Jourttol o/ V~,.errx.iinrii,rr?. 1976 Vol 27. pp 405-408 Pergamon Press Printed

in

Great Britain.

IMMUNOCHEMICAL STUDIES OF THE INTERACTION BETWEEN MYELIN BASIC PROTEIN AND s-t 00 PROTEIN’

s. P. MAHADIK, L. GRAFand M. M. RAPPORT Division of Neuroscience. New York State Psychiatric Institute and Departments of Biochemistry and Pathology. Columbia University College of Physicians and Surgeons, New York, NY 10032, U.S.A. (Rrcriced 30 Decrrnher 1975. Accepted 12 February 1976)

Abstract-The formation of a complex between myelin basic protein and S-100 protein was detected from the change in migration of S-100 protein on immunoelectrophoresis. A degree of specificity for the interaction was shown by two observations: ( I ) two other pure acidic proteins. 111-111-2 and bovine serum albumin. did not show it and (2) complex formation was dependent on specific ions. either C:i2+ (10 mhf) or M n Z + ( I mM). Mg”. Ba”. and Li’ had no effect. Nan-specific interactions between S-100 protein and other basic molecules (histones. polylysine) are not dependent on specific ions such as Ca” and Mn”. The complex was stable at physiological salt concentrations and contained 3 mol of basic protein per mol of S-100 protein. Complex formation was also detected from the alteration of migration rate of S-100 protein in polyacrylamide gels. Serological activity (complement-fixation) of S-100 protein with anti-S-100 serum was reduced in the complex by 30%.

THEPRESENT era in research on myelin basic protein (MBP) in relation to experimental demyelinating disease was initiated by the report of KiEs et al. (1958)

ing interest depending on whether they interfered with tests for encephalitogenic or immunologic activity, but they would seem to deserve more careful attenthat a collagen-like compound isolated from bovine tion. In this paper we wish to report an interaction spinal cord displayed encephalitogenic activity in guinea pigs. Since then. through the efforts of many between MBP and S-100 protein. a specific protein for the nervous system present in the mammalian investigators (for review, see CARNEGIE & DUNKLEY, 1975) the encephalitogen was isolated. identified as CNS. This interaction appears to depend on the cona basic protein. and its primary structure determined. formational state of MBP. of S-100 protein, or both. At present, research on MBP is directed toward two Evidence for a degree of specificity for the reaction goals: ( I ) determining its role in myelin structure and is that two other pure acidic proteins do not show (2) evaluating the encephalitogenic and immunologi- it and that complex formation is dependent on specical activities of fragments which represent parts of fic cations. The complex is stable at physiological salt its structure. These considerations tend to overlook concentrations and contains 3 mol of MBP per mol another important property of MBP, namely its inter- of S-100 protein. The demonstration of complex formation was based principally on the alteration of imaction or combination with other proteins. Although such interactions have been observed, munoelectrophoretic properties of the acidic proteins. they have not been carefully documented. Since the Since antibodies against such proteins in the nervous proteins might have been acidic in nature. it was system are difficult to prepare. we have thus far been probably considered that interactions with MBP did able to establish the specificity of the MBP-S-100 not involve any process more specific than coulombic protein interaction by comparing it only with two attraction between acidic and basic substances. How- other proteins for which antisera were available. ever, the interaction of MBP with serum r-globulin namely, 111-111-2 and bovine serum albumin. Neither (MCPHERSON el al.. 1970) or /&lipoprotein (OFFNER of these proteins reacted with MBP under the specific c’t a / . , 1974) do not involve molecules that are es- conditions required for the reaction with S-100 propecially acidic. Such reactions have elicited only pass- tein. There has been a preliminary report of this work (MAHADIK et a/., 1974). ’ This work was supported in part by grants from the National Multiple Sclerosis Society and the National Cancer Institute (CA 10903) MATERIALS AND METHODS Ahhrrciutioris used: MBP, myelin basic protein; EDTA, ethyle.nediaminetetraacetic acid; TEMED. N,N.N’.N’Chrmicals. Ammonium sulfate (enzyme grade) R B S tetramethylethylenediamine; EGTA. ethylene glycol-his(@ obtained from Schwartz-Mann. DEA E-ccllulosc (Cellcs-D) was purchased from Bio-Rad. Acrylamide. his-acrylamide aminoethyl ether)-N.N’-tetra-acetic acid 405

