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Glycosylation of cholinesterase forms in brain from normal and dystrophic Lama2dy mice
Neuroscience Letters 226 (1997) 45–48
Glycosylation of cholinesterase forms in brain from normal and dystrophic Lama2dy mice M. Teresa Moral-Naranjo, Juan Cabezas-Herrera, F. Javier Campoy, Cecilio J. Vidal* Departamento de Bioquı´mica y Biologı´a Molecular A. Edificio de Veterinaria, Universidad de Murcia, Apdo 4021, 30071 Murcia, Spain Received 28 February 1997; revised version received 20 March 1997; accepted 20 March 1997
Abstract Differences in the oligosaccharides attached to acetyl- (AChE) and butyrylcholinesterase (BuChE) forms in brain from control and merosin-deficient Lama2dy dystrophic mice were investigated by means of their interaction with agarose-immobilized lectins. Asymmetric AChE, hydrophilic and amphiphilic AChE and BuChE tetramers, and amphiphilic AChE and BuChE monomers were identified in brain. All ChE forms were strongly adsorbed to the lectins concanavalin A (Con A), Lens culinaris (LCA) and Triticum vulgaris (WGA), and poorly so to Ricinus communis agglutinin (RCA), suggesting that the oligosaccharides in AChE or BuChE subunits are similarly processed regardless of their state of polymerization. The lack of differences in the interaction of lectins with homologous AChE and BuChE forms in normal and dystrophic tissue indicates that, in contrast to ChEs forms in skeletal muscle, the dystrophic condition does not disturb the processing of the oligosaccarides of brain enzyme forms. 1997 Elsevier Science Ireland Ltd. Keywords: Acetylcholinesterase; Butyrylcholinesterase; Mouse brain; Glycosylation
Cholinesterases (ChEs) are polymorphic enzymes which hydrolyze acetylcholine and other choline-esters with great efficiency [4,10,18]. Two types of ChEs have been characterized in vertebrate tissues, acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BuChE, EC 3.1. 1.8). AChE and BuChE are encoded by separate genes [18], and differ in substrate specificity and reactivity with selective inhibitors [10]. Both AChE and BuChE are found in a range of molecular forms which are classified into collagen-tailed asymmetric (A) forms and globular (G) components. The AChE gene generates several mRNAs by alternative splicing. These mRNAs encode at least two types of catalytic subunits: the H subunits generate glycolipid-anchored dimers, and the T subunits produce other molecular forms [10]. Asymmetric AChE forms predominate at the neuromuscular junction, whereas globular ChE components are distributed throughout many tissues and fluids, where they exist as amphiphilic (GA) and hydrophilic (GH) species [10]. The amphiphilic AChE tetramers (GA4 ) from mammalian brain consist of two pairs of subunits, * Corresponding author. Tel.: +34 68 307100, ext. 2911; fax: +34 68 364147/363963; e-mail: [email protected]
one pair being disulfide-bonded and the other two subunits linked by disulfide bridges to a hydrophobic 20-kDa polypeptide anchor [2,5]. Whether the above structural motive applies to the amphiphilic BuChE tetramers is unknown. The hydrophilic AChE and BuChE tetramers (GH4 ) are made up by two pairs of non-covalently-bonded subunits, each pair being disulfide-bonded [7]. In certain tissues, the AChE dimers and monomers (GA2 , GA1 ) bear C-terminal linked glycosylphosphatidylinositol residues for membrane attachment, but some isoforms are resistant to phospholipase C, reflecting heterogeneity in the amphiphilic domain [1,17]. Cholinesterases are glycoproteins, and the mouse cDNAs encoding AChE and BuChE subunits show three and seven N-glycosylation sequences [13]. It is known that oligosaccharidic residues in AChE forms are processed in a stepwise manner, reflecting the transit of the enzyme through the secretory pathway [14]. Specific oligosaccharide-processing functions have been localized in early (cis-medial) and late (trans) Golgi regions. Thus, the incorporation of GlcNAc occurs in the early Golgi, whereas the addition of galactose takes place in the more distal cisternae. Because the presence of these sugar residues in glycoproteins can in
0304-3940/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940 (97 )0 0249-8
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M.T. Moral-Naranjo et al. / Neuroscience Letters 226 (1997) 45–48
many cases be detected by the appropriate lectin, it is possible to follow the processing of oligosaccharides in a glycoprotein by assaying its interaction with lectins. Any such interaction with concanavalin A (Con A), Triticum vulgaris (WGA) or Ricinus communis agglutinin (RCA) would provide indirect evidence for the addition of mannose, GlcNAc, or terminal galactose to the protein. The observation of differences in the interaction of homologous ChE forms extracted from normal or dystrophic skeletal muscle with lectins [3] prompted us to investigate the possible differential processing of the oligosaccaridic residues of ChEs in normal and dystrophic mouse brain. Phenotypically normal 129B6F1J (+/?) and dystrophic
Fig. 2. Interaction of brain BuChE forms with lectins. ChEs were extracted and incubated with Agarose-immobilized lectins. BuChE components were separated by sedimentation analysis, and characterized as described in Fig. 1. Note the much lower BuChE activity compared with the AChE activity in S2.
