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Bestenbalt products were used
in the following studies:
Metallothionein induces a regenerative reactive astrocyte phenotype
via JAK/STAT and RhoA signalling pathways
Y.K.J. Leunga, M. Pankhurst a, S.A.
Dunlop b, S. Ray a, J. Dittmann a, E.D. Eaton
a, P. Palumaa c, R. Sillard c, M.I. Chuah
a, A.K. West a and R.S. Chung a.
a) Menzies Research Institute, University of Tasmania,
Private Bag 58, Hobart, Tasmania 7001, Australia
b) School of Animal Biology, University of Western Australia, Nedlands, Western
Australia 6907, Australia
c) Department of Gene Technology, Tallinn Technical University. Akadeemia tee
15, Tallinn 12618, Estonia
Journal:
Experimental Neurology, Vol 221 (1), 98-106, 2010.
Abstract: Following central nervous system injury, astrocytes
rapidly respond by undergoing a stereotypical pattern of molecular and
morphological alterations termed “reactive” astrogliosis. We have reported
previously that metallothioneins (MTs) are rapidly expressed by reactive
astrocytes and that their secretion and subsequent interaction with injured
neurons leads to improved neuroregeneration. We now demonstrate that exogenous
MT induces a reactive morphology and elevated GFAP expression in cultured
astrocytes. Furthermore, these astrogliotic hallmarks were mediated via JAK/STAT
and RhoA signalling pathways. However, rather than being inhibitory, MT induced
a form of astrogliosis that was permissive to neurite outgrowth and which was
associated with decreased chondroitin sulphate proteoglycan (CSPG) expression.
The results suggest that MT has an important role in mediating permissive
astrocytic responses to traumatic brain injury.
Keywords: Traumatic brain injury; Astrogliosis; Regeneration
___________________________________
Metallothionein II-A improves healing after a burn wound
Morellini N.1,2,3, Giles N. 3,4, Rea S.
3,5,6, King C. 1,2, Dunlop S. 1,2, Beazley L.
1,2, West A. 7, Wood F. 1,5,6, Fear M. 3
1School of Animal Biology, University of Western
Australia,
2Western Australian Institute for Medical Research, Australia,
3The McComb Research Foundation, Perth, Australia,
4Department of Anatomy and Human Biology, University of Western Australia,
5Royal Perth Hospital, Perth, Australia,
6Princess Margaret Hospital for Children, Perth, Australia,
7NeuroRepair Group, Menzies Research Institute, University of Tasmania
The epidermal barrier prevents infection and dehydration. Its
rapid repair is essential after injury. Severe injuries often result in scarring
and life-long functional deficits, the outcome worsening with longer times to
heal. We investigated the potential of Metallothionein II-A (Apo-MT-IIA:
Bestenbalt, Tallinn, Estonia, rabbit-derived, >98% pure by HPLC in zinc sulphate
solution, PBS, pH 7.4), a naturally occurring small cysteine-rich protein, to
accelerate healing after burn wounds. In vitro assays of a human
keratinocyte cell line (HaCaT) indicated that at 1 g/ml and 2 g/ml MT-IIA
significantly increased cell proliferation (p<0.05). Annexin V and propidium
iodide FACS analysis of keratinocytes with increasing amount of MTII-A reduced
the percentage of cells undergoing apoptosis in response to both a UV insult and
to Staurosporine in a dose-dependent manner with the effect at 1 g/ml reducing
apoptosis by >50% p<0.05)). After a full thickness burn to the dorsal skin of
adult mice, immunohistochemistry revealed that endogenous MT-I/II expression
increased in basal keratinocytes during healing. Increases are seen in the wound
margin at early stages (3 and 7 days; p<0.05) and in its centre by 11 days
(p<0.05). Topical administration of exogenous MT-IIA immediately post-burn
accelerated the return of MTI/II expression to normal values by day 14 in the
wound margin (Normal: 39 ± 3% vs MT: 49 ± 7%, p>0.05; vs PBS: 68 ± 7%, p<0.05)
and improved healing as assessed by reduced epidermal thickness (MTII-A: 45 ± 4
μm vs control: 101 ± 19 μm, p<0.05) and faster wound closure at Day 3
post-injury 8.9 mm ± 0.27 mm in controls compared to 7.1 mm ± 0.7 mm in treated
wounds, by day 7 5.8 mm ± 0.98mm in controls versus 3.6 mm ± 1.0 mm in treated
wounds, p<0.05).. Our data suggest that MT-IIA may prove a valuable therapeutic
for patients with burns and other skin injuries.
