Journal of Neurockrnistry. 1978. Vol. 30. pp. 401411. Pergamon Press. Printed in Great Britain

IMMUNOCHEMICAL AND BIOCHEMICAL STUDIES DEMONSTRATING THE IDENTITY OF A BOVINE SPINAL CORD PROTEIN (SCP) AND A BASIC PROTEIN OF BOVINE PERIPHERAL MYELIN (BF) GLADYS E. DEIBLER, BERNARDF. DRISCOLL and MARIANW. KIES Section on Myelin Chemistry, Laboratory of Cerebral Metabolism, National Institute of Mental Health, Bethesda, MD 20014, U.S.A. (Received 30 March 1977. Accepted 21 July 1977)

\

Abstract-A protein extracted from bovine peripheral myelin (BF) and a protein extracted from bovine spinal cord (SCP) have been shown to be identical: the proteins cross-react immunochemically with each other but not with highly purified CNS myelin basic protein. Neither BF nor SCP have anti-encephalitogenic activity. Their electrophoretic behavior is the same at three different pH values. Their apparent molecular weight by sodium dodecyl sulfate-gel electrophoresis is 13,800 550. The amino acid compositions of the proteins are essentially identical. BF and SCP each contain 2 cysteine residues and have valine at the C terminus. The 23 major tryptic peptides are identical on peptide maps. Circular dichroic analyses yield essentially identical curves, which, when computed by best-fit curve analysis, indicate that each has 0% a helix and a large percentage of B structure.

SEVERAL proteins unique to the CNS and the peripheral nervous system (PNS) have been isolated and characterized in recent years. One of these, CNS myelin basic protein, has been studied intensively because of its ability to induce experimental allergic encephalomyelitis (EAE), an autoimmune disease involving the CNS (reviewed by K m , 1973). Another is SCP, a spinal cord protein described by YO & MACPHERSON (1972). Although the authors first identified this protein in spinal cord, they reported that it was also found in peripheral nerve. SCP appears not to induce an autoimmune reaction but was reported to protect guinea pigs against the encephalitogenic activity of CNS myelin basic protein (MACPHERSON & Yo, 1973). Proteins which appeared to be unique to PNS myelin were described by BROSTOFF& EYLAR(1972) and UYEMURA et al. (1972); see also BROSTOFF et al. (1975) and KITAMURAet al. (1975). Each group assigned its own nomenclature to the same group of proteins-Po, PI and P2(BROSTOFF& EYLAR,1972) are equivalent to BR, BM and BF (KITAMURA et al., 1975). Only BF (P2) will be considered in this study. Abbreviations used: BF, ‘basic fast’ protein extracted from bovine peripheral nerve myelin (= P2);BP, myelin basic protein isolated from bovine CNS; CD, circular dichroism; CFA, complete Freund’s adjuvant (with mycobacteria); IFA, incomplete Freund’s adjuvant (without mycobacteria); CPA and CPB. carboxypeptidases A and B; diamide, azodicarboxylic-bis-dimethylamide;EAE, experimental allergic encephalomyelitis; SCP, bovine spinal cord protein; SCP-PN, same protein as SCP but extracted from bovine spinal roots; SDS, sodium dodecyl sulfate; SEBC, saline extract of bovine cord. 401

This report presents data which demonstrate the identity of BF, the peripheral myelin basic protein of KITAMURA et al. (1975). and SCP, the spinal cord protein of Yo & MACPHERSON(1972). Both preparations were kindly supplied us by the respective investigators. SCP-PN, which was isolated from spinal roots by MACPHERSON et a/. (1976) was included in the study. Also presented are some new physicochemical data on the structure of the two proteins and the results of our biological experiments which did not confirm the antiencephalitogenic activity pre& YO, 1973). viously reported for SCP (MACPHERSON A preliminary report of the, present study has been published (KIESet al., 1975). MATERIALS AND METHODS Materials. All chemicals used were reagent grade. Azodicarboxylic acid-bidimethylamide (diamide) was obtained from Vega Fox, Tucson, AZ.Dansyl amino acids, pyridine (sequanal grade), thioglycolic acid and ninhydrin were from Pierce Chemical Co., Rockford. IL; carboxypeptidases A (Code: COADFP) and B (Code: COBDFPX trypsin (Code: TRTPCK), and ashymotrypsin, Worthington Biochemical Corp., Freehold, NJ; pancreatic RNase (5 x cryst, salt-free)and cytochrome c, Mann Research Laboratories, Inc., New York, NY. Chymotrypsinogen A (bovine pancreas, Type 11) was from Sigma Chemical Co., St Louis, MO; Trasylol (aprotinin, 10,ooO KIU/ml) was from FBA Pharmaceuticals, Inc., New York, NY; Seakem agarose, Marine Colloids, Inc., Rockland, ME; TLC chromatographic sheets, Eastman Kodak Co.,.Rochester, NY; veronal buffer (B-21 Beckman Instruments, Inc., Palo Alto, CA; Minicon concentrators, Amicon Corp., Lexington, MA.