Fig. 1. Adsorption of brain AChE components to immobilized lectins. The tissue was extracted (10% w/v) with 1 M NaCl, 50 mM MgCl2, 10 mM Tris, pH 7 (saline buffer, SB) containing a mixture of antiproteinases [12] to obtain the supernatant S1 with the loosely-bound ChEs, and the pellet reextracted with 1% w/v Triton X-100 and antiproteinases in saline buffer (saline-Triton buffer) to detach the tightly-bound ChEs (S2). Fractions S1 and S2 were recovered by centrifugation at 100 000 × g, 1 h, and separately incubated with Sepharose 4B (control, C), or with immobilized Con A, LCA, WGA, and RCA. ChE-lectin complexes were removed by centrifugation and the unbound enzyme forms were separated by sedimentation analysis on 5–20% linear sucrose gradients made in SB with 0.5% Brij 96. The tubes were centrifuged at 150 000 × g, for 18 h at 4°C. AChE and BuChE activities were measured in the collected fractions using a microtiter assay with appropriate substrates and selective inhibitors [12]. ChE activity is given in arbitrary units, and each unit refers to an increase of 0.001 absorbance units/ml and per min. The individual ChE forms were identified by their sedimentation coefficients [12], using B-galactosidase (G, 16.0S), catalase (C, 11.4S) and alkaline phosphatase (P, 6.1S) as sedimentation markers. Note that most of the AChE was released with Triton X-100. AChE and BuChE were fully bound to Con A- and LCA-Agarose. The experiments carried out with ChEs extracted from normal or dystrophic mouse brain provided the same results both as regards the molecular form profiles and the extent of lectin interaction.
