Allergic encephalomyelitis: A comparison of the encephalitogenic A1 protein from human and bovine brain

ARCHIVES

OF

Allergic

BIOCHEMISTRY

AND

Encephalomyelitis: Al

138, 606-613 (1970)

BIOPHYSICS

Protein YUKI

A Comparison from

Human

OSHIR02

The Salk Institute, Received

January

and

AND

of the Encephalitogenic Bovine

Brain’

E. H. EYLAR3

San Diego,

California

26, 1970; accepted

March

92112 25, 1970

The chemical and biological properties of the human encephalitogenic basic protein was compared to that from bovine brain. Both proteins are highly basic and contain approximately 25 net positive charges at pH 6. They each contain one residue of tryptophan per mole but no cysteine. The electrophoretic migration in polyacrylamide gel at pH 4.3 and 8.0 was almost identical. The sedimentation coefficient was estimated to be 1.34 S, and their molecular weight 18,00&19,006 daltons with the human protein possibly larger. The optical rotatory dispersion studies suggested a random-coil structure; no or-helix or p-form was indicated. The encephalitogenic activity of the two proteins measured in the guinea pig was approximately the same. Their ability to combine with antibody prepared against the bovine protein was similar as determined by the hemagglutination-inhibition test. Both proteins were found to induce a significant delayed skin reaction in guinea pigs regardless of whether the human or bovine protein was used to produce sensitized cells. Thus, the physical, chemical, and biological properties of these two basic proteins are similar; the amino acid sequence is not identical, however, as revealed by the tryptic peptide maps.

It is now well established that the antigen in central nervous system (CNS) tissue responsible for induction of experimental allergic encephalomyelitis (EAE) is a basic protein present in myelin (l-11). Although EAE can be considered a model for study of autoimmune disease, questions arise about the relationship of EAE to human demyelinating disease such as multiple sclerosis. It appeared of interest, therefore, to prepare basic protein, referred to previously as the Al protein (1) from human CNS tissue and compare it with the more widely studied bovine Al protein (1, 3, 4). A previous report from this laboratory described a method of preparation for highly purified Al protein from bovine brain and 1 This research was supported in part by a grant from U. S. Public Health Service (NBO8268-01) and in part by the Salk Institute. 2 U. S. Public Health Service Postdoctoral Trainee (FR-O5595-03). 3 U. S. Public Health Service Career Development Awardee (l-K4-Al-8848-01).

spinal cord using ion-exchange chromatography (2). This study shows that the same method can be applied to the purification of the human brain Al protein. Some physical, chemical, and immunological properties of these proteins are compared. The human brain basic protein is estimated to have a molecular weight of approximately 18,00019,000, and a conformation approximated by a random coil. The similarity of this protein to that from bovine brain is shown. MATERIALS

AND

METHODS

Preparation of Al protein. Fresh bovine brain was obtained from a local slaughter house and immediately frozen with liquid Nz. With human brain material a period of 6-12 hr elapsed before freezing. The detailed method of preparation of bovine Al protein is described elsewhere (2). Briefly, the frozen brain material was blended in methanol-chloroform (1:2) to remove most lipids, extracted at pH 1.7, passed through a DEAE column, precipitated in 45% ammonium sulfate, and fractionated on Bio-Rex 70 (Bio-Rad Lab., Calif.) or Cellex-P (Bio-Rad Lab., Calif.). The 606

