Wilson K.W. Luk

The Assembly of Proline-rich Membrane Anchor (PRiMA)-linked Acetylcholinesterase Enzyme GLYCOSYLATION IS REQUIRED FOR ENZYMATIC ACTIVITY BUT NOT FOR OLIGOMERIZATION

Acetylcholinesterase (AChE) anchors onto cell membranes by a transmembrane protein PRiMA (proline-rich membrane anchor) as a tetrameric form in vertebrate brain. The assembly of AChE tetramer with PRiMA requires the C-terminal “t-peptide” in AChE catalytic subunit (AChET). Although mature AChE is well known N-glycosylated, the role of glycosylation in forming the physiologically active PRiMA-linked AChE tetramer has not been studied. Here, several lines of evidence indicate that the N-linked glycosylation of AChET plays a major role for acquisition of AChE full enzymatic activity but does not affect its oligomerization. The expression of the AChET mutant, in which all N-glycosylation sites were deleted, together with PRiMA in HEK293T cells produced a glycan-depleted PRiMA-linked AChE tetramer but with a much higher Km value as compared with the wild type. This glycan-depleted enzyme was assembled in endoplasmic reticulum but was not transported to Golgi apparatus or plasma membrane.

Molecular Assembly and Biosynthesis of Acetylcholinesterase in Brain and Muscle: the Roles of t-peptide, FHB Domain, and N-linked Glycosylation

Acetylcholinesterase (AChE) is responsible for the hydrolysis of the neurotransmitter, acetylcholine, in the nervous system. The functional localization and oligomerization of AChE T variant are depending primarily on the association of their anchoring partners, either collagen tail (ColQ) or proline-rich membrane anchor (PRiMA). Complexes with ColQ represent the asymmetric forms (A12) in muscle, while complexes with PRiMA represent tetrameric globular forms (G4) mainly found in brain and muscle. Apart from these traditional molecular forms, a ColQ-linked asymmetric form and a PRiMA-linked globular form of hybrid cholinesterases (ChEs), having both AChE and BChE catalytic subunits, were revealed in chicken brain and muscle. The similarity of various molecular forms of AChE and BChE raises interesting question regarding to their possible relationship in enzyme assembly and localization. The focus of this review is to provide current findings about the biosynthesis of different forms of ChEs together with their anchoring proteins.

N-linked glycosylation of dimeric acetylcholinesterase in erythrocytes is essential for enzyme maturation and membrane targeting.

Acetylcholinesterase (AChE) is well‐known for its cholinergic functions in the nervous system; however, this enzyme is also found in other tissues where its function is still not understood. AChE is synthesized through alternative splicing as splicing variants, with isoforms including read‐through (AChER), tailed (AChET) and hydrophobic (AChEH). In human erythrocytes, AChEH is a glycophosphatidylinositol‐linked dimer on the plasma membrane. Three N‐linked glycosylation sites have been identified in the catalytic domain of human AChE. Here, we investigate the roles of glycosylation in assembly and trafficking of human AChEH. In transfected fibroblasts, expression of AChEH was able to mimic the function of the dimeric form of AChE on the erythrocyte membrane. A glycan‐depleted form was constructed by site‐directed mutagenesis. By comparison with the wild‐type AChEH, the mutant had a much lower enzymatic activity and a much higher Km value. In addition, the mutant was dimerized in the endoplasmic reticulum, but was not trafficked to the Golgi apparatus. The results suggest that the glycosylation may affect AChEH enzymatic activity and trafficking, but not dimer formation. The present findings indicate the significance of N‐glycosylation in controlling the biosynthesis of the AChEH dimer form.