(dystrophin-positive, merosin-negative, Lama2dy) mice were purchased from Jackson Memorial Lab. (Bar Harbor, ME, USA). Brain cholinesterases were released, and the interaction between lectins and the enzyme components investigated as described in Fig. 1. Details regarding mouse brain AChE and BuChE activities, sedimentation profiles of ChE forms in the soluble fractions S1 and S2, and hydrophilic or amphiphilic behavior of the various AChE and BuChE forms have been given in a previous report [12]. About 15–20% of the AChE was recovered in the saline extract (S1) and the rest required detergent for solubilization (S2). Sedimentation analysis of AChE incubated with lectin-free Sepharose 4B (control) revealed GH4 (47%), GA2 + GA1 (35%), GA4 (12%) and A12 (6%) AChE forms in the saline extract, and GA4 (84%) and GA2 + GA1 (16%) in the Triton X-100-soluble fraction (Fig. 1). The AChE forms were fully adsorbed to Con A and Lens culinaris (LCA), mostly to WGA, and very few to RCA (Figs. 1 and 3). No significant differences between normal and dystrophic brain were observed when the distribution of AChE forms or their interaction with the lectins were investigated, meaning that, unlike in muscle [3], dystrophy has no effect on the processing of the sugar residues linked to AChE forms in brain. Since all AChE forms displayed a high WGA-reactivity and a low binding to RCA, it is likely that they follow similar glycosylation processes: asymmetric, tetrameric and most monomeric AChE would transit the early-Golgi,
M.T. Moral-Naranjo et al. / Neuroscience Letters 226 (1997) 45–48
acquiring WGA reactivity, and pass through the late-Golgi (without acquisition of RCA-reactivity), before being exported by secretory vesicles to the plasma membrane for attachment or secretion. The finding that AChE dimers and tetramers in PC12 cells are co-secreted by the regulated secretory pathway [19] might indicate that the various enzyme forms in brain are co-transported. The similar pattern of interaction with lectins displayed by the salt-soluble AChE forms in mouse brain and serum [3] probably indicates that whatever the origin of the serum molecules may be, the oligosaccharides attached to the AChE species in the brain saline fraction and in serum are similarly processed. The presence of GH4 , G2 and G1 AChE in mouse brain and serum might indicate that the brain GH4 forms travel with the GA4 , GA2 and GA1 components toward the plasma membrane, the amphiphilic tetramers and a certain amount of the GA2 and GA1 forms being bound to the membrane, whereas GH4 AChE and a fraction of the light forms are released to the external milieu. A comparison of the results concerning the interaction of WGA with AChE monomers and dimers in mouse tissues revealed that the components in brain and serum strongly bound to WGA, whereas most of the isoforms in muscle failed in doing so [3]. These results confirm the differential glycosylation of the homologous ChE forms in several organs, including muscle and nervous tissue, of a particular animal [8,9,11,21]. Extra- and intracellular asymmetric AChE forms in mouse muscle differ in their interaction with RCA, since only the external forms bind to RCA (Vidal et al., unpublished results). This lack of interaction between asymmetric AChE species and the lectin might indicate that they are membrane-bound rather than linked to extracellular proteins. The adsorption of brain asymmetric AChE to phenyl-Agarose [12] strongly supports the presence of hydrophobic domains in these enzyme forms, although the exact nature of the hydrophobic anchor remains to be elucidated. Whether these domains are involved in membrane attachment deserves further investigation. As regards BuChE, 35–40% of the enzyme was released from brain with a saline buffer, and the rest with a Triton X100-containing buffer. Abundant GH4 (38%) and GA2 + GA1 (50%), and less GA4 (12%) forms were identified in S1, and GA4 (48%), GA2 + GA1 (44%) and GH4 (8%) in S2 (Fig. 2). The full set of BuChE components were recognized by Con A and LCA, most of them by WGA and very few by RCA (Fig. 2 and 3). A high fraction of GH4 , GA4 and GA2 + GA1 BuChE forms in S1 or S2 bound to WGA, but only the G4 forms showed a certain capacity to react with RCA. The BuChE forms released from normal or dystrophic mouse brain displayed great similarity as regards the enzyme forms profiles and patterns of lectin interaction. The partial adsorption of BuChE tetramers with WGA and RCA suggests that brain probably contains several types of G4 forms which differ in their oligosaccaridic residues. Most of the tetrameric BuChE forms are WGA-reactive and RCA-unreactive both in mouse brain and skeletal
47
Fig. 3. Binding of brain ChEs to lectins. The histograms show the average values of the results concerning the adsorption of individual enzyme forms to the agglutinins of Figs. 1 and 2. The percentage of lectin interaction was calculated considering the relative content of each enzyme form in the supernatants S1 and S2.. Cholinesterase activity in control samples was taken as the 100% value, and the percentage of lectin binding of each enzyme form was calculated from the difference between the activity in control and lectin supernatants. Results are mean of five experiments with normal mouse brain, the SE being less than 10% of the mean values. No significant differences were observed in the interaction between lectins and the homologous ChE forms released from normal and dystrophic mouse brain.