___________________________________
Simultaneous iron, zinc, sulfur and phosphorus speciation analysis of barley
grain tissues using SEC-ICP-MS and IP-ICP-MS
Daniel P. Persson, Thomas H. Hansen, Kristian H. Laursen, Jan K. Schjoerring and
Søren Husted
Journal: Metallomics, 2009, 1, 418 -
426.
Abstract: The increasing prevalence of iron (Fe) and zinc (Zn)
deficiencies in human populations worldwide has stressed the need for more
information about the distribution and chemical speciation of these elements in
cereal products. In order to investigate these aspects, barley grains were
fractionated into awns, embryo, bran and endosperm and analysed for Fe and Zn.
Simultaneously, phosphorus (P) and sulfur (S) were determined since these
elements are major constituents of phytic acid and proteins, respectively,
compounds which are potentially involved in Fe and Zn binding. A novel
analytical method was developed in which oxygen was added to the octopole
reaction cell of the ICP-MS. This approach greatly improved the sensitivity of
sulfur, measured as 48SO+. Simultaneously, Fe was measured as 72FeO+, P as
47PO+, and Zn as 66Zn+, enabling sensitive and simultaneous analysis of these
four elements. The highest concentrations of Zn, Fe, S and P were found in the
bran and embryo fractions. Further analysis of the embryo using SEC-ICP-MS
revealed that the speciation of Fe and Zn differed. The majority of Fe co-eluted
with P as a species with the apparent mass of 12.3 kDa, whereas the majority of
Zn co-eluted with S as a 3 kDa species, devoid of any co-eluting P. Subsequent
ion pairing chromatography of the Fe/P peak showed that phytic acid
(myo-inositol-1,2,3,4,5,6-hexakisphosphate: IP6) was the main Fe binding ligand,
with the stoichiometry Fe4(IP6)18. When incubating the embryo tissue with
phytase, the enzyme responsible for degradation of phytic acid, the extraction
efficiency of both Fe and P was doubled, whereas that of Zn and S was
unaffected. Protein degradation on the other hand, using protease XIV, boosted
the extraction of Zn and S, but not that of Fe and P. It is concluded that Fe
and Zn have a different speciation in cereal grain tissues; Zn appears to be
mainly bound to peptides, while Fe is mainly associated with phytic acid.
___________________________________
Metallothionein Treatment Attenuates Microglial Activation and Expression of
Neurotoxic Quinolinic Acid Following Traumatic Brain Injury
R. S. Chung1, Y. K. Leung1, C. W. Butler1,
Y. Chen2, E. D. Eaton1, M. W. Pankhurst1, A. K.
West1 and G. J. Guillemin2
(1) NeuroRepair Group, Menzies Research Institute,
University of Tasmania, Private Bag 58, Hobart, TAS, 7001, Australia
(2) Centre for Immunology, University of New South Wales, Sydney, 2052,
Australia
Journal:
Neurotoxicity Research, Volume 15, Number 4 / May, 2009.