406

S. P. MAHI\DIK. L. GRAFand M. M.

RAi'POKT

40 mA constant current. Fractions of I nil were collected. and N . N . ~ ' . N ' - t e t r a m e t h ~ I r t h ~ l e n e d i a m (TEMFD). ine A and. from their absorbance at !YO nm. pooled approprigrade. were purchased froii Eastman Organic Chemicals. ately. Thsse were concentrated. dialyzed. and freeze-dried. Tris (base). both cnr)me and bulfer grades. and gltcine These fractions were numbered from 1 t o 9. fraction 1 Hcre obtained from Schuartz-Maiiii, All other reagents being the tirst to cmerge (fastest migration), uere of A grade. Prorriri rro~nrnc.lntirre.In thc absence of a functional basis Prc.pururioii o/ \oliih/t, pr.otr,iris. All operations were confor designating the individual proteins. we have adapted ducted at 5 C . Fresh borine brains were ohtained from the slauehtcrhouss packed in ice. Connective tissue Has the scheme introduccd by M m i ~ r(19721 to the thrce-step mcthod or purification described. To avoid confusing the removed and most of the blood was washed out with two schemes. Roman numerals are used for t h u first SO d n e . Brain tissur' (XH) gl was cut into pieces of about steps and Arabic for the third. Thus ;ti1 indibidual protein I cm3 and then homogenized (Sorvall Omniniixcr. 80 V.. such as S-100 would appear in our nomenclature ;I\ 11160 s.) with 2 bolumes of cold sodium- povdssium phosphate IV-I. This ~ i nonicnclaturu e ~ w i l l he confined its f:tr ;IS buffer (IS 0.1. pH 7.4. prepared by mixing 22.6 ml of 0.5 M-KH,PO., and 59.0 ml of 0.5 M - N ~ I ~ H P O and , diluting possihle to protein5 that have not ytt been idcntificd A S cnrresponding to those of other investigators to 1 1). The homogen;ite was further homogeiii7ed in a Waring blender for 3 min iind then centriluged in the cold ProrriJis. The soluble acidic proteins used in the followat 45.000 y for I20 min. The supematant solution was ing studies are 111-IV-I identified as S-100 protein (from then fractionated according to the following three-step pro- its reactions with antiserum to S-I00 and comparison with a sample of S-100 protein obtained through the kindness cedure involving ammonium sulfate precipitation. ion exchange chromatograph). and preparatibe polyacryla- of Dr. MOON): 111-111-4. identified as bovinc serum alhuniin from the reactions of antiscrum to 111-111-4 with pure niide gel electrophoresis. .4rii~1iori;ur?i strlfirre precipirtrtio!i. The clear supernatant bovine serum proteins; and 111-111-2 for which ii l11oilon a s brought to pH 7.5 with I hf-NaOH. Powdered specific antiserum was obtained. ammonium sulfate was added in portions with constant. Myelin basic protein (MBP) was purified I'i-om hovine gentle stirring to 30?, saturation. the pH being maintained brain according to the procedure of OSHIXO& € \ L A X at 7.5 by dropwise addition of 1 ht-NaOH. Stirring was (1970). Calf thymus histone (Fraction I l l ) was obtained continued for I S min. and the precipitate (fraction 1) was from Worthington Biochemicals. and polylysirie from Ncw then collected by centrifugation (1S.oM) y ; 60 min). The England Nuclear Corp. .4ria/yricu/ po/j,ucrr/umiile qel t~/rc~fropho~rc.i\.. All gels clear supernatant was then brought to 60"" saturation i n a similar fashion. The precipitate (fraction 11) was collected (1 x I10 mm) were prepared with a gel mixturc containing b j centrifugation as before. and the supernatant was Tris-glycinc buffer. 60 ITIM (pH 8.6); acrylamide. 7.5",,: hisbrought to IOO", saturation. The pH was then lowered acrylamide. 0.37",; TEMED. 0.075"" and ammonium perto 4.0 by dropwise addition of glacial acetic acid. The sus- sulfate. 