muscle [3], whereas the isoforms in mouse [3] and human serum [20] show high RCA reactivity. Consequently, it is unlikely that the mouse serum BuChE tetramers come from brain, although if they did they would represent a reduced fraction of the isoforms secreted from other tissues. Amphiphilic and hydrophilic BuChE tetramers have been identified in human brain [16] and brain tumors [15,17], although their physiological significance is uncertain [6]. Finally, amphiphilic AChE dimers and monomers, and amphiphilic BuChE monomers in mouse brain display similar patterns of interaction with lectins, suggesting that their oligosaccharides undergo parallel processing before export to the cell membrane. Summarizing, mouse brain synthesizes asymmetric AChE forms, globular amphiphilic and hydrophilic tetramers and amphiphilic AChE dimers and monomers, all of which display similar patterns of lectin interaction. The results suggest that, irrespective of their final destination, the attached oligosaccharidic residues in the enzyme subunits are processed in a similar manner. In addition, the tissue produces amphiphilic and hydrophilic BuChE tetramers and amphiphilic dimers and monomers. The small fraction of RCA-reactive GH4 BuChE forms may be destined to the external milieu, whereas the bulk of GA4 , GA2 and GA1 , which are WGA-reactive and RCA-unreactive, are probably exported to the surface membrane. Neither the composition of brain AChE and BuChE forms nor the glycosylation of individual ChE components in brain is affected by muscular dystrophy.
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M.T. Moral-Naranjo et al. / Neuroscience Letters 226 (1997) 45–48
M.T. Moral-Naranjo is a recipient of a scholarship from the Ministerio de Educacio´n y Ciencia of Spain. This research was supported by the Fondo Investigaciones Sanitarias of Spain (Grant 95/1756). [1] Bon, S., Rosenberry, T.L. and Massoulie´, J., Amphiphilic, glycophosphatidylinositol-specific phospholipase C (PI-PLC)-insensitive monomers and dimers of acetylcholinesterase, Cell Mol. Neurobiol., 11 (1991) 157–172. [2] Boschetti, N. and Brodbeck, U., The membrane anchor of mammalian brain acetylcholinesterase consists of a single glycosylated protein of 22 kDa, FEBS Lett., 380 (1996) 133–136. [3] Cabezas-Herrera, J., Moral-Naranjo, M.T., Campoy, F.J. and Vidal, C.J., G4 forms of acetylcholinesterase and butyrylcholinesterase in normal and dystrophic mouse muscle differ in their interaction with Ricinus communis agglutinin, Biochim. Biophys. Acta, 1225 (1994) 283–288. [4] Chatonnet, A. and Lockridge, O., Comparison of butyrylcholinesterase and acetylcholinesterase, Biochem. J., 260 (1989) 625–634. [5] Ferna´ndez, H.L., Moreno, R.D. and Inestrosa, N.C., Tetrameric (G4) acetylcholinesterase: structure, localization and physiological regulation, J. Neurochem., 66 (1996) 1335–1346. [6] Layer, P.G., Nonclassical roles of cholinesterases in the embryonic brain and possible links to Alzheimer disease, Alzheim. Dis. Assoc. Disord., 9 (1995) 29–36. [7] Liao, J., Boschetti, N., Mortensen, V., Jensen, S.P., Koch, C., Norgaard-Pedersen, B. and Brodbeck, U., Characterization of salt-soluble forms of acetylcholinesterase from bovine brain, J. Neurochem., 63 (1994) 1446–1453. [8] Liao, J., Heider, H., Sun, M.C. and Brodbeck, U., Different glycosylation in acetylcholinesterases from mammalian brain and erythrocytes, J. Neurochem., 58 (1992) 1230–1238. [9] Liao, J., Norgaard-Pedersen, B. and Brodbeck, U., Subunit association and glycosylation of AChE from monkey brain, J. Neurochem., 61 (1993) 1127–1134. [10] Massoulie´, J., Pezzementi, L., Bon, S., Krejci, E. and Vallette, F.M., Molecular and cellular biology of cholinesterases, Prog. Neurobiol., 41 (1993) 31–91.