Acetylcholinesterase Protein Level Is Preserved in the Alzheimer's Brain

Acetylcholinesterase (AChE) is a key enzyme in the cholinergic nervous system and is one of the most studied proteins in the field of Alzheimers disease (AD). Moreover, alternative functions of AChE unrelated with the hydrolysis of acetylcholine are suspected. Until now, the majority of investigations on AChE in AD pathology have been focused on the determination of its enzymatic activity level, which is depleted in the AD brain. Despite this overall decrease, AChE activity increases at the vicinity of the two hallmarks of AD, the amyloid plaques and the neurofibrillary tangles (NFT). In fact, AChE may directly interact with Aβ in a manner that increases the deposition of Aβ to form plaques. In the context of protein–protein interactions, we have recently reported that AChE can interact with presenilin-1, the catalytic component of γ-secretase, influencing its expression level and also its activity. However, the alteration of AChE protein in the AD brain has not been determined. Here, we demonstrated by Western blotting and immunohistochemistry that a prominent pool of enzymatically inactive AChE protein existed in the AD brain. The potential significance of these unexpected levels of inactive AChE protein in the AD brain was discussed, especially in the context of protein–protein interactions with β-amyloid and presenilin-1.

Three N-Glycosylation Sites of Human Acetylcholinesterase Shares Similar Glycan Composition

Acetylcholinesterase (AChE; EC 3.1.1.7) is a glycoprotein possessing three conserved N-linked glycosylation sites in mammalian species, locating at 296, 381, and 495 residues of the human sequence. Several lines of evidence demonstrated that N-glycosylation of AChE affected the enzymatic activity, as well as its biosynthesis. In order to determine the role of three N-glycosylation sites in AChE activity and glycan composition, the site-directed mutagenesis of N-glycosylation sites in wild-type human AChET sequence was employed to generate the single-site mutants (i.e., AChETN296Q, AChETN381Q, and AChETN495Q) and all site mutant (i.e., AChET3N→3Q). The mutation did not affect AChE protein expression in the transfected cells. The mutants, AChET3N→3Q and AChETN381Q, showed very minimal enzymatic activity, while the other mutants showed reduced activity. By binding to lectins, Con A, and SNA, the glycosylation profile was revealed in those mutated AChE. The binding affinity with lectins showed no significant difference between various N-glycosylation mutants, which suggested that similar glycan composition should be resulted from different N-glycosylation sites. Although the three glycosylation sites within AChE sequence have different extent in affecting the enzymatic activity, their glycan compositions are very similar.

Characterization of acetylcholinesterase in Hong Kong oyster (Crassostrea hongkongensis) from South China Sea.

Acetylcholinesterase (AChE) activity has been used to evaluate the exposure of mollusk bivalves to organophosphates, carbamate pesticides, and heavy metals. Crassostrea hongkongensis is a Hong Kong endemic oyster, and has a high commercial value along the coastal area of South China. The use of this species as a bio-indicator of the marine environment, and the use of AChE activity measurements in tissues of C. hongkongensis require prior characterization of AChE in this species. Here, we report that gill tissue contains the highest AChE activity in C. hongkongensis, and that the molecular form of AChE is most likely to be a dimeric form. In addition, the effect of the pesticide acephate on AChE activity in the gill of C. hongkongensis was analyzed, and the mean inhibition concentration (IC50) value was determined. This study suggests that AChE activity in the gill tissue of C. hongkongensis might be used as a biomarker in monitoring organophosphate contamination in the marine fauna of South China.

Expression of cAMP-responsive Element Binding Proteins (CREBs) in Fast- and Slow-twitch Muscles: A Signaling Pathway to Account for the Synaptic Expression of Collagen-tailed Subunit (ColQ) of Acetylcholinesterase at the Rat Neuromuscular Junction

