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The structure of the terminal regions of the encephalitogenic basic protein from bovine myelin
ARCHIVES
OF
HIOCHEMISTRY
The Structure
AND
BIOPHYSICS
of the Terminal Protein GEORGE
136, 324-333 (1969)
Regions from
Bovine
A. HASHI!.W
AND
Basic
Myelin’ E. H. EYLAR3
San Diego, California
The Salk Institute, Received
of the Encephalitogenic
June 6, 1969; accepted
August
19, 1969
Treatment of the bovine encephalitogen (Al protein) with cyanogen bromide resulted in complete cleavage of the two methionyl-alanine linkages present in the protein, giving rise to three peptides (CBl, CB2, and CB3). Although the reaction mixture retained full encephalitogenic activity, located exclusively in peptide CB2, the antibody-combining activity was lost. Neither peptide CBl nor CB2 was active in the passive hemagglutination inhibition test; and peptide CB2 was negative in the Ouchterlony test. It was concluded that encephalitogenic site of the Al protein does not coincide with the site involved in interaction with antibody. Peptide CBl contains N-acetylalanine, and thus occupies the N-terminal region of the Al protein. The amino acid sequence of peptide CBl was determined from tryptic, chymotryptic, and peptic peptides. The complete sequence is: N-Ac-Ala-Ser-Ala-Gln-Lys-Arg-Pro-Ser-Gln-ArgSer-Lys-Tyr-Leu-Ala-Ser-Ala-Ser-Thr-Hser-OH. Thus, the first methionine residue of the Al protein is located at position No. 26 from the N-terminal acetylalanine. The internal peptide (CBZ), containing approximately 141 amino acids, has an N-terminal alanine residue and contains the only tryptophan found in the original protein. The amino acidsequence of peptide CB3, H-AlaArg-Arg-OH, is identical with the C-terminal sequence obtained for the Al protein, and reveals the location of the second methionine residue at position No. 4 from the C-terminal arginine. The C-terminal region was further clarified by treating the Al protein and peptide CB2 with carboxypeptidase. The sequence was found to be: -His-Phe-Met-Ala-Arg-Arg-OH. The C-terminal sequence of -Met-Ala-Arg-Arg-OH was also established for encephalitogenic Al proteins derived from human, monkey, dog, and guinea pig brain.
The basic encephalitogenic protein from myelin of the central nervous system is of interest because it induces experimental allergic encephalomyelitis (EAE), an autoimmune demyelinating disease, and because of its role as a membrane protein (14). 1 Supported by U. S. Public Health Grant NB 0826801 and The National Foundation. 2 U. S. Public Health Postdoctoral Fellow. Present address: The Continental Research Institute, 25 Cedar St., New York, N. Y. 3 Recipient of a Research Career Development Award, U. S. Public Healt,h Service. 324
The isolated protein appears homogeneous by polyacrylamide gel and immunoelectrophoresis (4) and accounts for approximately 30 % of the total myelin protein (4,7). It has a molecular weight of approximately 16,400 daltons, behaves as a random coil, and is highly resistant to denaturation (5). The protein, however, is highly susceptible to proteolytic digestion (6). Previous studies (8, 9) have focused attention on the amino acid sequence around the tryptophan-containing region of 16 residues which appears to contain the major encephalitogenic de-
ENCEPHALITOGENIC
terminant. The present study, part of a program (10, 11) to elucidate the complete amino acid sequence of the protein, describes the terminal regions of the molecule where the two methionine residues are located. EX.PERIMENTAL
PROCEDURE
Al proiein encephalitogen. Procedures for the isolation and characterization of the Al protein from bovine spinal cord were described elsewhere (4, 5). For the studies reported here, the Al protein from human, monkey, bovine, dog, and guinea pig brain were purified by Cellex-P column chromatography (12), and appeared homogeneous by ultracentrifugation and polyacrylamide gel electrophoresis at pH 4.5. Treatment with cyanogen bromide. Oxidation and cleavage of the C-methionyl bond in the Al protein was performed in either 70yo formic acid or 0.15 N HCl at 25” water bath for 24 hr (13). Cyanogen bromide in either solvent was added to the protein solution (20 mg/ml) to obtain a 100-400 molar excess over methionine. Separation of peptides. After incubation with cyanogen bromide for 24 hr, the reaction mixture was dried by flash evaporation