Abstract: The kynurenine pathway has been implicated as a major component
of the neuroinflammatory response to brain injury and neurodegeneration. We
found that the neurotoxic kynurenine pathway intermediate quinolinic acid (QUIN)
is rapidly expressed, within 24 h, by reactive microglia following traumatic
injury to the rodent neocortex. Furthermore, administration of the astrocytic
protein metallothionein attenuated this neuroinflammatory response by reducing
microglial activation (by approximately 30%) and QUIN expression. The
suppressive effect of MT was confirmed upon cultured cortical microglia, with 1
μg/ml MT almost completely blocking interferon–gamma induced activation of
microglia and QUIN expression. These results demonstrate the
neuroimmunomodulatory properties of MT, which may have therapeutic applications
for the treatment of traumatic brain injury.
Keywords: Traumatic brain injury - Neuroinflammation - Neuron-glia
interactions
___________________________________
Potential Role of a-Synuclein and Metallothionein in Lead-Induced Inclusion
Body Formation
Peijun Zuo*, Wei Qu*, Ryan N. Cooper*, Robert A. Goyer*,
Bhalchandra A. Diwan and Michael P. Waalkes*
* Inorganic Carcinogenesis Section, Laboratory of
Comparative Carcinogenesis, National Cancer Institute at the National Institute
of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
Basic Research Program, SAIC-Frederick, Inc., NCI at Frederick, Maryland 21702
Journal:
Toxicological Sciences 2009 111(1):100-108;
Abstract: Lead (Pb) produces aggresome-like inclusion
bodies (IBs) in target cells as a toxic response. Our prior work shows
metallothionein (MT) is required for this process. We used MT-I/II double
knockout (MT-null) and parental wild-type (WT) cell lines to further explore the
formation process of Pb-induced IBs. Unlike WT cells, MT-null cells did not form
IBs after Pb exposure. Western blot of cytosol showed soluble MT protein in WT
cells was lost during Pb exposure as IBs formed. Transfection of MT-I into
MT-null cells allowed IBs formation after Pb exposure. Considering Pb-induced
IBs may be like disease-related aggresomes, which often contain alpha-synuclein
(Scna), we investigated Scna expression in cells capable (WT) and incapable
(MT-null) of producing IBs after Pb exposure. Scna protein showed poor basal
expression in MT-null cells. Pb exposure increased Scna expression only in WT
cells. MT transfection increased Scna transcript to WT levels. In WT or MT-transfected
MT-null cells, Pb-induced Scna expression rapidly increased and then decreased
over 48 h as Pb-induced IBs were formed. A direct interaction between Scna and
MT was confirmed ex vivo by antibody pulldown assay where the proteins
coprecipitated with an antibody to MT. Pb exposure caused increased
colocalization of MT and Scna proteins with time only in WT cells. In WT mice
after chronic Pb exposure Scna was localized in renal cells containing forming
IBs, whereas MT-null mice did not form IBs. Thus, Scna could be component of Pb-induced
IBs and, with MT, may play a role in IBs formation.
Key Words: lead; inclusion bodies; alpha-synuclein; metallothionein;
MT-null.
___________________________________
Redefining the Role of Metallothionein within the Injured
Brain
EXTRACELLULAR METALLOTHIONEINS PLAY AN IMPORTANT ROLE
IN THE ASTROCYTE-NEURON RESPONSE TO INJURY.
Roger S. Chung1, Milena Penkowa2, Justin Dittmann1, Carolyn E. King3, Carole
Bartlett3, Johanne W. Asmussen2, Juan Hidalgo4, Javier Carrasco4, Yee Kee J.
Leung1, Adam K. Walker1, Samantha J. Fung1, Sarah A. Dunlop3, Melinda
Fitzgerald3, Lyn D. Beazley3, Meng I. Chuah1, James C. Vickers1, and Adrian K.