The gene encoding the collagen-tailed subunit (ColQ) of acetylcholinesterase (AChE) contains two distinct promoters that drive the production of two ColQ mRNAs, ColQ-1 and ColQ-1a, in slow- and fast-twitch muscles, respectively. ColQ-1a is expressed at the neuromuscular junction (NMJ) in fast-twitch muscle, and this expression depends on trophic factors supplied by motor neurons signaling via a cAMP-dependent pathway in muscle. To further elucidate the molecular basis of ColQ-1as synaptic expression, here we investigated the expression and localization of cAMP-responsive element binding protein (CREB) at the synaptic and extra-synaptic regions of fast- and slow-twitch muscles from adult rats. The total amount of active, phosphorylated CREB (P-CREB) present in slow-twitch soleus muscle was higher than that in fast-twitch tibialis muscle, but P-CREB was predominantly expressed in the fast-twitch muscle at NMJs. In contrast, P-CREB was detected in both synaptic and extra-synaptic regions of slow-twitch muscle. These results reveal, for the first time, the differential distribution of P-CREB in fast- and slow-twitch muscles, which might support the crucial role of cAMP-dependent signaling in controlling the synapse-specific expression of ColQ-1a in fast-twitch muscles.

Characterizations of Cholinesterases in Golden Apple Snail (Pomacea canaliculata)

Cholinesterases (ChEs) have been identified in vertebrates and invertebrates. Inhibition of ChE activity in invertebrates, such as bivalve molluscs, has been used to evaluate the exposure of organophosphates, carbamate pesticides, and heavy metals in the marine system. The golden apple snail (Pomacea canaliculata) is considered as one of the worst invasive alien species harmful to rice and other crops. The ChE(s) in this animal, which has been found recently, but poorly characterized thus far, could serve as biomarker(s) for environmental surveillance as well as a potential target for the pest control. In this study, the tissue distribution, substrate preference, sensitivity to ChE inhibitors, and molecular species of ChEs in P. canaliculata were investigated. It was found that the activities of both AChE and BChE were present in all test tissues. The intestine had the most abundant ChE activities. Both enzymes had fair activities in the head, kidney, and gills. The BChE activity was more sensitive to tetra-isopropylpyrophosphoramide (iso-OMPA) than the AChE. Only one BChE molecular species, 5.8S, was found in the intestine and head, whereas two AChE species, 5.8S and 11.6S, were found there. We propose that intestine ChEs of this snail may be potential biomarkers for manipulating pollutions.

Erythropoietin regulates the expression of dimeric form of acetylcholinesterase during differentiation of erythroblast

Acetylcholinesterase (AChE; EC 3.1.1.7) is known to hydrolyze acetylcholine at cholinergic synapses. In mammalian erythrocyte, AChE exists as a dimer (G2) and is proposed to play role in erythropoiesis. To reveal the regulation of AChE during differentiation of erythroblast, erythroblast‐like cells (TF‐1) were induced to differentiate by application of erythropoietin (EPO). The expression of AChE was increased in parallel to the stages of differentiation. Application of EPO in cultured TF‐1 cells induced transcriptional activity of ACHE gene, as well as its protein product. This EPO‐induced event was in parallel with erythrocytic proteins, for example, α‐ and β‐globins. The EPO‐induced AChE expression was mediated by phosphorylations of Akt and GATA‐1; because the application of Akt kinase inhibitor blocked the gene activation. Erythroid transcription factor also known as GATA‐1, a downstream transcription factor of EPO signaling, was proposed here to account for regulation of AChE in TF‐1 cell. A binding sequence of GATA‐1 was identified in ACHE gene promoter, which was further confirmed by chromatin immunoprecipitation (ChIP) assay. Over‐expression of GATA‐1 in TF‐1 cultures induced AChE expression, as well as activity of ACHE promoter tagged with luciferase gene (pAChE‐Luc). The deletion of GATA‐1 sequence on the ACHE promoter, pAChEΔGATA‐1‐Luc, reduced the promoter activity during erythroblastic differentiation. On the contrary, the knock‐down of AChE in TF‐1 cultures could lead to a reduction in EPO‐induced expression of erythrocytic proteins. These findings indicated specific regulation of AChE during maturation of erythroblast, which provided an insight into elucidating possible mechanisms in regulating erythropoiesis.