Al

PROTEIN

encephalitogenic Al protein from human brain was prepared by the same procedure. The yield of human Al protein was approximately the same as bovine Al protein (2). Polyacrylamide gel electrophoresis. Discontinuous disc electrophoresis at pH 4.3 described by Reisfeld et al. (12) was routinely used. Electrophoresis was also run at pH 9.8 (13). Histological and clinical assay of EAE activity. The EAE activity, routinely assayed in guinea pigs, was evaluated 8-25 days after injection of 3.3- to 100~pg quantities of test material in complete Freund’s adjuvant (CFA). Details of this assay method have been described elsewhere (1). Passive hemagglutination inhibition (PHI). Rabbit antiserum was produced using Al protein from bovine spinal chord as antigen (1). Using this antiserum and sensitized chicken red blood cells, the minimal amount of Al protein that inhibited hemagglutination was determined. The details of this method bave been described (1). Delayed skin test. Guinea pigs were injected in the usual manner with human or bovine Al protein (33 rg) in CFA. After 9-10 days, the animals were injected with Al protein in 0.15 M NaCl, and the delayed-type skin reaction was evaluated as described previously (1). N-terminal amino acid determination. The direct Edman degradation technique (14) and dansylation procedure was used (15) for determination of N-terminal residues. For the Edman procedure 0.1-0.5 mole of protein was used; 0.0050.02 pmole of protein was tested in the dansylation procedure. UltracentrZfugation. A Spinco Model E ultracentrifuge was used at 20.0 f 0.5”. The sample contained 0.8-6.0 mg/ml protein in 0.20 M NaCl and 0.20 M acetic acid (pH 2.6), and was dialyzed against solvent overnight at 4” before centrifugation. For sedimentation velocity studies, plain and wedge cells with one chamber were run at 59,780 rpm; pictures of Schlieren patterns were taken at 32-min intervals. For the long-column equilibrium sedimentation (16) the Yphantis cell with six chambers was used. The solvent was the same as above. The centrifugation was performed at 29,410 rpm for 16 hr. This method had previously been used for bovine spinal cord Al protein using lysozyme as a standard for the technique (17). For the short-column method (18) the same Yphantis cell and the same solvent was used. Centrifugation was performed at 20,410 rpm for 3 hr. A synthetic boundary double-sector cell with 0.15 ml of protein solution in one chamber and 0.4 ml of bufier in the other were used to obtain the Schlieren pattern of zero-time concentration. Tryptophan and tyrosine determination. The

607

OF BRAIN

tryptophan and tyrosine contents were determined by the absorption method of Edelhoch (19) using a Zeiss spectrophotometer. Tryptophan content was also determined by the calorimetric method described by Barman and Koshland (20). As an alternative to the standard procedure, the HNB-Al protein was washed five times in acetone containing 0.02 N HCl. An aliquot was taken, adjusted to pH above 12, and the optical density at 410 rnp was measured. Amino acid analysis. The amino acid composition was determined by the method of Moore et al. (21). Approximately 0.16 mg of protein was dissolved in 1 ml of constant boiling HCl, flushed with Nz, and sealed under low pressure. It was incubated at 110” for a period of 24, 48, and 72 hr. It was analyzed with a Beckman amino acid analyzer. Serine and threonine were extrapolated to zero-time hydrolysis. Cysteine and methionine, which are susceptible to oxidation during hydrolysis, were oxidized fnst with performic acid to cysteic acid and methionine sulfone, respectively, before hydrolysis according to the method described by Hirs (22). The amide nitrogen was de.. termined aft,er hydrolysis in 1 N HCl for 3 hr at 100”. Peptide mapping. Digestion with trypsin was performed for 5 hr at 37”. The hydrolysis was carried out with 10 mg of protein and 0.2 mg of trypsin which had been treated by the method of Kosta and Carpenter (23), in a buffer of 0.10 M triethylamine carbonate at pH 8.0. Peptide mapping of the digestion mixture was carried out as described by Katz et al. (24). Optical rotatory dispersion (ORD). Optical rotation of the Al protein was measured with a Jasco ORD U5-5 spectropolarimeter over the wave length range of 200-260 rn+ The protein was 0.1 dissolved in 0.15 M NaCl at approximately mg/ml. The protein concentration was determined by the Lowry method (25). The optical rotation was measured with l-cm cells at room temperature. The difference between the sample and solvent solution was taken as the rotation due to the basic protein. In calculating the reduced mean residue rotation (26) the index of water. obtained from the International Critical Table (27), was used at each wavelength. RESULTS

Purity. The purity of the bovine and hubrain Al protein preparations was demonstrated by polyacrylamide-gel electrophoresis at pH 4.3 and 8.9 (Fig. 1). In each case, the preparations showed only a single band when examined at 10-100 pg per tube. Homogeneity was also indicated by the abman

60s

OSHIRO

AND

sence of detectable N-terminal amino acids using the Edman and dansylation procedures. The N-terminal amino acid of the bovine spinal cord Al protein is N-acetylalanine (28) ; it is highly probable that the Al proteins from the bovine and human brain are similarly acetylated at the N-terminal position. Biological activities. The results of the assay for EAE activity, the PHI tests, and the delayed skin tests are shown in Table I.

