Effect of spermine-derived AGEs on oxidative stress and polyamine metabolism

Article abstract of DOI:10.1039/c7ob01346a

Non-enzymatic glycation between proteins and carbohydrates, such as advanced glycation end products (AGEs), are naturally occurring compounds implicated in aging and numerous degenerative diseases. Methyl glyoxal (MG), which is an intermediate of the AGE biosynthetic pathway, is known to react with primary amines of proteins to create a wide range of AGE modifications, such as carboxyethyl lysine (CEL) and methylglyoxal-derived lysine dimer (MOLD). As a means to investigate and probe the ROS production pathways of AGEs, low molecular weight compounds carboxyethyl spermine (CES) and methylglyoxal-derived spermine dimer (MOSD) were synthesized, which replace lysine with another highly nucleophilic biological amine, spermine (SPM). Contrary to expectations, results show CES- and MOSD-induced oxidative stress proceeds through different pathways. As such, we have developed useful probes that can be used to better understand and investigate pathways related to acrolein-based oxidative stress and/or polyamine metabolic pathways.

Full text of DOI:10.1039/c7ob01346a

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Effect of spermine-derived AGEs on oxidative stress and
polyamine metabolism
Received 00th January 20xx,
Accepted 00th January 20xx
c
Ayumi Tsutsui,a,b Ambara R. Pradipta,b Shinobu Kitazume,c Naoyuki Taniguchi and Katsunori
Tanaka * b,d,e
DOI: 10.1039/x0xx00000x
Non-enzymatic glycation between proteins and carbohydrates, such as advanced glycation end products (AGEs), are
naturally occuring compounds implicated in aging and numerous degenerative diseases. Methyl glyoxal (MG), which is an
intermediate of the AGE biosynthetic pathway, is known to react with primary amines of proteins to create a wide range of
AGE modifications, such as carboxyethyl lysine (CEL) and methylglyoxal-derived lysine dimer (MOLD). As a means to
investigate and probe the ROS production pathways of AGEs, low molecular weight compounds carboxyethyl spermine
(CES) and methylglyoxal-derived spermine dimer (MOSD) were synthesized, which replace lysine with another highly
nucleophilic biological amine, spermine (SPM). Contrary to expectations, results show CES- and MOSD-induced oxidative
stress proceed through different pathways. As such, we have developed useful probes that can be used to better
understand and investigate pathways related to acrolein-based oxidative stress and/or polyamine metabolic pathways.
www.rsc.org/
Introduction
Protein
Protein
Advanced glycation end products (AGEs) are proteins that have
been glycated by aldose-based sugars (eg. glucose, ribose, etc),
which are heavily implicated in numerous degenerative diseases,
such as atherosclerosis,1 Alzheimer's disease,2, 3 type 2 diabetes,1 as
well as retinopathy,1, 4, 5 and nephropathy.1, 6, 7 The general pathway
of various AGEs first involve Schiff base formation (between the
aldehyde of an aldose motif and a primary amine of a protein),
followed by Amadori rearrangement, and subsequent oxidation
(Scheme 1).1, 8-13 This naturally occurring process is known to be
further accelerated under oxidative stress conditions.
