Tài liệu Báo cáo khoa học: Fish and molluscan metallothioneins A structural and functional

Fish and molluscan metallothioneins

A structural and functional comparison

Laura Vergani1

, Myriam Grattarola1

, Cristina Borghi2

, Francesco Dondero3 and Aldo Viarengo3,4

1 Department of Biophysical Sciences and Technologies, M. & O. University of Genova, Italy

2 Department of Biology, University of Genova, Italy

3 Department of Environmental & Life Science, University of Piemonte Orientale, Alessandria, Italy

4 Center on Biology and Chemistry of Trace Metals, University of Genova, Italy

Metallothioneins (MTs) are cytosolic polypeptides

found in almost all organisms, including vertebrates,

invertebrates, plants and bacteria [1]. They do not

appear to be essential for life, even though they are

involved in many pathways, such as sequestration of

toxic (Cd, Hg) or essential (Zn, Cu) metals, scavenging

of oxyradicals, inflammation, and infection [2].

MTs exhibit unusual primary sequence, lacking histi￾dines and aromatic residues, and their 3D structure is

unique [3,4]. Cysteines represent one-third of the total

amino acids and are distributed in typical motifs con￾sisting of CC, CXC or CXYC sequences [5]. The beha￾viour of MTs is dominated by the nucleophilic thiol

group reacting with electrophilic compounds, including

many alkylating agents and radical species [6]. Verteb￾rate MTs have a monomeric dumbbell shape, com￾posed of two globular domains connected by a flexible

linker consisting of a Lys-Lys segment. Each domain

contains a ‘mineral core’ enclosed by two large heli￾cal turns of the polypeptidic chain. The N-terminal

Keywords

absorbance spectroscopy; circular

dichroism; metal release; structure ⁄

function relationship; thermal stability

Correspondence

L. Vergani, Department of Biophysical

Sciences and Technologies, M. & O.

University of Genova, Corso Europa 30,

16132 Genova, Italy

Fax: +39 010 3538346

Tel: +39 010 3538404

E-mail: [email protected]

(Received 6 July 2005, revised 14

September 2005, accepted 26

September 2005)

doi:10.1111/j.1742-4658.2005.04993.x

Metallothioneins (MTs) are noncatalytic peptides involved in storage of

essential ions, detoxification of nonessential metals, and scavenging of

oxyradicals. They exhibit an unusual primary sequence and unique 3D

arrangement. Whereas vertebrate MTs are characterized by the well-known

dumbbell shape, with a b domain that binds three bivalent metal ions and

an a domain that binds four ions, molluscan MT structure is still poorly

understood. For this reason we compared two MTs from aquatic organ￾isms that differ markedly in primary structure: MT 10 from the inverteb￾rate Mytilus galloprovincialis and MT A from Oncorhyncus mykiss. Both

proteins were overexpressed in Escherichia coli as glutathione S-transferase

fusion proteins, and the MT moiety was recovered after protease cleavage.

The MTs were analyzed by gel electrophoresis and tested for their differen￾tial reactivity with alkylating and reducing agents. Although they show an

identical cadmium content and a similar metal-binding ability, spectro￾polarimetric analysis disclosed significant differences in the Cd7-MT secon￾dary conformation. These structural differences reflect the thermal stability

and metal transport of the two proteins. When metal transfer from Cd7-

MT to 4-(2-pyridylazo)resorcinol was measured, the mussel MT was more

reactive than the fish protein. This confirms that the differences in the pri￾mary sequence of MT 10 give rise to peculiar secondary conformation,

which in turn reflects its reactivity and stability. The functional differences

between the two MTs are due to specific structural properties and may be

related to the different lifestyles of the two organisms.

Abbreviations

MT, metallothionein; GST, glutathione S-transferase; PAR, 4-(2-pyridylazo)resorcinol.

6014 FEBS Journal 272 (2005) 6014–6023 ª 2005 The Authors. Journal compilation ª 2005 FEBS