A simple method for the isolation of multiple sclerosis specific brain antigens

85

Clinica Chimica Acta, 101 (1980) 85-92 @ Elsevier/North-Holland Biomedical Press

CCA 1245

A SIMPLE METHOD FOR THE ISOLATION SPECIFIC BRAIN ANTIGENS

S.C. RASTOGI The Neurochemical

(Received

OF MULTIPLE

SCLEROSIS

* and J. CLAUSEN Institute,

Radmandsgade

58, 2200

Copenhagen

(Denmark)

May 31st, 1979)

Summary A method for isolation of two multiple sclerosis(MS)-specific brain antigens [3] is described. The brain cytosol was passed through a Con A-Sepharose column and the glycoproteins containing MS-specific antigens (MSG) were eluted with 50 mmol/l methyl-a-D-glucopyranoside, pH 6. By fractionation of MSG on DEAE-cellulose, a fraction containing MS-specific antigens (MSGB) was eluted in 0.25 mol/l NaCI, after prior elution of other proteins with distilled water. The MS specific antigens in the MSG2 fraction were traced and characterized by crossed immunoelectrophoresis and isoelectric focusing. A quantitative determination of proteins eluted in MSG2 revealed that the 2 MSspecific antigens had been purified 10 000 times. Using this method to isolate the two MS-specific antigens of autopsy brains, they were found to be present in 7/7 MS brains and absent in 9/9 non-MS brains studied. The method described in the present communication may be used both for the partial purification of the MS-specific antigens as well as for the tracing of the specific antigens in different topographical areas of the MS brains.

Introduction By means of crossed immunoelectrophoresis (CI) using antibodies raised by immunization of rabbits with multiple sclerosis non-MS brain cytosol, it was possible to trace two MS-specific antigens in all six MS brains studied but not in seven non-MS autopsy brains [l--3]. The antigens were partially purified, more than 3000 times, by means of stepwise millipore and molecular filtration followed by DEAE-cellulose column chromatography. The molecular weights of the antigens were found to be lo’-lo6 and the isoelectric points of the antigens * To whom correspondence

should be addressed.

86

were in the range pH 3.5-6.0. However, the nature of the antigens remained unknown. The antigens were precipitated from the specific antigen-containing fraction eluted from the DEAE-cellulose column (referred to as Peak 2 antigens) at the interval 0.84-1.68 mol/l (NH,)*S04. This fraction specifically stimulated MS lymphocytes to form active E-rosettes among lymphocytes from patients with neurological diseases [ 41. A minimum of 15-20 g of brain tissue is required for the partial purification of MS-specific brain antigens by the method described earlier [1,3]. Thus, that is not suitable for tracing the specific antigens in different parts of the brain, or to confirm our findings on coded samples where the sample available is restricted to 1-5 g. Therefore, in the present communication a method has been developed for the isolation of the MS-specific brain antigens which employs classical chromatographic techniques and results in the purification of the antigens by more than 10 000 times. Materials and methods Chemicals Concanavalin A-bound Sepharose (Con A-Sepharose) was purchased from Pharmacia Fine Chemicals, Uppsala (Sweden). Agarose A45 was obtained from 1’Industrie Biologique, France; DEAE-cellulose was from Sigma, U.S.A.; methyl-oc-D-glucopyranoside was a product of Calbiochem, U.S.A., and all other chemicals were of the highest obtainable purity from E. Merck, F.R.G. Brains The present study included seven MS and nine non-MS brains. Six MS and seven non-MS brains have been described previously [ 31. The last MS brain was from a man (61 years) who had very slowly progressive MS for 30 years before he died. One of the new non-MS brains was from a man who died of hepatic coma and the other was from a 35 year-old alcoholic woman. All the three new brains were processed fresh (before freezing). Isolation of brain cy tosol Cytosol from 3 g of brain was prepared in 15 ml 0.15 mol/l NaCl [3]. The cytosol was dialysed (4°C) overnight against 0.1 mol/l Na-acetate buffer (containing 1 mmol/l MnC12, 1 mmol/l MgCl*, 1 mmol/l CaCl,, 1 mol/l NaCl and 0.01% merthiolate, pH 6.0. Immunization of rabbits and isolation of antibodies Immunization of rabbits with brain cytosol [2] and isolation from anti-cytosol serum [ 31 have been described previously.