NH
N
CH
Protein
NH2
CH2
HC
O
C
O
HC OH
HO CH
HC OH
HO CH
HC OH
HO CH
Glucose derived AGEs
HC OH
HC OH
CH2OH
HC OH
HC OH
CH2OH
HC OH
CH2OH
D
-Glucose
Amadori compound
Schiff base
OH
OH
OH
OH
O
HC
C
O
O
HO2C
Arg + MG
Cys
N
N
N
NH
S
CH3
HN
HN
Cys
Orn
Arg-Pyrimidine
Orn
THP
Methyl glyoxal
SCEC
OH
Lys
O
Oxidative stress is defined as the presence of reactive oxygen
species (ROS), such as hydrogen peroxide (H2O2) and free radicals,
that can elicit harmful processes (eg. lipid peroxidation) within
biological systems. If not properly detoxified by catalase,14-17 H2O2
can react with Fe2+ to give strongly oxidizable hydroxyl radicals (-
OH).15, 16 One naturally occurring source of H2O2 is the polyamine
metabolic pathway.18
Arg
Lys + Arg
Orn
Lys
HN
Lys
CEL
N
H
N
N
Lys
N
Lys
MOLD
N
H
MODIC
O
NH
CO2H
NH2
2H
N
CO2H
NH2
NH2
H
N
CO2H
NH2
H
N
N
N
N
N
O
N
N
H
O
MG- H1
MG- H2
MG- H3
Scheme 1. General pathway for AGE formation. Nδ-(5-hydroxy-4,6-
dimethylpyrimidine-2-yl)-l-ornithine (Arg-Pyrimidine); Nδ-(4-carboxy-4,6-
dimethyl-5,6-dihydroxy-1,4,5,6-tetrahydropyrimidine-2-yl)-L-ornithine
(THP); S-carboxyethyl cysteine (SCEC); 2-ammonio-6-({2-[4-ammonio-5-
oxido-5-oxopentyl)amino]-4-methyl-4,5-dihydro-1H-imidazol-5-
a.Department of Agricultural and Life Science, Faculty of Agriculture, Shinshu
university, 8304 Minami-Minowa-mura, Kamiina-gun, Nagano 399-4598, Japan.
b.Biofunctional Synthetic Chemistry Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi ,
Saitama 351-0198 , Japan. E-mail: kotzenori@riken.jp
ylidene}amino)hexanoate (MODIC); 1,3-di(Nε-lysino)-4-methyl-imidazolium
(MOLD);
imidazolon-2-yl)-L-ornithine
Nε-(1-carboxyethyl)lysine
(MG-H1);
(CEL);
Nδ-(5-hydro-5-methyl-4-
2-amino-5-(2-amino-5-hydro-5-
methyl-4-imidazolon-1-yl)-pentanoic acid (MG-H2); 2-amino-5-(2-amino-4-
hydro-4-methyl-5-imidazolon-1-yl)pentanoic acid (MG-H3).
c. Disease Glycomics Team, Systems Glycobiology Research Group, Global Research
Cluster, RIKEN, 2-1 Hirosawa, Wako-shi , Saitama 351-0198 , Japan.
d.Biofunctional Chemistry Laboratory, A. Butlerov Institute of Chemistry, Kazan
Federal University, 18 Kremlyovskaya Street, Kazan 420008, Russia.
e. Japan Science and Technology Agency-PRESTO, 2-1 Hirosawa, Wako-shi, Saitama
351-0198, Japan.
Polyamines, such as spermine (SPM), are biological amines
known to have a role in various life processes. Due to their intrinsic
nucleophilicity, polyamines are also important precursors to various
metabolites. One notable example is the reaction of polyamines
with acrolein to produce 8-membered 1,5-diazacyclooctanes,19, 20
† Footnotes relaꢀng to the ꢀtle and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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Considering that polyamines are highly reactive nucleophiles, and
spermine conjugates are involved in oxidative stress processes,19-21
in this report, low molecular AGE compounds that structurally
mimic CEL and MOLD were synthesized. In the place of lysine,
spermine was reacted with MG to produce 2-carboxyethyl spermine
(CES) and methylglyoxal-derived spermine dimer (MOSD) (Scheme
2). Due to literature examples of biological pathways where AGEs
can induce ROS production,29 the goal of this study was to examine,
through cell-based assays, the ability of CES and MOSD to induce
production of oxidative stress substances, such as -OH, acrolein, and
H2O2.
which was previously shown by our group to inhibit amyloid-β-
peptide fibrillization.21 In the polyamine metabolic pathway, by-
products of polyamine catabolism by polyamine oxidase (PAO) are
H2O2 and 3-aminopropanal. Studies have also shown that 3-
aminopropanal can undergo further degradation to produce
acrolein.22, 23
Acrolein is
a compound that is characterized by a highly
electrophilic α,β-unsaturated aldehyde. Due to its potential to
alkylate proteins, acrolein has shown higher levels of cytotoxicity
compared to H2O2.24 There are, however, cases where acrolein-
based metabolites have shown beneficial bioactivities, such as the
1,5-diazacyclooctanes mentioned earlier.
