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Evolution of acetylcholinesterase and butyrylcholinesterase in the vertebrates: an atypical butyrylcholinesterase from the Medaka Oryzias latipes.

Pezzementi L, Nachon F, Chatonnet A - PLoS ONE (2011)

Bottom Line: It is effectively inhibited by physostigmine, typical of all ChEs.However, although the atypical BChE is efficiently inhibited by the BChE-specific inhibitor ethopropazine, it is not by another BChE inhibitor, iso-OMPA, nor by the AChE-specific inhibitor BW284c51.The atypical BChE is found as a glycophosphatidylinositol-anchored (GPI-anchored) amphiphilic dimer (G(2) (a)), which is unusual for any BChE.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Birmingham-Southern College, Birmingham, Alabama, United States of America. lpezzeme@bsc.edu

ABSTRACT
Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are thought to be the result of a gene duplication event early in vertebrate evolution. To learn more about the evolution of these enzymes, we expressed in vitro, characterized, and modeled a recombinant cholinesterase (ChE) from a teleost, the medaka Oryzias latipes. In addition to AChE, O. latipes has a ChE that is different from either vertebrate AChE or BChE, which we are classifying as an atypical BChE, and which may resemble a transitional form between the two. Of the fourteen aromatic amino acids in the catalytic gorge of vertebrate AChE, ten are conserved in the atypical BChE of O. latipes; by contrast, only eight are conserved in vertebrate BChE. Notably, the atypical BChE has one phenylalanine in its acyl pocket, while AChE has two and BChE none. These substitutions could account for the intermediate nature of this atypical BChE. Molecular modeling supports this proposal. The atypical BChE hydrolyzes acetylthiocholine (ATCh) and propionylthiocholine (PTCh) preferentially but butyrylthiocholine (BTCh) to a considerable extent, which is different from the substrate specificity of AChE or BChE. The enzyme shows substrate inhibition with the two smaller substrates but not with the larger substrate BTCh. In comparison, AChE exhibits substrate inhibition, while BChE does not, but may instead show substrate activation. The atypical BChE from O. latipes also shows a mixed pattern of inhibition. It is effectively inhibited by physostigmine, typical of all ChEs. However, although the atypical BChE is efficiently inhibited by the BChE-specific inhibitor ethopropazine, it is not by another BChE inhibitor, iso-OMPA, nor by the AChE-specific inhibitor BW284c51. The atypical BChE is found as a glycophosphatidylinositol-anchored (GPI-anchored) amphiphilic dimer (G(2) (a)), which is unusual for any BChE. We classify the enzyme as an atypical BChE and discuss its implications for the evolution of AChE and BChE and for ecotoxicology.

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Velocity sedimentation analysis of the molecular forms of recombinant O. latipes BChE.HIS extracts from COS-7 cells transfected with cDNA for BChE were sedimented on gradients prepared in presence (•) and absence (○) of Triton X-100 as described in Materials and Methods. Data are presented as the fraction of total BChE activity on the gradient as a function of sedimentation coefficient.
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pone-0017396-g007: Velocity sedimentation analysis of the molecular forms of recombinant O. latipes BChE.HIS extracts from COS-7 cells transfected with cDNA for BChE were sedimented on gradients prepared in presence (•) and absence (○) of Triton X-100 as described in Materials and Methods. Data are presented as the fraction of total BChE activity on the gradient as a function of sedimentation coefficient.

Mentions: ChEs exist in various homomeric and heteromeric molecular forms depending, in part, on the nature of their carboxyl termini. Since the amino acid sequence of the atypical BChE indicates an H-type C-terminus, we performed velocity sedimentation on sucrose gradients in the presence and absence of the non-ionic detergent Triton X-100 to determine the molecular forms of the recombinant enzyme produced in vitro by COS-7 cells. The extract contains G2a forms on the basis of the sedimentation coefficient (5.25±0.10 S; Mean±SE, N = 6) and its shift to higher values in the absence of detergent due to aggregation of the enzyme (8.96±0.05; Mean±SE, N = 6) (Fig. 7). Digestion of intact COS-7 cells with phosphatidylinositol-specific phospholipase C (PIPLC) releases ∼80% of the surface enzyme activity. Spontaneous release of activity into the supernatant during incubation in the absence of PIPLC was ∼10% (Fig. 8). These data indicate that the G2a produced is a glycophosphatidylinositol-anchored (GPI-anchored) form. It is unusual for any BChE to be found as a GPI-anchored form


Evolution of acetylcholinesterase and butyrylcholinesterase in the vertebrates: an atypical butyrylcholinesterase from the Medaka Oryzias latipes.

