1. Protein glycation in the pathology of ageing: A review
Boris Shilov
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1 Introduction
Biological ageing is a complex process and a long-standing enigma both in the history of
humanity and of biology. According to Jin (2010), the theories of ageing can generally be
split into two categories: programmed theories and damage theories.
Programmed theories postulate that ageing is an inherent, genetic process in the lifecy-
cle of an organism. The theories in this category include those that postulate that certain
genes switch on and off over time and result in various deficits, endocrine theories that
posit that there is a biological clock that regulates ageing hormonally and immune theo-
ries, which postulate that the gradual degradation of the immune system eventually leads
to the array of diseases whose risk increases drastically with old age. This also includes
the intriguing Hayflick limit theory, which posits that telomeres, the end regions of the
linear Eukariotic DNA, shorten with each successive division and, unless restored, a limit
is eventually reached and cellular division rendered impossible, or extremely damaging as
sections of coding DNA become lost.
Damage theories postulate that some form of damage to molecular structures of cells
results in ageing, with accumulation of damage over time leading to progressively worsened
outlook and eventually death. This includes wear and tear theory, where some vital com-
ponents of the cell, such organelles and long-term structural proteins accumulate damage
over time and eventually stop serving their function. Cross-linking theory which posits
that crosslink formation between proteins, DNA, lipids and other substances leads to a de-
crease in their function. Rate of living theory that posits that the lifespan of an organism
is inversely proportional to its basal metabolic rate. Free radical theory, one of the most
publicised, posits that free-radical species formed during normal metabolism may react
with various cellular components to cause damage. Further, DNA damage theory posits
that organisms continually incur damage to their DNA that, although mostly repaired,
accumulates over time, especially in mitochondria.
It is generally agreed that there is some substance to all of these theories, and that
they likely interact in complex ways to give the end result that organisms age (Jin 2010).
In this review, we will focus on how protein glycation affects the pathology of ageing. In
contrast to glycosylation, which is enzymatically mediated and serves a useful regulatory
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2. Boris Shilov
function in the cell in most cases, glycation is a spontaneous, non-enzymatic reaction that
occurs at a slow but constant rate in physiological conditions. Glycation may result in the
formation of advanced glycation end (AGE) products, a very diverse class of molecules that
bind proteins, DNA and lipids and may result in denaturation, breaks, cross-linking and
participate in undesirable side reactions. AGEs are very difficult for the body to degrade,
and some persist for the entire lifespan on an organism. Thus the accumulation of AGE
products in the body has been of considerable interest to ageing researchers recently (Frye
et al. 1998). We will see how AGEs are involved in diseases that result from ageing, with
particular focus on Diabetes mellitus as one of the most studied diseases the incidence of
which correlates with ageing.
2 Chemical aspects
Glycation is the non-enzymatic attachment of sugars to proteins or lipid molecules, where
the carbonyl group of a reducing sugar spontaneously reacts with the amino group of an
amino acid or other molecule. Lysine residue glycation plays an especially important role
in the pathology of ageing due to the abundance of this residue in structural proteins with
slow turnover rates, such as collagen (Ansari et al. 2011).
2.1 The Maillard reaction
The Maillard reaction is the major AGE-producing reaction at physiological conditions.
It can be divided into an initiation, propagation and termination steps, which account for
the generation of a variety of intermediates. Fig. 1 shows the generalised initiation step
for the reaction, leading to the formation of Amadori product that can either be converted
to Nε-carboxymethyl-lysine through oxidative fragmentation or proceed to form a number
of reactive dicarbonyl compounds. Fig. 2 shows the propagation stage where the Amadori
product isomerises to form either 1,2-eneaminol or 2,3-eneaminol. 1,2-eneaminol proceeds
to form 3-deoxyosone, whilist 2,3-eneaminol may react to form either 1-deoxyosone or 4-
deoxyosone. The deoxyosones may then decompose to form a variety of secondary products
in what is referred to as the termination step. These are the advanced glycation end
products themselves, and include a large variety of possible structures such as pentosidine,
pyrroles, furans, pyrimidines, pyrazines, imidazoles and many others (Belitz et al. (2009),
Monnier (1990)).
2.2 Methylglyoxal and the glyoxalase system
According to Thornalley (1996), methylglyoxal is a product of the conversion of glyceraldehyde-
3-phosphate (G3P) to dihydroxyacetonephosphate in glycolysis, and since this reaction may
proceed nonenzymatically, the major source of methylglyoxal in cells is the spontaneous
breakdown of G3P. This generates very small amounts, but occurs in every cell in the
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3. 2.3 The polyol pathway Boris Shilov
body, and over time would pose an unacceptable mutagenicity risk if methylglyoxal is not
properly deactivated. This is accomplished via the glyoxalase system.