West1
1)NeuroRepair Group, Menzies Research Institute, University of Tasmania, Private
Bag 58, Hobart, Tasmania 7001, Australia,
2)Section of Neuroprotection, Faculty of Health Sciences, University of
Copenhagen, Copenhagen DK2200, Denmark,
3)School of Animal Biology, University of Western Australia, Nedlands, Western
Australia 6907, Australia, and
4)Institute of Neurosciences and Department of Cellular Biology, Physiology and
Immunology, Animal Physiology Unit, Faculty of Sciences, Autonomous University
of Barcelona, Bellaterra, Barcelona 08193, Spain
Journal:
Journal of Biological Chemistry, Vol. 283, Issue 22, 15349-15358, May 30,
2008.
Abstract: A
number of intracellular proteins that are protective after brain injury are
classically thought to exert their effect within the expressing cell. The
astrocytic metallothioneins (MT) are one example and are thought to act via
intracellular free radical scavenging and heavy metal regulation, and in
particular zinc. Indeed, we have previously established that astrocytic MTs are
required for successful brain healing. Here we provide evidence for a
fundamentally different mode of action relying upon intercellular transfer from
astrocytes to neurons, which in turn leads to uptake-dependent axonal
regeneration. First, we show that MT can be detected within the extracellular
fluid of the injured brain, and that cultured astrocytes are capable of actively
secreting MT in a regulatable manner. Second, we identify a receptor, megalin,
that mediates MT transport into neurons. Third, we directly demonstrate for the
first time the transfer of MT from astrocytes to neurons over a specific time
course in vitro. Finally, we show that MT is rapidly internalized via the cell
bodies of retinal ganglion cells in vivo and is a powerful promoter of axonal
regeneration through the inhibitory environment of the completely severed mature
optic nerve. Our work suggests that the protective functions of MT in the
central nervous system should be widened from a purely astrocytic focus to
include extracellular and intra-neuronal roles. This unsuspected action of MT
represents a novel paradigm of astrocyte-neuronal interaction after injury and
may have implications for the development of MT-based therapeutic agents.
___________________________________
Exogenous metallothionein-IIA promotes accelerated healing after a burn wound
Natalie M. Morellini1,2,3; Natalie L. Giles3,4;
Suzanne Rea3,5,6; Katharine F. Adcroft3,4; Sian Falder3,5,6;
Carolyn E. King1,2; Sarah A. Dunlop1,2; Lyn D. Beazley1,2;
Adrian K. West7; Fiona M. Wood3,5,6; Mark W. Fear3
1. School of Animal Biology, University of Western Australia,
Crawley, WA, Australia,
2. Western Australian Institute for Medical Research, Perth, WA, Australia,
3. The McComb Research Foundation, Perth, WA, Australia,
4. Department of Anatomy and Human Biology, University of Western Australia,
Crawley, WA, Australia,
5. Royal Perth Hospital, Perth, WA, Australia,
6. Princess Margaret Hospital for Children, Perth, WA, Australia, and
7. NeuroRepair Group, Menzies Research Institute, University of Tasmania,
Hobart, Tas., Australia
Journal:
Wound Repair and Regeneration, Volume 16 Issue 5, Pages 682 - 690, 2008.
ABSTRACT: Severe injury to the epidermal barrier often results in
scarring and life-long functional deficits, the outcome worsening with a number
of factors including time taken to heal. We have investigated the potential of
exogenous metallothionein IIA (Zn7-MT-IIA), a naturally occurring small cysteine-rich
protein, to accelerate healing of burn wounds in a mouse model. Endogenous
MT-I/II expression increased in basal keratinocytes concurrent with
reepithelialization after a burn injury, indicating a role for MT-I/II in wound
healing. In vitro assays of a human keratinocyte cell line indicated that,
compared with saline controls, exogenous Zn7-MT-IIA significantly increased cell
viability by up to 30% (p<0.05), decreased apoptosis by 13% (p<0.05) and
promoted keratinocyte migration by up to 14% (p<0.05), all properties that may
be desirable to promote rapid wound repair. Further in vitro assays using
immortalized and primary fibroblasts indicated that Zn7-MT-IIA did not affect
fibroblast motility or contraction (p>0.05). Topical administration of exogenous
Zn7-MT-IIA (2 μg/mL) in vivo, immediately postburn accelerated healing, promoted
faster reepithelialization (3 days: phosphate-buffered saline (PBS), 8.9±0.3 mm
diameter vs. MT-I/II, 7.1±0.7 mm; 7 days: PBS 5.8±0.98 mm vs. MT-I/II, 3.6±1.0
mm, p<0.05) and reduced epidermal thickness (MT-I/II: 45±4 μm vs. PBS: 101±19 μm,
p<0.05) compared with controls. Our data suggest that exogenous Zn7-MT-IIA may
prove a valuable therapeutic for patients with burns and other skin injuries.