[11] Me´flah, K., Bernard, S. and Massoulie´, J., Interactions with lectins indicate differences in the carbohydrate composition of the membrane-bound enzymes acetylcholinesterase and 5′-nucleotidase in different cell types, Biochimie, 66 (1984) 59–69. [12] Moral-Naranjo, M.T., Cabezas-Herrera, J. and Vidal, C.J., Molecular forms of acetyl- and butyrylcholinesterase in normal and dystrophic mouse brain, J. Neurosci. Res., 43 (1996) 224–234. [13] Rachinsky, T.L., Camp, S., Li, Y., Ekstro¨m, T.J., Newton, M. and Taylor, P., Molecular cloning of mouse acetylcholinesterase: tissue distribution of alternatively spliced mRNA species, Neuron, 5 (1990) 317–327. [14] Rotundo, R.L., Thomas, K., Porter-Jordan, K., Benson, R.J.J., Fernandez-Valle, C. and Fine, R.E., Intracellular transport, sorting and turnover of acetylcholinesterase, J. Biol. Chem., 264 (1989) 3146– 3152. [15] Sa´ez-Valero, J., Poza-Cisneros, G. and Vidal, C.J., Molecular forms of acetyl- and butyrylcholinesterase in human glioma, Neurosci. Lett., 206 (1996) 173–176. [16] Sa´ez-Valero, J., Tornel, P.L., Mun˜oz-Delgado, E. and Vidal, C.J., Amphiphilic and hydrophilic forms of acetyl- and butyrylcholinesterase in human brain, J. Neurosci. Res., 35 (1993) 678–689. [17] Sa´ez-Valero, J. and Vidal, C.J., Monomers and dimers of acetylcholinesterase in human meningioma are anchored to the membrane by glycosylphosphatidylinositol, Neurosci. Lett., 195 (1995) 101–104. [18] Schwarz, M., Glick, D., Loewenstein, Y. and Soreq, H., Engineering of human cholinesterase explains and predicts diverse consequences of administration of various drugs and poisons, Pharmacol. Ther., 67 (1995) 283–322. [19] Schweitzer, E.S., Regulated and constitutive secretion of distinct molecular forms of acetylcholinesterase from PC12 cells, J. Cell Sci., 106 (1993) 731–740. [20] Tornel, P.L., Sa´ez-Valero, J. and Vidal, C.J., Ricinus communis agglutinin I reacting and non-reacting butyrylcholinesterase in human cerebrospinal fluid, Neurosci. Lett., 145 (1992) 59–62. [21] Treskatis, S., Ebert, C. and Layer, P.G., Butyrylcholinesterase from chicken brain is smaller than that from serum: its purification, glycosylation and membrane association, J. Neurochem., 58 (1992) 2236–2247.
Glycosylation of cholinesterase forms in brain from normal and dystrophic Lama2dy mice M. Teresa Moral-Naranjo, Juan Cabezas-Herrera, F. Javier Campoy, Cecilio J. Vidal* Departamento de Bioquı´mica y Biologı´a Molecular A. Edificio de Veterinaria, Universidad de Murcia, Apdo 4021, 30071 Murcia, Spain Received 28 February 1997; revised version received 20 March 1997; accepted 20 March 1997
Abstract Differences in the oligosaccharides attached to acetyl- (AChE) and butyrylcholinesterase (BuChE) forms in brain from control and merosin-deficient Lama2dy dystrophic mice were investigated by means of their interaction with agarose-immobilized lectins. Asymmetric AChE, hydrophilic and amphiphilic AChE and BuChE tetramers, and amphiphilic AChE and BuChE monomers were identified in brain. All ChE forms were strongly adsorbed to the lectins concanavalin A (Con A), Lens culinaris (LCA) and Triticum vulgaris (WGA), and poorly so to Ricinus communis agglutinin (RCA), suggesting that the oligosaccharides in AChE or BuChE subunits are similarly processed regardless of their state of polymerization. The lack of differences in the interaction of lectins with homologous AChE and BuChE forms in normal and dystrophic tissue indicates that, in contrast to ChEs forms in skeletal muscle, the dystrophic condition does not disturb the processing of the oligosaccarides of brain enzyme forms. 1997 Elsevier Science Ireland Ltd. Keywords: Acetylcholinesterase; Butyrylcholinesterase; Mouse brain; Glycosylation