of antibodies

Column chromatography All chromatographic studies were performed using the LKB-fraction collector type Ultrarack No. 7000 equipped with UVICORD type 8301A (LKB Ltd., Stockholm, Sweden). Con A-sepharose column chromatography Ten ml dialysed cytosol (approx. 60 mg protein)

was passed through

a Con

87

A-Sepharose column (0.9 X 15 cm). The column was then washed with 100 ml (20 ml/h) Na-acetate buffer, pH 6.0 (as mentioned before). The glycoproteins were then eluted with 50 ml of 50 mmol/l methyl-cu-D-glucopyranoside in Naacetate buffer. The glycoprotein fraction (G) was concentrated (4°C) to 1 ml by vacuum dialysis using a SM 13200 Sartorius Membrane (excluding molecules up to a mol. wt. of 20 000). The concentrated material was dialysed (4°C) against 2 1 redistilled water overnight and stored frozen prior to further fractionation by the DEAE-cellulose column - no precipitate was formed by the dialysis against redistilled water. DEAE-cellulose column chromatography The concentrated, dialysed glycoprotein fraction (MSG) isolated from the Con A-Sepharose column was applied on a DEAE-cellulose column (0.8 X 20 cm). The isolation of a fraction containing MS-specific antigens (MSGB) was then carried out as described before [3]. Briefly, the column was first washed with 50 ml redistilled water. A fraction containing the MS-specific antigens was then eluted with 0.25 mol/l NaCl. The corresponding control fractions of nonMS autopsy brains (KG2) were isolated in the same way. Crossed immunoelectrophoresis Crossed immunoelectrophoresis of 30-40 pg MSG2 or KG2 was performed in 1% agarose, 0.05 mol/l sodium barbitone buffer, pH 8.5, (5 ml gel on 7 X 7 cm’ glass plate) [ 21. The following experiments were performed: (a) MSG2 and KG2 were electrophoresed using both anti-MS cytosol and anti-control cytosol. The antibody-containing gel contained 45 ~1 anti-cytosol serum/cm3 gel. (b) MSG2 and KG2 were electrophoresed on the same plate (Tandem CIE). (c) Various concentrations of MSG2 and KG2 (15-100 pg) were electrophoresed using anti-MS cytosol pre-absorbed with various amounts of non-MS/MS cytosol (see below). The same antigens were also electrophoresed employing anti-non-MS cytosol pre-absorbed with various amounts of non-MS/MS cytosol. 600 ~1 anti-MS cytosol antibodies (or anti-non-MS cytosol antibodies) were incubated with 50 ~1 (500-3000 pg protein) non-MS cytosol (or MS cytosol) for 1 h. The immunoprecipitate formed was removed by centrifugation at 10 000 X g for 30 min. After electrophoresis unprecipitated antibodies were removed by pressing and washing the gel twice with 500 ml 0.6% NaCl (15 min), followed by washing with redistilled water. The dried gel was stained with Coomassie Brilliant Blue and de-stained with methanol/acetic acid/water (45 : 10 : 45). Isoelectric focusing Isoelectric focusing of MSG2 and KG2 on commercial polyacrylamide gel plates containing ampholines (pH range 3.5-9.5) was performed as described before [3]. Isoelectric focusing of MSG2 and KG2 was also performed in pH range 4-6.5. Electrophoretic conditions for the isoelectric focusing at pH 4-6.5 were the same as for the pH 3.5-9.5, except for the electrode solutions. The anode electrode solution used was 0.1 mol/l glutamic acid in 0.5 mol/l ortho-phosphoric acid and the cathode electrode solution was 0.1 mol/l p-alanine. Staining and destaining of gels was as described before [ 31.