0.035",,. Electrode buffer was Tris-glycine, 60 mM pension was stirred gently overnlght and the precipitate (pH 8.2). Gels were prerun for 1 hr at 2 mA per gel. (fraction I l l ) collected h\ centrifugation ( I 5.000 y: 60 min). Thc electrode buffer was replaced with fresh huffcr. and samples containing bromophenoi blue as markcr were The precipitate was suspended in SO to 100 ml of applied. Electrophoresis was continued at 1 mA per gel sodium-potassium phosphate buffer (IS 0.01. pH 7.4). and the pH was then adjusted to 7.4 by adding I M - N ~ O H . at room temperature with circulating tap water for cooling. Electrophoresis was continued until the dye reached 1 cm After dialysis overnight against 2 x 21 of this phosphate buffer. insoluble material was removed by centrifugation. from the anodic end. Gels were stained with Ainido Black DEAE-crllirlosr chromuutoyruphj. Fraction 111 (approx (0.5':,;1 Amido Black. methanol and 7"b acetic acid1 for 1 h and destaincd in ii diffusion destaincr containing 500 mg) was placed on a column of 100 ml of packed 7y, acetic acid. DEAE-cellulose cquilibrated with sodium-potassiom phosWhen the effect of cations was studied. they wcre pre~eiit phate buffer (IS 0.01. pH 7.4). Individual fractions of 10 in (he gels and electrode huffer (at the same conccntr;ition ml were collccted and their absorbance was determined as in the samples) during both prerun and elecirophorcsis. at 280 nm. Appropriate tuhes were combined. A first frac.41rtisuro. Rabbits (1.5 to 4 kg) werc inimuniscd with protion (111-1) was eluted with buffer. A second fraction (III-II) teins according to the following protocol of inptioiis: 0.5 was ohtained by elution with a lincar gradient of NaCl mg of protein in Frcund's complctc adjuvant into thc footin equilibrating buffer (500 ml with n o NaCl mixing with pads: after a 4 wcek interval. repeated; after n two week 500 ml of 0.3 M - N ~ C Iand ) appeared between 0.1 and 0.2 interval. 1.0 mg of protein in adjuvant. I.M.: repeated. M-NaCI A third (111-Ill) and a fourth (111-IV) fraction were Three weeks after the final inoculation. the rabbits wci-c eluted with a pH gradient obtained with 0.5 M-sodium acebled by cardiac puncture. The serum was collected and tate buffer. pH 5.5. Fraction 111-Ill appeared immediately stored without preservative at - 20 C. In this way specific and fraction 111-IV appeared at pH 6.1. These fractions were concentrated I n an Amicon membrane concentration antisera were obtained to S-100 protein, 111-111-2. and I I I 111-4 as judged by Ouchterlony diffusion tests against cell and then dialyzed overnight against 1 I of 60 mM-glycine-Tris buffer, pH 8.6. many brain protein fractions. Prepurutirr ye/ rlrctrophorrsis. The instrument used was Inirmrrio/Ogica/ UllU/y,$i.\, ~ ~ J r i p / L ' J l l ~ fJi .l Yi l i ~ t o l l . Tests Were carried out at a sensitivity level of four 50",, units ol comthe Buchler Polyprep-100. A 40 ml gel column was preplement as described previously ( R A P P O R T & GRAr. 1367). pared with 7.5"" acrylamide gel in the same way as tube gels (described below). The upper electrode hiifler was 200 I~n~~t~r~iui~/r~c~~,.opllo,.t.sis. Microslides ( 1 x 1 in) werc covered with 3 ml of I " , agarose (special gradu A. CalbiomM-g\yClne-TriS, pH X.6. The gel was prerun with 2 ml of bromphrnol blue in S",, sucrose at 60 inA constant cur- chem) in electrode buffer. A troiigli (2 x 50 mm) *:IS cut in the center. the gel being left in placc. and wells of 20 rent. Whcn thc dye emerged. protein fractions from the pl capacity were placed on either side. 5 nini distant. After DEAE cellulose column (50-75 mg in 10 ml containing lo", sucrose) were applied and the run was continued at positioning the slide (Buchler electrophoresia apparatus1 10