Cholinesterases (ChEs) are polymorphic enzymes which hydrolyze acetylcholine and other choline-esters with great efficiency [4,10,18]. Two types of ChEs have been characterized in vertebrate tissues, acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BuChE, EC 3.1. 1.8). AChE and BuChE are encoded by separate genes [18], and differ in substrate specificity and reactivity with selective inhibitors [10]. Both AChE and BuChE are found in a range of molecular forms which are classified into collagen-tailed asymmetric (A) forms and globular (G) components. The AChE gene generates several mRNAs by alternative splicing. These mRNAs encode at least two types of catalytic subunits: the H subunits generate glycolipid-anchored dimers, and the T subunits produce other molecular forms [10]. Asymmetric AChE forms predominate at the neuromuscular junction, whereas globular ChE components are distributed throughout many tissues and fluids, where they exist as amphiphilic (GA) and hydrophilic (GH) species [10]. The amphiphilic AChE tetramers (GA4 ) from mammalian brain consist of two pairs of subunits, * Corresponding author. Tel.: +34 68 307100, ext. 2911; fax: +34 68 364147/363963; e-mail: [email protected]
one pair being disulfide-bonded and the other two subunits linked by disulfide bridges to a hydrophobic 20-kDa polypeptide anchor [2,5]. Whether the above structural motive applies to the amphiphilic BuChE tetramers is unknown. The hydrophilic AChE and BuChE tetramers (GH4 ) are made up by two pairs of non-covalently-bonded subunits, each pair being disulfide-bonded [7]. In certain tissues, the AChE dimers and monomers (GA2 , GA1 ) bear C-terminal linked glycosylphosphatidylinositol residues for membrane attachment, but some isoforms are resistant to phospholipase C, reflecting heterogeneity in the amphiphilic domain [1,17]. Cholinesterases are glycoproteins, and the mouse cDNAs encoding AChE and BuChE subunits show three and seven N-glycosylation sequences [13]. It is known that oligosaccharidic residues in AChE forms are processed in a stepwise manner, reflecting the transit of the enzyme through the secretory pathway [14]. Specific oligosaccharide-processing functions have been localized in early (cis-medial) and late (trans) Golgi regions. Thus, the incorporation of GlcNAc occurs in the early Golgi, whereas the addition of galactose takes place in the more distal cisternae. Because the presence of these sugar residues in glycoproteins can in
0304-3940/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940 (97 )0 0249-8
46
M.T. Moral-Naranjo et al. / Neuroscience Letters 226 (1997) 45–48
many cases be detected by the appropriate lectin, it is possible to follow the processing of oligosaccharides in a glycoprotein by assaying its interaction with lectins. Any such interaction with concanavalin A (Con A), Triticum vulgaris (WGA) or Ricinus communis agglutinin (RCA) would provide indirect evidence for the addition of mannose, GlcNAc, or terminal galactose to the protein. The observation of differences in the interaction of homologous ChE forms extracted from normal or dystrophic skeletal muscle with lectins [3] prompted us to investigate the possible differential processing of the oligosaccaridic residues of ChEs in normal and dystrophic mouse brain. Phenotypically normal 129B6F1J (+/?) and dystrophic
Fig. 2. Interaction of brain BuChE forms with lectins. ChEs were extracted and incubated with Agarose-immobilized lectins. BuChE components were separated by sedimentation analysis, and characterized as described in Fig. 1. Note the much lower BuChE activity compared with the AChE activity in S2.
