Acetylcholinesterase (AChE) is known to be crucial in the nervous system for its cholinergic functions. Interestingly, AChE exists in other tissues and is reported to involve in the development of different cells. By alternative splicing, AChE is synthesized in different isoforms, including tailed (T), read-through (R) and hydrophobic (H) forms. AChE_H form is a glycophosphatidylinositol (GPI)-linked dimer presents abunduntly on plasma membrane of mammalian erythrocyte; but the function of which is largely unknown. Here, we aimed to evaluate the functional role of AChE during erythrocyte maturation and to reveal the assembly of the GPI-linked AChE dimer.

Transgenic mice with ACHE gene depletion were employed in the study to investigate the role of AChE in blood cell formation. AChE knoc...[ Read more ]

Acetylcholinesterase (AChE) is known to be crucial in the nervous system for its cholinergic functions. Interestingly, AChE exists in other tissues and is reported to involve in the development of different cells. By alternative splicing, AChE is synthesized in different isoforms, including tailed (T), read-through (R) and hydrophobic (H) forms. AChE_H form is a glycophosphatidylinositol (GPI)-linked dimer presents abunduntly on plasma membrane of mammalian erythrocyte; but the function of which is largely unknown. Here, we aimed to evaluate the functional role of AChE during erythrocyte maturation and to reveal the assembly of the GPI-linked AChE dimer.

Transgenic mice with ACHE gene depletion were employed in the study to investigate the role of AChE in blood cell formation. AChE knock-out mice were found to suffer normocytic anemia. In the erythrocyte of ACHE^-/- mice, the amount of hemoglobin, especially α globin, was found to be markedly reduced. In addition, the number of erythrocyte and the hematocrit of the mutant mice were also significantly lowered. This arouse our interest to probe the role of AChE in erythropoietic differentiation, i.e. the maturation of erythroblasts (erythrocyte precursors) into erythrocytes.

Upon induction of erythroblast-like cells (TF-1) to differentiate into a mature stage by erythropoietin (EPO), AChE transcript, as well as the enzymatic activity, was increased significantly along with the erythroid protein genes, e.g. α and β globins, transcription factor GATA-1. In the cultures, AChE_H was the dominant form of AChE over the others. In addition, the expression of AChE in erythroblast during EPO-induced differentiation was mediated by PI3K/Akt/GATA-1 cascade, a downstream of EPO receptor signaling. In cultured TF-1 cells, the teatment of PI3K inhibitor and knock-down of GATA-1 reduced the expression of AChE. By over-expression or transient knock-down approaches, the manipulation of AChE expression in cultured TF-1 cells led to significant changes of the expressions of GATA-1, α and β globins. In parallel, we found that AChE and GATA-1 could affect the expression of each other. Furthermore, the GPI-linked AChE was shown to be an interacting partner of EPO receptor, as reviewed from the co-immunoprecipitation studies. These results suugested that AChE could have a regulatory role in erythropoietic differentiation, possibly through interaction with EPO receptor. We propose that the interaction between AChE and EPO receptor could be mediated by scalfold protein. In this way, EPO receptor could be stabilized to the lipid-raft to amplify downstream signaling for driving erythropoiesis.

In cultured TF-1 cells, the proportion of AChE bearing mature glycan was increased during EPO-induced differentiation. To search for the role of glycosylation in this GPI-linked AChE, the cDNA encoding AChE_H was transiently transfected into HEK293T cells. The recombinant GPI-linked AChE was functionally expressed in dimeric form and heavily glycosylated, similar as that in erythrocytes. The function of glycosylation was studied by site-direct mutagenesis, in where AChE_H glycosylation mutant showed complete removal of glycans. To reveal the functions of glycosylation, kinetic studies showed that the glycan-deprived AChE had a higher K_m value than that of the wild type enzyme. Interestingly, the glycosylation mutant could still form AChE dimer in the cells, but mostly retained in endoplasmic reticulum (ER). These results suggested that the glycosylation of AChE_H affected the catalytic activity of enzyme as well as the protein trafficking, in particular during the assembly process from ER to plasma membrane; however, the formation of dimer was not affected.

The present study indicated the significance of AChE in the function and maturation of erythrocyte and the importance of N-linked glycosylation in controlling the biosynthesis of GPI-linked AChE form. The findings on the role of AChE in erythropoietic differentiation and its assembly mechanism could provide an insight for elucidating other possible mechanisms in the regulation of erythropoiesis.