Role of trehalose phosphate synthase and trehalose during hypoxia: from flies to mammals
Department of Pediatrics and Neuroscience, Albert Einstein College of Medicine, Bronx, New York, USA
* Author for correspondence (e-mail: Ghaddad{at}aecom.yu.edu)
Accepted 9 June 2004
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Summary |
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Key words: trehalose, glucose, hypoxia, anoxia, trehalose phosphate synthase
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Introduction |
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In this review, we capitalize on some of our most recent work on an invertebrate, Drosophila melanogaster, and focus on the role of some chaperones in hypoxic injury or survival. We will review how trehalose, a disaccharide, works in general, its role in Drosophila and in mammalian cells.
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Trehalose: molecular properties |
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This naturally occurring disaccharide is widespread throughout the
biological world. Elbein summarized the distribution of 1,1-trehalose in over
80 species representing plants, algae, fungi, yeasts, bacteria, insects and
other invertebrates (Elbein,
1974). From the wide variety of species that have been shown to
contain trehalose, it seems likely that trehalose may be present in many other
organisms.
In the animal kingdom, trehalose was first reported in insects, where it is
present in the hemolymph and also in larvae or pupae. In the adult insect, the
levels of trehalose fall rapidly during certain energy-requiring activities,
such as flight, indicating a role for this disaccharide as a source of glucose
for energy (Elbein et al.,
2003). From our studies we found that the metabolic pool of
trehalose was active in Drosophila (discussed later). Trehalose is
not found in higher species (mammals), even though trehalase has been found in
significant amounts in the small intestine and other organs of various species
(Richards et al., 2002
).
Synthesis and degradation
The best studied pathway for the biosynthesis of
,
-1,1-trehalose is that involving the enzyme trehalose-phosphate
synthase (TPS1), which catalyzes the transfer of glucose from UDP-glucose to
glucose-6-phosphate to produce trehalose-6-phosphate plus UDP; then
trehalose-6-phosphate is hydrolyzed to trehalose by trehalose-6-phosphate
phosphatase (TPS2). This reaction was first described in yeast
(Cabib and Leloir, 1958
) and
has since been demonstrated in numerous organisms, including insects
(Murphy and Wyatt, 1965
) and
plants (Eastmond et al., 2002
;
Vogel et al., 2001
). Trehalase
is an enzyme that specificly hydrolyzes trehalose, yielding glucose.
Trehalose levels may vary greatly in certain cells depending on the stage
of growth, the nutritional state of the organism, and the environmental
conditions. In insects, trehalose is a major sugar in the hemolymph and thorax
muscles and is consumed during flight. Trehalose is also an important
component in fungal spores, where trehalose hydrolysis is a major event during
germination and can serve as a source of carbon
(Elbein et al., 2003). In
mycobacteria, trehalose can be incorporated into glycolipids and therefore
acts as a structural component (Elbein and
Mitchell, 1973
).
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Function of trehalose |
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Heat, dehydration and oxidant stress
Organisms have evolved various mechanisms for adaptation to adverse
environmental conditions such as lack of water (dehydration) and high
temperature. For example, a chironomid, Polypedilum vanderplanki
Hint., is the largest multicellular animal known to tolerate almost complete
dehydration without damage. Cryptobiotic larvae show extremely high thermal
tolerance from 270°C to +106°C and can recover soon after
prolonged dehydration of up to 17 years
(Hinton, 1960).
One of the mechanisms for the tolerance of Polypedilum
vanderplanki to extreme conditions is that larvae can rapidly accumulate
a large amount of trehalose (18% of dry body mass)
(Watanabe et al., 2002). Soto
et al. (1999
) examined the
effects of trehalose-6-phosphate (trehalose-6P) synthase overexpression on
resistance to several stresses in cells of S. pombe transformed with
a plasmid bearing the tps1 gene, which codes for
trehalose-6-phosphate synthase, under the control of the strong
thiamine-repressible promoter. Upon induction of trehalose-6-phosphate
synthase, the elevated levels of intracellular trehalose correlated not only
with increased tolerance to heat shock but also with resistance to other
stresses such as freezing and thawing, dehydration and osmostress, and toxic
levels of ethanol (Soto et al.,
1999
).