FIG.5. Interaction of MBP with S-100 protein studied by electrophoretic migration on polyacrylamide gels. 7 1/2% gel, pH 8.6 (see Methods). All gels contain S-100 protein (25 pg). A. No additions B. +MBP, 75 pg. No complex in absence of CaCI,. C. +MgCI,, 10 mM. Mg slows migration of S-100 protein. D. +MgCI,, 10 mM + MBP, 75 p g . No complex formation. E. +CaCI,, 5 mM. Marked slowing of migration of S-100 protein with formation of multiple bands. F. +CaCI,, 10 mM. Migration of S-100 protein further slowed; bands become too diffuse for resolution. G. t CaCI,, 10 mar + MBP, 7 5 pg. Complex formation with very slow migration. FIG.6. Non-interaction of MBP with acidic proteins: 111-111-2 protein and bovine serum albumin.

Myelin basic protein-S-100 ~ t samples l

containing 5 to 10 pg of protein were placed the wells. Contact between slides and electrode buffer \\as made with strips of paper (Whatman No. 3). Electrophoresis was started at 2 mA per slide and continued as appropriate For the pH and the protein interaction under \ttidy. When complete, the gel from the trough was i-moved and replaced with 75 111 of antiserum. Slides were placed in a storage box and left overnight at room temperature to allow precipitation arcs to develop. in

RESULTS

protein interaction

407

TABLE1 SLROLOCICAL ACTIVITY OF

TEIN-s-lOO-CaZ’

M Y E L I ~ BASIC PROCOMPLtX WITH A\TISkKL‘M TO s-100

Antiserum 2041

2044

Sample

Unitsimg S-100

s-100 4- cd2’(10 mM) S-100 + MBP ( I 2 ) Ca’‘ S-100 + MBP ( 1 3 )

6000 4000

5900 1300

I00 70

4000

4200

69

+

“,,Activity

f Cd”

Iiiitn~inorlc~ctrophori~sis

The interaction between S-100 protein and MBP in the absence of cations was relatively weak at pH X.6 (Fig. la). This is shown by the long line of precipitate with anti-S-100 serum extending from the position of S-100 alone to that of the complex. In the presence of Ca” (10 mM) or M n Z + ( I mM) a stable complex was obtained at this pH. and all the S-100 appeared in the complex (Fig. 1 b and lc, respectively). Mg”, Ba”. and Li+ (10 mM) did not stabilize the complex. Removal of ions with EDTA or EGTA pi-evented complex formation (Fig. Id). The complex formed in the presence of Ca” (10 mM) was stable in the presence of 120 mM-KCI or -NaCl (Fig. 2a and 2b), but some dissociation took place at 200 mM (Fig. 2c and 2d). At lower pH values some dissociation of complex was detectable; this was found at pH 7.4 with both phosphate and Tris-maleate buffers (Fig. 3. a-d). In Tris-maleate at pH 7.4 the S-100 protein displayed two forms; these migrated similarly but differed in their interactions with antibody to S-100 and apparently in their ability to complex with M B P (Fig. 3d). When the quantity of MBP relative to S-100 was increased from 0 to 3.9 by wt. it was found (Fig. 4) that all of the S-100 was present in the complex at a value of about 3.0. Since the molecular weight of MBP I S 18.500 (EYLAK.1970) and that of S-100 pro& LEVINE.1969). a weight tein is 71.300 (DANNICS ration of 3.0 corresponds to a molar ratio of 3.4. When larger amounts of MBP were present. it caused the complex to migrate slightly towards the cathode.

