides†
Cite this: DOI: 10.1039/c4ob02536a
a
b
a
a
Meder Kamalov, Paul W. R. Harris, Christian G. Hartinger, Gordon M. Miskelly,
b,c,d,e
a,b
Garth J. S. Cooper
and Margaret A. Brimble*
Emerging evidence indicates that levels of advanced glycation end-products (AGEs) correlate with age-
and diabetes-related organ damage and may play a causative role in such damage. Increased chelation of
Cu(II) ions appears to play an important role in this process, however, the precise relationship between
formation of AGEs and accumulation of Cu(II) is yet to be determined. The interaction between AGEs and
Cu(II) has been investigated using a collagenous peptide that has been site-specifically modified by a key
AGE. Potentiometric titration showed that introduction of this AGE increased the capacity of the host-
peptide to bind Cu(II). This result was confirmed by mass spectrometric characterisation of the AGE-
modified peptide-Cu(II) system.
Received 4th December 2014,
Accepted 20th January 2015
DOI: 10.1039/c4ob02536a
www.rsc.org/obc
and the resulting hyperglycaemia lead to tissue Cu(II) excess
remains unresolved.
Introduction
Divalent copper is an integral component of cuproproteins.
This disbalance in the metabolism of Cu(II) could be
These proteins serve a number of physiological functions that mediated by advanced glycation end-products (AGEs). AGEs
are critical for the growth, development, and fitness of the are a family of derivatives of naturally occurring amino acids
1
human organism. A prolonged severe imbalance in Cu(II) that form when sugars and sugar degradation products modify
6
metabolism leads to serious debilitating conditions, such as protein side-chains. Such AGE formation is irreversible and
Menkes disease in cases of Cu(II) deficiency and Wilson’s AGE-modification of proteins in blood-vessel walls is a wide-
2
,3
disease in cases of Cu(II) excess.
An imbalance in Cu(II) spread phenomenon in diabetes. AGE accumulation is closely
metabolism on a smaller scale is also believed to play an linked to the pathogenesis of vasculopathy and organ damage
6
important role in the pathogeneses of a number of ageing- in nonclinical models and in diabetic patients. Levels of AGEs
4
and diabetes-related conditions. The precise cause and effects and the extent of disbalance of Cu(II) metabolism both
of this imbalance are yet to be determined.
increase with the advanced age and increase markedly with
7
,8
Elevated levels of chelatable Cu(II) in tissues are observed in the onset of diabetes mellitus.
nonclinical models of diabetes, whereas studies of urinary
The relationship between AGEs and Cu(II) appears to lead
copper excretion have provided evidence for systemic copper to a self-reinforcing and destructive cycle. Treatment of dia-
4
excess in diabetic patients. Because Cu(II) is highly redox betic rats with a copper chelator has significantly reduced the
9
active it can contribute to diabetic tissue damage by generating levels of not only Cu(II) but also of AGEs and AGE-precursors.
5
reactive oxygen species. The mechanism by which diabetes This result is consistent with the concept that excess Cu(II)
plays an important role in AGE formation. AGEs, in turn, are
believed to produce the pathological Cu(II)-binding sites in
a
tissues, thus further enhancing the systemic excess of Cu(II).
Yet direct evidence for Cu(II)-binding by protein-bound AGEs is
School of Chemical Sciences, The University of Auckland, 23 Symonds Street,
Auckland, New Zealand. E-mail: m.brimble@auckland.ac.nz
b
10
Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland,
missing.
3
c
Symonds Street, Auckland, New Zealand
ε
A key member of the AGE family, N -carboxymethyllysine
School of Biological Sciences, The University of Auckland, 3 Symonds Street,
Auckland, New Zealand
(
CML, Fig. 1), accumulates in organ tissues and its levels corre-
d
late with the severity of ageing-related conditions, such as catar-
Centre for Advanced Discovery and Experimental Therapeutics, Central Manchester
1
1
University Hospitals, NHS Foundation Trust, and the School of Biomedicine,
The University of Manchester, Manchester, UK
act and heart disease. Because of its structure, CML has the
potential to effectively chelate Cu(II) ions via the N-donor atom
e
Department of Pharmacology, Division of Medical Sciences, University of Oxford,
12
(
the ε-amine) and the neighbouring carboxylate. However, the
Oxford, UK
†
effect of CML formation on the overall metal binding capacity
Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4ob02536a
of a host peptide or protein has not been reported.
