Molecular detection of methionine in rat brain using specific antibodies

NEURgSCIDiCE ELSEVIER

NeuroscienceLetters185(1995)147-150

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Molecular detection of methionine in rat brain using specific antibodies A l i A m a r a a, M o n i q u e C o u s s e m a c q b, M i c h e l Geffard",* aLaboratoire d'lmmunologie et Pathologic, Universit~ de Bordeaux 11, BP 66, 146 rue L~o Saignat, 33076 Bordeaux Cedex, France bLaboratoire de Neurochirurgie Exp~rimentale et Neurobiologie, Universit~ de Bordeaux 11, BP 48, 146 rue IMo Saignat, 33076 Bordeaux Cedex, France

Received 29 July 1994; revisedversionreceived 12 December1994; accepted 12 December1994

Abstract

In order to study the localization of methionine in rat brain, an immunological approach was developed by raising antibodies directed against this amino acid. Methionine was conjugated to bovine serum albumin (BSA) or human serum albumin (HSA) via glutaraldehyde. The conjugates were then reduced by sodium borohydride and injected alternately into rabbits. Antibody affinity and specificity were evaluated using an adapted ELISA method, by competition experiments between conjugated methionine and related conjugated compounds, pre-incubated with anti-methionine antibodies diluted at 1/20 000. The resulting cross-reactivity ratios, calculated at half-displacement, showed that glutaraldehyde-methionine conjugate (methionine-G-BSA) was the best recognized compound. Non-reduced methionine conjugate (methionine=G=BSA) and the related-conjugated molecules such as homocysteine, homocysteic acid, cysteine, cystathionine and glutamate were not recognized at all. Antibodies to methionine were directed against a glutaraldehyde-methionine epitope and their very high affinity and specificity made them reliable tools for molecular detection of methionine in rat brain. Using purified antibodies diluted at 1/20 000, motoneurons were found to be the most methionineimmunoreactive cell bodies in glutaraldehyde-fixed rat brain sections. Keywords: Methionine; Glutaraldehyde; Antibody; ELISA; Immunohistochemistry; Motoneurons

Methionine (Met) is an essential amino acid for the normal growth and the development of mammals. Met participates in several biological processes, including protein synthesis; the different steps of transmethylation pathway; the biosynthesis of homocysteine and cysteine; the metabolism of choline, folates and vitamin B12, and in the formation of polyamines (spermidine and spermine) [6,14]. Quantitative autoradiographic methods were used for evaluating local rates of Met and studying its incorporation into cerebral proteins [ 10,13]. However, up until now, no direct visualization of the Met itself has been described. For this purpose, the production of specific antibodies (Ab) was attempted. The previous development of Ab directed against conjugated catecholamines [4,15], indolealkylamines [8], amino acids [2,3,5] and more recently conjugated glutathione [1] enabled us to raise Ab directed against conjugated Met, using experimental conditions similar to those previously described for other conjugated small-sized molecules. The present * Corresponding author, Tel.: +33 57 57 16 58; Fax: +33 56 24 12 09.

report describes the first visualization of Met in rat brain using specific Ab. To be immunogenic, Met (Met, Sigma) was conjugated via glutaraldehyde (G, Merck) to carrier protein. LMet (4 mg) was dissolved in 2 ml of a 1.5 M sodium acetate solution (pH 8). Two microliters of 3H-labeled compound (NEN, specific activity 250 mCi/mM) were added in order to control each step of the immunogen synthesis. Then, 400/tl of a 0.5 M G solution and 1 ml of 15 mg bovine serum albumin (BSA, Sigma) or human serum albumin (HSA, Sigma) dissolved in a 1.5 M sodium acetate solution were rapidly and successively added. The reaction time lasted 2 min at room temperature. The coupling reaction was over when the mixture became yellow in color. Then, 200/tl of 0.1 M sodium borohydride solution (Fluka) was added to reduce the double bonds. The mixture became nearly colorless and was then dialyzed against 0.01 M sodium phosphate buffer (pH 7.6) for 48 h. Beta counting (Wallac 1409 DSA, Pharmacia) was performed on 100-/tl aliquots, both before and after dialysis in order to calculate the concen-

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148 BIBo

A. Amara et al. I Neuroscience Letters 185 (1995) 147-150

. ~

~,S6,,7, S, 9,10,11

2

-14

Conjugates coated on well-plates

OD read at 492 nm

Met-G-BSA Met-AG-BSAa Met-CDI-BSAb S-Adenosyl-methionine-G-BSA HomocysteicAcid-G-BSA Homocysteine-G-BSA Glutamate-G-BSA Cysteine-G-BSA Cystathionine-G-BSA G-BSA

0.995 0.061 0.042 0.082 0.192 0.154 0.035 0.096 0.022 0.02

3

0.5

0,1

Table 1 Binding of anti-conjugated Met Ab diluted at 1/20 000 on Met and related conjugates

. -13

. -12

.

