Lecture about a large family of proteins that can bind metals and protect against oxidative stress damage on our cells.

1. Metallothionein: Induction, isolation, and identification
a-domain structure of Sea Urchin (Strongylocentrotus
purpuratus) Cd7-Metallothionein A.
Riek et al., J. Mol. Biol. (1999) 291, 417-428.

2. Acknowledgements
• Dr Gunnar Nordberg, Professor, Dept of
Environmental Medicine, Umeå University
• Dr Taiyi Jin, former WHO Fellow at the
Dept of Environmental Medicine, Umeå
University
• Dr Monica Nordberg, Associate Professor,
Institute of Environmental Medicine
(IMM), Karolinska Institute

3. Outline
• Characteristics of metallothionein:
Occurrence, structure, functions
• Induction: Observed and experimental
• Isolation of MT from rat liver
• Identification
• Future research possibilities

4. Characteristics of metallothionein(1)
(Kojima, Meth Enzymol. 205, 1991, 8-10; Nordberg, Talanta 46, 1998, 243-254.)
• MTs have been isolated from and classified in groups by
the following species: vertebrates, molluscs, crustaceans,
echinodermata, diptera, nematodes, ciliata, fungi,
prokaryotes, and plants.
• Mw = 6000 - 7000, 61 amino acids
• 20 Cys (30%), N-acetylmethionine, C-alanine, no
aromatics, no His
• Tertiary structure with 2 metal clusters (a and b)
• Metal content: 5-10% (w/w) of Cd, Zn, Cu, Hg
• UV absorption: Zn 225 nm, Cd 250 nm, Cu 275 nm, Hg
300 nm

5. Margoshes and Vallee, J. Am. Chem. Soc. 1957, 79, 4813.

6. Characteristics of metallothionein(2)
• Induced synthesis by Cd and Zn but also many other
factors (e.g. hormones, growth factors, inflammatory
agents, vitamins, antibiotics, cytotoxics, and stress-
producing conditions)
• No disulfide bonds
• Heat stability (important for isolation procedure)
• Cytoplasmic localization
• Isoforms (5 mammalian subfamilies)
• Chromosomal localization: Mouse - 4 genes on
chromosome 8, Human - at least 16 genes on chromosome
16.
• Functions: Transport, storage, metabolism, and
detoxification of metals; radical scavenger(?)

7. Schematic presentation of the mechanism of cadmium nephrotoxicity.
Mt = metallothionein; alb = albumin.
Nordberg and Nordberg, 1985.

8. Kägi and Nordberg, 1979.

9. Mode of distribution of cysteinyl residues.
Kägi and Nordberg 1979

10. Cd-Cys coordination in human liver Cd2-MT-2.
Top: 3-metal thiolate cluster. Bottom: 4-metal thiolate cluster.
Arabic numerals: residue position in the amino acid sequence. Roman numerals: Metal sites.
Kägi, Meth. Enzymol. 1991, 205, 613-626.

11. Stereo view of human liver Cd7-MT-2.
Top: Amino-terminal domain; Bottom: Carboxyl-terminal domain
Metal
position
Cysteine
side chain

12. (a) The a-domain of sea urchin MTA
comprising the residues 2-36
and a four-metal/11 Cys cluster.
3D-structures based on NMR data (Cd7-MTA from sea urchin)
(b) The b-domain of MTA comprising
residues
37-64 and a three-metal/nine Cys cluster.
Riek et al., J. Mol. Biol. (1999) 291, 417-428.

13. Stereo view of superpositions for best fit of
the metal ions of sea urchin MTA and human
MT-2.
(a) The a-domain; (b) the b-domain. The
polypeptide
backbone of the sea urchin MTA is drawn as a
yellow spline function through the C a positions,
and a corresponding presentation of the
backbone of the human MT-2 is shown in cyan.
The metal ions are colored red for sea urchin
MTA and blue for human MT-2. The terminal
amino acid residues of the domains are
identified with the one-letter amino acid code
and the residue number.
Riek et al., J. Mol. Biol. (1999) 291, 417-
428.
Sea urchin MTA vs. Human MT-2

14. Induction of metallothionein
• Many factors can induce MT, but experimentally, metals
have been used most commonly.
• Example of induction of rat MT: Repeated sc doses (5
days) of 0.5 - 2.0 mg/kg CdCl2 . (Jin and Nordberg, Acta
pharmacol. et toxicol. 1986, 58, 137-143.)
• For investigation of Cd-induced proteinuria, a protocol
with CdCl2 pretreatment as above plus a subsequent single
sc CdMT challenge dose is employed (Jin, Nordberg, and
Nordberg; unpublished.).
• Interactions between Cu, Zn, and Cd on Cd toxicity has
been studied by Cu and/or Zn pretreatment prior to a
CdMT injection (Liu, Jin, Nordberg, Sjöström, Zhou,
Toxicol. Appl. Pharmacol. 1994, 126, 84-90.)