Results and Discussion
Two unique AGE modifications that are of interest to this work
are 2-carboxyethyl lysine (CEL) and methylglyoxal-derived lysine
dimer (MOLD), which are formed when 1 and 2 equivalents of lysine,
respectively, react with methylgloxal (MG).25 MG is an intermediate
derived from D-glucose known to be important in the AGE
biosynthetic pathway (Scheme 1).25 Due to its high reactivity
(compared to glucose), MG is crucial for the formation of numerous
AGE compounds. Although the correlation between ROS levels and
MG concentration has been reported both in animals and cultured
cells,26-28 the effects of two AGEs (MOLD or CEL) on ROS or H2O2
production are still not clear.
The synthesis of the spermine-based CES and MOSD compounds are
detailed in Scheme 3. For CES (Scheme 3a), tri-N-Boc-spermine 1
was reacted with ethyl 2-bromopropionate to give tri-N-Boc-CES 2,
which through subsequent deprotection by 5M NaOH and 4N HCl
gave CES hydrochloride. For MOSD (Scheme 3b), MOSD
hydrochloride was synthesized by deprotection of hexa-N-Boc-
MOSD 3 with 6N HCl in dioxane-H2O (1:1). In this study, two
synthetic routes for hexa-N-Boc-MOSD 3 were pursued. One route,
which closely mimics the biological glycation reaction, relies on
incubating MG with 2 equivalents of tri-N-Boc-SPM 1. Although
MOLD has been synthesized in a similar manner between N-Boc-
Lys-OH and MG in literature,30 the reaction mechanism is still not
entirely clear. To obtain hexa-N-Boc-MOSD 3 through a more
classical synthetic approach, a route based on a previous synthetic
scheme of MOLD was pursued.31 Starting from 3-N-Boc-amino-1-
propanol 4, tri-N-Boc-SPM bromide 7 can be obtained in 7 steps.
Subsequent reaction with 4(5)-methyl imidazole can then give hexa-
N-Boc-MOSD 3. One issue with this synthetic route, however, is that
reaction with 4(5)-methyl imidazole gave many by-products with
inconsistent yields (16%-trace). In contrast, the glycation-mimicking
reaction discussed earlier produces more consistent yields (25%).
O
OH
H
H
CO H
2
HO C
2
N
H N
N
2
N
N
H
H
NH
2
CES
CEL
H
CO H
2
N
N
NH
2
H
N
N
NH
2
N
N
NH
H
2
N
N
NH
2
CO H
2
H
MOLD
MOSD
Scheme 2. Proposed modification of lysine-based CEL and MOLD to By comparison of the NMR spectrum, mass spectrometry and HPLC
spermine-based CES and MOSD.
data of hexa-N-Boc-MOSD 3 produced by both methods (Figure S1
HCl
OH
A)
O
OEt
O
Boc
Boc
H
N
i
ii, iii
BocHN
N
BocHN
N
H N
2
N
NH
2
N
N
H
N
H
N
H
Boc
Boc
1
2
CES
B)
H
Boc
N
N
N
NH
2
N
NHBoc
Boc
iv
v
H
Boc
BocHN
N
N
N
N
N
N
NH
2
HCl
NH
Boc
H
N
Boc
N
1
N
H
N
NHBoc
2
Boc
N
3
MOSD
xiii
Boc
BocHN
OH
BocHN
N
Br
4
Boc
7
vi-viii
xii
6
Boc
N
Boc
N
ix-xi
BocHN
BocHN
OH
N
OH
Boc
5
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Scheme 3. i) Ethyl bromoacetate, CH3CN, reflux, 70%; ii) 5N NaOH, MeOH-H2O, Amberlite IR-120B; iii) 4N HCl, Dioxane-H2O, quant. (2 steps); iv)
methylglyoxal, dioxane, 100°C, 28%; v)6N HCl, Dioxane-H2O, 81%; vi) PPh3, CBr4, toluene, 76%; vii) 4-amino-1-butanol, CH3CN, 54%; viii) (Boc)2O, CaCO3,
MeOH, 97%; ix) PPh3, CBr4, toluene, 75%; x) 3-amino-1-propanol, CH3CN, 77%; xi) (Boc)2O, CaCO3, MeOH, 79%; xii) PPh3, CBr4, toluene, 62%; xiii) 4(5)-
methylimidazole, CH3CN, trace.