FIG. 1. Polyacrylamide gel electrophoresis of human (1,3) and bovine (2, 4) Al protein preparations. The first two were run at pH 4.3 and the last two at pH 8.9.

EYLAR

The proteins from both sources are potent encephalitogens. In the 30 animals tested with each protein, however, it was noted that a slightly higher incidence of clinically positive animals were found with the bovine protein. Within the limits of the PHI test both proteins inhibited the hemagglutination to a similar degree. In the skin test, expressed as the area of erythema in millimeters, both proteins are positive in animals sensitized with either the human or bovine Al protein. Thus, cross-reactivity exists between the protein from one species and the cells sensitized by the other species. It was found routinely, however, the strongest delayed skin reaction occurred with the same protein as used in sensitization. The human and bovine Al proteins are indistinguishable on the basis of these tests. Sedimentation coejicients. The variation of sedimentation rate with protein concentration is shown in Fig. 2. Conversion of s values to ~20,~ was made by using the formula given by Svedberg and Pederson (29). The partial specific volume was assumed to be 0.721 ml/g based on the composition of the bovine Al protein (17). An acidic pH was used since these proteins tend to aggregate and even precipitate in phosphate buffer at neutral pH. The ~~0,~value, obtained by ex-

TABLE A COMPARISON

Source

Bovine

Human

I

OF THE ENCEPHALITOGENIC AND ANTIBODY-COMBINING FROM HUMAN AND BOVINE BRAIN EAE assay”

100.0 &g) s/10 10.0 9/10 3.3 6/10 100.0 9/10 10.0 7/10 3.3 5/10

PHI teat*

0.04-0.1

0.1-0.25

bg)

ACTIVITIES

OF THE Al PROTEINS

Skin testC Bovide

HUIllall

16 (mm)

11 (mm)

11

13

a The results are expressed as a ratio of those animals showing clinical and/or histological signs of EAE over the total tested for any given dose. * The PHI test is expressed as the amount of material which just inhibits the agglutination of four hemagglutinating doses of antibody prepared in rabbits against the bovine Al protein. e The skin test was performed 9-10 days after injection of 33 pg of human or bovine Al protein in CFA. The delayed skin reaction was evaluated by the area of erythema in millimeters which developed 24 hr after 20 pg of protein in 0.15 M NaCl was injected intradermally (1). Injections of bovine serum albumin or saline produced no discernible reaction; an area of erythema less than 6 mm was taken as negative.

Al

PROTEIN

The results are summarized in Table III. The gradual increase in molecular weight observed from the top of the cell toward the bottom indicated a minor degree of aggregation. Also, the average molecular weight value appeared to increase when the protein concentration was increased. Therefore, it seems appropriate to take the lower molecular weight values for both Al proteins. Similar results were obtained with the bovine spinal cord Al protein (17) where a value of 16,400 was found. Using the short-column equilibrium sedimentation method (lti), the calculated molecular weights were 20,100, and 19,400 for bovine and human brain basic proteins, respectively. Tryptophan and tyrosine contents. The results are summarized in Table III. The molecular weight seems to agree with the values obtained by other methods. The ratio of tyrosine to tryptophan was found to be close to 4: 1 and 3:1, respectively, for the human and bovine Al proteins (Table III).

trapolation to zero concentration, was 1.34 S for both bovine and human Al proteins. Molecular weights by equilibrium sedimentation. From the long-column sedimentation pattern, the molecular weight of bovine Al protein was calculated at 26 positions (16).

CONCENTRATION

(mg/ml

)

2. Sedimentation coefficients of human (0) and bovine (0) Al proteins. The protein was dissolved in 020 M NaCl and 0.20 M acetic acid at pH 2.6 and dialyzed in the same buffer overnight at 4”. Centrifugation was performed at 20.0 f 0.5” at 59,780 rpm. FIG.

