Metallothioneins (MTs)

Discovery.

First identified in 1960 by Kagi and Vallee who isolated

the protein from equine renal cortex.

Noted that the majority of the Cd in the organ was

associated with a small cytoplasmic protein.

Isolation of protein showed that it could also bind other

metals such as Zn and Cd.

Unusual absorbance profile with high 254/280nm ratio

when metals were bound.
 High 254 absorbance due to metals associated
with cysteine residues forming a mercaptide
bond. Amino acid analyses showed that
approximately 30% of the amino acids are
cysteines

AA sequencing showed cysteines arranged in 3

motifs:

Cys-X-Cys
Cys-X-X-Cys
Cys-Cys
Where X is any amino acid
 Associated metals were acid labile and could be
removed at pH of below 5 although Cu was totally
removed only at lower pH values. Loss of metal was
accompanied by a decrease in absorption at 254 and
an increase at 220nm.

Now known to occur in nearly all eucaryotic cells

although in plants it is replaced by a functionally
analogous class of polypeptides called
phytochelatins.
 Structure
Small in size. Comprised of a single polypeptide with no
carbohydrate moieties attached.

Molecular weight estimations by size exclusion HPLC,

MALDI-MS and AA sequencing indicate a molecular weight
in the 6,000 7,000 range indicating a polypeptide of
approximately 55-70 amino-acids in length.
 X-ray crystallography has indicated that the protein in
mammals is dumb-bell shaped with the metals being
coordinated via multiple thiolate groups in the center
of each dumbbell to form two metallo-clusters. -
domain holds 3 atoms (Zn/Cd) and domain holds 4
(Zn/Cd)

Cysteine-metal co-ordination can be variable

depending upon the metals being bound.

Cd/Zn normally tetrahedrally coordinated

Cu either tetrahedrally or trigonally coordinated

Ag appears to have trigonal co-ordination.

o Titration studies have shown metals have differing
affinities for the protein:

Hg 2+ > Ag1+> Cu 1+> Cd2+ > Zn2+

Stability constants
1022 1018 5x1012

Some evidence of both in vivo and in vitro

displacement of metals in accordance with the above
hierarchy.

Stoichiometry of metal binding appears to vary

according to the metal in Question.
 In fully metal saturated molecule:

Zn = 7 g.atoms
Cd = 7 g.atoms
Cu = 7 or 13 g.atoms increasing affinity and change in co-ordination.
Ag = 13 g.atoms
Hg = 20-21 g.atoms

Metals can occur simultaneously and in mixed clusters

Zn and Cd often associated

Cu and Ag often associated
 Metallothionein
Zn or Cd Metallothionein

" $
13
7 6
24 5

Highly conserved and ubiquitous

Supergene Family with various isoforms and variants
Low molecular weight 6-14 K.Da
No enzymatic Activity
High cysteine content (30%)
Ability to bind a number of metals Hg>Ag>Cu(I)>Cd>Zn>Co
Induced synthesis by metals, (oxidative stress, hormones)
Measurement and identification of MT

No enzymatic activity therefore cannot undertake

classical enzyme activity measurements to quantify
the protein (function).

Chromatography. Can use separatory techniques to

purify and quantify MT content in an organism.
 Protocols for Isolation and Analysis of
Metallothioneins

Isolation

Homogenize tissue

Centrifuge 100,000g for 1 hour.

Heat cytosol to 65oC (MT very heat stable)

Respin and reconstitute pellet in buffer

Slowly add 70% acetone/alcohol to supernatant and chill to 20oC

Respin and reconstitute pellet in buffer

Purification of metallothionein

Size exclusion chromatography

Ion exchange chromatography

Detect MT by associated metals using FAA, GAA,

ICP-OES, ICP-MS
Techniques for Detection and Quantification

Polographic techniques that quantify SH content upon

acidification. Problems with glutathione and other SH
compounds such as free cysteine and other proteins rich
is cysteine.

DTNB colorometric assay for heat denatured cytosol.

Again free cysteine has to be removed by ultrafiltration
or some other procedure

Attempts at antibody production to produce an

ELISA/Western assay have been marginally successful
due to lack of antigenicity
 Methods for Detection and Quantification
Radioactive 203Hg Saturation Assay

o Acidify sample to below pH 6.0 and excess 203Hg in

presence of excess hemoglobin

o Raise pH >7.5 and incubate for 30 mins

o Add hemoglobin in XS and Spin 1 hour at 100,000g

o Analyze Supernatant and Quantify 203Hg Content.

o Assume a fully saturated molecule with a

stoichiometry of 21 atom Hg/mol.
 Evolution of MT:
Antiquity, Invariability and Duplicity ?
Highly conserved and ubiquitous
Found as a supergene family
4 major isoforms in humans (16 genes)
Often show similar promoters/patterns of expression
 Numerous isoforms of the protein with same
apparent molecular weight but different PIs

Indicative of a supergene family that probably

originated by unequal X over during meiosis.
Confirmed by in-situ hybridizations with
radioactive cDNA clones.
 MT Nomenclature and Classification.