at room temperature and passed through a column (86 X 1.9 cm) containing equal portions of Sephadex G-25 and G-75. The peptides were eluted with 3Ooj, acetic acid; 7.0 ml per tube was collected. The elution of the peptides was monitored by measuring the absorption at 235 and 280 rnp. The symmetric peak eluting immediately aft’er the void volume of the column was divided into two fractions, tubes 10-25 and 26-40. Further fractionation of material from tubes 2640 was achieved by gel filtration on Sephadex G-10 and G-25 column (105 X 3.2 cm) in 0.1 M acetic acid. Amino acid analysis. The amino acid composition of the peptides was determined as described elsewhere (5). Tryptophan was estimated by the Spies and Chambers procedure (14) described previously (11). Gel electrophoresis. Polyacrylamide-gel electrophoresis (4) was performed at pH 4.5. Generally, 50 pg Al protein was applied; in the case of peptides 10&200 rg was used. Hi’gh voltage electrophoresis. Electrophoresis at pH 4.7 was carried out for 1 hr at 2500 V using Whatman 3MM filter paper. After electrophoresis and air drying, the paper was stained with 0.370 ninhydrin in acetone. For preparative highvoltage electrophoresis, the peptides were identified using guide strips and eluted from the paper with water. Paper chromatography. Chromatography for
BASIC
PROTEIN
325
16-24 hr in n-butanol:acetic acid:water (4:1:5 v/v/v). Hydrazinolysis. Peptide material was first dried in the desiccator at 60” using a vacuum pump; 0.1 ml of 97% anhydrous hydrazine was added and the solution was heated at 100” in Teflon-lined screw-capped test tube for 10 hr (15). Prior to amino acid analysis, the mixture was dried in vacuum, taken up in water, and washed two times with n-heptane and once with ethylacetate. Amino acid sequence studies. Both the direct Edman method (16) and the subtractive Edman combined with the dansylation techniques (17) were used. Enzyme digestion. Treatment with trypsin, chymotrypsin, and pronase was performed at 37” in 0.1 M triethylamine bicarbonate buffer at pH 8.0. An enzyme-to-protein weight ratio of 1:50 was used. For C-terminal sequence, carboxypeptidases A and B were used; generally, carboxypeptidase A was added first to a solution of peptide in 0.05 M KHC03 at pH 7.6; after 15 min at 25”, carboxypeptidase B was added. Samples were removed at timed intervals, lyophilized, and redissolved in acetate buffer pH 2.2 for amino acid analysis. Enzyme to protein weight ratio of 1:25 was used in the case of both enzymes. Identification of the N-terminal blocking group. After hydrazinolysis, the mixture was dried. The hydrazone derivative of the N-blocking group was identified by paper chromatography (18). Acetylalanine was identified directly by paper chromatography (19), and by gas chromatography of the silyl derivative of the peptide. Immunology and EAE assay. The peptide mixture and pure peptides from the cyanogen bromide-treated Al protein were mixed with complete Freund’s adjuvant and tested for encephalitogenic activity at l&100 pg in guinea pigs as described elsewhere (4). The reactivity of the peptides with the antibody, prepared in rabbits and against the Al protein, was measured by both the Ouchterlony 1 and the passive hemagglutination inhibition test (4). In the latter case, four hemagglutinating doses of rabbit antiserum were used along with tanned chicken erythrocytes to which Al protein was adsorbed. RESULTS
Treatment of the Al protein with cyanogen bromide in 70% formic acid or 0.15 N HCl for 24 hr resulted in complete oxidation and cleavage of the C-methionyl bonds. No trace of the original Al protein was detected by polyacrylamide-gel electrophoresis of the reaction mixture at pH 4.5. Instead, a single band was seen; it moved slightly but dis-
326
HASHIM
tinctly faster than the Al protein. Highvoltage electrophoresis, however, resolved the reaction mixture into three components which are referred to as peptides CBl, CB2, and CB3. All three peptides move toward the cathode on high-voltage electrophoresis at pH 4.7 (Fig. 1). Peptide CB3, the fastest moving peptide, migrates at the same rate as lysine. Purificatim of peptides CBl, CBS, and CBS. After gel filtration of the reaction mixture on a Sephadex G-25 and G-75 column, the peptides eluted in a symmetrical peak immediately after the void volume. The peak was divided into two fractions; tubes
AND
EYLAR 2.5
TUBE
NO.