Fig. 1. Adsorption of brain AChE components to immobilized lectins. The tissue was extracted (10% w/v) with 1 M NaCl, 50 mM MgCl2, 10 mM Tris, pH 7 (saline buffer, SB) containing a mixture of antiproteinases [12] to obtain the supernatant S1 with the loosely-bound ChEs, and the pellet reextracted with 1% w/v Triton X-100 and antiproteinases in saline buffer (saline-Triton buffer) to detach the tightly-bound ChEs (S2). Fractions S1 and S2 were recovered by centrifugation at 100 000 × g, 1 h, and separately incubated with Sepharose 4B (control, C), or with immobilized Con A, LCA, WGA, and RCA. ChE-lectin complexes were removed by centrifugation and the unbound enzyme forms were separated by sedimentation analysis on 5–20% linear sucrose gradients made in SB with 0.5% Brij 96. The tubes were centrifuged at 150 000 × g, for 18 h at 4°C. AChE and BuChE activities were measured in the collected fractions using a microtiter assay with appropriate substrates and selective inhibitors [12]. ChE activity is given in arbitrary units, and each unit refers to an increase of 0.001 absorbance units/ml and per min. The individual ChE forms were identified by their sedimentation coefficients [12], using B-galactosidase (G, 16.0S), catalase (C, 11.4S) and alkaline phosphatase (P, 6.1S) as sedimentation markers. Note that most of the AChE was released with Triton X-100. AChE and BuChE were fully bound to Con A- and LCA-Agarose. The experiments carried out with ChEs extracted from normal or dystrophic mouse brain provided the same results both as regards the molecular form profiles and the extent of lectin interaction.
(dystrophin-positive, merosin-negative, Lama2dy) mice were purchased from Jackson Memorial Lab. (Bar Harbor, ME, USA). Brain cholinesterases were released, and the interaction between lectins and the enzyme components investigated as described in Fig. 1. Details regarding mouse brain AChE and BuChE activities, sedimentation profiles of ChE forms in the soluble fractions S1 and S2, and hydrophilic or amphiphilic behavior of the various AChE and BuChE forms have been given in a previous report [12]. About 15–20% of the AChE was recovered in the saline extract (S1) and the rest required detergent for solubilization (S2). Sedimentation analysis of AChE incubated with lectin-free Sepharose 4B (control) revealed GH4 (47%), GA2 + GA1 (35%), GA4 (12%) and A12 (6%) AChE forms in the saline extract, and GA4 (84%) and GA2 + GA1 (16%) in the Triton X-100-soluble fraction (Fig. 1). The AChE forms were fully adsorbed to Con A and Lens culinaris (LCA), mostly to WGA, and very few to RCA (Figs. 1 and 3). No significant differences between normal and dystrophic brain were observed when the distribution of AChE forms or their interaction with the lectins were investigated, meaning that, unlike in muscle [3], dystrophy has no effect on the processing of the sugar residues linked to AChE forms in brain. Since all AChE forms displayed a high WGA-reactivity and a low binding to RCA, it is likely that they follow similar glycosylation processes: asymmetric, tetrameric and most monomeric AChE would transit the early-Golgi,
M.T. Moral-Naranjo et al. / Neuroscience Letters 226 (1997) 45–48
acquiring WGA reactivity, and pass through the late-Golgi (without acquisition of RCA-reactivity), before being exported by secretory vesicles to the plasma membrane for attachment or secretion. The finding that AChE dimers and tetramers in PC12 cells are co-secreted by the regulated secretory pathway [19] might indicate that the various enzyme forms in brain are co-transported. The similar pattern of interaction with lectins displayed by the salt-soluble AChE forms in mouse brain and serum [3] probably indicates that whatever the origin of the serum molecules may be, the oligosaccharides attached to the AChE species in the brain saline fraction and in serum are similarly processed. The presence of GH4 , G2 and G1 AChE in mouse brain and serum might indicate that the brain GH4 forms travel with the GA4 , GA2 and GA1 components toward the plasma membrane, the amphiphilic tetramers and a certain amount of the GA2 and GA1 forms being bound to the membrane, whereas GH4 AChE and a fraction of the light forms are released to the external milieu. A comparison of the results concerning the interaction of WGA with AChE monomers and dimers in mouse