___________________________________
Simple Method for Identification of Metallothionein Isoforms in Cultured
Human Prostate Cells by MALDI-TOF/TOF Mass Spectrometry
Rongying Wang,†‡ Donald A. Sens,† Amy Albrecht,† Scott
Garrett,† Seema Somji,† Mary Ann Sens,† and Xiaoning Lu*†‡
Department of Pathology, and Proteomics Core Facility,
School of Medicine and Health Science, University of North Dakota, Grand Forks,
North Dakota 58202
Journal:
Anal. Chem., 2007, 79 (12), pp 4433–4441.
Abstract: The present paper describes a rapid method for
identification and characterization of human metallothionein (MT) isoforms in
complex cell cultures using high-resolution matrix-assisted laser desorption/ionization
time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF). In the proposed
method, the sample preparation of MTs from cultured cells is both simple and
fast. It is accomplished by trypsin cleavage of cell proteins into small peptide
species, the majority of which are subsequently removed by gel filtration using
beads with an exclusion limit of 4000 Da. In contrast to most cell proteins, MTs
remain intact (undigested) upon being treated with trypsin, being excluded by
the gel beads and thus recovered by low-speed centrifugation. To identify the
protein constitutes of the MT preparation, the MT sample is divided into two
parts, one for intact protein accurate mass measurement, the other for tryptic
digestion followed by MS and MS/MS analyses. In the latter case, the MT proteins
are denatured by the addition of EDTA which strips heavy metals from MTs and
renders them susceptible to tryptic digestion. The obtained accurate mass with
the unique peptide sequences of each MT isoform allows for unambiguous
identification of MT isoforms in the prepared mixture. The method has been
applied to RWPE-1 cells derived from normal human prostate epithelium. Four MT
isoforms, 1E, 1G, 1X, and 2A, have been confidently identified, being primarily
acetylated at N-termini. These results are in agreement with the expression of
MT mRNAs in RWPE-1 cells determined by real-time reverse-transcription
polymerase chain reaction (RT-PCR).
___________________________________
Metallothionein-IIA promotes neurite growth via the megalin receptor
Melinda Fitzgerald (1) , Pia Nairn (1),
Carole A. Bartlett (1), Roger S. Chung (3), Adrian K. West (3) and Lyn D.
Beazley (1, 2)
(1) Experimental and Regenerative Neurosciences, School of Animal Biology,
University of Western Australia, Hackett Drive, Crawley, 6009, WA, Australia
(2) Western Australian Institute of Medical Research, University of Western
Australia, Hackett Drive, Crawley, 6009, WA, Australia
(3) Neurorepair Group, Menzies Research Institute, University of Tasmania,
Private Bag 58, Hobart, TAS, 7001, Australia
Journal: Experimental Brain Research, Volume 183,
Number 2 / November, 2007, p. 171-180
Open:
Entire document (Subscription to
the journal required).
Abstract: Metallothionein (MT)-I/II has been shown to be neuroprotective
and neuroregenerative in a model of rat cortical brain injury. Here we examine
expression patterns of MT-I/II and its putative receptor megalin in rat retina.