Intcractioris qf other acidic protrins with M B P . histones. and polyljsine. Studies of the possible interactions of MBP with two other acidic proteins for which antisera were available. 111-111-2 protein and bovine serum albumin. showed that no alteration was produced in their electrophoretic migrations by M B P either in the absence or presence of Ca” (Fig. 6). Confirmatory observations were made by polyaerylamide gel electrophoresis. With histones and polylysine all three acidic proteins. namely. s-100.111-111-2. and bovine serum albumin, showed complex formation even in the absence of added divalent cations.

DISCUSSION

The observations reported in this paper show that S-100 protein and myelin basic protein form a com-

plex in the presence of either Ca” or Mn”. That this complex formation shows a degree of specificity is attested to by the following Facts: ( I ) two other pure acidic proteins, bovine serum albumin and III111-2, did not show it and (2) it was dependent on specific’divalent ions. either Ca’+ or M n 2 + . Mg’+ and B a 2 + had n o effect. Chelators such as EDTA and EGTA prevented complex formation. The optimal concentration of Ca” was 10 times higher than the optimal concentration of Mn’+. Non-specific interactions between acidic proteins and basic molGc4 drcrrophoresis ecules such as polylysine and histones d o not depend Using 7.57, acrytamide gels at pH 8.6, i t was found o n the presence of Ca2+ and Mn”. Three mol of that S-100 formed a complex with MBP in the pres- myelin basic protein reacted with I mol of S-100 procnce of Ca’ (10 mM). but no complex formed either tein to form the complex. i n the presence of Mg’+ (10 mM) or in the absence It seems likely that the mechanism underlying comof added cations (Fig. 5). S-100 protein dissociated plex formation involves a change in conformation of into several components in the presence of Ca”. This either one or both proteins caused by the Ca” o r rt rrl. (1969) shoncd that the S-100 Mn’+. CALISSANO IS readily seen at 5 mM (gel F). Each component appeared to form stable complexes with M B P (gel protein undergoes a conformational chanse in the G 1. presence of Ca” leading to the appearance..uf multiple forms that are separable on 7.5”, acrylamide gcls. Sc~iolo~~icul uctirirj We have confirmed this observation and have also When the serological activity of S-100 was com- shown that the fluorescence of S-100 protein is pared with that of thecomplex (Table I),it was found enhanced b? Ca’-, n-hereas the fluorescence of MBP that at a weisht ratio of 2 or 3 for M B P to S-100. is enhanced only b? Mn’* (PrRmiAL & M A I ~ A ~ I K . the activity with 2 different antisera was reduced 1974). These conformational changes are not produced by Mg”. ahout 300;. +

408

S. P. MAHADIK. L. GRAFand M. M. RAPPORI

The interaction between MBP and S-100 protein may depend on the accessibility of hydrophobic regions in the S-100 protein molecule. It has been found MOOR^. 1972) by means of the fluorescent probe 8-anilinonaphthalene-sulfonic acid that n o hydrophobic sites are exposed on the surface of S-100 protein in the absence of Ca”. As a result of conformational changes in the presence of Ca2’ (10 mM). hydrophobic sites capable of binding this probe are detectable. However. the dissociation of the complex at high salt concentration suggests that charged group interactions are also involved. The reduction in serological activity of S-100 by formation of the complex with MBP indicates that in the complex some antigenic sites on the S-100 protein are probably covered or sterically blocked. It will be of considerable interest to determine whether this is also true for MBP. It is possible that specific protein-protein interactions in the CNS are far more general than presently thought. This probably cannot be convincingly demonstrated until a reliable set of specific antibody reagents is available for decisive identification of individual proteins. However it should be borne in mind that the interaction was first detected by gel electrophoresis alone. although this method is considerably less sensitive than immunochemical techniques. Inasmuch as the function of most of the proteins in the CNS still remains to be established, the detection of specific interactions is one of the most promising methods of looking for such functions.

Ackiio~ledyemenfs-We wish to thank Mrs. ANNA KORand Mr. YUNG-YU HUANGfor expert technical

ENOVSKY

assistance.