MT family
share a particular set of sequence specific
characters.
Members of a family can belong to only one family
and are thought to be evolutionary related.
The amino acid sequence is alignable with that of all
members.
A common and exclusive sequence pattern, a
profile and a phylogenetic tree can therefore be
connected with each family.
Each family is identified by its number and its
taxonomic range. eg. Family 1 : vertebrate MTs.

Classification based upon both alignment of bases on DNA coding for the
protein and the upstream regulatory sequences
CIS-ACTING SEQUENCES:

Metal Regulatory Elements (MRE).

Present in a variable number of copies.

Consensus sequence is the 15mer nucleotide

5CTNTGCRCNCGGCCC 3

Where: R is a purine and N any base

central highly conserved core sequence is required

for the binding of protein factors.

5TGCRCNC 3
The numbers of MREs and their positions on the coding
( ) or on non-coding strand ( ) in 5'UT-region of
vertebrate MT genes correlate with subgroup specificity.
Comparisons of MT from different organisms shows evidence for
punctuated equilibria for evolution.

Yeast

Sea urchin

Invertebrates (Molluscs and Arthropods)

Vertebrates
Yeast:

Responds to Cu/Ag only and not Zn and Cd

Unusual stoichiometry of metal binding (7 cys and 6 atoms of Cu)

Rich in Cysteine but approximately half content of other MTs (7-12 cys)

Coded for by CUP1 gene that has simple MREs (termed UAScup1).
Sea Urchin.

Responds to Zn , Cu and Cd etc.

Molecule has two domains that appear to be inverted repeats

Other Invertebrates and Vertebrates.

Two domain protein which have characteristic metal clusters.

 Molluscan cDNA PCR Product and Resulting
Amino Acid Sequence
Nucleotide Sequence

TCTTCAGTTTTCGGAGCAGGATGCACGGACGTGTGCAA
GCAGACGCCATGCGGCTGTGCCACCTCGGGCTGTAAC
TGCACGGACGACTGCAAGTGTCAGTCATGCAAATACGG
AGCGGGTTGCACGGACACATGCAAGCAGACACCATGTG
GGTGTGGCAGCGGGTGCAACTGTAAGGAGGACTGTCG
CTGTCAGAGCTGTTCCACCGCCTGCAAGTGTGCGGCTG
GAAGCTGCAAGTGCGGCAAGGGATGCACAGGGCCAGA
CAGCTGCAAGTGTGACCGATCGTGCTCCTGCAAATAA

Amino Acid Sequence

SSVFGAGCTDVCKQTPCGCATSGCNCTDDCKCQSCKY 10 C-X-C
GAGCTDTCKQTPCGCGSGCNCKEDCRCQSCSTACKCA 8 C-X-X-C
AGSCKCGKGCTGPDSCKCDRSCSCK
 Metallothionein Induction
Transcriptionally inducable by a number of different metals

Different metals induce in different organisms

Yeast : Cu and Ag but not Zn or Cd

Molluscs : Cd, Ag and Cu but poorly with Zn

Fish : Cd, Ag, Cu and Zn

 DNA bindingZn sensor

"
$ COO -
Zn 2+ 2+
Zn > 10-9 M

Cd, Hg, Ag, Au, NH 3+

Metallothionein

High stability KaZn 1013M-1

Binds and Detoxifies metal
Switches MT gene off
The Transacting Factor.

Majority of information is from yeast although some

studies have been conducted on fish. Very little
information on marine invertebrates

In yeast transacting factor gene is found on

chromosome 7: termed ACE 1 gene.

Sequence for gene shows 3 ORFs that could code

for 22/24 or 25K.Da proteins.
 ORF 1 produces a protein with an amino-acid sequence
that contains 12 cysteines at amino-terminal end having
Cys X Cys and Cys X X Cys motifs.

Binding and positive activation shown by footprinting

and reporter assays on MRE cis-locus. Similar factors
have now been found, identified and sequenced in fish.

Specificity of metals causing induction can be different

to specificity in MT metal binding
DEMONSTRATION OF FIRST TRANSGENIC ORGANISM
 Function of Metallothinein ? Still Looking
Ubiquitous therefore considered to be very important

Multiple Knock out causes problems with development and

metal toxicity but not death in mammals.

Gene amplification by recombinant DNA technology has shown

that it can cause metal resistance to toxic effects of Cd and Cu.

Lack of inducibility by Cd in yeast has been used as an

argument for a role in metal homeostasis.

High affinity of MT for metals has been used as an argument for

detoxification and against ability to donate metals to apo-
enzymes for activation.

However, recent evidence shows it Is a redox sensitive protein

and affinity for metals can be altered by redox status of the cell
(ie GSH and GSSG balance).