402

E. DEIBLER, BERNARDF. DRISCOLL and MARIANW. KIES GLADYS

serum. were incubated with -0.01 nmol of '2SI-labelled Preparation ofCNS and P N S proteins. All proteins used er a/. (1974). Goat antiin this study were prepared from bovine tissues. Prep- antigen, as described by DRISCOLL arations of myelin basic protein (BP) were isolated from rabbit y-globulin was used to precipitate labelled antigeneither spinal cord or brain by the batch procedure of antibody complexes. Electrophoretic analyses. Standard analyses were carried DEIBLER et a / . (1972). Preparations from each tissue (spinal cord and brain) were further purified by ion-exchange out at pH 2.4, pH 7.0 and pH 10.6 in 5% (w/w) polyacrylet a/., 1971). chromatography at alkaline pH (DEIBLER & MARTENSON. amide gels containing 8 M-urea (MARTENSON 1973). a procedure which removes impurities and separates For determination of apparent molecular weight, electrothe protein into its five differently charged components. phoresis was carried out in 1O",b(w/w) polyacrylamide gels containing 0.1% (w/v) sodium dodecyl sulfate (SDS) and Two samples of purified BP were prepared from brait+ & RUECKERT, one of which consisted of components 1-3 and another 0.1 M-sodium phosphate. pH 7.2 (DUNKER & DEIBLER, 1975). The electrophoretic which consisted only of component I. This chromato- 1969; MARTENSON graphic procedure was also used to obtain a fraction from mobilities of all proteins were measured relative to chymothe cord BP which contained the contaminant that cross- trypsinogen A (mol. wt. 25.800). Molecular weight stanreacted immunochemically with SCP and BF. dards were cytochrome c (12.400). RNase (13.700), n-chyPeripheral nerve protein (BF). prepared from spinal root motrypsin B chain (13,900) and a-chymotrypsin C chain myelin was a gift from Dr. K. UYEMURA. Spinal cord pro- (10,200). Six samples (15 pg) of each standard were run, tein (SCP) prepared from the saline extract of spinal cord. and the averages of the relative mobilities were used to was a gift from Dr. C. MACPHERSON. The saline extract plot the standard curve. Relative mobilities of BF (l5pg) of bovine cord (SEBC) was prepared in our laboratory and SCP (I5pg) were determined in parallel electroas described by Yo & MACPHERKIN (1972). SEBC was dia- phoresis in the same manner as the standards. lyzed against H 2 0 at 4°C and lyophilized. Spinal cord proAmino acid andyses. Proteins were hydrolyzed for 22 h et al., 1976) at ll0"C in constant-boiling HCI in an evacuated desictein from spinal roots (SCP-PN) (MACPHERSON was also a gift from Dr. C. M A C ~ E R S O N . & BYNUM. 1967). Tryptophan was detercator (DREYER Protection experiments. Strain 13 guinea pigs were mined by hydrolysis in the presence of 4% (v/v) thioglycolic & SASKI,1969). Hydrolyzates equivalent preimmunized with SCP. BF or brain BP in incomplete acid (MATSUBARA Freund's adjuvant (IFA) and subsequently challenged with to 60-100pg of lyophilized protein were analyzed for the brain BP in complete Freund's adjuvant (CFA). Details usual amino acids on a Beckman 121C amino acid anaof timing, amounts and route of injections are given in lyzer equipped with a Beckman Systems AA Integrator. the footnotes to Table I. The schedules were those sugOne hydrolyzate. equivalent to 375pg of BF. was anagested by Dr. C. MACPHERSON (personal communication). lyzed for methylated basic amino acids on a column Antibody induction. Antibodies to batch-purified BP (0.9 x 2Ocm) of DCdA resin (Durrum Chemical Corp.. from bovine cord were induced in two adult New Zealand Palo Alto, CA). The starting buffer and temperature were white rabbits by the following procedure: BP in physiolo- 0 . 3 5 ~sodium citrate, pH 5.80 and 28'C. respectively. At gic saline (2mdml) was emulsified with an equal volume 70 min, the buffer was changed to 0.35 N-sodium citrate, of CFA containing 85 parts of mineral oil, 15 parts of pH 4.70 and the temperature was raised to 56°C. The Arlacel A and heat-killed mycobacteria (H3,Rr). 10 mg/ml. buffer flow rate was 70 ml/h throughout the entire analysis. All injections were given intracutaneously. The initial injec- Elution times of the standard amino acids to the nearest tion of 0.5 ml contained 0.5 mg of BP and 2.5 mg of myco- min were as follows: lysine, 62; N'-monomethyllysine, 70; bacteria. Nine subsequent injections (3 times weekly for N'-dimethyllysine, 75; N'-trimethyllysine, 78; histidine, 81 ; 3 weeks) consisted of 0.5 mg of BP in IFA. Five and one- ammonia, 94; NC,NC-dimethylarginine. 145; NC,N'Chalf weeks after the last BP/IFA injection the rabbits were dimethylarginine. 152; Nc-monomethylarginine, 173; and again given a single injection of 0.5mg of BP in CFA. arginine. 180. After a further lapse of 3 months during which no signifiPerformic acid oxidations and end-group analyses. cant amount of antibody was produced they were injected Samples of BF (1.5 mg), SCP (1.4 mg) and RNase (1.5 mg) with lOmg of BP again in CFA. This injection induced were oxidized simultaneously at 0" in the dark for 16 h high levels of precipitating antibody in both rabbits. with 1 ml of performic acid as described by WIKEHART & Neither rabbit succumbed to allergic encephalomyelitis. A LEES(1973). The samples were diluted with 25 ml of H 2 0 similar schedule was used to induce antibodies to purified (4°C).frozen and lyophilized. The dry oxidized proteins were brain BP (components 1-3) in two other rabbits. no longer soluble in H 2 0 . Therefore, they were dissolved Immunodifluion. The agarose gel used for immunodiffuin 0.01 N HCI (l.Omg/ml), divided into 3 equal aliquots sion was made by dissolving 1 g of agarose in 90ml of and lyophilized. One of the aliquots was used for total 0.01 M-veronal buffer, pH 8.6, plus 10 ml of Trasylol solu- amino acid analysis. The other two were incubated with tion. Hot agarose (2.5ml) was pipetted onto each micro- carboxypeptidase A (CPA) and carboxypeptidase B (CPB) scope slide (25 x 75 mm). The slides were allowed to cool essentially as described by DEIBLER et a / . (1975). The reacand were stored for at least 2 h at 4°C before use. Patterns tion was stopped in one aliquot after 10min and in the for immunodiffusion were cut with a Boskamp gel cutter. other after 120 min by the addition of 0.2 ml of 0.5 M-acetic The distance between wells was 8 mm (center to center), acid, which brought the pH below 4. Performic acid oxiand the well volume was 10 pl. Antisera were concentrated dation was also carried out as described by Hws (1967) 4 times with a Minicon concentrator, and antigens were for 4 h at 4°C but with 2ml of performic acid per mg used in concentrations of 1 &lop1 unless otherwise speci- of protein. The same results were obtained by both profied. Diffusion was carried out for 24-48 h at 4°C. cedures. Radioimmunoassay. The binding of BF and purified brain N-Terminal analyses were carried out as described elseBP (components 1-3) to antiserum induced with batch- where (DEIBLER et al., 1975). purified cord BP was determined by radioimmunoassay. Tryptic digests and peptide maps. BF (1.5 mg) and SCP Fifty pI of antiserum, neat or diluted with normal rabbit (1.5mg) were each mixed with 30pg of trypsin which had