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Results Optimization of isolation procedures A 3000-times purification of two MS-specific brain antigens was achieved in a fraction isolated from MS brain cytosol by means of stepwise molecular filteration followed by DEAE-cellulose column chromatography (MS peak 2) [3]. The MS Peak 2 was subjected to further purification by means of ammonium sulphate precipitation. The two MS-specific antigens were thus traced in Peak 2 precipitated between 0.84 and 1.68 mol/l ammonium sulphate (pH 6.8). This salting out gave rise to a further 50-times purification, but this schedule of purification could not be used for small brain samples (1-3 g). Furthermore, the molecular filterations of lo-15 ml cytosol followed by DEAE-cellulose chromatography is inconvenient and the yield of the Peak 2 fraction obtained is negligible. The isolation of MS-specific antigens directly by the ammonium sulphate precipitation of cytosol failed (probably due to proteinase activation during precipitation or precipitation of antigen-antibody complex formed between the antigens and corresponding IgG-antibodies present in the MS tissues). However, the precipitation of MS-specific antigens by ammonium sulphate from the chromatographic fraction (MS Peak 2) may indicate that they may be proteins. Assuming that the MS-specific antigens may be glycoproteins, MS Peak 2 was subjected to affinity chromatography for the further purification of MS-specific brain antigens. The MS Peak 2 was loaded onto a Con A-Sepharose column and after washing out non-glycoproteins, glycoproteins were eluted by methyl-oc-D-glucopyranoside or methyl-a-D-mannopyranoside at pH 5.5 and pH 6.0. Thus, the MSspecific antigens were eluted and maximum yield was obtained when 50 mmol/l methyl-cu-D-glucopyranoside at pH 6.0 was used for elution. When these conditions were employed, the glycoproteins isolated from crude MS brain cytosol (MSG) were found to contain the two MS-specific antigens. Further purification of MSG performed by DEAE-cellulose column chromatography to isolate MSG2 fraction (corresponding to MS Peak 2 fraction) revealed that MSG2 was purer than MS Peak 2 fraction (see later). The method hereafter has been employed on l-3 g tissue samples from seven MS brains and nine non-MS brains and found to be satisfactory, i.e. the yield of MSG2 was enough for characterization by crossed immunoelectrophoresis, isoelectric focusing and the active-E-rosette test. Quantitation of glycoproteins A quantitative estimation of the cytosol glycoproteins (MSG) eluted from the Con A-Sepharose column and glycoprotein fraction 2 (MSGB) isolated from DEAE-cellulose column in terms of protein eluted is described in the Table. The yields of both MSG and MSG2 were significantly higher than those of KG and KG2 respectively (p < 0.025 and p < 0.001 respectively, Student’s t-test). Isolation of the specific antigens in MSG2 (262 1.18protein) from 3 g wet brain shows that the antigens are purified more than 10 000 times. Characterization of MS-specific antigens Isoelectric focusing of MSG2 and KG2 at pH range 3.5-9.5

revealed that the

TABLE

89

I

QUANTITATION OF THE YIELDS OF CRUDE GLYCOPROTEIN FRACTION (G) AND GLYCOPROTEIN FRACTION 2 (GZ) ISOLATED FROM MULTIPLE SCLEROSIS (MS) AND CONTROL (IO BRAIN CYTOSOL (60 mg PROTEIN, APPROXIMATELY 3 g WET WEIGHT) G was isolated

by means of Con A-Sepharose

column

and G2 by DEAE

cellulose

column

chromatography

of G). Brain type

No. of brains used

Total G isolated (Mean + S.D.)

MS K

I 9

2.50 + 0.32 2.04 t 0.22 P < 0.025

L recovery MS cytosol K cytosol

mg protein

Total G2 isolated (Mean f S.D.)

fig protein

262.5 i 53.8 141.3 + 30.7 P < 0.001

from

% of G2 isolated MSG KG

0.44 0.24

4.2 3.4 from

p values are calculated

10.5 6.9 for Student’s

t test. 4.0

i-

1 .----

PH

K

MS

Fig. 1 (a and b). (a) Isoelectric focusing of MSG2 (MS) and KG2 (K) at pH 3.5-9.5. Fifteen ~1 (50 pg protein) MSG2 and KG2 were applied and electrophoresis was performed (1O’C) at P = 30 W. c’ = 1200 V and Z = 30 mA. The proteins were fixed by floating the gel in sulphosalicylic acid (3.5%) and trichloroacetic acid (11.5%) dissolved in 33% methanol. The gels thereafter were stained (6O’C) with 0.115% Coomassie Brilliant Blue R-250 dissolved in destaining solution (ethanol/acetic acid/water, 2518167). The area between the arrows for MS pattern is different from K. (b) Isoelectric focusing of MSGP (MS) and KG2 (K) at pH 44.5. 15 ~1 (88 fig) MSGP and KG2 were applied on the gel abd the electrophoresis was performed as described in (a). The two bands in MS-pattern indicated by arrows. were absent in K.