. -I1

. -10

. -9

.

. -8

1 -7

-6

-5

losc Fig. 1. Displacement curves established with ELISA tests from competition between Met-G-BSA coated on well-plates and Met-G-BSA and each related conjugate, previously incubated with anti-conjugated Met Ab (final dilution 1/20 000) for 16 h at 4°C: (1) Met-G-BSA; (2) homocysteine-G-BSA;(3) Met=G=BSA; (4) S-adenosyl-methionine-GBSA; (5) homocysteic acid-G-BSA; (6) cysteine-G-BSA;(7) cystathionine-G-BSA; (8) glutamate-G-BSA;(9) uncoupled Met; (10) Met-CDIBSA; and (11) Met-AG-BSA. C is the molar concentration of competitor. BIB 0 is the ratio between the absorbance with (B) and without (B0) competitor. Each point drawn for the displacement curves represents the average of three experiments. For each reading, standard deviations are not shown as they were too small. tration of conjugated Met. The concentration of carrier protein was evaluated by measuring optical density (OD) at 280 nm. Molar coupling ratios (moles of Met per mole of carrier protein) were calculated and ranged between 10 and 15. Two rabbits were immunized according to Geffard et al.'s protocol [8]. Eve1~j 15 days, they were alternately injected with 2 0 0 # g of Met conjugate having either BSA or HSA as carrier protein in an emulsion containing 500#1 of 0.15 M NaCI and 500#1 of Freund's complete adjuvant (Difco). The antisera were usually collected 8 days after each injection and purified by immunoadsorption with the carrier proteins [2,8,15]. The purified supernatant was used both for E L I S A and immunohistochemical studies. S-Adenosyl-L-methionine (Sigma), D,L-homocysteine (Sigma), L-cysteine (Sigma), L-glutamate (Merck), homocysteic acid (Sigma) and L-cystathionine (Sigma) were conjugated via G to BSA under the same experimental conditions as those used for conjugated Met. All these conjugates were reduced by a 0.1 M sodium borohydride solution. A part of the Met conjugate was not reduced and was denoted Met=G=BSA. Molar coupling ratios (moles of hapten per mole BSA) were calculated and found to be similar to that for Met-G-BSA. Tests of Ab avidity and specificity were performed on E L I S A plates as previously described for other conjugated small-size molecules [ 1 5]. The well-plates were filled with 200 ~1 of a solution of Met conjugate or G-BSA at 1 lag/ml in 0.05 M carbonate buffer (pH 9.6) for 16 h at 4°C. Then, they were washed with phosphate-buffered saline (PBS) containing 0.05% Tween 20 plus 0.2% BSA (buffer A) and left for I h at 37°C to fully saturate the well-plates and prevent non-

a Met linked to BSA with glutaric anhydride. b Met linked to BSA with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide. specific binding of antisera. The well-plates were then rinsed twice with PBS 0.05% Tween 20. Purified and diluted antisera (1/20 000) were pre-incubated for 16 h at 4°C with or without conjugated compounds (Met-G-BSA, Met=G=BSA, S-adenosyl-methionine-G-BSA, homocysteine-G-BSA, glutamate-G-BSA, cysteine-G-BSA, homocysteic acid-G-BSA and uncoupled Met) in buffer A for 16 h at 4°C. Then they were applied to well-plates coated with Met-G-BSA or G-BSA for 2 h at 37°C. Again, the well-plates were washed twice with PBS-0.05% Tween 20 and filled with 200#1 of 1/10 000 goat anti-rabbit immunoglobulin G labeled with horseradish peroxidase (Diagnostic Pasteur) diluted in PBS-0.05% Tween 20 containing 0.1% BSA. After incubation for 1 h at 37°C, the well-plates were washed three times with PBS-0.05% Tween 20. Peroxidase was assayed as described by Amara et al. [1]. The reaction was stopped by the addition of 50 #1 of a 4 M H2SO 4 solution per well, and the optical density (OD) was read at 492 nm with a Multiscan spectrophotometer (MR 610 Dynatech). Experimental values Table 2 Specificity of anti-conjugated Met Ab Compounds

Cross-reactivity ratioa

Methionine-G-BSA methionine=G=BSA Met-AG-BSA Met-CDI-BSA S-Adenosyl-methionine-G-BSA Homocysteine-G-BSA Homoeysteicaeid-G-BSA Cystathionine-G-BSA Cysteine-G-BSA Glatamate-G-BSA Uncoupled Met

1 >1000 >10000 >10000 > 10000 > 1000 > 10000 >10000 >10000 >10000 > 10000

a Met-G-BSA concentration/unconjugated or conjugated analogs at half displacement.

A. Amara et al. / Neuroscience Letters 185 (1995) 147-150

were corrected by deducting blank values obtained from well-plates coated with G-BSA. The most immunoreactive conjugate, Met-G-BSA, showed a displacement between 10-12 and 10-6 M. The half-displacement occurred at 7.5 x 10 -1° M, indicating a very high Ab affinity, slightly superior to that found for conjugated amino acid antisera [2,3,5]. In agreement with previous results [8,9], uncoupled Met exhibited no immunoreactivity. The related-conjugates, S-adenosylmethionine-G-BSA, homocysteine-G-BSA, homocysteic acid-G-BSA, cysteine-G-BSA, cystathionine-G-BSA and glutamate-G-BSA were not recognized at all (Fig. 1, Tables 1, 2). These results demonstrate that conjugated Met can induce highly specific Ab exhibiting no detectable cross-reactivity with other related compounds. Therefore, the S - C H 3 moiety of Met appeared to be the most immu-

149

nodominant part of this neutral amino acid. When Met amino or carboxylic group was linked to BSA, respectively, with glutaric anhydride [7] (Met-AG-BSA) or 1ethyl-3-(3-dimethylaminopropyl) carbodiimide [ 11 ] (MetCDI-BSA), our antisera did not recognize this antigenic presentation of Met, suggesting that anti-Met Ab did not interfere with Met involved in protein structure. Although the non-reduced Met conjugate (Met=G=BSA) was not so well recognized as the reduced one (Met-G-BSA), sodium borohydride must be used as reducing agent to obtain the best immunodetection of Met in biological samples. For the immunohistochemical procedure, male and female Sprague-Dawley rats (250-380 g) were perfused through the aorta with 500 ml of a 0.5 M G solution in 0.1 M phosphate buffer saline (pH 7.5). The brains were removed, post-fixed for 1 h in 0.5 M G solution and then

Fig. 2. Light micrographs of vibratome sections of glutaraldehyde-fixed rat brain after immunostaining with anti-conjugated Met Ab diluted at 1/20 000. In the mesencephalon and pons, multipolar cell bodies (large arrows) exhibited an intense immunoreactivity. Note the presence of other Metimmunoreactive cell bodies (small arrows). (A) Motoneurons (large arrows) are visible at low magnificationin mesencephalic trigeminal nerve nucleus (large cell bodies; low magnification view, 250x). Scale bar = 40/~m. (B) Immunostainingof the ventral parabrachial nucleus (small cell bodies) near locus coeruleus, LC (low magnificationview, 125x ). Scale bar = 80/.*m.

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A. Amara et al. I Neuroscience Letters 185 (1995) 147-150

washed thoroughly with 0.05 M Tris-NaCl buffer (TBS) (pH 7.5). Sections (50/~m thick transverse or sagittal sections) were cut with a Lancer vibratome, placed in TBS, reduced with a 0.1 M sodium borohydride solution, washed, and then processed for immunohistochemistry using the peroxidase/antiperoxidase (PAP) method. Following incubation with 3% non-specific serum in TBS for 1 h at room temperature, the sections were incubated overnight at 4°C in TBS containing 1% non-specific serum, 0.02% Triton X-100, and subsequently diluted anticonjugated Met Ab (final dilution, 1/20 000). The sections were then washed, incubated for 1 h at 37°C with swine to rabbit immunoglobulin 1/400 (Dakopatts, Z196), washed again and incubated in rabbit PAP complex diluted at 1/2000 (Dakopatts, Zl13), re-washed and incubated with 0.25 mg/ml of diaminobenzidine (Sigma, ND 5637) containing 3 mg/ml ammonium nickel sulfate (Aldrich) in Tris-NaCl (pH 7.6), with 0.025% H20 2 for 15 min. After three washes, the sections were mounted on slides, dried, coverslipped, and observed by light microscopy (Nikon Microphot-Sea). Rat brain sections displayed different staining of several cell bodies, indicating an ubiquitous distribution of cellular Met (Fig. 2). Three types of specific immunoreactivity were found in G-fixed rat brain sections: (i) a faint staining of numerous cell bodies; (ii) a more intense immunoreactivity in some rat brain structures such as locus coeruleus (Fig. 2B) which may be related to high incorporation rate of Met found by previous authors [12,13] ; and (iii) a surprising intense staining was seen in motoneurons (Fig. 2A). Yet, Met incorporation has not been found high in pons nuclei [13]. A few motoneurons present in mesencephalon, pons and spinal cord (data not shown) were found to be highly immunoreactive. Since acetylcholine is an important neurotransmitter in motoneurons on the one hand and Met is involved in the metabolism of choline [6] on the other hand, the Met immunoreactivity could be predicted. For example, Fig. 2 shows a very intense staining of multipolar cell bodies in mesencephalic trigeminal nerve and ventral parabrachial nuclei near locus coeruleus. To evaluate the staining specificity, Ab to conjugated Met were incubated for 2 h at 37°C with Met conjugate and each of the derivative conjugates (hapten concentration varying from 10-7 to 10.-9 M) used for characterization of Ab affinity and specificity. Each mixture was then centrifuged at 10 000 x g for 20 min and the supernatant was used for immunohistochemistry. All types of immunoreactivity disappeared only after pre-adsorption of anticonjugated Met Ab with Met-G-BSA. Pre-absorption with other related compound,; did not affect the staining (data not shown), indicating a specific binding of Ab in rat brain sections, and consequently reliable localization of Met. In conclusion, this study describes the preparation