Introduction of trehalose into plant and mammalian cells using transgenic
techniques increases resistance to drought and desiccation. For example, Garg
et al. (2002) overexpress
Escherichia coli trehalose biosynthetic genes (otsA and
otsB) as a fusion gene under the control of stress-dependent
promoters in rice. The transgenic rice plants accumulated trehalose at levels
310 times that of the nontransgenic controls. Compared with
nontransgenic rice, several independent transgenic lines exhibited sustained
plant growth, less photo-oxidative damage, and more favorable mineral balance
under salt, drought and low-temperature stress conditions
(Garg et al., 2002
). Similar
results were obtained with human fibroblasts that had the otsA and
otsB genes inserted and expressed. These cells could be maintained in
the dry state for up to 5 days, as compared with control cells that were very
sensitive to drying (Guo et al.,
2000
). Trehalose at 50, 100 and 200 mmol l1
protected corneal epithelial cells in culture from death by desiccation
(Matsuo, 2001
).
Tissue injury due to oxidant species is present in many clinical settings,
such as hypoxic pulmonary hypertension, ARDS (acute respiratory distress
syndrome) and coronary heart disease. Though there is no evidence that
trehalose can reduce oxidant injury in mammalian cells, a large body of
evidence has been collected in Candida albicans and S.
cerevisiae, indicating that trehalose could be a promising free radical
scavenger. Growing Candida albicans cells from trehalose-deficient
mutant were extremely sensitive to severe oxidative stress exposure
(H2O2), while in wild-type cells
H2O2 exposure induced intracellular accumulation of
trehalose and a higher survival rate after the same exposure. Exposure of
Saccharomyces cerevisiae to a mild heat shock (38°C) or to a
proteasome inhibitor (MG132) induced trehalose accumulation and markedly
increased the viability of the cells upon exposure to a free
radical-generating system (H2O2/iron). When cells were
returned to normal growth temperature (28°C) or MG132 was removed from the
medium, the trehalose content and resistance to oxygen radicals decreased
rapidly. Providing trehalose exogenously enhanced the resistance of mutant
cells to H2O2, and trehalose accumulation was found to
reduce oxidative damage to amino acids in cellular proteins
(Benaroudj et al., 2001).
Tps1 and trehalose synthesis in Drosophila
We used yeast tps1 cDNA to blast the Drosophila database,
and found a gene with a 2427 bp open reading frame; this Drosophila
gene was found to be 30% similar to yeast tps1. Drosophila cDNA
library screening was performed using part of the sequence as a probe. We
cloned this gene, and its amino acid sequence was compared to yeast TPS1 and
TPS2. Drosophila TPS1 shows 29.7% identity to S. cerevisiae
TPS1 and 17.4% and 22.5% identity to S. cerevisiae TPS2 and S.
lepidophylla TPS1, respectively. Our experiment of overexpression of the
gene in Drosophila and mammalian cells confirmed that its function is
in synthesis of trehalose using UDP-glucose and glucose-6-phosphate as
substrate (Chen et al., 2002,
2003
).
Overnight-fasted flies were exposed to a solution of [113C]glucose
for 2 h, and fly heads were subjected to measurement of 12C- and 13C-labeled
trehalose using NMR. We found that 40.4% of the total trehalose was labeled at
13C, confirming that the trehalose pool is metabolically active
(Chen et al., 2002).
Trehalose protects Drosophila and mammalian cells from hypoxic and anoxic injury
The ability of organisms to sustain O2 deprivation is limited.
Irreversible injury may occur to mammalian tissues within 510 min of
severe hypoxia or ischemia. However, Drosophila can tolerate a
complete N2 atmosphere for up to 4 h, after which they totally
recover. One of the mechanisms that contributes to the survival of
Drosophila is the profound decline in metabolic rate during periods
of low environmental O2 levels
(Haddad et al., 1997;
Haddad and Ma, 2001
).