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MS-specific antigens may correspond to proteins with isoelectric points in the pH range 5.0-6.5 (Fig. la). This is in agreement with previously reported isoelectric points (3.5-6.0) of the MS-specific brain antigens [ 31. Isoelectric focusing of MSG2 and KG2 at narrow pH range demonstrated the presence of at least two bands in MSG2 which were absent in KG2 (Fig. lb). This experiment also revealed that they have isoelectric points between 5.2 and 6.2. Confirmation of the specific nature of the two antigens was achieved by Tandem CIE of MSG2 and KG2 where anti-MS antibodies used were absorbed with non-MS cytosol before mixing with agarose. Tandem CIE of MSG2 (15-100 I.rg), employing neutralised anti-MS antibodies, showed only two immunoprecipitation arcs in all experiments, irrespective of the concentration of non-MS cytosol (0.5-3 mg protein/600 ~1) used for neutralization of antiMS antibodies (Fig. 2a and b). Similar experiments employing anti-non-MS cytosol antibodies neutralized with non-MS/MS cytosol or anti-MS cytosol antibodies neutralized with MS cytosol revealed no immunoprecipitation arcs. By means of the chromatographic methods described, the two MS-specific antigens were traced in the G and G2 fraction of all seven MS brains studied. but not in any of the nine non-MS brains studied.

. r

Fig.

2a.

For

text

see p. 91.

1

:

i’:

Fig. 2 (a and b). (a) Tandem crossed immunoelectrophoresis of MSG2 and KG2. 40 @g proteins. KSG2 (hole A) and MSG2 (hole B), were electrophoresed from left to right at 250 V (1O’C) for 1.5 h. The electrophoresis in the second direction (bottom to top) was performed in the gel-containing anti-MS brain cytosol antibodies neutralized with non-MS cytosol (2 mg non-MS brain cytosol protein/600 yl anti-MS brain cytosol antibodies). 1 and 2 represent the two MS-specific brain antigens. (b) Tandem crossed immunoelectrophoresis of 60 &g KSC2 (hole A) and MSG2 (hole B). Electrophoretic run in the first direction 3 h. The antibody-containing gel for the second direction electrophoresis contained anti-MS brain cytosol antibodies (600 ~1) which were neutralized with 1.5 mg non-MS brain cYtOso1. 1 and 2 represent the two MS-specific brain antigens.

Discussion The purification of the MS-specific brain antigens in the present study has been monitored by crossed ~munoeiectrophoresis of MS/non-MS antigens versus both anti-MS cytosol antibodies and anti-non-MS antibodies and by isoelectric focusing. The G2 fractions isolated from MS brain cytosol have been found to contain two antigens (present in 717 brains) which were absent in control brains (absent in 9/9 non-MS brains). Tandem crossed electroimmuno~ phoresis of the two MS-specific brain antigens, isolated by the present method and those described before [ 1,3], revealed that the specific antigens isolated by the two different methods were identical (unpublished results). Furthermore, the MSG.2 fraction stimulated active E rosette forming lymphocytes of only MS patients among patients with neurological diseases (unpublished results).

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Knowing that the MS-specific brain antigens can be precipitated by ammonium sulphate and isolated by affinity chromatography using Con A-Sepharose, they may be glycoproteins. However, further verification of the glycoprotein nature of the antigens will be further investigated, when they are in a purer form. It should be mentioned here that SDS-polyacrylamide gel electrophoresis has not yet shown clear differences between MSG2 and KG2. Although isoelectric focusing clearly demonstrated the presence of two extra bands of MSG2 pattern compared to KG2 pattern, the system is not suitable for glycoprotein staining. The isoelectric points of these bands are between pH 5.2 and 6.2 and they can be precipitated by ammonium sulphate at 0.84 to 1.68 mol/l. These properties of MS-specific antigens are similar to those of other glycoproteins. Results of the present study reveal that the MS-specific antigens are purified 10 000 times and they also indicate that further purification may be achieved by isoelectric precipitation of antigens from the MSG2 fraction. Chromatography of MS cytosol on the isoelectric-focusing column may also result in a purer preparation of specific antigens, but the quantities required will be too large. Until now, it has not been possible to separate the two MS-specific antigens from each other. Acknowledgements We thank Miss Mette Belter

for her skillful

technical

assistance.

References In Proceedings of the Meeting of the Intern. 1 Clausen. J.. Rastogi, S.C., Offner, H. and Konat. G. (1978) Multiple Sclerosis Societies, Sep. 14-18. Gijttingen. in press 2 Rastogi, S.C.. Clausen. J. and Fog, T. (1978) Acta Neural. Stand. 57. 438-442 Acta Neural. Stand. 59, 281-296 3 R&o& S.C.. Clausen, J., Offner, H., Konat, G. and Fog, T. (1979) 4 Offner. H.. Rastogi. S.C.. Konat, G. and Clausen. J. (1979) J. Neural. Sci. 42. 349-355