15. Isolation of MT from rat liver
(based on Liu, Jin, Nordberg,
Sjöström, Zhou, Toxicol. Appl.
Pharmacol. 1994, 126, 84-90.
UV spectrophotometry
at 230 nm (Zn-MT-complex),
250 nm (Cd-MT complex),
270 nm (Cu-MT complex) and
280 nm (aromatic amino acids*))
*One criteria of purity is a high A250/A280 ratio
(Nordberg, Nordberg, and Piscator,
Environ. Physiol. Biochem. 1975, 5, 396-403.)
Rats killded by cervical dislocation
Liver isolated
Liver tissue
Homogenization with Teflon homogenizer(at 5C, in 5ml 0.01 M Tris/HCl, pH 7.4)
Centrifugation (5C, 105,000 g, 1h)
Supernatants (cytosol)
Gel filtration (Sephadex G-75)
0.01M Tris/HCl, pH 8.6
Protein content determined by UV spectrophotometry
MT fractions
Heat treatment – boiling, 5 min
Supernatants (with heat-stable MT)
Determination of Zn at 213.9 nm
UV spectrophotometry as above
Heat treatment of MT fractions as above
Centrifugation (105,000 g, 1h)
Gel filtration (Sephadex G-50)0.01M Tris/HCl, pH 8.0
UV spectrophotometry and Zn determination as above

16. Chromatogram after final G-50 run (hepatic sample from mouse after chronic CdCl2
administration)
Nordberg, Nordberg, and Piscator, Environ. Physiol. Biochem., 1975, 5, 396-403.
MT-containing
peak:
High A250/A280
High Cd & Zn

17. Isolation of MT from other sources (other
tissues/cell suspensions)
• Isolation procedures commonly include G-75 and G-50
runs
• Precipitating agents (ethanol, acetone) sometimes used
before gel filtration (alternative to heat treatment)
• Isoforms can be separated by HPLC w/wo prior gel
filtration (ex: C18, 10mM Na2PO4 buffer, pH 7.0 : Same
buffer + 60% CH3CN; gradient system) (Richards, J.
Chromatogr., 1989, 482, 87-97.)

18. Identification of MT
• UV absorption as described
(220/250/275/280/300 nm)
• Metal concentration measured by AAS
• NMR structure determined on freeze-dried
fractions from gel filtration, resuspended in
Na2PO4 buffer + H2O/D2O + 2-
mercaptoethanol, Ar gassed

19. NMR spectrum of Cu7-MT from
S.Cervisiae
(Bertini, Hartmann, Klein, Liu, Luchinat, and Weser, Eur. J. Biochem., 2000, 267, 1008-1018.)

20. ”Astonishing facts about MT”
• Studies on fetuses have revealed the role of MT as a Cu- and Zn accumulating agent. Some suggest that
this is an important evolutionary role for the metalloprotein – to function as a reservoir and accumulator
of homeostatically important metals.
(Quaife, Hammer, Mottet, and Palmiter, Dev. Biol. 1986, 118, 549-555)
• Research into Alzheimer’s disease pointed to the fact that MT III, initially named GIF - growth
inhibitory factor, could selectively inhibit neuronal survival.
(Bogumil, Faller, Puntney, and Vasak, Eur. J. Biochem. 1996, 238, 698-705)
The association of MT III with Alzheimer’s has not been confirmed but it is known that mice
lacking MT III has an increased sensitivity to induced seizures.
(Miura and Naganuma, FEBS Letters 2000, 479, 146-148)
• There is a great number of factors that can induce the synthesis of MT. Apart from metal ions, inducers
include hormones, growth factors, cytokines, vitamins, antibiotics, cytotoxic agents, and also the
conditions of starvation, infection, inflammation and exposure to UV radiation.
(Kägi, Meth. Enzymol. 1991, 205, 613-626)
• All of these facts suggest more roles for MT than just metal handling. One popular suggestion is that
MT might protect against oxidative damage (Andrews, Biochem. Pharmacol. 2000, 59, 95-104).

21. Future research possibilities
• Study MT and its ”relatives” in a large number of organisms, plants as well as
mammals; Ubiquity indicates common important evolutionary role
• Induction and/or presence of MT forms can be an interesting indicator for
biological monitoring
• MT induction offers protection - so genetic engineering might help protect
susceptible individuals