Identity of SCP and BF proteins been treated with L-( I-tosylamido-2-pheny1)ethylchioromethyl ketone and the mixtures incubated for 16 h at 37°C in 0.1 M-NH~HCO,,pH 8.2. saturated with toluene. The reaction mixtures were frozen and lyophilized twice. Each digest (3o(t.4oopg in lop1 of H,O) was applied to TLC sheets (20 x 20cm) and subjected to electrophoresis toward the cathode for 100 min in pH 4.7 buffer consisting ofpyridine, acetic acid and H 2 0(1 :1:48, by vol). Ascending chromatography was run for 150min in a solvent mixture consisting of pyridine:acetic acid: butanol: H 2 0 (189:38:122:151,by vol). Several peptide maps of each protein were made and sprayed with Pauly, Sakaguchi and Ehrlich reagents to determine histidineltyrosine-, arginineand tryptophan-containing peptides (EASLEY,1965). Methionine- and cysteine-containing peptides were located by platinic iodide spray (EASLEY,1965), and the cysteine-containing peptides confirmed by fluorescence quenching (KARUSHet al., 1964). Polymerization reaction. BF, SCP and SCP-PN were inet a!. cubated with diamide as described by MARTENSON (1975) and subsequently subjected to electrophoresis at acid pH. In one experiment, SCP was reduced prior to the diamide treatment. The reduction was carried out by incubating 1.5 mg SCP 15 h at room temperature in 1 ml of a solution of 8 M-urea, 1% (v/v) 8-mercaptoethanol and 0.01 M-NH~HCO,.The incubation mixture was made pH 2 by the addition of 150 pi or I N HCI. The low molecular weight reactants were removed by gel filtration in 0.01 N HCI. T h e fractions which contained reduced SCP were combined, lyophilized and subsequently incubated with diamide as described above. Circular dichroic (CD) studies. Circular dichroic spectra were obtained on the Cary 60 spectropolarimeter with CD attachment Model 6001 at full scale sensitivity of 0.04 degrees and 0.5mm optical path length. Calculations of mean residue ellipticities were based on a mean residue weight of 1 1 1 obtained from the molecular weight (13,800) and the amino acid composition (124 residues) reported in this paper. All computations of best-fit analysis of the CD curves were carried out on an MLAB computer as described by KNOW& SHRAGER (1972).

403

RESULTS Protection experiments

All of the guinea pigs preimmunized with SCP or BF (Table 1) developed allergic encephalomyelitis (EAE) with a weight loss of more than l o g when challenged with BP in CFA. Preimmunization of the guinea pigs at different sites and for different lengths of time (see footnote, Table 1) appeared t o have no effect o n the efficacy of the protection. All ten guinea pigs preimmunized with SCP succumbed t o EAE when challenged with BP in CFA. Their disease index was not significantly different from the disease index of the control guinea pigs. Only BP in IFA protected the guinea pigs completely against EAE induction. Two guinea pigs preimmunized with BF survived beyond 21 days but both showed clinical signs of EAE, weight loss and had lesions characteristic of EAE. Other experiments which are not shown in Table 1 also failed t o demonstrate any significant protective effect derived from SCP immunization. Immunodiffusion analysis.

Antisera from two rabbits immunized with batchpurified cord BP gave precipitin lines with BF, SCP, SEBC and highly purified brain BP (component 1). A typical reaction is shown in Fig. la. BF and SCP gave lines of identity (i.e. there was complete fusion with no spur formation), whereas the purified brain BP precipitin line was completely independent of the other two lines. These reactions provided the first evidence that BF and SCP might be identical proteins. There was n o indication that either SCP or BF contained antigenic sites which cross-reacted with antigenic sites in BP. When SCP-PNwas substituted for SCP exactly the same results were obtained as shown in Fig. la.

TABLE1. PROTECTION EXPERIMENTS Pretreatment IFA (8 x 0.1 ml 1FA)t Brain BP (component 1)” (8 x 0.1 mg in IFA) BF (8 x 0.1 mg in 1FA)t SCP (8 x 0.1 mg in IFAH SCP (3 x 0.1 mg in [FA)$ 05 011

Clinical No. Pos./Total

Survival No. Survivedflotal

Disease index* Mean f SE.