Pezzementi L, Nachon F, Chatonnet A - PLoS ONE (2011)

Velocity sedimentation analysis of the molecular forms of recombinant O. latipes BChE.HIS extracts from COS-7 cells transfected with cDNA for BChE were sedimented on gradients prepared in presence (•) and absence (○) of Triton X-100 as described in Materials and Methods. Data are presented as the fraction of total BChE activity on the gradient as a function of sedimentation coefficient.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3045457&req=5

pone-0017396-g007: Velocity sedimentation analysis of the molecular forms of recombinant O. latipes BChE.HIS extracts from COS-7 cells transfected with cDNA for BChE were sedimented on gradients prepared in presence (•) and absence (○) of Triton X-100 as described in Materials and Methods. Data are presented as the fraction of total BChE activity on the gradient as a function of sedimentation coefficient.
Mentions: ChEs exist in various homomeric and heteromeric molecular forms depending, in part, on the nature of their carboxyl termini. Since the amino acid sequence of the atypical BChE indicates an H-type C-terminus, we performed velocity sedimentation on sucrose gradients in the presence and absence of the non-ionic detergent Triton X-100 to determine the molecular forms of the recombinant enzyme produced in vitro by COS-7 cells. The extract contains G2a forms on the basis of the sedimentation coefficient (5.25±0.10 S; Mean±SE, N = 6) and its shift to higher values in the absence of detergent due to aggregation of the enzyme (8.96±0.05; Mean±SE, N = 6) (Fig. 7). Digestion of intact COS-7 cells with phosphatidylinositol-specific phospholipase C (PIPLC) releases ∼80% of the surface enzyme activity. Spontaneous release of activity into the supernatant during incubation in the absence of PIPLC was ∼10% (Fig. 8). These data indicate that the G2a produced is a glycophosphatidylinositol-anchored (GPI-anchored) form. It is unusual for any BChE to be found as a GPI-anchored form

Bottom Line: It is effectively inhibited by physostigmine, typical of all ChEs.However, although the atypical BChE is efficiently inhibited by the BChE-specific inhibitor ethopropazine, it is not by another BChE inhibitor, iso-OMPA, nor by the AChE-specific inhibitor BW284c51.The atypical BChE is found as a glycophosphatidylinositol-anchored (GPI-anchored) amphiphilic dimer (G(2) (a)), which is unusual for any BChE.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Birmingham-Southern College, Birmingham, Alabama, United States of America. lpezzeme@bsc.edu

ABSTRACT
Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are thought to be the result of a gene duplication event early in vertebrate evolution. To learn more about the evolution of these enzymes, we expressed in vitro, characterized, and modeled a recombinant cholinesterase (ChE) from a teleost, the medaka Oryzias latipes. In addition to AChE, O. latipes has a ChE that is different from either vertebrate AChE or BChE, which we are classifying as an atypical BChE, and which may resemble a transitional form between the two. Of the fourteen aromatic amino acids in the catalytic gorge of vertebrate AChE, ten are conserved in the atypical BChE of O. latipes; by contrast, only eight are conserved in vertebrate BChE. Notably, the atypical BChE has one phenylalanine in its acyl pocket, while AChE has two and BChE none. These substitutions could account for the intermediate nature of this atypical BChE. Molecular modeling supports this proposal. The atypical BChE hydrolyzes acetylthiocholine (ATCh) and propionylthiocholine (PTCh) preferentially but butyrylthiocholine (BTCh) to a considerable extent, which is different from the substrate specificity of AChE or BChE. The enzyme shows substrate inhibition with the two smaller substrates but not with the larger substrate BTCh. In comparison, AChE exhibits substrate inhibition, while BChE does not, but may instead show substrate activation. The atypical BChE from O. latipes also shows a mixed pattern of inhibition. It is effectively inhibited by physostigmine, typical of all ChEs. However, although the atypical BChE is efficiently inhibited by the BChE-specific inhibitor ethopropazine, it is not by another BChE inhibitor, iso-OMPA, nor by the AChE-specific inhibitor BW284c51. The atypical BChE is found as a glycophosphatidylinositol-anchored (GPI-anchored) amphiphilic dimer (G(2) (a)), which is unusual for any BChE. We classify the enzyme as an atypical BChE and discuss its implications for the evolution of AChE and BChE and for ecotoxicology.

Show MeSH
Related in: MedlinePlus