tissues revealed that the components in brain and serum strongly bound to WGA, whereas most of the isoforms in muscle failed in doing so [3]. These results confirm the differential glycosylation of the homologous ChE forms in several organs, including muscle and nervous tissue, of a particular animal [8,9,11,21]. Extra- and intracellular asymmetric AChE forms in mouse muscle differ in their interaction with RCA, since only the external forms bind to RCA (Vidal et al., unpublished results). This lack of interaction between asymmetric AChE species and the lectin might indicate that they are membrane-bound rather than linked to extracellular proteins. The adsorption of brain asymmetric AChE to phenyl-Agarose [12] strongly supports the presence of hydrophobic domains in these enzyme forms, although the exact nature of the hydrophobic anchor remains to be elucidated. Whether these domains are involved in membrane attachment deserves further investigation. As regards BuChE, 35–40% of the enzyme was released from brain with a saline buffer, and the rest with a Triton X100-containing buffer. Abundant GH4 (38%) and GA2 + GA1 (50%), and less GA4 (12%) forms were identified in S1, and GA4 (48%), GA2 + GA1 (44%) and GH4 (8%) in S2 (Fig. 2). The full set of BuChE components were recognized by Con A and LCA, most of them by WGA and very few by RCA (Fig. 2 and 3). A high fraction of GH4 , GA4 and GA2 + GA1 BuChE forms in S1 or S2 bound to WGA, but only the G4 forms showed a certain capacity to react with RCA. The BuChE forms released from normal or dystrophic mouse brain displayed great similarity as regards the enzyme forms profiles and patterns of lectin interaction. The partial adsorption of BuChE tetramers with WGA and RCA suggests that brain probably contains several types of G4 forms which differ in their oligosaccaridic residues. Most of the tetrameric BuChE forms are WGA-reactive and RCA-unreactive both in mouse brain and skeletal
47
Fig. 3. Binding of brain ChEs to lectins. The histograms show the average values of the results concerning the adsorption of individual enzyme forms to the agglutinins of Figs. 1 and 2. The percentage of lectin interaction was calculated considering the relative content of each enzyme form in the supernatants S1 and S2.. Cholinesterase activity in control samples was taken as the 100% value, and the percentage of lectin binding of each enzyme form was calculated from the difference between the activity in control and lectin supernatants. Results are mean of five experiments with normal mouse brain, the SE being less than 10% of the mean values. No significant differences were observed in the interaction between lectins and the homologous ChE forms released from normal and dystrophic mouse brain.
muscle [3], whereas the isoforms in mouse [3] and human serum [20] show high RCA reactivity. Consequently, it is unlikely that the mouse serum BuChE tetramers come from brain, although if they did they would represent a reduced fraction of the isoforms secreted from other tissues. Amphiphilic and hydrophilic BuChE tetramers have been identified in human brain [16] and brain tumors [15,17], although their physiological significance is uncertain [6]. Finally, amphiphilic AChE dimers and monomers, and amphiphilic BuChE monomers in mouse brain display similar patterns of interaction with lectins, suggesting that their oligosaccharides undergo parallel processing before export to the cell membrane. Summarizing, mouse brain synthesizes asymmetric AChE forms, globular amphiphilic and hydrophilic tetramers and amphiphilic AChE dimers and monomers, all of which display similar patterns of lectin interaction. The results suggest that, irrespective of their final destination, the attached oligosaccharidic residues in the enzyme subunits are processed in a similar manner. In addition, the tissue produces amphiphilic and hydrophilic BuChE tetramers and amphiphilic dimers and monomers. The small fraction of RCA-reactive GH4 BuChE forms may be destined to the external milieu, whereas the bulk of GA4 , GA2 and GA1 , which are WGA-reactive and RCA-unreactive, are probably exported to the surface membrane. Neither the composition of brain AChE and BuChE forms nor the glycosylation of individual ChE components in brain is affected by muscular dystrophy.
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