515

015

8.3 f 0.7

015

515

515

215 015 015

0 7.1 f 1.1 8.4 f 0.4 8.4 f 0.5 8.3 0.4 8.0 f 0.5

515

515 313 515

013 015

* Seventy of disease based on a scale of 0 to 10. 0 = no clinical or histologic signs; 10 = maximum possible clinical and histologic disease. +The first four pretreatment injections were given in the right flank, the next two in the left flank and the last two in the foot pads. A ninth injection was given in the left flank at the same time the guinea pigs were injected intracutaneously over the sternum with 0.1 mg brain BP (component 1) in 0.1 ml CFA containing 0.1 mg mycobacteria. $All three pretreatment injections were given in the right foot pad. A fourth injection of SCP in IFA was divided in two parts, one-half injected into the right foot pad, the other half into the skin at the right of the sternum. At the same time the animals were challenged with 0.05mg brain BP (component 1 ) in CFA, one-half injected into the left foot pad, the other half into the skin at the left of the sternum. $Controls were injected with 0.1 mg brain BP (component 1) in CFA intracutaneously over the sternum. // Controls were injected with 0.05 mg brain BP (component 1) in CFA. one-half injected into the left foot pad. the other half into the skin at the left of the sternum.

404

GLADYS E. DEIBLER, BERNARDF. DRIWLLand MARIANW.KIES

In contrast to the above, antisera from rabbits immunized with purified brain B P (components 1-3) gave a precipitin line only with purified brain BP (component 1) and not with B F or S C P (Fig. lb). The saline extract of bovine cord (SEBC) formed precipitin tines which fused with the lines of SCP and BF (Fig. la) but reacted so weakly with anti-brain BP that the reaction may not be visible in either Fig. l a or b. It should be noted that a large amount of SEBC (125pg) was required to give the reaction shown in Fig. la. The fact that BF and SCP reacted with an antiserum induced with batch-purified cord BP but not with an antiserum induced with purified brain BP suggested that the cord BP preparation was contaminated with SCP (BF). This was verified by subjecting the batch-purified cord BP to chromatography on carboxymethyl cellulose at high pH (DEIBLER & MARTENSON, 1973). The following fractions were collected for immunochemical analysis: 1, breakthrough peak; 2, small peak immediately following the breakthrough peak; and 3, a fraction which just preceded the peak corresponding to the least basic of the BP components. (The remaining column fractions consisted of the more basic components of BP.) All fractions were tested for the presence of an antigen which would cross-react with BF and SCP but only Fraction 3 contained such an antigen. The immunologic identity of this antigen with BF is shown in Fig. 2a. From the density of the precipitin lines in Fig. 2a one can estimate that 5Opg of Fraction 3 contains TABLE2. AMINO

ACID COMPOSITION OF

BF, SCP AND SCP-PN

BF*

SCP' (mol/lO mol leucine)

SCP-PNt

18.3

17.9 0.3 6.4 13.2 11.8 7.3

18.3 0.1 6.4 13.3 12.3

14.1

13.6 2.9 or 2.28 1.3 10.9 6.6 9.4 2.6 6.1 10.0

Amino acid Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Half cystine Glycine Alanine Valine Methionine Isoleucine Leucine ' Tyrosine Phenylalanine Tryptophan Cysteic acid Methionine sulfone N terminus C-terminal residues11

approx 1 pg of a protein or peptide which cross-reacts with BF. The amount of protein in Fraction 3 is equivalent to 5% of the total protein recovered from the column. Therefore, the contaminant represents -0.1% of the total weight of the batch-purified cord BP. The inner diffuse precipitin lines seen in both Fig. 2a and b are due to BP or fragments of BP in Fraction 3. The antiserum which gave precipitin lines (Figs. l a and 2a) with SCP, BF and purified brain BP (component 1) was analyzed by radioimmunoassay for its ability to bind 'ZSI-labelledBP and '2sI-labelled BF. By serial dilution it was estimated that 1 ml of neat serum bound approx 10nmol of BP and 20nmol of BF, i.e. there appeared to be more antibody to the contaminant than to the major protein. It was possible to estimate the amount of contaminant actually present in the batch-purified cord BP by inhibiting the binding of 12sI-labelled BF with unlabelled batchpurified cord BP. The amount of contaminant found by this procedure (0.087%) agreed well with the estimate of 0.1% obtained by comparing the precipitin lines. Polyacrylamide gel electrophoresis. Both BF and SCP, when subjected to electrophoresis towards the cathode at pH 2.4, pH 7.0 and p H 10.6 (Fig. 3) showed identical electrophoretic mobilities and sirnilar banding patterns. In other experiments not shown in Fig. 3 SCP-PN behaved identically to SCP and BF. B P had a much greater electrophoretic mobility and more complex banding pattern a t pH 10.6 than

0.0

6.6 13.1 12.1 7.1 13.4 2.9 or 2.2% 0.7 9.1 5.0

9.4 2.7 6.1 10.0 1.8 4.7

0.97

1.8$ 2.4% Blocked -Lys-Val (0.76) (1.01)

2.8 or 2.11 0.9 9.3 5.4 9.3 2.5 5.9 10.0 1.8 4.8 1.17 2.0$ 2.4$ ?

-Lys-Val (0.86) (0.98)

7.1

I .8

4.7 N.D. 2.44 1.99: ?

-Lys-Val (1.20) (1.17)

Except where indicated the results are averages of 6 independent analyses of the native proteins.

t Based on one analysis.

$ Four separate performic acid oxidations. c; Single performic acid oxidation. 11 C-terminal after performic acid oxidation, 2 h incubation with CPA and CPB.