At neonatal stages, MT-I/II was present in retinal ganglion cells (RGCs) but not
glial or amacrine cells; megalin was present throughout the retina. Whilst
MT-I/II was absent from adult RGC in normal animals and after optic nerve
transection, the constitutive megalin expression in RGCs was lost following
optic nerve transection. In vitro MT-IIA treatment stimulated neuritic growth:
more RGCs grew neurites longer than 25 μm (P < 0.05) in dissociated retinal
cultures and neurite extension increased in retinal explants (P < 0.05). MT-IIA
treatment of mixed retinal cultures increased megalin expression in RGCs, and
pre-treating cells with anti-megalin antibodies prevented MT-IIA-stimulated
neurite extension. Our results indicate that MT-IIA stimulates neurite outgrowth
in RGCs and may do so via the megalin receptor; we propose that neurite
extension is triggered via signal transduction pathways activated by the NPxY
motifs of megalin’s cytoplasmic tail.
Keywords: Metallothionein - Retinal ganglion cells - Neuroregeneration
___________________________________
Metal binding of metallothionein-3 versus
metallothionein-2: lower affinity and higher plasticity
Peep Palumaa (1), , Indrek Tammiste (2), Keiu Kruusel (1), Liina Kangur (1),
Hans Jörnvall (3) and Rannar Sillard (3)
(1) Department of Gene Technology, Tallinn Technical University Akadeemia tee
23, 12618 Tallinn, Estonia
(2) National Institute of Chemical Physics and Biophysics, Akadeemia tee 23,
12618 Tallinn, Estonia
(3) Department of Medical Biochemistry and Biophysics, Karolinska Institutet,
S-171 77 Stockholm, Sweden
Journal: Biochimica et
Biophysica Acta (BBA) - Proteins & Proteomics. Volume 1747, Issue 2, 14 March
2005, Pages 205-211
Abstract:
Mammalian metallothioneins (MTs) are involved in cellular metabolism of zinc
and copper and in cytoprotection against toxic metals and reactive oxygen
species. MT-3 plays a specific role in the brain and is down-regulated in
Alzheimer's disease. To evaluate differences in metal binding, we conducted
direct metal competition experiments with MT-3 and MT-2 using electrospray
ionization mass spectroscopy (ESI-MS). Results demonstrate that MT-3 binds Zn2+
and Cd2+ ions more weakly than MT-2 but exposes higher metal-binding capacity
and plasticity. Titration with Cd2+ ions demonstrates that metal-binding
affinities of individual clusters of MT-2 and MT-3 are decreasing in the
following order: four-metal cluster of MT-2>three-metal cluster of
MT-2≈four-metal cluster of MT-3>three-metal cluster of MT-3>extra metal-binding
sites of MT-3. To evaluate the reasons for weaker metal-binding affinity of MT-3
and the enhanced resistance of MT-3 towards proteolysis under zinc-depleted
cellular conditions, we studied the secondary structures of apo-MT-3 and
apo-MT-2 by CD spectroscopy. Results showed that apo-MT-3 and apo-MT-2 have
almost equal helical content (approximately 10%) in aqueous buffer, but that
MT-3 had slightly higher tendency to form α-helical secondary structure in TFE–water
mixtures. Secondary structure predictions also indicated some differences
between MT-3 and MT-2, by predicting random coil for common MTs, but 22%
α-helical structure for MT-3. Combined, all results highlight further
differences between MT-3 and common MTs, which may be related with their
functional specificities.
Keywords: MT-3; Alzheimer's disease; ESI-MS; Secondary structure
prediction; CD spectroscopy
Abbreviations: MT, metallothionein; DTT, dithiothreitol; TFE,
2,2,2,-trifluoroethanol; CD, circular dichroism; ESI MS, electrospray ionization
mass spectroscopy
___________________________________
Metal binding to brain-specific metallothionein-3 studied
by electrospray ionization mass spectrometry.
Palumaa P (1), Eriste E (2), Kruusel K (1),
Kangur L, (1) Jörnvall H (2), Sillard R (2).