FIG. 2. The gel filtration pattern of peptides CBl, CB2, and CB3 is shown using a Sephadex G-10 (lower half) and G-25 column (105 X 3.2 cm). The peptides were eluted with 30% acetic acid and 8 ml per tube was collected; optical density at 280 rnr (--a--); optical density at 235 rnp (+).
FIG. 1. The high-voltage electrophoresis pattern at pH 4.7 of peptides from the Al protein treated with cyanogen bromide: (1) the reaction mixture; (2) peptide CB2; (3) peptide CBl; (4) peptide CB3; (5) lysine.
10-25, containing only peptide CB2 as shown by high-voltage electrophoresis, and a second fraction from tubes 2640 which contained a mixture of all three peptides as revealed by high-voltage electrophoresis. Further fractionation of the peptide mixture (tubes 2640) by gel filtration on Sephadex G-10 and G-25 (Fig. 2) produced peptide CB2 in tubes 3047, peptide CBl along with traces of CB2 in tubes 48-57, and peptide CB3 along with traces of CBl in tubes 58-75. It can be seen that this latter fraction does not have a significant optical density at 280 rnp. Further purification of peptides CBl and CB3 from tubes 48-57 was achieved by preparative high-voltage electrophoresis using the system shown in Fig. 1. Recovery and purity of isolated peptides.
ENCEPHALITOGENIC
THE
Amino
acid
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methioninec Isoleucine Leucine Tyrosine Phenylalanine Trypt0pha.n Total
BASIC PROTEIN
TABLE I Aailr~o ACID COMPOSITIONS Peptide
Alb
Protein 13 10 17 10 9 19 10 12 24 14 4 2 3 10 3 8 1 169
327
CBl
CB2
CB3
2 0 2 0 1 5 2 1 0 4
11 9 13 10 6 14 10 12 23 9
0 1 1 0 0 20
(t) 2 8 2 6 1 141
0 0 2 0 0 0 0 0 0 1 0 0 0 0 0 0 0 3
Pl
2 0 2 0 0 3 2 1 0 2 0 0 0 0 1 0 0 13
Tl
1 0 0 0 0 1 1 0 0 2 0 0 0 0 0 0 0 5
T2
T3
0 0 2 0 0 1 1 1 0 0 0 0 0 0 0 0 0 5
1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 2
T4
0 0 0 0 1 2 0 0 0 2 0 1 0 1 1 0 0 8
Cl
1 0 0 1 0 1 0 0 0 0 0 0 0 0 1 0 0 4
C2
1 0 1 0 0 1 1 1 0 0 0 0 0 0 0 0 0 5
Ccc
1 0 1 0 0 1 0 0 0 0 0 0 0 0 1 0 0 4
QThe number of amino acid residues per molecule is computed assuming one leucine or tyrosine residue for peptide CBl; two tyrosine or isoleucine residues for peptide CB2; one alanine residue for peptide CB3; one tyrosine or two alanine residues for peptide Pl; one lysine or one glutamic acid residue for peptide Tl; one proline or one glutamie acid residue for peptide T2; one Ieucine or one tyrosine for peptide T4; one lysine or one tyrosine for peptides Cl and C3; one lysine or one proline residue for peptide C2. b The number of amino acid residues per Al protein molecule was calculated from data assuming three tyrosine and one tryptophan residue, and molecular weight values near 18,OOOdaltons (12). This value is higher than previous estimates of 16,400 daltons and 142 residues (5). c Methionine coIltent of peptide CBl was determined as homoserine. Traces of homoserine and homoserine lactone were detected in peptide CB2.
Peptides CBl, CB2, and CB3 appeared homogeneous by high-voltage eleetrophoresis (Fig. 1) and end-group analysis. Peptide CB2 also appeared as a single band on polyacrylamide gel electrophoresis. Based on dry weight, 42 mg of peptide CBl, 238 mg of peptide CB2 and 2 mg of CB3 were obtained. Based on the original protein used, these values represent 11, 63, and 0.5% respectively, or a total recovery of approximately 7.5%. Size and composition of peptides CBI , CBZ, and CBS. Peptide CBl contains 20 amino acid residues, assuming 1.0 mole of leucine or proline per mole of peptide (Table I), with a calculated molecular weight of 2168. Peptide CBl contains 1 tyrosine, 2 glutamic acid residues, and a lysine to arginine ratio
of 1.O. In contrast to our earlier findings (lo), peptide CBl does not contain histidine or glycine. Peptide CB2 contains a representative of all of the amino acids, including the only tryptophan residue found in the Al protein. Based on the best fit to the amino acid analysis, it was calculated that peptide CB2 contains approximately 141 amino acid residues. As seen in Table I, good agreement exists between the sum of the amino acid residues of peptides CBl, CB2, and CB3 with the analysis of the Al protein. The composition of the Al protein shown in Table I represents a revision from a previous analysis (12). Peptide CB3, the smallest of the three peptides, contains 1 alanine and 2 arginine residues.