405

FIG. 1. Immunodiffusion of SCP, BF. SEBC, and brain BP (component I ) with 2 different antisera. (a) Center well: antiserum from a rabbit injected with batch-purified cord BP. ( I ) I pg of SCP; (2) I pg of BF; (3) 1 pg of brain BP (component 1); (4) I p g of SCP; (5) 1 pg of BF; (6) 125 pg SEBC. (b) Center well: antiserum from a rabbit injected with brain BP (components 1-3). Wells ( I ) to (6) as in (a). FIG. 2. Detection of BF cross-reacting antigen in fraction 3 of batch-purified cord BP. (a) Center well: antiserum as in Fig. I(a). (1) 1 pg of BF; (2) 20pg of Fraction 3; (3) I pg of BF; (4) 5 0 s of Fraction 3 ; (5) I pg of BF; (6) lOpg of Fraction 3. (b) Center well: antiserum as in Fig. I(b). Wells (1) to (6) same as in (a).

FIG.3. Electrophoretic analyses of BF ( I ) . SCP (2) and hatch-purified cord BP (3) on polyicrylarnide gets containing 8 M-Urel. Origin ar top of gel. cathode at bottom. (a) Electrophoresis a1 pH 2.4. 25 pg of each protein: (h) electrophoresis al pH 7.0. 501c.g of each protein: (c) electrophoresis at pH 10.6. 50pg of each protein. Arrows explained in text.

8

P

407

f

,_ +?

4

. .

:.

r.

r

C FIG.4. Electrophoretic analyses of SCP and BF in polyacrylamide gels containing SDS. Gel A : 15 pg each of SCP and chymotrypsinogen A. ( I ) Polymer of SCP; (2) chymotrypsinogen A ; (3) SCP; (4) SCP-peptide (?). Gel B : 15 pg each of BF and chymotrypsinogen A. ( I ) Polymer of BF; (2) chymotrypsinogen A ; (3) BF. Gel C: 15 jig each of chymotrypsinogen A (mol. wt., 25.800). RNase (13,700) and cytochrome c (12.400). (2) chymotrypsinogen A ; (3) RNase; (4) cytochrome c.

408

6 A

B

I

2

3

4

FIG. 6. Electrophoretic patterns of diamide-treated proteins. A. Control: 2 m g of BF/ml of 8 ~ - u r e a ~ ) . 0 5 ~ - N H , H CpH O ~ ,8.3, incubated 1 h at 25°C. B. Experimental: Same as A but with 10mol of diamide/mol of protein added. Reactions stopped by the addition of an equal vol of 8 M-urea-l M-acetic acid. 100 pg of BF applied to each gel and electrophoresis carried o u t at pH 2.4, in the presence of 8 M-urea for 2-1/2 h. FIG. 7. Electrophoretic patterns of diamide-treated proteins. Control and experimental conditions the same as in Fig. 6 except different 'amounts of protein applied to the gels. ( I ) Control: 50 pg of BF; (2) control: 5 0 p g of reduced SCP; (3) experimental: 50pg of treated BF; (4) experimental: IOOpg of treated SCP which was reduced prior to this experiment.

Identity of SCP and BF proteins

the other two proteins. At the lowest pH (Fig. 3a), where most of the protein carboxyl groups were uncharged, the electrophoretic pattern of each of the three proteins consisted of one major band. BF and SCP did not migrate as far as BP and have one faint band, probably a polymer of the proteins (Fig. 3a). Batch-purified cord BP also has a polymer and other minor contaminants. None of the minor bands in BP are BF or SCP. At pH 7.0, where amide differences might be detected, the electrophoretic patterns of BF and SCP consisted of two distinct bands and a fainter third band (see arrows, Fig. 3b). At pH 10.6 (Fig. 3c) where BP exhibits 5 differently charged comet al., 1975), BF and SCP each ponents (DEIBLER showed 3 bands very close to the origin (see arrows). Most preparations of BF and SCP did not migrate either anodically or cathodically at pH 10.6, possibly because of the loss of 1 or 2 amide groups. Therefore, the isoelectric points of BF and SCP were estimated to be close to pH 10.6. The apparent molecular weight of BF and SCP was determined by electrophoresis in SDS gels. Thirteen gels containing SCP and 11 gels containing BF were run to determine their mobilities relative to chymotrypsinogen A. The average mobility for SCP was 1.527 and 1.531 for BF. The margin of error in these determinations was more than the difference (0.004) of the two averaged relative mobilities for BF and SCP. Therefore, it could be concluded that BF and SCP have the same apparent molecular weight (13,800 f 550). An example of the electrophoretic patterns is shown in Fig. 4. The same pattern was obtained for SCP (Fig. 4, gel A) whether or not the protein had been incubated in 1% /3-mercaptoethanol for 1 h at 45°C in 4 M-urea prior to electrophoresis. However, the band seen in BF (gel B, position 1) was less prominent after /3-mercaptoethanol treatment. Position 1 corresponds to 27,600, the size expected for a dimer of BF or SCP. The band at position 4 in gel A, which might be SCP peptide, was never present in BF. The excellent separation of proteins according to size achieved by this technique is shown in gel C. RNase at position 3 (mol. wt. 13,700) is clearly separated from cytochrome c at position 4 (12, 400).