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Potentiometric analysis
Both peptides 1 and 2 feature carboxylic acid, hydroxyl, and
amine functional groups. The protonation constants of pep-
tides 1 and 2 were measured by potentiometrically titrating
acidic solutions of peptides with NaOH (results summarised
in Table 1). Peptide 1 can be considered a pentaprotic acid
with five ionisable groups in the measurable pH range (2–12),
i.e., two acids, an amine, and the two phenol groups of tyro-
sine side-chains. In contrast to peptide 1, six ionisable groups
are present in peptide 2, which has an additional carboxylic
acid group. The protonation constants determined for these
Fig. 1 Peptide-bound CML.
Peptide complexes of metal ions have been extensively
investigated as model systems for metalloprotein structure and
20
groups were in good agreement with literature data. The
presence of the extra carboxylic acid appeared to have altered
the pK values of the remaining acids in the peptide (Table 1).
a
1
3
function. Herein we report our initial studies on the Cu(II)
binding properties of peptides site-specifically modified by
CML. Since collagen is the major target of glycation and the
telomere region of collagen has access to transition metals
1
4,15
in vivo,
modified collagen telopeptides were employed as
glycation targets.
Results and discussion
Peptide synthesis
The binding behaviour between peptides 1 and 2 (Fig. 2) as
ligands and Cu(II) was investigated via potentiometry and mass
spectrometry.
The peptide sequence YGYDEKSTGGISVP was used to
model the telomere region of collagen and was derived from
1
6
human type I α1 collagen. Peptide 1 was synthesised via
automated solid phase peptide synthesis following the pro-
1
7
cedure described previously. Peptide 2 was synthesised via
1
8
an on-resin amino alkylation strategy, where Fmoc-Lys(Ns)-
OH was incorporated using analogous conditions as for
peptide 1 (Scheme 1). Upon completion of the assembly of
peptide 3 but prior to its cleavage, the N-terminus of the resin-
bound peptide was acetylated with acetic anhydride, and the
resulting peptide was subjected to alkylation with tert-butyl
bromoacetate in the presence of DIPEA to afford peptide 4. Scheme 1 Synthesis of CML-containing peptide 2. Reagents and con-
The nosyl group on peptide 4 was then smoothly deprotected ditions: (a) Fmoc-SPPS (i) 20% piperidine in DMF, (ii) Fmoc-AA-OH,
1
9
HBTU, DIPEA, (iii) steps (i) and (ii) repeated until the desired sequence is
built; (b) (i) N-terminal Fmoc removal: 20% piperidine in DMF, (ii) Ac O,
DIPEA; (c) tert-butyl bromoacetate, DIPEA, DMF, rt, overnight; (d) (i) Ns
using 2-mercaptoethanol and DIPEA (15 min), and the result-
ing peptide cleaved from the resin with TFA–TIS–H
38 : 1 : 1). The crude peptide was purified by HPLC to afford 2
2
2
O
(
removal: 2-mercaptoethanol, DBU, DMF, rt, 15 min, (ii) peptide release:
in 30% overall yield with >98% purity.
TFA–TIS–H O, rt, 2 h.
2
Fig. 2 Structures of the target peptides 1 and 2.
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Table 1 Stepwise protonation constants for peptides 1 and 2 and their
stability constants with Cu(II) at 298.1 K and I = 0.1 M (NaCl)
1
2
H
H
H
H
H
HL
CuH
CuH
6
5
4
3
2
L
L
L
L
L
—
2.06(6)
3.63(6)
4.84(7)
9.10(3)
9.66(2)
10.07(6)
4.01(5)
8.62(2)
11.6(6)
2.23(7)
5.93(7)
9.30(4)
10.29(4)
10.33(2)
3.33(9)
—
3
L
L
2
CuHL
—
a
Equipped with the pK values for the individual functional
groups of the peptides, we then measured the stability con-
stants of complexes of peptides 1 and 2 with Cu(II) (summarised
in Table 1). Only one species of a metal complex could be fitted
to the experimental titration curve obtained for the peptide
1
/Cu(II) system, i.e. CuH L (Table 1). The stability constant
3
3
obtained for CuH L agrees with previously reported values for
individual amino acid side-chain groups present in the peptide
2
1
and shows that peptide 1 does not strongly bind Cu(II).