and tl/e immunochemical characteristics of antibodies to glutaraldehyde-conjugated Met. These new tools will facilitate the study of the distribution of this essential amino acid in the nervous system as well as in other tissues. This work received financial support from Fondation pour la Recherche M6dicale, Pads and IDRPHT, Talence (France). [1] Amara, A., Coussemacq, M. and Geffard M., Antibodies to reduced glutathione, Brain Res., 659 (1994) 237-242. [2] Campistron, G., Buijs, R. and Geffard, M., Specific antibodies against aspartate and their immunoeytoehemicalapplication in the rat brain, Brain Res., 365 (1986) 179-184. [3] Campistron,G., Buijs, R. and Geffard, M., Glyeine neurons in the brain and spinal cord. Antibody production and immunocytochemical localization, Brain Res., 376 (1986) 400-405. [4] Chagnand, J.L., Mons, N., Tuffct, S., Grandier-Vazeilles, X. and Geffard, M., Monoclonal antibodies against glutaraldehyde-conj ugated dopamine, J. Neurochem., 49 (1987) 487-494. [5] Chagnaud LL., Campistron, G. and Geffard, M., Monoclonal antibody directed against glutaraldehyde-conjugated glutamate and immunoeytochemicalapplications in the rat brain, Brain Res., 481 (1989) 175-181. [6] Finkdstein J.D., Methionine metabolism in mammals, J. Nutr. Biochem., 1 (1990) 228-237. [7] Geffard, M., Puizillout, J.J. and Delaage, M.A., A single radioimmunologieai assay for serotonin, N-aeetylserotonin, 5-methoxytryptamine and melatonin, J. Neurochem., 39 (1982) 12711277. [8] Geffard, M., Dulluc, J. and Rock, A., Antisera against the indolealkylamines: tryptophan, 5-hydroxytryptophan,5-hydroxytryptamine, 5-methoxytryptophan and 5-methoxytrypamine tested by an enzyme-linked immunosorbent assay method, J. Neurochem., 44 (1985) 1221-1228. [9] Geffard, M., McRae-Degueuree,A., and Souan, M.L., lmmunoeytochemical detection of acetylcholine in the rat central nervous system, Science., 229 (1985) 77-79. [10] Grange, E., Gharib, A., Lepetit, P., Guilland, J., Sarda, N. and Bobillier, P., Brain protein synthesis in the conscious rat using L[35S]methionine: relationship of methionine specific activity between plasma and precursor compartment and evaluation of methionine metabolic pathways, J. Neuroehem., 59 (1992) 14371443. [11] Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988, p. 84. [12] Lepetit, P., Touret, M., Grange, E., Gay, N. and Bobillier, P., Inhibition of methionine incorporationinto brain proteins after the systemic administration of p-chlorophenylalanine and L-5hydroxytryptophan,Eur J. Pharmacol., 209 (1991) 207-212. [13] Lestage, P., Gonon, M., Lepetit, P., Vitte, P.A., Debilly, G., Rossato, C., Lecestre, D. and Bobillier, P, An in vivo kinetic model with L-[35S]methionine for the determination of local cerebral rates for methionine incorporation into protein in the rat, J. Neuroehem., 48 (I 987) 352-363. [14] Long,M., Weir, D. and Scott, J., Source of methyl groups in brain and nerve tissue in the rat, J. Neurochem., 52 (1989) 377-380. [15] Mons, N. and Geffard, M., Specific antisera against the cateeholamines: L-3,4-dihydroxyphenylalanine,dopamine, noradrenaiine, and octopamine tested by an enzyme-linked immunosorbent assay, J. Neurochem., 48 (1987) 1826-1833.