Tài liệu Báo cáo khoa học: Physico-chemical characterization and synthesis of neuronally active

MINIREVIEW

Physico-chemical characterization and synthesis of neuronally active

a-conotoxins

Marion L. Loughnan and Paul F. Alewood

Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia

The high specificity of a-conotoxins for different neuronal

nicotinic acetylcholine receptors makes them important

probes for dissecting receptor subtype selectivity. New

sequences continue to expand the diversity and utility of

the pool of available a-conotoxins. Their identification

and characterization depend on a suite of techniques

with increasing emphasis on mass spectrometry and micro￾scale chromatography, which have benefited from recent

advances in resolution and capability. Rigorous physico￾chemical analysis together with synthetic peptide chemistry

is a prerequisite for detailed conformational analysis and to

provide sufficient quantities of a-conotoxins for activity

assessment and structure–activity relationship studies.

Keywords: a-conotoxins; Conus; peptide synthesis; post￾translational modifications; sulfotyrosine.

Classification, primary structure and biology

of a-conotoxins

Cone snails are a group of hunting gastropods that

incapacitate their prey, which consists of worms, molluscs

or fish, by envenomation. Conotoxins from the venom of

cone snails are small disulfide-rich peptide toxins that act at

many voltage-gated and ligand-gated ion channels. They

can be grouped according to their molecular form into

several superfamilies, each defined by characteristic arrange￾ments of cysteine residues (not necessarily a single pattern),

and characteristic highly conserved precursor signal

sequence similarities. Individual conopeptide families

within a superfamily are denoted by Greek letters and

contain peptides that have a particular disulfide framework

and target homologous sites on a particular receptor [1].

Each of the characterized conopeptides is named using

a convention that indicates the activity (Greek letter), the

source species from which the peptide was first isolated

(Arabic letter(s)), the disulfide framework category (Roman

numeral) and the order of discovery within that category

(Arabic capital letter) [1]. For example a-AuIB belongs to

the a-conotoxin family and was the second peptide, B, with

that framework, I, isolated and reported from Conus aulicus

[1,2]. The names of some conotoxins deviate from this

nomenclature convention because their discovery preceded

its formulation. Hence some a-conotoxin names do not

conform to the alphabetical identifier system used to

indicate order of discovery of peptides with a specified

disulfide framework from the venom of any one species. The

framework identifiers I and II are both used in reference

to disulfide frameworks of the A superfamily without

distinction.

The A superfamily is so far comprised of the K+ channel

blocking jA familiy and the a and aA families, which

together with the w family act at the nicotinic acetylcholine

receptor (nAChR). No aA or w conopeptides have been

reported to block neuronal nicotinic receptors with high

affinity. Rather, they are generally muscle-specific nicotinic

receptor antagonists [1]. The a-conotoxins fall into two

categories depending on whether they act at muscle￾type or neuronal-type receptors. The neuronally active

a-conotoxins are the focus of this minireview.

The known a-conotoxins consist of 12–19 amino acids.

Most a-conopeptides have four cysteine residues and the

general sequence GCCXmCXnC. The disulfide connectivity

is between alternate cysteine residues (I-III, II-IV).

The numbers of amino acid residues encompassed by the

second and third cysteine residues (m) and the third and

fourth cysteine residues (n) are the basis for a further

division into several structural subfamilies (a3/5, a4/3, a4/6

and a4/7) [1,3,4]. For example a4/6-AuIB belongs to the 4/6

disulfide loop size subgroup of the a-conotoxin family. The

neuronally active a-conotoxins are typically from the a4/7,

a4/6 and a4/3 subfamilies (Table 1). Peptides from the most

abundant a4/7subfamily are typically 16 residues in length

and range from
1600 to
1900 Da in mass. However

there have been recent additions to this subfamily in which

Correspondence to P. F. Alewood, Institute for Molecular Bioscience,

The University of Queensland, Brisbane, QLD 4072, Australia.

Fax: + 61 73346 2101, Tel.: + 61 73346 2982,

E-mail: [email protected]

Abbreviations: c-CRS, c-carboxylation recognition sequence;

nAChRs, nicotinic acetylcholine receptors; RT, retention time;

PTM, post-translational modification; TPST, tyrosyl-protein

sulfotransferase; TCEP, tris(2-carboxyethyl)phosphine; M-biotin,

maleimide-biotin; NEM, N-ethylmaleimide; IAM, iodoacetamide.

(Received 22 January 2004, revised 16 March 2004,

accepted 6 April 2004)

Eur.J.Biochem. 271, 2294–2304 (2004)
FEBS 2004 doi:10.1111/j.1432-1033.2004.04146.x