Trehalose is present in flies at a concentration of 120 µg 30
mg1 whole fly tissue and is metabolically active
(Chen et al., 2002
). We
investigated whether trehalose plays an important role in protecting flies
against anoxic stress. We first cloned tps1 (the gene for
trehalose-6-phosphate synthase, which synthesizes trehalose), and examined the
effect of tps1 overexpression or mutation on the resistance of
Drosophila to anoxia. Upon induction of tps1, trehalose
levels increased, and this was associated with increased tolerance to anoxia
(Fig. 1). Furthermore, in
vitro experiments showed that trehalose reduced protein aggregation (such
as Na+/K+ATPase) caused by anoxia
(Fig. 2). To determine whether
trehalose can protect against anoxic injury in mammalian cells, we transfected
the Drosophila tps1 gene (dtps1) into human HEK-293 cells
using the recombinant plasmid pcDNA3.1()-dtps1 and obtained
more than 20 stable cell strains. Glucose starvation in culture showed that
HEK-293 cells transfected with pcDNA3.1()-dtps1
(HEK-dtps1) do not metabolize intracellular trehalose and,
interestingly, these cells accumulated intracellular trehalose during hypoxic
exposure. In contrast to HEK-293 cells transfected with pcDNA3.1()
(HEK-v), cells with trehalose were more resistant to low oxygen stress (1%
O2). To elucidate how trehalose protects cells from anoxic injury,
we assayed protein solubility and the amount of ubiquitinated proteins. There
were three times more insoluble protein in HEK-v cells than in
HEK-dtps1 cells after 3 days of exposure to low O2. The
amount of Na+/K+ATPase present in the insoluble proteins
dramatically increased in HEK-v cells after 2 and 3 days of exposure, whereas
there was no significant change in HEK-dtps1 cells. Ubiquitinated
proteins increased dramatically in HEK-v cells after 2 and 3 days of exposure
but not in HEK-dtps1 cells over the same period
(Fig. 3). Our results indicate
that increased trehalose in mammalian cells following transfection by the
Drosophila tps1 gene protects cells from hypoxic injury. The
mechanism of this protection is probably related to a decrease in protein
denaturation through proteintrehalose interactions (Chen et al.,
2002
,
2003
).
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Mechanisms of action of trehalose
Different proteins are expected to interact with cosolvent molecules in
varied ways depending on their physicochemical properties. However,
trehalose has been observed to provide protection to different proteins to
various extents and the efficacy of protection depends on the nature of the
protein. To understand the mechanism of action of trehalose in detail, Kaushik
and Bhat (2003) conducted a
thorough investigation of its effect on the thermal stability in aqueous
solutions of five well-characterized proteins differing in their various
physico-chemical properties. Among them, RNase A has been used as a model
enzyme to investigate the effect of trehalose on the retention of enzymatic
activity upon incubation at high temperatures. Trehalose was observed to raise
the stability temperature of RNase A by as much as 18°C and Gibbs free
energy by 4.8 kcal mol1. There is a decrease in the heat
capacity of protein denaturation in trehalose solutions for all the studied
proteins. An increase in free energy and a decrease in protein denaturation
values for all the proteins point toward a general mechanism of stabilization
due to the elevation and broadening of the stability curve (free energy
versus temperature). They further show that an increase in the
stability of proteins in the presence of trehalose depends upon the length of
the polypeptide chain. Their pH dependence data suggest that even though the
charge status of a protein contributes significantly, trehalose can be
expected to work as a universal stabilizer of protein conformation due to its
exceptional effect on the structure and properties of solvent water compared
with other sugars and polyols (Kaushik and
Bhat, 2003
). The continued presence of trehalose, however, can
interfere with refolding, demonstrating the importance of its rapid hydrolysis
following heat shock (Singer and
Lindquist, 1998
).
During the freeze-drying process, or on subsequent storage in the dry
state, the protein conformation may be changed, exposing highly reactive sites
that are prone to physical and chemical changes over prolonged periods
(Service, 1997). This could
lead to protein degradation, and to a loss in biological activity. Carpenter
and coworkers showed that sugars form hydrogen bonds with the protein in a
glassy matrix to maintain the protein's native conformation with reduced
mobility during lyophilization and storage
(Allison et al., 1999
). These
investigators have also shown that molecular compatibility between components
is an important factor for determination of their propensity to phase separate
and crystallize (Izutsu et al.,
1996
). The amorphous nature of trehalose is more `compatible' with
the protein than the crystalline nature of lactose, which could result in
phase separation of the protein and the sugar. Lam et al.
(2002
), studied protein
mobility in lyophilized proteinsugar powders using solid-state NMR, and
their results indicated that trehalose was `bound' to lysozyme while the
lactose phase separated during lyophilization and storage, which makes
trehalose a better protectant than lactose
(Lam et al., 2002
).
In summary, trehalose is an important protectant of protein integrity and seems to be important during stress. It is possible that some of the mechanisms that are important for recovery from stress are shared not only between organisms but also between various types of stresses. Trehalose is an example of how a disaccharide molecule can enhance protein integrity and limit protein degradation not only under heat stress and oxidant injury but also in anoxia. This is also an example of a mechanism that is helpful not only in Drosophila but in mammalian cells.
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Acknowledgments |
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