HASHIM
-(Ol-
3
4
5
0
AND
6
-
0°0
0 0
0
0
FIG. 3. The high-voltage electrophoresis at pH 4.7 and chromatography of peptides derived from peptide CBl after trypsin treatment: (1) lysine; (2) trypsin digest of peptide CBl; (3) chromatography of the slower migrating spot from electrophoresis showing peptides Tl (upper) and T4 (lower) ; (4) serine-lysine dipeptide; (5) chromatography of the faster migrating spot from electrophoresis showing peptides T2 (upper) and T3 (lower) ; (6) lysine.
Peptide C’Bl. Peptide CBl has an Nterminal block as demonstrated by the direct Edman and the dansylation procedures. Treatment with carboxypeptidase A for 2 hr released 0.7 moles homoserine along with 0.6 moles threonine, 0.5 moles serine, and 0.35 moles alanine per mole of peptide; the following sequence is suggested for the C-terminal region of this peptide: -AlaSer-Thr-Hser-OH. High-voltage electrophoresis of peptide CBl treated with trypsin revealed only two peptide bands (Fig. 3). These two bands were eluted and subjected to paper chromatography; each band from electrophoresis contained two peptides which were clearly separated by chromatography and are referred to as peptides Tl, T2, T3, and T4 (Fig. 3). Based on the amino acid analysis,
EYLAR
which is shown in Table I, these peptides appeared to be highly purified; the amino acid molar ratios varied within f5 % and no trace of other amino acids was seen. Three chymotryptic peptides were derived from peptide CBl and designated Cl, C2, and C3. These peptides were purified by electrophoresis alone. When examined by chromatography, they appeared homogeneous. Peptide Pl. The peptide was obtained from the pepsin digest of the Al protein obtained as described previously using Cellex-P column chromatography (8), and subsequently purified by paper chromatography. The N-terminal amino acid of peptide Pl was not detected by either the direct Edman or the dansylation technique. Carboxypeptidase A and B released 0.9 moles tyrosine, 0.8 moles lysine, 0.4 moles serine, and 0.2 moles arginine per mole of peptide Pl. These data suggest the following Cterminal sequence for peptide Pl: -ArgSer-Lys-Tyr-OH. Peptide T3. Amino acid analysis of this peptide revealed only two amino acids, serine and lysine. Based on the known specificity of trypsin, the sequence is SerLys-OH. Peptide T4. This peptide contains eight amino acid residues. Edman degradation gave the sequence H-Tyr-Leu-Ala-Ser-Ala-. Carboxypeptidase A released 0.9 moles homoserine, 0.7 moles threonine, 0.5 moles serine, and 0.5 moles alanine. Thus, the sequence of the final three residues is: (Ala, Ser)-Thr-Hser-OH. This sequence corresponds with the sequence of the C-terminal region of peptide CBl and thus positions peptide T4. Peptide Tl . Peptide Tl has an N-terminal block; no reaction was indicated by either the direct Edman or the dansylation procedure. It was concluded that this peptide is derived from the N-terminal region of peptide CBl. Treatment of peptide Tl with carboxypeptidase A and B liberated 1 mole of lysine per mole of peptide in the first 5 min. This was followed by 1 mole of glutamine and 0.5 moles alanine. Treatment with pronase released 0.9 mole each of lysine, glutamine, and alanine and 0.3 moles of serine per mole of peptide in addition t,o
ENCEPHALITOGENIC
peptide ‘1’1-1 which did not stain with ninhydrin. Peptide Tl-1 was obtained by eluting the anodic region of the electrophoretogram after high-voltage electrophoresis of the pronase digest. Hydrazinolysis of peptide Tl-1 gave alanine and serine in approximately equal molar ratios, suggesting the presence of equivalent amounts of two peptides. This was confirmed by paper chromatography of peptide Tl-1 which revealed two peptides after staining for monosubstituted amides (19). One of the spots was identical to Nacetylalanine with an R, value of 0.7. Further, gas chromatography of the silyl derivative of peptide Tl-1 gave two peaks, one corresponding to N-acetylalanine. The presence of acetate as the N-terminal block was further confirmed by identifying acetylhydrazide by paper chromatography after hydrazinolysis of peptide Tl-1. Moreover, the presence of an N-acetyl blocking group in peptides Tl, CBl, and PI, as well as the Al protein, was revealed by the identification of acetylhydrazide after hydrazinolysis. From these results, the following structure for peptide Tl is: N-Ac-Ala-Ser-Ala-GlnLys. Peptide T2. This peptide contains five amino acid residues. The Edman degradation procedure gave the sequence Arg-Prcof Ser. . . . From the known specificity trypsin and the amino acid analysis, the sequence of this peptide is H-Arg-Pro-SerGln-Arg-OH. Peptide Cl. Edman degradation of peptide Cl gave a Leu-Ala-Ser-Ala . . . sequence. Carboxypeptidase released 0.8 moles homoserine, 0.7 moles threonine, 0.5 moles serine, and 0.3 moles alanine per mole peptide giving the following sequence for peptide Cl, H-Leu-Ala-Ser-Ala-Ser-Thr-Hser-OH. Peptide C2. The direct Edman and dansylation procedures gave an H-Lys-ArgPro sequence; hydrazinolysis gave a Cterminal glutamic acid. Carboxypeptidase A and B treatment gave 0.7 moles glutamine and 0.2 moles of serine. The sequence of this peptide is H-Lys-Arg-Pro-Ser-Gln-OH. Peptide CS. An N-terminal Arg-Ser sequence was established by the dansylation procedure. Further, a C-terminal tyrosine was found by hydrazinolysis. Thus, the
BASIC
PROTEIN
329
sequence of this peptide is H-Arg-Ser-LysTyr-OH. The structure of peptide CBl. In common with the Al protein, peptide CBl has an N-terminal block; thus this peptide occupies the N-terminal region in the parent molecule. Since peptide CBl contains 20 residues, the methionine residue must be at position No. 20 from the S-terminal end of the Al molecule. The amino acid sequence of peptide CBl, shown in Table II, is constructed from the sequences of the peptides derived from trypsin, chymotrypsin, and pepsin treatment. The C-terminal region of the Al protein. Carboxypeptidases A and B were used to investigate the C-terminal region. When the Al protein was treated with these enzymes, as shown in Fig. 4, approximately 2 moles of arginine and 1 mole alanine/mole protein were rapidly released. Lesser amounts (0.5, 0.3, 0.2 moles) of methionine, phenylalanine, and histidine were subsequently released; less than 0.1 mole of valine, leucine, and isoleucine were observed. These data suggest a His-Phe-Met-Ala-Arg-Arg-OH sequence for the C-terminal region of the Al protein. Further clarification of this region of the Al protein comes from peptides CB2 and CB3. The peptide CB3, which gave alanine by the Edman degradation, has an Ala-ArgArg sequence which is identical with the last three residues of the Al protein and firmly establishes the structure of this region. The C-terminal region of peptide CB2, derived from the carboxypeptidase data shown in Fig. 4, has the sequence (Ileu, Val, Leu) -Arg-His-Phe. Thus, the region of the Al protein surrounding the second methionine residue appears to have a sequence of -Arg-His-Phe-Met-Ala-Arg-ArgOH. The absence of homoserine from peptide CB2 suggests that this amino acid is cleaved off during or after the reaction with cyanogen bromide. Using several preparations of peptide CB2, the amino acid analysis of the carboxypeptidase-treated material gave approximately 10 % of the expected homoserine or homoserine lactone. This result was veiified by hydrazinolysis of peptide CB2 which gave phenylalanine in high yield, approximately 10 times that of the homoserine. In order to evaluate the phylogenetic
HASHIM
330
AND EYLAR
TABLE THE AMINO Peptide
ACID
II
SEQUENCE
h-0. of residues
OF PEPTIDE
CBl
Sequence determineda
CBl 20 T4 8 Cl 7 Pl 13 c3 4 T3 2 T2 5 Tl 5 c2 5 Tl-1 1, 2 CBl (complete)
-Ala-Ser.Thr-Hser-OH H-Tyr-LetI-Ala-Ser-Ala-Ser-Thr-Hser-OH H-Leu-Ala-Ser.Ala-Ser-Thr-Haer-OH -Arg-Ser-Lys-Tyr-OH H-Arg-Ser.Lys-Tyr-OH H-&I-Lys-OH H-Am-Pro-Ser.Gin-Arp-OH N-4c-.ila-Ser-Ala-Gin-Lys-OH H-Lys-hrg-Pro-Ser.Gin-OH N-AC-Ala, N-AC-Ala-&r-OH N-Ac-Alrt-Ser-Ala-GIn-Lys-4rg-Pro-Ser-Gln-Ar~-Se~-Lys-Tyr-~u-Ala-Ser-Ala-Ser-Thr-Hser-OH
a The amino acid sequences shown represent that portion of the peptide for which the sequence w&s determined.
1.0
2 r” $ 0.5 P
15
30
45
60
75
90
MINUTES
0.0
15
30
45
60
MINUTES
FIG. 4. The moles of amino acid per mole protein released from the Al protein (left), and peptide CB2 (right) after treatment with carboxypeptidase at 25 and 37”, respectively. Samples were removed at indicated times and analyzed for amino acids.
of the sequence around the methionine in the C-terminal region, the Al proteins, isolated from several species (12), were chosen for study. The results shown in Table III reveal an identical sequence of -Phe-Met-Ala-Arg-Arg-OH for human, monkey, dog, and guinea pig, w well as the bovine material. Immunologic studies. The biological properties of peptides CBl and CB2 are compared to the Al protein in Table IV. In order to measure interaction with antibody, prepared against the Al protein, the inhibition of hemagglutination (PHI) and the
variation
immunodiffusion tests were used. The data reveal the absence of antibody-combining activity of both peptides CBl and CB2. However, the encephalitogenic activity of the Al protein appears to be fully retained in peptide CB2. In contrast, peptide CBl did not induce EAE when tested at 10-100 pg doses. Pyrrolidonecarboxylic acid. In order to evaluate the possible presence of pyrrolidonecarboxylic acid (PCA) at the N-terminal position of the Al protein, peptide CBl (2 pmoles), peptide Pl (2 pmoles) and the Al protein (0.5 pmole) were each incu-
ENCEPHALITOGENIC TABLE
III
THE -MOLES AMINO ACID/MOLE PROTEIN CARBOXYPEPTIDASE RELEASED BY TREATMENT OF ENCEPHALITOGENIC Al PROTEIN” Amino acid
Arginine Alanine Methionine Phenylalanine
Human
Monkey
Dog
Guinea pig
1.53 1.00 0.15 0.11
1.94 0.67 0.20 0.13
1.33 0.88 0.12 0.10
1.44 0.79 0.16 0.11
a Carboxypeptidases A and B were added together. The samples were removed for amino acid analysis after incubation at 37” for 1 hr. Other details are given in the text. TABLE
IV
THE IMMUNOLOGIC REACTIVITY OF THE PEPTIDES OBTAINED FROM CYANOGEN BROMIDE TREATMENT
Material
Pept,ide mixture Peptide Cl31 Peptide Cl32 Al protein
EAE activitp
Ouchterlonyb
17/20 o/10 9/10 18/20
Negative Negative Negative Positive
Passive hemagglutin&ion inhibitionC
1000 1000 1000 1.0
a The EAE activity was evaluated from both clinical and histologic criteria. The materials were tested at doses from 10-100 pg per guinea pig as described elsewhere (4). The figures give the ratio of guinea pigs with disease over those tested. b The Ouchterlony test (4) was performed using peptide concentrations from 0.5-10 mg/ml and observed 2 weeks. c The PHI test refers to the amount of material relative to the Al protein which inhibits hemagglutination by antiserum. The Al protein inhibits four hemagglutinating doses of antiserum at a level of 0.03-0.1 pg.
