Tài liệu Báo cáo khoa học: Restricted localization of proline-rich membrane anchor (PRiMA) of

Restricted localization of proline-rich membrane anchor

(PRiMA) of globular form acetylcholinesterase at the

neuromuscular junctions – contribution and expression

from motor neurons

K. Wing Leung, Heidi Q. Xie, Vicky P. Chen, Mokka K. W. Mok, Glanice K. Y. Chu, Roy C. Y. Choi

and Karl W. K. Tsim

Department of Biology and Center for Chinese Medicine, The Hong Kong University of Science and Technology, China

Acetylcholinesterase (AChE; EC 3.1.1.7) plays a cru￾cial role in terminating the synaptic transmission by

hydrolyzing the neurotransmitter acetylcholine at the

neuron-to-neuron synapses in the central nervous sys￾tem and at the neuromuscular junctions (NMJs) in the

peripheral nervous system. AChE exists in different

molecular forms. The formation of these molecular

forms depends on alternative splicing in the 3¢ region

of the primary transcript [1], which generates the

AChER (‘readthrough’), AChEH (‘hydrophobic’) and

AChET (‘tailed’) subunits, containing the same cata￾lytic domain but different carboxyl termini [1]. In

mammals, the AChER variant produces a soluble

monomer that is up-regulated in the brain during

stress [2]; the AChEH variant produces a glycosylphos￾phatidylinositol-linked dimer and is expressed in blood

cells; the AChET variant is the only subunit expressed

in the brain and muscle. The AChET subunits form

nonamphiphilic tetramers with a collagen tail as asym￾metric AChE (A4, A8 and A12) in muscle. In addition,

the AChET variant produces monomers (G1), dimers

(G2) and tetramers (G4). The amphiphilic tetramer

(G4) is linked with a proline-rich membrane anchor

(PRiMA) as a globular form of AChE (PRiMA-linked

Keywords

acetylcholinesterase; molecular form;

muscle fiber type; neuromuscular junction;

proline-rich membrane anchor

Correspondence

K. W. K. Tsim, Department of Biology,

The Hong Kong University of Science and

Technology, Clear Water Bay Road,

Kowloon, Hong Kong SAR, China

Fax: +852 2358 1559

Tel: +852 2358 7332

E-mail: [email protected]

(Received 21 November 2008, revised 11

March 2009, accepted 25 March 2009)

doi:10.1111/j.1742-4658.2009.07022.x

The expression and localization of the proline-rich membrane anchor

(PRiMA), an anchoring protein of tetrameric globular form acetylcholines￾terase (G4 AChE), were studied at vertebrate neuromuscular junctions.

Both muscle and motor neuron contributed to this synaptic expression

pattern. During the development of rat muscles, the expression of PRiMA

and AChET and the enzymatic activity increased dramatically; however,

the proportion of G4 AChE decreased. G4 AChE in muscle was recognized

specifically by a PRiMA antibody, indicating the association of this enzyme

with PRiMA. Using western blot and ELISA, both PRiMA protein and

PRiMA-linked G4 AChE were found to be present in large amounts in

fast-twitch muscle (e.g. tibialis), but in relatively low abundance in slow￾twitch muscle (e.g. soleus). These results indicate that the expression level

of PRiMA-linked G4 AChE depends on muscle fiber type. In parallel, the

expression of PRiMA, AChET and G4 AChE also increased in the spinal

cord during development. Such expression in motor neurons contributed to

the synaptic localization of G4 AChE. After denervation, the expression of

PRiMA, AChET and G4 AChE decreased markedly in the spinal cord, and

in fast- and slow-twitch muscles.

Abbreviations

AChE, acetylcholinesterase; AChR, acetylcholine receptor; BChE, butyrylcholinesterase; ChAT, choline acetyltransferase; GAPDH,

glyceraldehyde-3-phosphate dehydrogenase; GFAP, glial fibrillary acidic protein; NeuN, neuronal nuclei; NMJs, neuromuscular junctions;

PRiMA, proline-rich membrane anchor; SNAP-25, synaptosomal-associated protein 25.

FEBS Journal 276 (2009) 3031–3042 ª 2009 The Authors Journal compilation ª 2009 FEBS 3031