A novel species of cholinesterase in vertebrates : the discovery of PRiMA-linked AChE-BChE hybrid tetramer and its molecular assembly mechanism

Abstract

Vertebrates possess two types of cholinesterases (ChEs), acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). In the nervous system, these two enzymes are responsible for the hydrolysis of the neurotransmitter, acetylcholine. The functional localization and oligomerization of AChE and BChE depend on the association of their interacting partners, either collagen tail (ColQ) or p̲roline r̲ich m̲embrane a̲nchor (PRiMA). Complexes with ColQ represent the asymmetric forms in muscle, while complexes with PRiMA represent tetrameric globular forms (G4) mainly in brain and muscles. Apart from these traditional molecular forms, a ColQ-linked asymmetric form of hybrid ChE, having both AChE and BChE catalytic subunits, was revealed in chicken muscles. Based on this interesting finding, the physiological relevance of the hybrid ChEs was investigated here. Specifically, the existence of the PRiMA-linked hybrid ChE as well as its molecular assembly mechanism was extensively determined. A recombinant system in cultured HEK293T cells was firstly employed. The co-transfection of cDNAs encoding AChET, BChET and PRiMA produced a single type of PRiMA-linked AChE-BChE G4 hybrid. This G4 hybrid molecule consisted of an AChE dimer and a BChE dimer, i.e. [AChET]2-[BChET]2-PRiMA. However, other combination of AChET and BChET subunits in the G4 hybrid molecule was not found. Using AChE and BChE chimeric constructs, the dimer formation of AChET and BChET was found to depend on their catalytic domains, and the assembly of the catalytic subunits with PRiMA required the presence of the C-terminal t-peptides. Furthermore, the assembly of AChET and BChET with PRiMA to form tetramers occurred in endoplasmic reticulum (ER). During protein trafficking from ER to plasma membrane, both AChET and BChET attained glycosylation. The functional role of glycosylation on ChEs was demonstrated by the fact that the elimination of N-glycans on AChET did not affect the formation of PRiMA-linked G4 AChE or AChE-BChE G4 hybrid. The ChE glycosylation was shown to be required for the exportation of the G4 enzymes from ER to plasma membrane. In addition to the recombinant system, PRiMA-linked AChE-BChE G4 hybrid was also found to occur naturally in chicken nervous system. The expression of this hybrid enzyme underwent different developmental changes in brain and muscles. During the development, the expression of this G4 hybrid enzyme in brain was strongly increased, while that was gradually decreased in muscles, indicating possible functions of the hybrid enzyme in various developmental stages. At the cellular level, this G4 hybrid ChE was found to be in the synaptosome fractions of adult chicken brain, suggesting it might function in the synapses. The findings on the new species of PRiMA-linked ChE and its assembly mechanism could provide an insight for elucidating the possible function and regulation of different molecular forms of ChEs in vertebrate.

Date of Award	2011
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology