M.T. Moral-Naranjo

MOLECULAR FORMS OF ACETYL- AND BUTYRYLCHOLINESTERASE IN NORMAL AND DYSTROPHIC MOUSE BRAIN

In searching for possible differences in the composition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) forms in dystrophic brain, the distribution of various enzyme molecules in normal (NB) and dystrophic (DB) 129B6F1/J mouse brain has been investigated. The tissue was sequentially extracted with saline (S1) and with saline‐Triton X‐100 buffers (S2) to release soluble and membrane‐bound cholinesterases. About 15% of the AChE and 35% of the BuChE activities in NB were recovered in S1, and the rest in S2, G4, G2, and G1 AChE and BuChE forms were identified in the soluble fractions obtained from NB and DB. The shift in sedimentation values of the separated AChE and BuChE species in sucrose gradients made with and without detergents revealed the occurrence of hydrophilic (H) and amphiphilic (A) variants of cholinesterases in the extracts. The amphiphilic properties of the several AChE and BuChE molecules were analyzed by Triton X‐114 phase‐partitioning and by phenyl‐agarose chromatography. A12 (1%), G4A (72%), G4H (8%), and G2A + G1A (19%) AChE molecules, and G4A (34%), G4H (19%), and G2A + G1A (47%) BuChE forms, were identified in NB. The G4A AChE and BuChE isoforms differed in their interaction with Triton X‐114 and with a hydrophobic matrix. Neither the extent of cholinesterase solubilization, nor the distribution of individual enzyme forms, was significantly altered in DB. The lack of specific differences in the distribution of AChE and BuChE forms between NB and DB suggests that the biosynthetic pathway leading to the various enzyme forms is altered in muscle but not in dystrophic mouse brain.

G4 forms of acetylcholinesterase and butyrylcholinesterase in normal and dystrophic mouse muscle differ in their interaction with Ricinus communis agglutinin.

Differences in glycosylation between molecular forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in muscle and serum of normal and dystrophic mice have been studied by means of their adsorption to immobilized lectins. Application of a two-step extraction procedure, first with saline buffer, and second with saline buffer and Triton X-100, brought into solution most of the muscle AChE and BuChE activities. The AChE activity was five times greater than that of BuChE in normal (NM) and dystrophic muscle (DM). The AChE activity in the serum of dystrophic mice was twice that measured in control animals, but the BuChE activity remained almost unchanged. Both AChE and BuChE in muscle and serum bound completely to concanavalin A (Con A) and Lens culinaris agglutinin (LCA). A12, A8 and G4 AChE, but not the light G2 and G1 AChE forms, in NM and DM were completely adsorbed to wheat germ agglutinin (WGA). Similarly, G4 BuChE, but not the G2 and G1 forms, were associated to WGA. A high proportion of G4 and G1 AChE and G4 BuChE forms in mouse serum were fixed to WGA. Asymmetric AChE in NM and DM reacted with Ricinus communis agglutinin (RCA) but the light AChE and BuChE forms in muscle and serum did not bind to the lectin. G4 AChE and G4 BuChE in NM were not recognized by RCA, but the isoforms in DM bound fully to the lectin. Serum G4 AChE from control or dystrophic mice did not react with RCA, but G4 BuChE was fixed to the lectin. Since RCA is specific for galactose, the results suggest that in dystrophic muscle galactose is incorporated early in G4 AChE and this affects the level of the functional tetramers destined for insertion in the plasma membrane.

Expression of cholinesterases in human kidney and its variation in renal cell carcinoma types

Despite the aberrant expression of cholinesterases in tumours, the question of their possible contribution to tumorigenesis remains unsolved. The identification in kidney of a cholinergic system has paved the way to functional studies, but details on renal cholinesterases are still lacking. To fill the gap and to determine whether cholinesterases are abnormally expressed in renal tumours, paired pieces of normal kidney and renal cell carcinomas (RCCs) were compared for cholinesterase activity and mRNA levels. In studies with papillary RCC (pRCC), conventional RCC, chromophobe RCC, and renal oncocytoma, acetylcholinesterase activity increased in pRCC (3.92 ± 3.01 mU·mg−1, P = 0.031) and conventional RCC (2.64 ± 1.49 mU·mg−1, P = 0.047) with respect to their controls (1.52 ± 0.92 and 1.57 ± 0.44 mU·mg−1). Butyrylcholinesterase activity increased in pRCC (5.12 ± 2.61 versus 2.73 ± 1.15 mU·mg−1, P = 0.031). Glycosylphosphatidylinositol‐linked acetylcholinesterase dimers and hydrophilic butyrylcholinesterase tetramers predominated in control and cancerous kidney. Acetylcholinesterase mRNAs with exons E1c and E1e, 3′‐alternative T, H and R acetylcholinesterase mRNAs and butyrylcholinesterase mRNA were identified in kidney. The levels of acetylcholinesterase and butyrylcholinesterase mRNAs were nearly 1000‐fold lower in human kidney than in colon. Whereas kidney and renal tumours showed comparable levels of acetylcholinesterase mRNA, the content of butyrylcholinesterase mRNA was increased 10‐fold in pRCC. The presence of acetylcholinesterase and butyrylcholinesterase mRNAs in kidney supports their synthesis in the organ itself, and the prevalence of glycosylphosphatidylinositol‐anchored acetylcholinesterase explains the splicing to acetylcholinesterase‐H mRNA. The consequences of butyrylcholinesterase upregulation for pRCC growth are discussed.

Glycosylation of acetylcholinesterase forms in microsomal membranes from normal and dystrophic Lama2dy mouse muscle.

Abstract: The distribution and glycosylation of acetylcholinesterase (AChE) forms in vesicles derived from sarcoplasmic reticulum of normal muscle (NMV) were investigated and compared with those from dystrophic muscle vesicles (DMV). AChE activity was similar in NMV and DMV. Most of the AChE in NMV and half in DMV were released with Triton X‐100. Asymmetric (A12) and globular hydrophilic and amphiphilic (GH4, GA4, GA2, and GA1) AChE species occurred in NMV and DMV, the lighter forms being predominant. The percentage of GH4 and GA4 decreased in DMV. A fraction of the AChE that could not be extracted with detergent was detached with collagenase. Most of the detergent‐released A12 AChE from NMV and nearly half in DMV failed to bind to Ricinus communis agglutinin (RCA‐I). Conversely, the collagenase‐detached isoforms bound to RCA, revealing that asymmetric AChE associated with internal membranes or basal lamina differed in glycosylation. Moreover, nearly half of GA4 AChE in DMV and a few in NMV bound to RCA. Most of the RCA‐unreactive GA4 forms in NMV come from sarcolemma. The results indicate that dystrophy induces minor changes in the distribution and glycosylation of AChE forms in internal membranes of muscle.

Muscular dystrophy with laminin deficiency decreases the content of butyrylcholinesterase tetramers in sciatic nerves of Lama2dy mice

The Lama2dy mouse, a model of congenital muscular dystrophy (CMD) by merosin deficiency (MCMD), shows muscle degeneration and dysmyelination of peripheral nerves. Although it has been reported that MCMD reduces acetylcholinesterase (AChE) activity of mouse sciatic nerve, no information is available regarding its action on butyrylcholinesterase (BuChE). Amphiphilic BuChE monomers (G(1)(A), 39%), dimers (G(2)(A), 18%), and tetramers (G(4)(A), 33%), along with hydrophilic tetramers (G(4)(H), 10%), were identified in mouse sciatic nerve. It also contained abundant G(4)(A) (75%) and less G(1)(A), G(2)(A), G(4)(H) and A(12) AChE components. In dystrophic nerves, the BuChE activity increased 2-fold but the proportion of the G(4)(A) form dropped from 33% to 10%. AChE activity decreased and the composition of enzyme forms was unaffected. Lectin interaction studies showed that, in contrast to skeletal muscle, the defect of merosin did not greatly alter the glycosylation of nerve cholinesterases. The anomalous synthesis of BuChE forms in dystrophic nerve may be related with peripheral neuropathy of MCMD.

The level of aryl acylamidase activity displayed by human butyrylcholinesterase depends on its molecular distribution

Butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE) display both esterase and aryl acylamidase (AAA) activities. Their AAA activity can be measured using o-nitroacetanilide (ONA). In human samples depleted of acetylcholinesterase, we noticed that the ratio of amidase to esterase activities varied depending on the source, despite both activities being due to BuChE. Searching for an explanation, we compared the activities of BuChE molecular forms in samples of human colon, kidney and serum, and observed that BuChE monomers (G(1)) hydrolyzed o-nitroacetanilide much faster than tetramers (G(4)). This fact suggested that association might cause differences in the AAA site between single and polymerized subunits. This and other post-translational modifications in BuChE subunits probably determine their level of AAA activity. The higher amidase activity of monomers could justify the presence of single BuChE subunits in cells as a way to preserve the AAA activity of BuChE, which could be lost by oligomerization.

Molecular properties of acetylcholinesterase in mouse spleen

The presence of acetylcholinesterase (AChE) mRNA and activity in the tissues and cells involved in immune responses prompted us to investigate the level and pattern of AChE components in spleen. AChE activity was higher in mouse spleen (0.46 +/- 0.13 micromol of acetylthiocholine split per hour and per mg protein) than in muscle or heart, but lower than in brain. The spleen was essentially free of butyrylcholinesterase (BuChE) activity. About 40% of spleen AChE was extracted with a saline buffer, and a further 40% with 1% Triton X-100. Sedimentation analyses, the splitting of subunits in AChE dimers, phosphatidylinositol-specific phospholipase C (PIPLC) exposure, and phenyl-agarose chromatography showed that hydrophilic (G1H, 43%) and amphiphilic AChE monomers (G1A, 36%), as well as amphiphilic dimers (G2A, 21%), occurred in spleen. All these molecules bound to fasciculin-2-Sepharose, although the extent of binding was higher for G1H (77%) than for G1A (63%) or G2A (48%) forms. Differences in the extent to which wheat germ lectin (WGA) adsorbed with AChE of mouse spleen and of erythrocyte allowed us to discard the blood origin of spleen AChE activity. A 62 kDa protein was labeled in spleen samples using antibodies against human AChE. The protein was attributed to AChE monomers since its size was the same, regardless of whether disulfide bonds were reduced or not. Since cholinergic stimulation modulates proliferation/maturation of lymphoid cells, AChE may be important for regulating the level of acetylcholine (ACh) in the neighborhood of cholinergic receptors (AChR) in spleen and other lymphoid tissues.

Human butyrylcholinesterase components differ in aryl acylamidase activity

Abstract Apart from its esterase activity, butyrylcholinesterase (BuChE) displays aryl acylamidase (AAA) activity able to hydrolyze o-nitroacetanilide (ONA) and its trifluoro-derivative (F-ONA). We report here that, despite amidase and esterase sites residing in the same protein, in human samples depleted of acetylcholinesterase the ratio of amidase to esterase activity varied depending on the source of BuChE. The much faster degradation of ONA and F-ONA by BuChE monomers (G1) of colon and kidney than by the tetramers (G4) suggests aggregation-driven differences in the AAA site between single and polymerized subunits. The similar ratio of F-ONA to butyrylthiocholine hydrolysis by serum G1 and G4 forms support structural differences in the amidase site according to the source of BuChE. The changing ratios of amidase to esterase activities in the human sources probably arise from post-translational modifications in BuChE subunits, the specific proportion of monomers and oligomers and the variable capacity of the tetramers for degrading ONA and F-ONA. The elevated amidase activity of BuChE monomers and the scant activity of the tetramers justify the occurrence of single BuChE subunits in cells as a means to sustain the AAA activity of BuChE which otherwise could be lost by tetramerization.

Characterization of acetylcholinesterase and butyrylcholinesterase forms in normal and dystrophic Lama2dy mouse heart

In searching for possible differences in acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) forms of dystrophic heart, the properties of ChE species in normal (NH) and dystrophic Lama2dy mouse heart (DH) were investigated. BuChE predominated over AChE. Loosely‐ and tightly‐bound ChEs were released with saline (extract S1) and saline‐Triton X‐100 buffers (S2). About 50% of AChE, and 25% of BuChE, in NH or DH was measured in S1, and the rest in S2. Asymmetric AChE forms A12 (15%) and A8 (11%), globular hydrophilic G  4H (8%), amphiphilic G  4A (15%), and G  2A + G  1A (51%) AChE species, and BuChE forms G  4H (13%), G  4A (3%), and G  2A  + G  1A (84%) were identified in NH and DH. Most of the asymmetric and G  4A AChE species were bound to Triticum vulgaris (WGA) or Ricinus communis (RCA) agglutinins. About half of G  4H and G  2A  + G  1A AChE were bound to WGA, and less (10%) to RCA. Variable amounts of G  4H  + G  4A (60%), and G  2A  + G  1A (75%) BuChE bound to WGA, and 50 and 10% to RCA. The lack of structural differences between ChE species in NH and DH indicates that, in contrast to the ChE forms in mouse skeletal muscle, the biosynthesis of ChE components in heart is not disturbed by dystrophy. J. Neurosci. Res. 56:295–306, 1999.

Hydrolysis of acetylthiocoline, o‐nitroacetanilide and o‐nitrotrifluoroacetanilide by fetal bovine serum acetylcholinesterase

Besides esterase activity, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) hydrolyze o‐nitroacetanilides through aryl acylamidase activity. We have reported that BuChE tetramers and monomers of human blood plasma differ in o‐nitroacetanilide (ONA) hydrolysis. The homology in quaternary structure and folding of subunits in the prevalent BuChE species () of human plasma and AChE forms of fetal bovine serum prompted us to study the esterase and amidase activities of fetal bovine serum AChE. The kcat/Km values for acetylthiocholine (ATCh), ONA and its trifluoro derivative N‐(2‐nitrophenyl)‐trifluoroacetamide (F‐ONA) were 398 × 106 m−1·min−1, 0.8 × 106 m−1·min−1, and 17.5 × 106 m−1·min−1, respectively. The lack of inhibition of amidase activity at high F‐ONA concentrations makes it unlikely that there is a role for the peripheral anionic site (PAS) in F‐ONA degradation, but the inhibition of ATCh, ONA and F‐ONA hydrolysis by the PAS ligand fasciculin‐2 points to the transit of o‐nitroacetalinides near the PAS on their way to the active site. Sedimentation analysis confirmed substrate hydrolysis by tetrameric 10.9S AChE. As compared with esterase activity, amidase activity was less sensitive to guanidine hydrochloride. This reagent led to the formation of 9.3S tetramers with partially unfolded subunits. Their capacity to hydrolyze ATCh and F‐ONA revealed that, despite the conformational change, the active site architecture and functionality of AChE were partially retained.