bated with a purified pyrrolidonyl peptidase preparation from Pseutiomonas jluorescens (20). After incubation at 28” for 16 hr, under conditions which release PCA from fibrinopeptides, the mixture was examined for end groups by the Edman method and for PCA by low-voltage electrophoresis (pH 4.1). No trace of PCA or an uncovered N-terminal amino acid was found. DISCUSSION
The present study confirms the presence of two methionine residues as previously
BASIC
PROTEIN
331
reported for the bovine Al protein (5) ; cleavage of the C-methionyl bonds with cyanogen bromide gave three peptides. These results are at variance with Carnegie (21) who reported only 1 methionine residue for a human encephalitogenic protein, and only 2 peptides after cyanogen bromide treatment. The action of carboxypeptidase reported here demonstrates the presence of a second methionine residue in the C-terminal region in both bovine and human Al proteins. The strategic location of the methionine residues facilitated the determination of the amino acid sequence in the Nand C-terminal regions of the Al molecule. One methionine residue is present at position No. 20 from the N-terminal end and the other at position No. 4 from the C-terminal end. Previous studies revealed a dislocation between the antibody-combining sites of the Al protein and the encephalitogenic determinant. Pepsin treatment (6), for example, rapidly destroys the antibody-combining activity (using the Ouchterlony test) whereas the encephalitogenic determinant was maintained in a peptide (peptide E) whose sequence has been determined (8, 9). The influence of the cyanogen bromide treatment on the immunologic properties of the Al protein, therefore, is of particular interest. It is clear that the encephalitogenic activity is unaffected; the reaction mixture is as potent as the untreated Al protein. Of the three peptides produced, only peptide CB2 induces EAE; full activity appears to be retained in this portion of the molecule. Although the claim has been made that peptide CBl induces histologic lesions characteristic of EAE at high relative doses (21), we found no evidence that peptide CBl was encephalitogenic either by clinical or histologic criteria. By contrast, peptide CB2, which contains the inner 141 residues, appears as active as the Al protein. These data are consistent with a previous finding of a major encephalitogenic determinant located in a small region of 16 residues containing the single tryptophan residue. This region is located within peptide CB2; it would be anticipated, therefore, that this large peptide would be highly encephalitogenie in view of the absence of appreciable
332
HASHIM
AND
secondary or tertiary structure in the Al molecule. In contrast to the full encephalitogenic activity of peptide CB2, the antibody-combining activity as measured by the PHI and Ouchterlony tests is lost. It is apparent that cleavage of the C-methionyl bonds disrupts one or more of the sites involved in interserum. Beaction with hyperimmune cause of the random, unfolded nature of the polypeptide chain, it is likely that one or more of the combining sites of the Al molecule is in the proximity of one (or both) of the methionine residues, and is directly affected by the cyanogen bromide cleavage. It is possible, however, that this treatment, indirectly influences the antigen-antibody interaction by inducing conformational changes in the remaining large peptide CB2 which partially masks the antigenic site(s). Peptide CBl, derived from the N-terminal region of the Al protein, is a basic peptide with four positive charges (two- lysine and two arginine) and only one negative charge (C-terminal carboxyl). This region also contains one of the three tyrosine residues present in the Al protein. It is noteworthy that this peptide has N-acetylalanine at the terminal position; the unreactivity of the N-terminal amino group found previously in the Al protein is thereby resolved. No evidence was found for the presence of pyrrolidonecarboxylic acid in the N-terminal position. The presence of N-acetylalanine as the terminal residue of the Al protein is of interest