The speciation diagram for the complex formation between
Cu(II) and peptide 1 showed that the complexation started at
pH 3, peaked at pH 5.5, but largely vanished at pH 7 due to
2
competition by precipitation of Cu(OH) (Fig. 3A). In vivo, type I
α1 collagen, from which the telopeptide sequence was taken, is
predominantly exposed to an environment of pH ca. 7 and
2
2
therefore little metal binding by telopeptide 1 is expected.
The titration curve of the CML telopeptide 2 in the presence
of Cu(II) was significantly different from that of the telopeptide Fig. 3 Species distribution diagrams for titration of peptides 1 (A) and 2
B) with NaOH in presence of Cu(II).
(
1
. Three metal complex species could be fitted to the titration
curve, i.e., CuHL, CuH L, and CuH L. The species distribution
2
3
diagram indicated that the first complexation to form CuH3L
started at pH >3.0 and peaked at pH 4.12, where it accounted
for 40% of all Cu(II) present in solution (Fig. 3B). The com-
suited for studies on ligand binding interactions due to the soft-
ness of the ionisation process. The interaction between Cu(II)
ions and the synthetic telopeptides 1 and 2 at different pH
levels was probed using ESI-MS by recording the mass spectra
of peptides 1 and 2 in negative ion mode both in the absence
and in the presence of Cu(II). For all ESI-MS experiments
described here, the peptide concentration was 3.5–5 μM.
As often observed for peptides, the negative ESI mass
spectra of peptides 1 and 2 featured a number of singly and
multiply charged ions due to deprotonation. The most abun-
plexation to form CuH L started at pH 3.09 before the for-
2
3
mation of CuH L was complete and peaked at pH 5.85, where
it accounted for 87% of all Cu(II) present in solution. In paral-
lel, the third complexation to form CuHL started at pH 4.52
and was the dominant complex type at pH 8, the highest in
this experiment. Importantly, at pH 7.00 the copper complexes
CuH L and CuHL accounted for 96% of all Cu(II) species
2
present in solution, i.e., the majority of the available copper
was bound by the CML-modified collagen telopeptide 2.
While the dramatic difference between the titration of pep-
tides 1 and 2 in the presence of Cu(II) is notable, the key
problem with such potentiometric studies of metal–peptide
interaction is the complex nature of the equilibria under investi-
gation. Furthermore, the information on the species formed is
limited and therefore such data needs to be complemented by
other characterisation methods such as mass spectrometry.
dant peaks were assigned to the respective doubly negative
2−
[M − 2H] ions, and therefore this region of the mass spec-
trum was selected for data analysis (results summarised in
Table 2). The peak at m/z 755.36 in the spectrum of 1 at pH 7.3
2−
was attributed to the [1 − 2H] ion, while the peak at m/z
2−
7
92.32 was identified as the [1 + K + Cl − 2H] ion. Analo-
gously, the mass signal at m/z 784.35 in the spectrum of 2 at
2−
pH 6.9 was attributed to the [2 − 2H] ion, while the signal at
2−
m/z 795.34 was assigned to the [2 + Na − 3H] ion.
The ESI-mass spectrum of peptide 1 in the presence of
Cu(II) at pH 6.7 showed no additional peaks that could be
Mass spectrometry
Electrospray ionisation-mass spectrometry (ESI-MS) was used assigned to Cu adducts but a NaCl adduct was detected at m/z
to complement the potentiometric data. ESI-MS is perfectly 784.34 (Table 2, Fig. 4A). In order to study the effect of the pH
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Table 2 Experimental (m/z) and theoretical (m/zcalc) m/z (z = 2) values copper complex ion of the composition [2 + Cu − 4H]2−,
for peptides 1 and 2 and the identified Cu(II) adducts
whereas the signal at m/z 833.81 was identified as a peptide–
2−
copper complex ion of the composition [2 + Cu + Cl − 3H]
.