REFERENCES CARNEGIE P. R. & DUNKLEY P. R. (1975) Adc. in Neurochem. 1. 95-135. CALISSANO P.. MOOREB. W. & FKII.SI:NA. (1969) Biochemistry. Easton 8. 43184326. L. (1969) Biochem. hiophys. RPS. DANNIES P. S. & LEVINE Commun. 37. 587-592. EYLARE. H. (1970) Proc. t1ahl. Acad. Sci., U.S.A. 67. 1425-143 I , KIES M. W., ALVORDE. C. & Row2 E. (1958) J . Nrurochem. 2. 261-264. MCPHERSON T. A., MARCHALON~S J. J. & LENNON V. (1970) Immunology 19. 929-933. S. P.. GRAFL. & RAPPORTM. M. (1974) Fedn. MAHADIK Proc. Fedn. Am. Socs. exp. Biol. 33. 777. MOOREB. W. (1972) Inr. Rev. Neurohiol. 5. 215-225. OFFNERH., CLAUSEN J. & FOG T. (1974) Acra neurol, Scand. 50. 221-226. OSHIRO U. & EYLARE. H. (1970) Arch. Biochem. hiophys. 188. 392-396. PERUMAL A. S. & MAHADIK S. P. (1974) Abstracts. fourth Annual Meeting of Society for Neuroscience. St. Louis, Missouri. p. 372. RAPPORT M. M. & GRAFL. (1967) in Methods in Immunology and Immunochemistry, (WILLIAMS C. A. & CHASE M. W.,eds.) Vol. 1, pp. 187-196. Academic Press, New York.

FIG. 1. Interaction of MBP with S-100 protein: effect of Ca”, Mn”, EDTA, EGTA. Veronal buffer, pH 8.6. Trough contains anti-S-100 serum, 75 pl. ( a t u p p e r : S-100 protein, 5 pg. Lower: Same + MBP. 15 p g . (bF U p p er : S-100, 5 p g + CaCI,, 10 mM. Lower: same + MBP, 15 p g . ( c t U p p e r : S-100, 5 p g + MnCI,. I mM. Lower: same MBP. 15 p g . ( d FU p p er : S-100, 5 p g + MBP, 15 p g + EDTA. I mM. Lower: S-100. 5 p g + MBP, 15 fig + EGTA, I mM. FIG. 2. Interaction of MBP with S-lo0 protein: effect of KCI and NaCI. Veronal buffer, pH 8.6. Trough contains anti-S-100 serum, 75 pl. All wells contain S-100 protein, 5 p g + CaCI,. 10 mM. (a F U ppe r, KCI, 120 mM. Lower: Same + MBP, 15 p g . ( b t U p p e r , NaCI, 120 mM. Lower: same MBP. 15 pg. ( cb Up p er , KCl, 200 mM. Lower: same + MBP. 15 pg. ( d t U p p e r NaCI, 200 mM. Lower: same + MBP. 15 p g . FIG. 3. Interaction of MBP with S-100 protein: effect of pH. Trough contains anti-S-100 serum, 75 pl. All upper wells contain S-100 protein, 5 pg CaCI,, 10 mM, and all lower wells contain, in addition, MBP, 15 pg. A11 buffer concentrations are 50 mM. (aFVerona1 buffer, pH 8.6; (b)-acetate buffer, pH 6.4: ( ctp h o sp h ate buffer, pH 7.4; (dtTris-maleate buffer, pH 7.4. FIG.4. Interaction of MBP with S-100 protein: effect of increasing the weight ratio of MBP to S-100 protein. Trough contains anti-S-100 serum, 75 p l . All wells contain S-100 protein, 5 pg and CaCI,, 10 mM. Weight ratios of MBP to $100 protein are as follows: ( a b u p p e r , 0; Lower, 0.187; ( b t U p p e r , 0.375; Lower, 0.75; (cbUpper, 1.5; Lower, 3.0; (d) Upper, 3.2; Lower, 3.9

+

+

+

Immunochemical studies of the interaction between myelin basic protein and S-100 protein.

Jourttol o/ V~,.errx.iinrii,rr?. 1976 Vol 27. pp 405-408 Pergamon Press Printed in Great Britain. IMMUNOCHEMICAL STUDIES OF THE INTERACTION BETWEEN...
1MB Sizes 0 Downloads 0 Views