-

Amino acid composition BF and SCP (from bovine spinal cord) had approximately the same amino acid composition (Table 2) when each analysis was normalized to 10 leucines. All SCP preparations contained a small amount of histidine. which indicated roughly 0.2% contamination with other proteins containing histidine. On the basis of a mol. wt. of 14,300 (calculated from the amino acid composition, Table 2), both proteins contained two cysteines, one tryptophan and probably only two prolines. Without performic acid oxidation before acid hydrolysis of the proteins, the recovery of cystine was low and proline was high. However, amino acid analysis after performic acid oxidation of

409

BF and SCP demonstrated that there were indeed two cysteines (Table 2, cysteic acid) and only two prolines present. SCP-PN (from bovine spinal roots) had approximately the same amino acid composition as BF and SCP. One analysis for methylated basic amino acids was done on a large amount of BF (375 pg). No methylated lysines, histidines or arginines were found, and n o histidine was present. End group analyses. Dansylation of SCP (4Opg) and SCP-PN (4Opg) followed by acid hydrolysis of the proteins showed the same 9 N-terminal residues (alanine, threonine, serine, tyrosine, lysine, phenylalanine, valine, leucine and glutamic acid). Therefore, a question mark was used for the N terminus of these proteins in Table 2. Dansylated BF (40jig and 80 pg) showed no N terminus (Table 2). To check for the presence of free amino acids in the samples, the entire procedure was repeated with and without hydrolysis of SCP and SCP-PN. Before hydrolysis, there were no fluorescent spots that coincided with the standard dansylated amino acids. After hydrolysis of the dansylated SCP and SCP-PN, the same 9 amino acids were identified. Before performic acid oxidation of BF and SCP, C-terminal analysis with CPA and CPB yielded only 0.25 mol of valine/mol of protein. A parallel analysis of purified brain BP (component 1) for comparison gave quantitative results for its C-terminal residues (-Ala-Arg-Arg).After performic acid oxidation of BF, SCP and SCP-PN, 10 min incubation of the proteins with CPA and CPB yielded 0.66-0.84mol of valine and W.5mol of lysine/mol of protein. The 2 h incubations (Table 2) of the proteins gave quantitative results for the C-terminal residue (valine) and almost quantitative recovery of the penultimate residue (lysine). SCP and SCP-PN consistently yielded over 0.4mol of leucine and 0.4mol of threonine/mol of protein in addition to valine and lysine. This was probably due to contamination by one or two other proteins since it could not be attributed to endopeptidase activity in the CPA and CPB. No C-terminal residues other than valine and lysine were found in the BF analyses. Parallel C-terminal analyses of RNase (performic acid oxidized) yielded 0.95mol of valine/mol of protein in 10min and quantitative yields for the last three C-terminal residues (-Ala-SerVal) in 120min. Peptide maps Both BF and SCP were subjected to tryptic digestion and peptide maps were prepared under identical conditions. Twenty-three peptides found in BF were also present in SCP. The latter had 8 extra peptides (dashed lines, Fig. 5 ) ; 5 of these were in the same regon of the map as the 23 identical peptides; 3 others had much lower electrophoretic and/or chromatographic mobilities. The 8 arginine-containing peptides and 2 cysteine-containing peptides were identical in the two proteins. Only 2 peptides from

410

GLADYS E. DEIBLER, BERNARD F. DRISCOLL and MARIANW.KIES r-..

structure (A. Stone, unpublished data) for comparison. BF had 0% a helix, 71 f 15% /3 conformation and 29 & 11% unordered structure. SCP had 0% a helix, 74 15% /3 structure and 26 7% unordered structure. DISCUSSION

The protection experiments which we have described were carried out in Strain 13 guinea pigs, but earlier experiments with NIH outbred guinea pigs gave similar results. The BP used to challenge the protected animals was column purified BP (component 1) from bovine brain. The SCP preparations C used in our attempts to prevent EAE were the same Y E ’ preparations described in other sections of this paper FIG.5 . Superposition of peptide maps of BF and SCP. but we do not know whether they were the p r e p Identical peptides are shown by solid lines; extra peptides arations used by Yo & MACPHERSONin their 1972 in SCP are indicated by broken lines. studies. We have no good explanation for our failure the BF protein reacted with Pauly’s reagent. Since to confirm their results. However, we d o not believe BF contained no histidine (Table 2) these were the use of highly inbred animals nor the fact that assumed to be tyrosine-containing peptides. The same a different preparation of BP was used for challenge 2 peptides were present on the SCP map. O n the could account for the observed differences. It is possother hand, SCP, which contained 0.3 mol/mol of his- ible that some preparations of SCP contained a small tidine. had more than 2 Pauly-reacting spots. These amount of degraded BP which was very effective at were all found in the extra peptides on the SCP map. preventing subsequent induction of EAE. Our data Three peptides containing methionine were identified indicate that only BP or encephalitogenic fragments in each protein map but only one spot was common of BP are capable of preventing EAE (DRISCOLLer a!., 1976). We cannot suggest any logical reason why to both maps. Polymerization with diamide

The presence of at least 2 cysteine residues in the BF molecule was confirmed by the formation of multiple polymeric forms when the protein was treated with diamide, a reagent known to oxidize free -SH groups (Kosow~xer a/., 1969). Fifteen polymeric forms were clearly visible on the acid gels of diamide treated BF (Fig. 6b). The same result was obtained with two different preparations of BF. O n the other hand, four attempts to polymerize SCP and SCP-PN under identical conditions were unsuccessful. However, when SCP was reduced prior to polymerization, four distinct polymeric forms could be seen on the gel, which migrated the same distance as the dimer, trimer, tetramer and pentamer of BF (Fig. 7).

Circular dichroism of RF and SCP C D curves of BF and SCP (Fig. 8) were almost identical. The small variations between the two curves could readily be attributed to instrumental variations, since the two CD spectra were not obtained consecutively. Both C D spectra showed a negative peak at 217nm, a positive peak at 196nm and a cross-over at approx 204nm, all characteristic of the CD curve of poly-L-lysine in /3 conformation. Computations were carried out by the method of best fit analysis of the experimental curves (LITOWSKY et a/., 1972) with standard curves for poly-L-lysine in the a helix form (TIMASIEFF& GORBUNOFF, 1967), /3 conformation (Greenfield & Fasman, 1969) and unordered

190

200

210

220

Wavelength,

230 240

250

260

nm

FIG.8. CD spectra of BF (-) and SCP (---). 0.5 mg of B F / d of H 2 0 , pH 5.1 ; 0.5 mg of SCP/ml of H,O, pH 6.0.