Peptide
Adduct
m/z
m/zcalc
The mass spectrum of the CML-modified peptide 2 com-
2
1
1
1
2
2
2
2
2
[1 − 2H] −
755.36
792.32
784.34
784.35
795.34
792.82
814.82
833.81
755.36 bined with CuCl at pH 5.4 displayed signals corresponding to
2
2
−
[1 + K + Cl − 2H]
792.32
784.34
784.35
the same three peptide–copper complexes observed at pH 7.0.
Notably, no adducts were observed at pH 3.2. This result is in
795.34 accordance with the potentiometric titration analysis, which pre-
2
−
[1 + Na + Cl − 2H]
2−
[2 − 2H]
[2 + Na − 3H]
[2 + Cu − 4H – CO
2
−
2
−
2
]
792.82
814.82
833.81
dicted only a small amount of copper complexation at pH 3.2.
2
−
[2 + Cu − 4H]
[2 + Cu + Cl − 3H]
2
−
The nature of the Cu–peptide species was further confirmed
by comparing the experimentally measured isotope ratios with
calculated isotopic distributions (Fig. 5). All of the calculated
isotope distributions closely match the experimental data. The
impact of the presence of Cu(II) is clearly visible in all three
isotopic distributions. In case of the signal at m/z 833.81 the
2
−
composition was identified as [2 + Cu + Cl − 3H] , as the
isotope distribution clearly shows the presence of a single Cl
in the dianion.
The results of this ESI-MS study of the copper binding by
telopeptides 1 and 2 confirm the conclusions from the poten-
tiometric study. The formation of strong copper–peptide 2
complexes was predicted by the potentiometric titration. Such
Cu(II) complexes were clearly visible in the ESI-MS spectra of
solution containing peptide 2 and Cu(II) at pH 7. ESI-MS
measurements at lower pH levels were also in agreement with
the potentiometric predictions. Thus, at pH 3.2 no copper-con-
taining species could be observed by ESI-MS. Not surprisingly,
analogous ESI-MS investigations of the lysine-containing CTP
1
did not show any copper-containing species.
Experimental
Peptide synthesis
SPPS was performed via the Fmoc strategy on Rink Amide
resin using a Biotage Alstra peptide synthesiser on 0.1 mmol
scale. The Fmoc group was deprotected with 20% piperidine
in DMF for 2 + 3 minutes at 60 °C. The coupling step was per-
formed with Fmoc-AA-OH (5 equiv.) in DMF (0.2 M), HBTU in
DMF (4.5 equiv., 0.45 M) and DIPEA (10 equiv.) for 5 min at
Fig. 4 Mass spectra in the range m/z 700–900 of peptides 1 (A) and 2
B) in presence of Cu(II) at various pH levels featuring double negatively
charged anions.
(
75 °C. The final Fmoc-group was removed and the amine was
on adduct formation, ESI-MS measurements of incubation mix- acetylated by Ac O in the presence of DIPEA at rt for 10 min.
2
tures containing 1 and CuCl were conducted at pH 5.9 and 2.9.
Resin-bound peptide 3 (0.1 mmol, 1 equiv.) was swollen in
2
However, neither spectrum showed any evidence of Cu coordi- dichloromethane (30 min), then in DMF (10 min), and
nation. A KCl adduct was observed at pH 5.9, whereas at pH 2.9 drained. A solution of tert-butyl bromoacetate (70 μl, 0.5 mmol,
both NaCl and KCl adducts were observed (Fig. 4A).
