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.

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

Outline

• Characteristics of metallothionein:

Occurrence, structure, functions

• Induction: Observed and experimental

• Isolation of MT from rat liver

• Identification

• Future research possibilities

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

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

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(?)

Schematic presentation of the mechanism of cadmium nephrotoxicity.

Mt = metallothionein; alb = albumin.

Nordberg and Nordberg, 1985.

Kägi and Nordberg, 1979.

Mode of distribution of cysteinyl residues.

Kägi and Nordberg 1979

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.

Stereo view of human liver Cd7-MT-2.

Top: Amino-terminal domain; Bottom: Carboxyl-terminal domain

Metal

position

Cysteine

side chain

(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.

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

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.)

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 5C, in 5ml 0.01 M Tris/HCl, pH 7.4)

Centrifugation (5C, 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

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

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.)

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

NMR spectrum of Cu7-MT from

S.Cervisiae

(Bertini, Hartmann, Klein, Liu, Luchinat, and Weser, Eur. J. Biochem., 2000, 267, 1008-1018.)

”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).

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