TABLE AMINO

ACID

ANALYSIS

609

OF BRAIN

OF

II

THE

BRAIN

BASIC

PROTEIN

HUIILUl Amino acid

LYS His Ammonias Arg Asp Thr Ser Glu Pro ‘JY Ala 34 CYS” Val Metb Is01 Leu Tyrc Phe Tryc

Moles per 4 moles of Tyr

12.2 11.0 6.07 17.2 11.6 8.0 17 9.7 11.3 25.6 12.7 0 4.30 1.90 3.90 8.10 4.00 9.00 0.95

Bovine Near;;t.;rhole

wT%h” 0

8.60 7.53 14.90 7.35 4.05 8.15 6.94 6.08 8.06 4.95 0 2.39 1.35 2.43 5.05 3.60 7.21

12 11 6 17 12 8 17 10 11 26 13 0 4 2 4 8 4 9

1.00

1

Moles per 3 moles Tyr

Weight (%)

Nearest whole number

12.7 9.58 7.12 16.7 10.1 6.9 15 9.6 11.7 23.9 13.7 0 2.64 2.03 2.57 9.50 3.00 8.03

9.32 7.50 14.77 6.92 4.34 8.43 7.14 6.56 7.85 5.52 0 1.46 1.50 1.68 6.06 2.87 6.74

13 10 7 17 10 7 15 10 12 24 14 0 3 2 3 10 3 8

0.93

1.03

1

a Ammonia content was determined by separate analysis after hydrolysis in 1 N HCl at 100” for 3 hr. b The cysteine was determined after oxidation with performic acid. The methionine was confirmed by measurement of methionine sulfone after oxidation. c Tyrosine and tryptophan were determined spectrophotometrically.

610

OSHIBO

AND

Amino acid composition. The results of the amino acid analysis are shown in Table II. Tyrosine was used as a basis for calculation of the amino acid composition since it was independently found to be 3 and 4 moles, respectively, per mole of the bovine and human Al proteins. The composition of these proteins are very similar and do not differ appreciably. The human Al protein appears to be slightly larger. It is of interest that the molecular weight, found by the addition of the constituent amino acids, is in good agreement with those from other methods as shown in Table III. These two proteins are distinctly basic and contain 40 residues of lysine, histidine, and arginine. Approximately g of the 20 glutamic and aspartic acid residues are in the amide form giving an excess of 25 positive charges. Thus, the composition of the Al proteins from brain is very similar to the analogous protein from bovine spinal cord. One of the characteristics of these basic proteins is the absence of cysteine. This was observed in a routine amino acid analysis at

EYLAIL

6 M HCI for 24 hr at 110”. It was confirmed when these Al proteins were oxidized with performic acid before amino acid analysis. The content of methionine was determined by two techniques: directly as methionine or as methionine sulfone after oxidation with performic acid. The methionine sulfone eluted from the amino acid analyzer between aspartic acid and threonine. A small amount of methionine sulfoxide was also observed. For both the human and bovine Al proteins, 2 moles of methionine were found by these two procedures. Tryptophan determined by HNB. The bovine and human proteins were treated with HNB in order to determine the number of tryptophan residues. The equivalent weight per mole of HNB was 17,400 for the bovine protein and 18,200 for the human protein. These results establish the presence of 1 mole of tryptophan per mole of protein since the molecular weights from this method are in agreement with those from sedimentation data. Peptide mappiTy. The peptide maps of the bovine and human Al proteins treated by

0

0 (b)

t-1

(-) t

<3’ CHROMATOGRAPHY 3. The peptide I’ denotes fluorescence FIG.

0 I,

CHROMATOGRAPHY

4

maps of the tryptic digest of the human (a) and the bovine (b) Al proteins. when viewed under ultraviolet light; Y denotes a yellow ninhydrin reaclion.

Al PROTEIN

OF BRAIN

TABLE

611

III

MOLECULAR WEIGHTS OF THE BRAIN BASIC PROTEIN Hlllllan

Long-column sedimentation equilibriuma Long-column sedimentation equilibrium6 Long-column sedimentation equilibriuma Long-column sedimentation equilibriumb Short-column sedimentation equilibrium Tryptophan analysisC Tyrosineltryptophand Tryptophan analysise Amino acid analysis

Bovine

19,600 (4.90 mg/ml)

18,600 (3.56 mg/ml)

23,200 (4.90 mg/ml)

20,700 (3.56 mg/ml)

22,100 (5.88 mg/ml) 25,600 (5.88 mg/ml)

-

19,400 (4.90 mg/ml)

20,100 (3.56 me/ml)