5 equiv.) with DIPEA (175 μl, 1 mmol, 10 equiv.) in DMF (5 ml)
Addition of CuCl to the CML-containing peptide 2 at a was added in one portion and the reaction mixture shaken over-
2
molar ratio of 1 : 1.1 (peptide : Cu) caused the formation of night at rt to afford the peptide 4, which was washed with DMF.
ions that were not observed in the mass spectrum of 2 without Peptide 4 (0.1 mmol, 1 equiv.) was next swollen in dichloro-
CuCl
2
addition (Fig. 4B).
methane (30 min), then in DMF (10 min) and filtered. A solu-
Upon reaction of 2 with Cu(II) at pH 7.0 three copper con- tion of 1,8-diazabicyclo[5.4.0]undec-7-ene (90 μl, 0.6 mmol, 6
taining ions were observed (Fig. 4B). The signal at m/z 792.82 equiv.) and 2-mercaptoethanol (42 μl, 0.6 mmol, 6 equiv.) in
was assigned to the decarboxylated Cu-containing [2 + Cu − 4H DMF (5 ml) was added in one portion and the mixture shaken
2−
−
CO2] ion. The loss of CO is common and can be explained for 15 min, drained, and washed with DMF.
2
by degradation of one of the three acid residues present in the
The peptides were released from the resin with concomitant
system. The signal at m/z 814.82 was assigned to a peptide– removal of the side-chain protecting groups by treatment with
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Fig. 5 Comparison of the simulated (left) and measured (right) isotopic distributions of Cu-species observed in the mass spectra of 2 in presence of
CuCl at pH 7.0.
2
TFA–TIS–H
2
O (38 : 1 : 1, 5 ml) at rt for 2 h. Peptides were preci-
The ionic strength of all solutions was set at 0.1 M using
pitated with cold diethyl ether, isolated by centrifugation, NaCl. CuCl was the source of the metal ion. 0.01 M HCl was
2
washed in cold diethyl ether, dissolved in 1 : 1 MeCN–H O con- used in calibration of the pH electrode (E° = 408.9 V, slope
2
taining 0.1% TFA and lyophilised. The peptides were analysed factor = 0.995). Peptides 1 and 2 were dissolved in 0.01 M HCl
for purity by LCMS using a Zorbax C3 column (3.5 μ; 3 × at 0.1 M ionic strength and titrated with 0.189 M NaOH either
−1
1
50 mm; Agilent) at 0.3 ml min using a linear gradient. The with or without addition of the CuCl solution. The metal-to-
2
solvent system used was A (0.1% formic acid in H
2
O) and B ligand molar ratio in titrations in the presence of Cu(II) was
(0.1% formic acid in acetonitrile). Purification of the crude 1 : 1. Titrations were carried out under a nitrogen atmosphere
peptides was performed by semipreparative HPLC using a at 298 K using a total volume of 5–6 ml. 0.4–0.6 mM ligand
Gemini C18 column (10 μ; 250 × 10 mm; Phenomenex) at 5 ml concentration was used in all measurements. Starting with the
−
1
min using a shallow linear gradient. The solvent system fully equilibrated initial titration solution (pH 2–3, 25 °C), a
used was A (0.1% TFA in H O) and B (0.1% TFA in known volume of base was added and the solution allowed to
2
acetonitrile). The resulting purified peptides were analysed re-equilibrate. The potential was then recorded. This procedure
by the LCMS system used for crude peptide analysis. was repeated until the full pH range of interest was covered.
The purities were determined from the integrated areas of
the peaks corresponding to peptide 1 (13.1 min) and to tiometric data by using Hyperquad. The species distribution
Stability constants (log β) were determined from the poten-
2
3
peptide 2 (12.7 min).
diagrams were prepared from the determined log β values
using HySS.
