Title

Posttranslational mechanisms for regulating the activity of human choline acetyltransferase

Date of Award

1997

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Molecular and Cellular Pharmacology

First Committee Member

William L. Strauss, Committee Chair

Abstract

Synthesis of the neurotransmitter acetylcholine is catalyzed by the enzyme choline acetyltransferase (ChAT). Homeostatic mechanisms in the cholinergic nerve terminal control the rate of acetylcholine synthesis over a wide range of physiological conditions such that levels of transmitter are not limiting in signal transduction. Two post-translational processes were examined as possible mechanisms for regulating ChAT activity in human cholinergic neurons: amino-terminal cleavage of a proenzyme, and interaction with a previously unidentified protein produced by alternative splicing of the human ChAT gene. To test whether the 70-kDa human ChAT enzyme is produced by precursor processing, a cDNA clone containing all exons of the human gene was constructed. Although both 70-kDa ChAT and a larger ChAT protein were detected in cells transfected with the full-length clone, no processing of the larger molecule was observed. Moreover, introducing a 5$\sp\prime$-frame-shift mutation in the ChAT cDNA did not affect expression of 70-kDa ChAT despite abolishing translation of the larger protein. Consistent with the possibility that expression of the larger protein was an artifact of over-expression in transfected cells, only 70-kDa ChAT was detected in a human cholinergic neuroblastoma cell line.Alternative splicing of the human ChAT gene encodes both 2500-nt and 6000-nt transcripts (Lorenzi et al. 1992a). In vitro translation and transfection of cultured cells demonstrated that the 2500-nt mRNA encodes human ChAT. The 6000-nt mRNA was shown to encode a 27-kDa protein in the human CNS which does not possess ChAT activity, and does not represent a degradation product of ChAT. The possibility that the 27-kDa protein is capable of altering enzyme activity was tested using recombinant ChAT gene products. The 27-kDa protein was shown to produce significant changes in ChAT activity in vitro using both purified and crude protein preparations. Using immunohistochemical techniques, it was further possible to demonstrate that the 27-kDa protein is co-expressed with ChAT in cholinergic neurons of the human CNS. These data suggest that the 27-kDa protein generated by alternative splicing of the human ChAT gene is capable of affecting ChAT activity in vivo, and therefore could contribute to the regulation of acetylcholine synthesis in human cholinergic neurons.

Keywords

Biology, Molecular; Biology, Neuroscience; Chemistry, Biochemistry

Link to Full Text

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