20,200 3.78 18,200 18,300

17,000 2.93 17)400 17,700

a Average values of upper four positions. b Overall javerape values of 26 positions. c Molecular weight was calculated from spectrophotometric data on assumption that there is only one tryptophan residue per molecule. d The molar ratio was determined by spectrophotometric method. e Based on the reaction with HNB. trypsin are shown in Fig. 3a and b. The tryptic digest of the bovine Al protein showed 30 distinct spots which are equal to the moles of lysine and arginine per mole of the protein. The peptide map of the human

basic protein was similar to that of the bovine; a few peptides, however, were distinctly different. Molecular weiyht. In Table III, the molecular weight values determined by physical and chemical methods are listed. There is good agreement between these values; the molecular weight of the human protein averages slightly higher than the bovine protein. These values are somewhat higher than the 16,400 daltons found for the Al protein from bovine spinal cord, and it now appears that this latter -value was underestimated. Optical rotatory dispersion. As shown in Fig. 4 there is no trough at 233 rnp, which would indicate the presence of a-helix or P-form

(30) in either

of the protein

prepara-

tions. The optical rotation was also studied in 1.5 M NaCl, in 0.1 M Tris at pH 7.3, and in HCl solution at pH 1.4. The ORD in these solutions did not change significantly from the curves shown in Fig. 4. The curves resemble a part of the figure reported by

+2,000 0

r< -2,000 z -4,000 -6,000 200

220

240

260

X(mp)

FIG. 4. The reduced mean residue rotation, [m’], of the human (H) and bovine (a) basic proteins. The optical rotation was measured in 0.15 M NaCl solution at room temperature.

Sarker and Doty (30) for random-coil structure of poly-L-lysine. DISCUSSION

It can be concluded from this study that the Al proteins derived from bovine and human brain are nearly identical in physical, chemical and biological properties and composi;on, and strongly resemble the Al protein from bovine spinal cord. Prominent features of this protein are its strongly cationic character, absence of cysteine, and open conformation as shown by optical rotatory dispersion, viscosity (17)) susceptibility to proteolytic digestion (31), and resistance

612

OSHIRO

AND

to denaturation (17). It is this property which accounts for the presence of a diseaseinducing site which, in the terminology of Sela (32), can be considered a sequential determinant, i.e., due to the primary structure of a short region of the polypeptide chain which, in this case, is a nine-residue section (or less) surrounding the single tryptophan residue (33). Based on the similar encephalitogenic properties of the bovine and human Al protein, it appears that the highly specific requirements of amino acid sequence of the encephalitogenic determinant found for the bovine protein must also exist in the human protein. It is highly probable, therefore, that the chemical basis for induction of encephalitogenic disease in man will be the same as for EAE if, indeed, the Al protein is primarily responsible. In view of the open conformation of the Al molecule, it is probable that the specificity of the antibody-combining site is due to primary structure rather than to a folded orientation of the polypeptide chain; the cross reactivity of the bovine and human proteins suggests nearly identical sites. A comparison of the amino acid sequences of the bovine and human proteins will answer this question. From both physical and chemical methods, it appears that the molecular weights of the human and bovine Al protein are nearly identical, 18,00&19,000 daltons. This value is higher than that reported by Eng et al. (11) who found 8,900-12,000 daltons for the protein which they extracted with salt and acid from normal human brain. It is unlikely that such wide variation is due to a variety of basic proteins in myelin since it was previously demonstrated (1) that over 85 % of the basic protein of myelin is due to the Al protein. Rather, it appears that the variable molecular weights obtained from different laboratories arise from proteolysis that may occur in situ (1) or during acid extraction (34). A peptide (lo), encephalitogenic in rabbits, of 4800 daltons derived from human brain is very likely a degradation product of the acid-extraction step. It is evident that the human and bovine Al proteins are similar by all criteria used in this study. Close examination of the amino acid analyses, however, indicate a