-
ROS detection (including detection of OH, H2O2, and NOS, but not
O2-), the ROS-IDTM Total ROS detection kit (Enzo) was employed.
For H2O2 detection, the OxiVision Green™ hydrogen peroxide
sensor kit (ATT Bioquest) was used. Finally, acrolein detection was
performed with an azide-based reagent previously developed in our
laboratory.32
Scheme 4. Metabolism of spermine by APAO and SMO. N1-Acetyltransferase
(SSAT); N1-Acetylpolyamine oxidase (APAO); Spermine oxidase (SMO);
Flavin-adenine-dinucleotide (FAD).
To compare with CES and MOSD, the controls AGE, SPM+MG, and
separate sample runs of SPM and MG were also tested: AGE refers
to a mixture of CES, MOSD, and other AGEs products formed from
the pre-incubation of SPM and MG for 17 h before cell addition,
while SPM+MG refers to SPM and MG mixed at the time of cells
addition.
Typically, HeLa cell culture requires high concentrations of glucose
in order for normal growth. However, to avoid glycation of
spermine with glucose, media containing minimal levels of glucose
is required. As an initial study, the minimal glucose concentration
that could support cell growth overnight was examined (Figure S3),
leading to the choice of assay media to contain 1 mM D-glucose
without FCS.
ROS detection results are shown in Figure 1a and Figure S4. In
general, ROS species appear to be produced more than acrolein or
H2O2. ROS detection reagents were added after 15 min incubation
times, followed by fluorescence intensity detection after 45 min.
Pyocyanine is used as a positive control for ROS production. In all
samples under study (AGE, SPM+MG, MOSD, CES, SPM-only, MG-
only), an increase of ROS production was detected. This correlates
well with a previous report confirming ROS production through MG
addition.33,34
Figure 1. Detection of oxidative stress substances in HeLa cells incubated
with CES, MOSD, and corresponding controls. A buffer blank was added to
cells for the control sample. Fluorescent cell imaging was obtained with a
fluorescence microscope (KEYENCE BZ-X710, Japan) and intensities were
quantified by Image J software (NIH, USA). HeLa cells concentration were
adjusted to 30,000 cells/well in
a 8 well slide dish. Final compound
concentrations were 1 mM, except in the cases of Pyocyanine (200 μM),
acrolein (10 μM), and H2O2 (50 μM). Depending on the assay, different
excitation and emission wavelengths were employed: 470 nm/525 nm for
ROS detection, 545 nm/605 nm for Acrolein detection, and 470 nm/525 nm
for H2O2 detection.
In literature, acrolein production is widely associated with
spermine metabolism.35 Through two competing pathways,
spermine can be either metabolized by spermine oxidase (SMO) to
produce 3-aminopropanal, or by N1-acetyl polyamine oxidase
(APAO) to give 3-acetamidopropanal.23 Although both enzymes
produce H2O2, only SMO-produced 3-aminopropanal can be
converted to acrolein (Scheme 4).35 Considering these metabolic
pathways, the addition of spermine and its derivatives to cell
cultures is expected to lead to an increase in acrolein levels. In
Figure 1b and Figure S5, acrolein detection assay results are shown.
Observations show that CES addition produces more acrolein than
with spermine addition. As such, it is likely that the high acrolein
and S2), it can be confirmed that the glycation-mimicking reaction
produces the desired glycated compound.
With the synthesized CES and MOSD compounds, cell-based
assays were performed to gauge their ability to stimulate oxidative
s t r e s s s u b s t a n c e s ( R O S , H 2 O 2 a n d a c r o l e i n ) i n
HeLa cell lines. We tried various cell lines, but HeLa cells were
strong enough to survive to perform the designed experiments, i.e.,
under oxidatively stressed conditions, by exposures of AGEs.
Qualitative analysis of oxidative stress substances can be performed
through the use of commercial, fluorescence-based assay kits. For
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levels observed for the AGE and SPM+MG samples are due to not
only SPM but also CES formed either through pre-incubation or
intracellularly. The most significant observation from this data,
however, is that in contrast to every other sample, MOSD addition
consistently causes
a roughly 2 fold reduction in acrolein
production. Further time-course studies (Figure S7) of acrolein
production following MOSD addition show that cells do not produce
acrolein for 2-24 h. As a result of this data, it can be speculated that
the acting metabolic enzyme/pathway between MOSD and CES are
different. To complete the oxidative stress substance detection
study, H2O2 assay results are shown in Figure 1c and Figure S6.
Observations show clear production of H2O2 following MOSD and
CES addition.
(B) MOSD not recognized by SMO/PAO
To indirectly identify the metabolic pathway of CES, the effect of
external factors on CES-induced acrolein production was
investigated (Figure 2). In theory, inhibition of the pathway
responsible for CES metabolism should lead to decreased levels of
acrolein.
Scheme 5. Proposed metabolic pathways of CES and MOSD in cells.
Polyamine oxidase (PAO); Spermine oxidase (SMO); N1-Acetylpolyamine
oxidase (APAO); Flavin-adenine-dinucleotide (FAD).
As a collective result of this data, it can be advocated that CES-
induced acrolein production proceeds through a multi-faceted
approach (Scheme 5a). Naturally, CES is a likely substrate for
SMO/PAO, which directly leads to acrolein. As a secondary path,
CES can also be a substrate of APAO, which will produce the
metabolite spermidine. Subsequent PAO activity can further
metabolize spermidine to also give acrolein. Another significant
result of this work is that the compound MOSD unexpectedly
causes a reduction in acrolein, which indirectly suggests that it is
not a SMO/PAO substrate. It can be speculated that another
pathway may be responsible for its metabolism, likely through an
oxidative pathway (Scheme 5B). In this manner, acrolein would not
be produced as observed.
Figure 2. Changes in AGE- and spermine-induced levels of acrolein with AG
and GSH supplementation. A buffer blank was added to cells for the control
sample. HeLa cells concentration were adjusted to 30,000 cells/well in a 8
well slide dish. Final compound concentrations were 1 mM, except in the
case of H2O2 (50 μM). Final concentrations of acrolein and ROS inhibitors, AG
and GSH, was 10 mM.
Conclusions
With this work, two low molecular weight AGEs compounds (CES
and MOSD) were synthesized and examined through cell-based
assays for their ability to induce production of oxidative stress
substances. Results indicate that CES is likely a substrate of the
spermine metabolic pathway enzymes SMO/PAO, which as a by-
product, generates acrolein. It can be further suggested that CES
can also be metabolized by APAO, leading to spermidine and
eventual acrolein production via PAO. On the other hand, the highly
unusual activity of MOSD to halt and reduce acrolein production in
cells (by as much as twofold compared to control cells) spurns a
debate whether it is a substrate for an unknown metabolic
pathway. It can even be speculated whether AGEs, like MOSD, play
previously unknown roles in metabolic pathways. Overall, the
results of this work help to open the door for future research to
better understand and investigate pathways related to acrolein-
based oxidative stress and/or polyamine metabolic pathways. For
example, CES-derivatives can be explored for their greater acrolein-
production properties compared to normal polyamines, and AGEs
like MOSD can be used to probe other biological pathways in search
for other AGE-related activities.
As such, this study looked closely at the effects of amino guanidine
(AG). Besides being a known glycation inhibitor, AG is also a
SMO/PAO enzyme inhibitor.36 Therefore, if CES is part of the
polyamine metabolic pathway, AG addition should decrease
acrolein production. Also used in this study was glutathione (GSH),
which is a well known ROS inhibitor.24 In Figure 2, cellular acrolein
levels following CES incubation with and without AG and/or GSH
addition is shown. As expected, AG and/or GSH addition
significantly decreased acrolein levels for all CES-containing samples
(CES-only, AGE, or SPM+MG). As a consequence, it can be strongly
suggested that CES is a likely SMO/PAO substrate in the polyamine
metabolic pathway.
(A) Proposed mechanism of CES-induced acrolein production
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25. G. Vistoli, D. De Maddis, A. Cipak, N. Zarkovic, M. Carini and
G. Aldini, Free Radical Res., 2013, 47, 3-27.
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