Identity of SCP and BF proteins

41 1

pure SCP or BF should have any protective effect ent preparations of SCP and one preparation of against BP challenge, since it is generally agreed SCP-PN. The C-terminal residues of BF after perfor(UYEMUP-A et a/., 1972; BROSTOFF et al., 1974; MAC- mic acid treatment were found to be -Lys-Val. In PHERSON & Yo, 1973) that SCP (BF, Pz) is not ence- addition to these residues, leucine and threonine were also liberated by CPA and CPB from SCP. The trypphalitogenic. The observation that a PNS-specific protein (BF) tic peptide maps were identical except for 8 extra p e p is identical to a protein isolated from spinal cord tides on the SCP map. Both proteins were polymer(SCP) suggests that the BF protein is not specific to ized in the presence of diamide, but SCP had to be the peripheral nervous system or the SCP which was reduced before the diamide treatment was effective. We conclude from these results that the two proisolated from cord came from the intradural roots which had not been completely removed from the teins are the same. Any difference noted appeared to be related either to the presence of impurities in SCP cord It should be noted that Yo & M A C ~ E R S O N (1972) had originally reported the presence of SCP which were not present in BF or to a limited amount in spinal roots although the method of preparation of degradation in SCP preparations Except for the and chemical characteristics of the antigen did not presence of histidine in the SCP preparations, the suggest its identity with BF. The antiserum which amount of contamination or degradation was not gave the first hint of identity of the two proteins (SCP extensive enough to alter the amino acid composition and BF) was induced with batch-purified BP prepared or molecular weight as determined by SDS gel elecfrom frozen spinal cords that had been obtained com- trophoresis. It did, however, show up as multiple mercially. These cords had not been stripped of their N-terminal groups, extra C-terminal residues and meninges and may have contained significant extra peptides on the tryptic maps. The procedure amounts of intradural roots. However, RAINE(1976) used in the SCP isolation (salt extraction of whole has reported that there is a small amount of peri- tissue) was more apt to preserve enzymatic activity pheral myelin in apparently normal spinal cord. The and lead to limited proteolytic digestion than the BF amount, though very small, may be sufficient to isolation procedure (acid extraction of peripheral account for the traces of SCP (BF) which were nerve myelin). The different behavior of the two protein prepdetected in our preparation of batch-purified cord BP. Although both BF (SCP) and CNS myelin basic arations with respect to diamide polymerization must protein have high isoelectric points, the latter is much have been due to differences in their numbers of free more basic than the former and should have been S H groups. BF was polymerized readily without completely separated from it by ion-exchange prior reduction, whereas SCP could only be polymerchromatography. When chromatography of batch- ized after reduction. Many more polymers were purified cord BP was carried out, the fraction which clearly visible in the BF pattern than in the SCP patcontained the contaminant (Fraction 3) was obtained tern. In the latter only the dimer, trimer, tetramer from the shoulder preceding the peak of BP (com- and pentamer of the undegraded protein were seen. ponent 5). This is the least basic of the BP com- In addition, there were many faint intermediate bands ponents and is the one most difficult to obtain in in the electrophoretic pattern of polymerized SCP not pure form. None of the other column fractions had found in the electrophoretic pattern of polymerized BF. These faint bands could have been polymers of reactions of identity with BF and SCP. From the amount of Fraction 3 recovered from partially degraded SCP present in small amounts in batch-purified cord BP and the amount of this frac- the original preparation. The isolation conditions tion required to give a precipitin line comparable to could readily explain oxidation of cysteine to cystine 1 pg BF it was estimated that the batch-purified cord in SCP4.e. long term exposure to pH values above BP contained -0.1% BF (SCP). This figure was 4 in the absence of a reducing agent. found by radioimmunoassay to be 0.087%. Even Evidence for some polymerization in both proteins though present in such small amounts, the contami- was observed in our SDS sizing experiments. Both nant induced roughly twice as much antibody as did SCP and BF patterns had a faint band (Fig. 4, posthe CNS basic protein itself. In fact, the trace of BF ition 1, gels A & B) with lower mobility than chymo(SCP) in batch-purified cord BP would never have trypsinogen A. This band was assumed to be a dimer been detected had it not been so highly antigenic. from its position on the gel, which corresponded to Chemical studies also demonstrated that BF and a molecular weight of -27,600. The fact that it did SCP were the same proteins: the electrophoretic be not disappear completely after &mercaptoethanol havior of the two in three different buffers (PH2.4, treatment meant that the reducing conditions were 7.0 and 10.6) was identical. Their apparent molecular not drastic enough for complete reduction of the weights on SDS gels were the same. The C D spectra SS-bonds. A faint band with the same mobility indicating 70% /I structure were identical. Their as cytochrome c'was visible on SCP but not BF patamino acid compositions were the same except for terns. This may have been the SCP-peptide described the presence of a small amount of histidine in SCP by Yo & MACPIERSON(1972). (0.3 mol/mol protein). No N-terminal residue was The chemical studies which confirmed the identity detected in BF whereas nine were found in two differ- of SCP and BF also led to several new observations

