Tài liệu Báo cáo khoa học: Acetylcholinesterase from the invertebrate Ciona intestinalis is capable

Acetylcholinesterase from the invertebrate

Ciona intestinalis is capable of assembling into

asymmetric forms when co-expressed with vertebrate

collagenic tail peptide

Adam Frederick1

, Igor Tsigelny2

, Frances Cohenour1

, Christopher Spiker1

, Eric Krejci3

,

Arnaud Chatonnet4

, Stefan Bourgoin1

, Greg Richards1

, Tessa Allen1

, Mary H. Whitlock1 and

Leo Pezzementi1

1 Department of Biology, Birmingham-Southern College, Birmingham, AL, USA

2 Department of Chemistry and Biochemistry, San Diego Supercomputer Center, University of California at San Diego, La Jolla, CA, USA

3 Institut National de la Sante´ et de la Recherche Me´dicale U686, Universite´ Paris Descartes, Biologie des Jonctions Neuromusculaires,

Paris, France

4 Institut National de la Recherche Agronomique, Montpellier, France

Keywords

acetylcholinesterase; asymmetric forms;

butyrylcholinesterase; Ciona intestinalis;

evolution

Correspondence

L. Pezzementi, Department of Biology,

Birmingham-Southern College, Box 549022,

Birmingham, AL 35254, USA

Fax: +1 205 226 3078

Tel: +1 205 226 4806

E-mail: [email protected]

Website: http://faculty.bsc.edu/lpezzeme/

Database

The nucleotide sequence and derived amino

acid sequence data reported for the AChE

from Ciona intestinalis are available in the

Third Party Annotation Section of the

DDBJ ⁄ EMBL ⁄ GenBank databases under the

accession no. TPA: BK006073. The align￾ment used to determine the phylogenetic

tree for vertebrate and invertebrate cholines￾terases presented here is deposited at the

EMBL-ALIGN database as ALIGN_001208

(Received 14 November 2007, revised 7

January 2008, accepted 15 January 2008)

doi:10.1111/j.1742-4658.2008.06292.x

To learn more about the evolution of the cholinesterases (ChEs), acetylcho￾linesterase (AChE) and butyrylcholinesterase in the vertebrates, we investi￾gated the AChE activity of a deuterostome invertebrate, the urochordate

Ciona intestinalis, by expressing in vitro a synthetic recombinant cDNA for

the enzyme in COS-7 cells. Evidence from kinetics, pharmacology, mole￾cular biology, and molecular modeling confirms that the enzyme is AChE.

Sequence analysis and molecular modeling also indicate that the cDNA

codes for the AChET subunit, which should be able to produce all three

globular forms of AChE: monomers (G1), dimers (G2), and tetramers (G4),

and assemble into asymmetric forms in association with the collagenic

subunit collagen Q. Using velocity sedimentation on sucrose gradients, we

found that all three of the globular forms are either expressed in cells or

secreted into the medium. In cell extracts, amphiphilic monomers (G1

a

)

and non-amphiphilic tetramers (G4

na) are found. Amphiphilic dimers (G2

a

)

and non-amphiphilic tetramers (G4

na) are secreted into the medium.

Co-expression of the catalytic subunit with Rattus norvegicus collagen Q

produces the asymmetric A12 form of the enzyme. Collagenase digestion of

the A12 AChE produces a lytic G4 form. Notably, only globular forms are

present in vivo. This is the first demonstration that an invertebrate AChE is

capable of assembling into asymmetric forms. We also performed a phylo￾genetic analysis of the sequence. We discuss the relevance of our results

with respect to the evolution of the ChEs in general, in deuterostome inver￾tebrates, and in chordates including vertebrates.

Abbreviations

a

, amphiphilic; AChE, acetylcholinesterase; AChEH, splice variant H; AChET, splice variant T; ATCh, acetylthiocholine; BTCh,

butyrylthiocholine; BuChE, butyrylcholinesterase; ChE, cholinesterase; ColQ, collagen Q; DEPQ, 7-[(diethoxyphosphoryl)oxy]-1-

methylquinolinium iodide; DTNB, 5-(3-carboxy-4nitro-phenyl)disulfanyl-2-nitro-benzoic acid; GPI, glycophosphatidylinositol; HIS buffer, high

ionic strength buffer; IC50, half maximal inhibitory concentration; LBA, long branch attraction; na, non-amphiphilic; PPII, polyproline II;

PRAD, proline-rich attachment domain; PRiMA, proline-rich membrane anchor; WAT, tryptophan (W) amphipathic tetramerization domain.

FEBS Journal 275 (2008) 1309–1322 ª 2008 The Authors Journal compilation ª 2008 FEBS 1309