2
4
Potentiometry
Mass spectrometry
2
Stock solutions of supporting electrolyte, CuCl , and peptide
ligands were prepared volumetrically from the analytical grade All mass spectra were collected with a Bruker microTOF mass
salts. Stock solutions of HCl and carbonate-free NaOH were spectrometer (Bruker Daltonics GmbH, Bremen, Germany). A
prepared from aqueous concentrates, and their concentrations syringe pump (KD Scientific) provided a stable liquid flow at
determined by titrating the base against a volumetrically pre- 3 μl min− to the fused silica spray capillary. This capillary pro-
pared potassium hydrogen phthalate standard solution. The truded a short distance from the end of a stainless steel tube,
1
acid was titrated against the base.
through which nitrogen gas was passed to assist in desolva-
The titration apparatus consisted of a 20 mL water-jacketed tion. The spray assembly was held at a potential of 3.2 kV to
glass titration vessel attached to a thermostated water bath and induce charging of the liquid droplets, which were directed
sitting on a magnetic stir plate. The burette and electrode were through a series of heated apertures into a vacuum chamber
clamped to the stir plate and attached to the Metrohm (dry gas 6 L min− , dry temperature 180 °C). Here they were
1
Titrando 907 titrator.
desolvated to form isolated ions, which were directed by lenses
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into the high vacuum region of the time-of-flight mass analy-
ser. All m/z values reported here correspond to the most promi-
nent mass ions within each isotopic cluster, and all calculated
ion masses are based on the monoisotopic atomic weights.
Peptide concentrations in the analysed solutions were 4–5 μM
and ligand-to-metal ratio was 1 : 1.1. Lowering of the pH was
accomplished by addition of conc. acetic acid. Spectra were
recorded in negative mode over 1.4 min and averaged.
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Conclusions
The majority of plasma Cu(II) is bound to ceruloplasmin, while
the remainder is bound to albumin, transcuprein, various pep-
tides and amino acids. It is known that diabetes mellitus causes
defective copper regulation, and elevated tissue levels of chelat-
able copper occur in nonclinical models of diabetes. A systemic
excess of copper has also been shown in diabetic patients.
8 D. Konukoğlu, G. D. Kemerli, T. Sabuncu and
H. H. Hatemi, Horm. Metab. Res., 2002, 34, 367.
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4
22
Accumulation of AGEs is a widespread phenomenon in dia- 10 A. K. Saxena, P. Saxena, X. Wu, M. Obrenovich, M. F. Weiss
betes and this accumulation is closely linked to the patho-
geneses of vasculopathy and organ damage. AGEs can modify
and V. M. Monnier, Biochem. Biophys. Res. Commun., 1999,
260, 332–338.
amino acid residues in proteins of the fibrous ECM. Therefore 11 N. Rabbani and P. Thornalley, Amino Acids, 2012, 42, 1087–
accumulation of AGEs is believed to make the ECM proteins 1096.
more susceptible to copper binding, thereby causing the dis- 12 S. T. Seifert, R. Krause, K. Gloe and T. Henle, J. Agric. Food
balance of copper. However, to date no evidence of the enhanced
copper binding by AGE-modified peptides has been reported.
Our investigation of the copper binding capacities of pep-
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13 K. L. Haas, A. B. Putterman, D. R. White, D. J. Thiele and
K. J. Franz, J. Am. Chem. Soc., 2011, 133, 4427–4437.
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pH dependent ESI-MS experiments. 28, 1297–1310.
These results warrant further investigations into properties 16 M.-L. Chu, W. de Wet, M. Bernard, J.-F. Ding, M. Morabito,
of AGE-copper complexes and their impact on the develop-
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various AGE-peptides, is currently underway.
M. A. Brimble, Chem. Commun., 2014, 50, 4944–4946.
8 M. Kamalov, S. Yang, P. W. Harris, G. J. Cooper and
M. A. Brimble, Synlett, 2014, 1835–1838.
9 T. M. Woods, M. Kamalov, P. W. Harris, G. J. Cooper and
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1
1
Acknowledgements
Authors would like to thank the Maurice Wilkins Centre for 20 T. Kowalik-Jankowska, M. Ruta, K. Wiśniewska and
Molecular Biodiscovery for generous financial support.
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2
2
2
Notes and references
1
T. Hirayama, G. C. Van de Bittner, L. W. Gray, S. Lutsenko
and C. J. Chang, Proc. Natl. Acad. Sci. U. S. A., 2012, 109, 24 L. Alderighi, P. Gans, A. Ienco, D. Peters, A. Sabatini and
228–2233. A. Vacca, Coord. Chem. Rev., 1999, 184, 311–318.
2
Org. Biomol. Chem.
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