EYLAR

slight difference in composition which is reflected in peptide mapping. It can be concluded, therefore, that the differences in amino acid sequence of the bovine and human proteins are not consequential with regard to the biological activities such as EAE induction, antibody-combining sites, and delayed skin reactivity. ACKNOWLEDGMENTS We are grateful for the encouragement of Dr. Jonas Salk throughout the course of this work, and acknowledge the able technical assistance of Mr. J. Jackson, and Mr. D. K. Miller for the photography. REFERENCES 1. EYLAR, E. H., SALK, J., BEVERIDGE, G., AND BROWN, L., Arch. Biochem. Biophys. 132, 34 (1969). 2. OSHIRO, Y., AND EYLAR, E. H., Arch. Biochem. Biophys. 138, 392 (1970). 3. NAKAO, A., DAVIS, W., AND ROBOZ-EINSTEIN, E., Biochim. Biophys. Acta 130, 163 (1966). 4. KIES, M., MURPHY, J., AND ALVORD, E. C., Fed. Proc. 19, 207 (1960). 5. WOLFGRAM, F., Ann. N. Y. Acad. Sci. 122,164 (1965). 6. MARTENSON, R., AND LEBARON, F., J. Neurothem. 13, 1469 (1966). 7. CARNEGIE, P., BENCINA, B., AND LAMOUREUX, G., Biochem. J. 106, 559 (1967). 8. CASPARY, E. A., AND FIELD, E., Ann. N. Y. Acad. Sci. 122, 182 (1965). 9. LUMSDEN, C., ROBERTSON, I)., AND BLIGHT, R., J. Neurochem. 13, 127 (1966). 10. KIBLER, R. F., AND SHAPIRA, R., J. BioE. Chem. 243, 281 (1968). 11. ENG, L. F., CHAO, F. C., GERSTL, B., PRATT, D., AND TAVASTSTJERNA, M. G., Biochemistry 7, 4455 (1968). 12. REISFELD, R., LEWIS, U., AND WILLIAMS, D., Nature 196, 281 (1962). 13. DAVIS, B. J., Ann. N. Y. Acad. Sci. 121, 494 (1964). 14. BLOMBACK, B., BLOMBACK, M., EDMAN, P., .~ND HESSEL, B., Biochim. Biophys. Acta 116, 371 (1966). in Enzymology” 15. GRAY, W. R., in “Methods (C. H. W. Hirs, ed.), Vol. 11, p. 469. Academic Press, New York (1967). of 16. ROBINSON, A., Thesis (Ph.D.), University California, San Diego (1968). 17. EYLBR, E. H., AND THOMPSON, M., Arch. Biochem. Biophye. 129, 468 (1969). 18. YPHANTIS, D., Ann. N. Y. Acad. Sci. 33, 586 (1960).

Al

PROTEIN

19. EDELHOCH, H., Biochemistry 6, 1948 (1967). T., AND KOSHLAND, D. E., J. Biol. 20. BARMAN, Chem. 242, 5771 (1967). 21. MOORE, S., SPECKMAN, D. H., AND STEIN, W. H., Anal. Chem. 30, 1185 (1958). 22. HIRS, C. H. W., in “Methods in Enzymology” (C. H. W. Hirs, ed.), Vol. 11, p. 59. Academic Press, New York (1967). F. H., J. Biol. 23. KOSTA, Y., AND CARPENTER, Chem. 239, 1799 (1964). 24. KATZ, A. M., DREYER, W. J., AND ANFINSEN, C. B., J. Biol. Chem. 234, 2897 (1959). 25. LOWRY, 0., ROSEBROUGH, N., FARR, A., AND RaNDaLL, R., J. Biol. Chem. 193,265 (1951). P., AND DOTY, P., Advan. Protein 26. URNES, Chem. 16, 401 (1961). 27. International Critical Table, ‘7, 13 (1930).

OF BRAIN

613

28. HASHIM, G., AND EYLAR, E. H., Biochem. Biophys. Res. Commun. 34, 770 (1969). 29. SVEDBERG, T., AND PEDERSON, K. O., in “The Ultracentrifuge,” p. 273. Oxford Univ. Press, London and New York (1940); Johnson Reprint Corp., New York (1959). 30. SARKER, P. K., AND DOTY, P., Proc. Nat. Acad. Sci. U. S. A. 66, 981 (1966). 31. HASHIM, G. A., AND EYLAR, E. H., Arch. Biochem. Biophys 129, 635 (1969). 32. SELA, N., Science 166, 1365 (1969). 33. EYLAR, E. H., CACCAM, J., JACKSON, J., WESTALL, F., AND ROBINSON, A. B., Science, in press. 34. NAK.40, A., DAVIS, W. J., AND EINSTEIN, E. It., Biochim. Biophys. Acta 130, 171 (1966).