I

412

GLADYS E. DEIBLER.BERNARD F. DRIKOLLand MARIAN W. KIES

regarding their nature. One was the presence of at least 2 residues of cysteine and the complete absence of histidine. In addition. we identified the C terminus as -Lys-Val. BROSTOFFet al. (1975) reported lysine as the C terminus for P2 which was the term they used for the BF protein. We obtained essentially theoretical recoveries of valine as the C-terminal residue and lysine as the penultimate residue after the molecule had been oxidized with performic acid to expose the C terminus to CPA and CPB activity. The resistance of SCP and BF to CPA and CPB activity is easily understood in the light of their observed conformations. Both proteins by CD analysis contained over 70% /I structure, the highest content of /I structure yet reported for a soluble globular protein whereas RNase contains only 38% /I structure (calculated from X-ray crystallography, KARTHAet al., 1967). Our CD results have been confirmed by UYEMURA et al. (1977). The electrophoretic analyses revealed the presence of some heterogeneity in BF and SCP, but less than that known to occur in the CNS basic protein. The amide content of P2 (BF) has been reported by BROSTOFF et a/. (1974) to be 14.3 & 1.5 mol/mol of protein, so it is possible that small differences in amide content could have accounted for the heterogeneity observed in these studies. We d o not believe that this charge heterogeneity which we observed in B F and SCP could explain the /I and y forms of SCP reported by Yo & MACPHERSON (1972) and MACPHERSON et al. (1976). We suspect that the two forms (y- and fi-SCP) are related instead to the state of oxidation of different preparations. Acknowledgements-The authors wish to thank the following people: Dr. K. UYEMURA for the samples of BF; Dr. C. MACPHERSON for the samples of SCP and SCP-PN; Dr. A. STONEfor the C D analyses and the interpretation of them; Dr. A. KRAMER for the tryptic digestion, peptide maps and SDS-gel electrophoresis; Mrs. M. BACONand Ms. M. ADAMSfor their technical assistance with the immunodiffusion analyses. This work was supported in part by a grant from the National Multiple Sclerosis Society.

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DEIBLERG. E.. MARTENSON R. E., KRAMERA. J., KIES M. W. & MIYAMOTO E. (1975) J. hiol. Chem. 250, 7931-7938. W. J. & BYNUME. (1967) in Methods in EnzymoDREYER logy (HIRSC. H.W., ed.) Vol. XI, pp. 32-39. Academic Press. New York. DRISCOLL B. F.. KIESM. W. & ALVORD E. C., JR. (1974) J. lmmunol. 112, 392-397. DRISCOLL 9. F., KIESM. W. & ALVORD E. C., JR. (1976) J. lmmunol. 117, 11&114. DUNKERA. K. & RUECKERT R. R. (1969) J. hiol. Chem. 244, 5074-5080. EASLEYC. W. (1965) Biochim. biophys. Acta 107, 386388. GREENFIELD N. & FASMAN C. D. (1969) Biochemistry 8, 410841 16. HIRS C. H. W. (1967) in Methods in Enzymology (HIRS C. H.W., ed.) Vol. XI. pp. 59-62. Academic Press. New York. D. (1967) Nature, Lond. KARTHAG., BELLOJ. & HARKER 213, 862-865. KARUSHF., KLINAMN. R. & MARKSR. (1964) Analyt. Biochem. 9. 1W114. KIES M.W. (1973) in Biology of Brain Dysfunction (GAULL G. E.. ed.) Vol. 2. pp. 185-224. Plenum Press, New York. KIESM. W.. DEIBLER G. E., KRAMER A. J.. MACPHERSON K. (1975) Trans. Abstr. 5th Int. Meet. C. F. & UYEMURA lnt. SOC.Neurochem, Barcelona, p. 419. KITAMURA K., YAMANAKAT. & UYEMURA K. (1975) Biochim. biophys. Acta 379, 582-591. KNOTTG. D. & SHRAGERR. J. (1972) in Computer Graphics: Proc. SIGGRAPH Computers in Medicine Symposium. Vol. 6, No. 4, pp. 138-151. ACM SIGGRAPH Notices. KOWWERN. S.,KOSOWERE. M. & WERTHEIM 9. (1969) Biochem. Biophys. Res. Commun. 31, 593-596. I., BLAUERG., ENLARDS. & BETHEIL J. J. (1972) LITOWSKY Biochemistry 11, 21762181. MACPHERSON C. F. C. & Yo S.-L. (1973) J. lmmunol. 110, 1371-1375. MACPHERSON C. F. C., ARMSTRONG H. & TAN0. (1976) J. rmmunol. 116, 227-231. MARTENSON R. E. & DEIBLERG. E. (1975) J. Neurochem. 24, 79-88. MARTENSONR. E., DEILILERG. E. & K r ~ sM. W. (1971) J. Neurochem. 18, 2417-2426. G. E. & KRAMERA. J. (1975) MARTENSON R. E., DEIBLER J. Neurochem. 24, 959-962. H.& SASKIR. M. (1969) Biochem. Biophys. MATSUBARA Res. Commun. 35, 175-181. RAINEC.S. (1976) J. Neurocytol. 5, 371-380. TIMASHEFF S. N. & GORBUNOFF M. J. (1967) Annual Reuiew of Biochem. (BOYERP. D., ed.) Vol. 36 I. pp. 13-53, Annual Reviews, Palo Alto, CA. UYEMURA K., KATO-YAMANAKA T. & KITAMURA K. (1977) J. Neurochem. 29, 6 1 4 8 . UYEMURA K.. TOBARI C., HIRANOS. & TSUKADA Y. (1972) J. Neurochem. 19, 2607-2614. WIKEHARTD. R. & LEES M. 9. (1973) J. Neurochem. 20, 1303-1315. Yo S.-L. & MACPHERSON C. F. C. (1972) J . Immunol. 109, 1009-1016.