Abstract
Acetylcholinesterase (AChE) is anchored onto cell membranes by the transmembrane protein PRiMA (proline-rich membrane anchor) as a tetrameric globular form that is prominently expressed in vertebrate brain. In parallel, the PRiMA-linked tetrameric butyrylcholinesterase (BChE) is also found in the brain. A single type of AChE-BChE hybrid tetramer was formed in cell cultures by co-transfection of cDNAs encoding AChE(T) and BChE(T) with proline-rich attachment domain-containing proteins, PRiMA I, PRiMA II, or a fragment of ColQ having a C-terminal GPI addition signal (Q(N-GPI)). Using AChE and BChE mutants, we showed that AChE-BChE hybrids linked with PRiMA or Q(N-GPI) always consist of AChE(T) and BChE(T) homodimers. The dimer formation of AChE(T) and BChE(T) depends on the catalytic domains, and the assembly of tetramers with a proline-rich attachment domain-containing protein requires the presence of C-terminal "t-peptides" in cholinesterase subunits. Our results indicate that PRiMA- or ColQ-linked cholinesterase tetramers are assembled from AChE(T) or BChE(T) homodimers. Moreover, the PRiMA-linked AChE-BChE hybrids occur naturally in chicken brain, and their expression increases during development, suggesting that they might play a role in cholinergic neurotransmission.
Highlights
In vertebrates, cholinesterases are widespread enzymes present in cholinergic and noncholinergic tissues, as well as in plasma and body fluids [1, 2]
The asymmetric form (A12) of AChE, purified by immunoaffinity chromatography from 1-day-old chicken pectoral muscles, was shown to contain AChE and BChE catalytic subunits in a 1:1 ratio [15]. The existence of this type of hybrid molecule was supported by several lines of evidence: (i) using anti-BChE antibody 7D11 in immunoprecipitation could precipitate AChE activity [15] and (ii) in the purified asymmetric AChE, three subunits corresponding to AChE, BChE, and ColQ were detected on the silver-stained SDS-PAGE gel [24]
We extend this study by showing the formation of PRiMA-linked AChE-BChE tetrameric hybrid molecules, both in transfected cells in culture and in intact chicken brain
Summary
Cell Cultures—A human embryonic kidney fibroblast cell line (HEK293T) was obtained from the American Type Culture Collection (Manassas, VA). BChE activity was assayed in a similar manner, except that the lysates were preincubated with 20 M BW284c51 (an inhibitor of AChE; Sigma) for 10 min, and the substrate was 0.625 mM butyrylthiocholine iodide (BTCh; Sigma). The assays for both enzymes were highly specific (supplemental Fig. S1A). Equal amounts of protein from cell lysates were scribed above Another method to subjected to sucrose density gradient analysis to analyze the molecular forms, as in A. Samples of tissue lysates with equal amounts of AChE activity from chicken cerebrum at different developmental stages were loaded on anti-AChE antibody-precoated ELISA plates. Other Assays—Protein concentrations were measured by the Bradford method [32] with a kit from Bio-Rad
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