The glyoxalase system converts methylglyoxal into D-lactate and S-D-lactoylglutathione.
It consist of the enzymes glyoxalase I and II. Glyoxalase I catalyzes the formation of S-
D-lactoylglutathione from hemithioacetal, which is nonenzymatically formed from methyl-
glyoxal and reduced glutathione. Glyoxalase II hydrolyses S-D-lactoylglutathione into D-
lactate and reforms reduced glutathione. Methyglyoxal may also be converted mostly to
hydroxyacetone by aldose dehydrogenase (Thornalley 1996).
Methyglyoxal’s mutagenic activity is complex. It can crosslink DNA strands in AT rich
regions, react with guanine and guanylate residues. It binds lysine and arginine residues in
proteins, preferring arginine. Methyglyoxal can create crosslinks between DNA and DNA
polymerase. This again occurs through reacting with a lysine residue (Murata-Kamiya &
Kamiya 2001).
2.3 The polyol pathway
Another way for AGE products to be generated in the cell is the polyol pathway. This is
activated when excess glucose enters the cell, beginning with the reduction of glucose to
sorbitol by aldose reductase, with the consumption of NADPH, and proceeding with sor-
bitol dehydrogenase, which converts sorbitol to fructose with the generation of NADH. The
fructose may then be converted to fructose-3-phosphate by 3-phosphokinase (Ramasamy
et al. 2005). 3-deoxyglucosone is generated in this reaction, a glucose-derived 3-deoxyosone,
which as previously mentioned may participate in a variety of reactions with proteins and
amino acids to generate pentosidine, pyralline and imidazolone. It is worth mentioning
that the rate of pentosidine generation is greatly accelerated by aerobic conditions, which
is the case in the cell (Niwa 1999).
The polyol pathway further complicates matters by the fact that fructose has been
shown to also generate AGEs and induce protein oxidation (Ramasamy et al. 2005).
3 Clinical aspects and implications
3.1 Diabetes mellitus
Although diabetes does not arise due to AGE products, the development of chronic hyper-
glycaemia leads to the occurrence of a multitude of diabetic complications, many caused by
increased AGE product generation. According to Thornalley (1996), intracellular hyper-
glycaemia is known to be the main mechanism of intra- and extracellular AGE generation.
Spontaneous oxidation of glucose to glyoxal and methylglyoxal formation are both more
pronounced as a result. The glyoxalase system that would usually detoxify these com-
pounds is overwhelmed in hyperglycaemia.
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4. 3.1 Diabetes mellitus Boris Shilov
AGE products contribute to the observable clinical symptoms of diabetes via cellular
damage, including the abnormal function of glycated proteins, abnormal interactions of
glycated matrix proteins with integrins, and reactive oxygen species (ROS) production by
macrophages that have bound AGEs. Macrophage binding is of particular importance in
diabetes-induced vascular disease and it has been shown that blockade of the receptor for
AGE (RAGE) on macrophages suppresses macrovascular disease (Brownlee 2001). Bind-
ing of AGE receptors on macrophages eliminates glycated proteins via receptor-mediated
endocytosis and lysosomal degradation (Thornalley 1996).
The role of RAGE in the pathogenesis of diabetes and many other diseases is interesting.
Hudson et al. (2002) reports that RAGE is a signalling receptor family that is itself part
of the immunoglobulin receptor superfamily, some classifying it as a pattern recognition
receptor. It has three immunoglobulin domains, two V type and one C type, having one
transmembrane region as well as a cytosolic signalling tail. RAGE seems to be expressed
in very low amounts normally, but diabetes upregulates RAGE expression in vascular
endothelium and smooth muscle, at least in animal models, which may contribute to the
chronic inflammation sometimes observed in diabetes (Hudson et al. 2002).
Some major complications of diabetes mellitus include nephropathy, neuropathy and
retinopathy. It has been found that, both in insulin-dependent and insulin-independent
diabetes, the amount of glycated haemoglobin and glyoxylase I activity both strongly
correlated with the appearance of these complications. Furthermore, increased incidence
of glycated proteins leads to their recognition by macrophages and monocytes, which in
turns leads to increase secretion of proinflammatory cytokines IL-1β and CSF-1, both impli-
cated to contribute to atherosclerosis, glomerulosclerosis and proliferative vitreoretinopathy
(Thornalley 1996).
Glycated proteins are broken down in the cell as part of normal protein turnover.
According to Moheimani et al. (2010), it is known that the balance of protein synthesis
and degradation is disrupted in diabetes and other AGE-involving diseases, and although
it has also been found that pathways that detoxify and remove modified proteins are also
upregulated in response to this, they do not appear to be able to compete efficiently with
the increased rate of AGE formation in the cell. When molecular repair mechanisms
such as refolding by chaperones fail, the proteins are designated to either by degraded
by the proteasome or processed through the endolysosomal system, with the proteasome
degradation pathway being the major one for intracellular proteins. The proteasome is
a multienzyme complex resembling a barrel (X-ray crystallography structure shown in
Fig. 3) that consists of a family of proteases called N-terminal nucleophile hydrolases, and
includes six active sites: two trypsin-like sites, two caspase-like sites and two chymotrypsin-
like sites. It is known experimentally that glycated bovine serum albumin is able to inhibit
the proteasome, specifically the chymotrypsin-like and caspase-like sites. Several reasons
for this inhibition have been proposed, such as the possibility of intraprotein crosslinks
that stabilise the structure, formation of crosslinks with the proteasome subunits and
modulation of the α subunit that facilitates substrate entry into the proteasome. Another
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5. 3.2 Alzheimer disease Boris Shilov
possible mechanism is the modulation of regulatory subunit conformation, which would
decrease proteasomal activity. It is interesting to note that only the long-term presence of
glycated protein seems to affect proteasome activity (Moheimani et al. 2010).
3.2 Alzheimer disease
Alzheimer disease is known to involve the deposition of insoluble β-amyloid protein ag-
gregates in neuronal cells. Studies suggest that AGE crosslinks may play a major role in
initiating the ”seed” required for these aggregates to form en masse (Vitek et al. 1994).
Damage results from several mechanisms, including ROS production from sugar autoxi-
dation, AGE-triggered interleukin-6 release and the subsequent immune reaction, leading
to lysis of nearby neurons through microglial overactivation. Microglia also posses RAGE
receptors, binding of which can lead to increase in oxidative stress. RAGE binding has also
been implicated in nonpathological neurite growth, and AGEs may interfere destructively
with this process. As mentioned previously, AGE-protein crosslinks lead to reduced pro-
teolytic susceptibility, which may contribute greatly to aggregate formation (M¨unch et al.
1997).
3.3 Rheumatoid arthritis
AGEs likely contribute to osteoporosis that is a major component of rheumatoid arthritis.
Hein (2006) asserts that AGEs are known to inhibit osteoblasts, cells that lay down the
extracellular matrix necessary to generate bone, and they are also known to increase bone
resorption by osteoclasts. Pentosidine is known to form collagen crosslinks, degrading the
mechanical properties necessary to maintain bone structure, and it has been found that
variation in pentosidine content partially accounts for differences in amount of strain bone
can sustain.
The modification of collagen in bone is hypothesised to be of major importance in
osteoporosis since collagen is a protein with an extremely low turnover. Accumulation of
AGE products in the bone matrix would presumably lead to hardening of the bone due to
denaturation of bone protein. However it is not currently clear if the increased incidence
of AGEs in bone is a cause of osteoporosis or an effect of it (Hein 2006).
4 Conclusion
AGEs remain poorly characterised as a group, despite intense research into their involve-
ment in disease. Further efforts are complicated by the heterogeneity of these compounds
and the difficulty in isolating them, and assays are available for few compounds, with pen-
tosidine and Nε-carboxymethyl-lysine being the best characterised AGE products. This
situation is similar for AGE receptors and their pathways, RAGE being the best charac-
terised receptor to date. Nonetheless, the field has seen steady progress and pharmaceu-
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6. REFERENCES Boris Shilov
ticals that reverse or inhibit glycation could prove to be a powerful treatment or way to
postpone the onset of later stages of disease (Singh et al. 2001).
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R NH2
Amine group
+ H C
C
H O
OH
R
Reducing sugar
R NH CH C
OH
R
Schiff base
R NH CH2 C
O
R
Amadori product
Fe2+
O2
R NH CH2 COOH
Nε-Carboxymethyl-lysine
Figure 1: The generalised initiation step of the Maillard reaction. The Amadori product
may proceed to the propagation step. The oxidative fragmentation reaction that gives rise
to Nε-carboxymethyl-lysine is irreversible. Adapted from Monnier (1990).
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9. REFERENCES Boris Shilov
R NH CH2 C
O
R
Amadori product
R NH2
Regenerated amine
+ CH3 C
O
C
O
R
1-deoxyosone
R NH2 + H C
O
C
O
CH2 R
3-deoxyosone
R NH CH2 C
O
C
O
CH2 R
4-deoxyosone
Figure 2: The generalised propagation step of the Maillard reaction. The Amadori product
isomerises to form either 1,2-eneaminol or 2,3-eneaminol (not shown), which proceed to
form different deoxyosones. The deoxyosones themselves are then free to assume a variety
of cyclic hemiacetal forms (not shown), and participate in termination reactions with a
variety of compounds. Adapted from Monnier (1990).
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10. REFERENCES Boris Shilov
Figure 3: The human 20S proteasome complexed with carfilzomib, a 28-meric protein
complex, viewed from the side. From Harshbarger et al. (2015)
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