(1) Department of Gene Technology, Tallinn Technical University, Ehitajate tee
5, EE-19086 Tallinn, Estonia.
(2) Department of Medical Biochemistry and
Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden.
Journal: Cell Mol Biol (Noisy-le-grand). 2003
Jul;49(5):763-768.
Abstract:
Metallothionein-3 (MT-3) is a brain-specific isoform of metallothioneins,
which is down-regulated in Alzheimer's disease (AD), inhibits the growth of
neurons in vitro, and differs from common MTs also in gene regulation. To
elucidate the differences in structure and function between MT-3 and common MTs,
Zn2+ and Cd2+ binding to MT-3 and MT-1 were studied using electrospray
ionization time of flight mass spectrometry (ESI TOF MS) at pH values between
7.5 and 2.7. The metal binding properties of MT-3 differ considerably from those
of MT-1. After reconstitution with a metal excess, metallated MT-3 exists as a
mixture of Zn7MT-3 (or Cd7MT-3, respectively) and several metalloforms with
stoichiometries below and above seven. In contrast, MT-1 exists as a single
Zn7MT-1 (or Cd7MT-1). Lowering of pH leads to a stepwise release of metals from
metallated MT-3, first from extra sites, then from the 3-metal cluster and
finally from the 4-metal cluster. At acidic pH values the 4-metal cluster of
MT-3 is slightly more stable than that of MT-1. The results demonstrate higher
structural plasticity, dynamics and metal binding capacity of MT-3 than of MT-1,
which makes MT-3 suitable as a zinc buffer-transfer molecule in zinc-enriched
neurons functioning at conditions of fluctuating zinc concentrations.
___________________________________
Brain-Specific Metallothionein-3 Has Higher Metal-Binding
Capacity than Ubiquitous Metallothioneins and Binds Metals Noncooperatively
Peep Palumaa, Elo Eriste, Olga Njunkova, Lesja Pokras, Hans Jörnvall, and
Rannar Sillard
Centre for Gene Technology, Tallinn Technical University, Ehitajate tee 5,
EE-19086 Tallinn, Estonia,
Department of Medical Biochemistry and
Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden,
National Institute of Chemical Physics and
Biophysics, Akadeemia tee 23, EE-12618 Tallinn, Estonia
Journal: Biochemistry 41(19), 6158-6163; 2002.
Abstract:
Zinc metabolism in the cells is largely regulated by ubiquitous small
proteins, metallothioneins (MT). Metallothionein-3 is specifically expressed in
the brain and is down regulated in Alzheimer's disease. We demonstrate by mass
spectrometry that MT-3, in contrast to common MTs, binds Zn2+ and Cd2+ in a
noncooperative manner and can also bind higher stoichiometries of metals than
seven. MT-3 reconstituted with seven metals exists in a dynamic equilibrium of
different metalloforms, where the prevalent metalloform is Me7MT-3, but
metalloforms with 6, 8, and even 9 metals are also present. The results from pH
and stability studies demonstrate that the heterogeneity of metalloforms
originates from the N-terminal -cluster, whereas the C-terminal -cluster of MT-3
binds four metal ions such as that of common MTs. Experiments with EDTA
demonstrate that the -cluster of ZnMT-3 has a higher metal transfer potential
than the -cluster of Zn7MT-2. Moreover, ZnMT-3 loses metals during
ultrafiltration. MT-3, reconstituted with an excess of Zn2+ or Cd2+, exists as a
dynamic mixture of metalloforms with higher than 7 metal stoichiometries (8-11).
Such forms of ZnMT-3 are unstable and decompose partly already during a rapid
gel filtration, whereas CdMT-3 forms are more stable. Extra metal ions may bind
to the -cluster region as well as to the carboxylates of MT-3. The specific
metal-binding properties of MT-3 could be functionally implemented for buffering
of fluctuating concentrations of zinc in zincergic neurons and for transfer of
zinc to synaptic vesicles.
___________________________________
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