in comparison with calf thymus histones. It appears that the “arginine-rich” histone (F3) also contains a terminal Nacetylalanine residue (22). Other fractions, however, such as the “slightly lysine-rich” histone, contain N-acetylproline (F2b) and N-acetylserine (F2al and F2a2) at the Nterminal position (23). It should be noted that the sequence of peptide CBl, derived from bovine Al protein, differs in several respects from that obtained by cyanogen bromide treatment from a human basic protein reported by Carnegie (21). The His-Gly sequence, reported for residues 11-12 (between the Arg-Ser linkage) of the human protein is deleted in the bovine protein. The bovine protein has a N-AcAla-Ser-Ala-Gln sequence, whereas the hu-
EYLAR
man protein was reported to have an N-AcAla-Ser-Gln sequence. Thus, it appears in the latter case that an alanine residue is deleted. Finally, the bovine protein has a Leu-Ala-Ser-Ala sequence compared to a Leu-Ala-Thr-Ala sequence in the human protein. Thus, a serine residue is replaced by a threonine residue. Aside from the three aforementioned differences, the human and bovine proteins appear identical. A considerable portion of the amino acid sequence in the C-terminal region was determined using carboxypeptidase on the Al protein and peptide CB2, giving: -(Leu, Val, Ileu)-Arg-His-Phe-Met-Ala-Arg-Arg-OH. The tripeptide CB3, Ala-Arg-Arg-OH, was split from this region by the cyanogen bromide treatment. No phylogenetic variation in the -Phe-Met-Ala-Arg-Arg-OH sequence was found in the species examined, which include human, monkey, bovine, dog, and guinea pig. ACKNOWLEDGMENTS We are very grateful to Dr. R. Doolittle, University of California, San Diego, for the pyrrolidonyl peptidase studies. We thank Mr. John Griesgraber and Mrs. Barbara Thompson for technical assistance, Dr. R. Sanchez for the gas chromatography, Mrs. Ruth Goldman for the amino acid analyses, Mr. D. K. Miller for the photography, and Mrs. Bessie Wood for typing the manuscript. REFERENCES 1. ALVORD, E. C., JR., in “The Central Nervous System,” Intern. Acad. Pathol. Monograph No. 9 Williams & Wilkins, Baltimore (1968). 2. NAKAO, A., DAVIS, W., AND RABOZ-EINSTEIN, E., Biochim. Biophys. Acta 130, 163 (1966). 3. KIES,M., Ann. iv. Y. Acad. Sci. l22,161 (1965). 4. EYLAR, E. H., SALK, J., BEVERIDGE, G., AND BROWN, L., Arch. Biochem. Biophys. 130, 34 (1969). 5. EYLAR, E. H., AND THOMPSON, M., Arch. Biochem. Biophys. 129, 468 (1969). 6. HASHIM, G. A., AND EYLAR, E. H., Arch. Biochem. Biophys. 129, 635 (1969). 7. ENG, L. F., CHAO, F. C., GERSTL, B., PRATT, D., AND TAWASTSTJERNA, M. G., Biochemistry 7, 4455 (1968). 8. HASHIM, G. A., AND EYLAR, E. H., Arch. Biochem. Biophys. 129, 645 (1969).
ENCEPHALITOGENIC 9. EYLAK, E. H., AND HASHIM, G. A., Proc. N&Z. Aca.d. Sci. U. S. 61, 644 (1968). 10. HASHIM, G. A., AND EYLAR, E. H., Biochem. Biophys. Res. Commun. 34, 770 (1969). 11. EYLAR, E. H., AND HASHIM, G. A., Arch. Biochem. Biophys. 130, 215 (1969). 12. Os~rno, Y., AND EYLAR, E. H., Arch. Biochem. Biophys. Submitted. 13. GROSS, E., in “Methods in Enzymology” (Cl. H. W. Him, ed.), Vol. 11, p. 238. Academic Press, New York (1967). 14. SPIES, J., AND CHAMBERS, O., Anal. Chem. 21, 1249 (1949). 15. AKAB~RI, S., OHNO, K., AND NARITA, K., Bull. Chem. Sot. Japan 26, 214 (1952). 16. BLOMBACK, B., BLOMBACK, M., EDMAN, P.
BASIC
17.
18. 19.
20. 21. 22. 23.
PROTEIN
333
AND HESSEL, B., Biochim. Biophys. Acta 116, 371 (1966). GRAY, W. R., in “Methods in Enzymology” (C. H. W. Him, ed.), Vol. 11, p. 469. Academic Press, New York (1967). ANDREAE, W. A., Can. J. Biochem. Physiol. 36, 71 (1958). MACEK, K., in “Paper Chromatography” (I. M. Hais and K. Macek, eds.), p. 784. Academic Press, New York (1963). DOOLITTLE, R. F., AND ARMENTROUT, R. W., Biochemistry 7, 516 (1968). CARNEGIE, P. R., Biochem. J. 111, 240 (1969). PHILLIPS, D. M. P., Biochem. J. 87,268 (1963). PHILLIPS, D. M. P., Biochem. J. 107, 135 (1968).
OF
HIOCHEMISTRY
The Structure
AND
BIOPHYSICS
of the Terminal Protein GEORGE
136, 324-333 (1969)
Regions from
Bovine
A. HASHI!.W
AND
Basic
Myelin’ E. H. EYLAR3
San Diego, California
The Salk Institute, Received
of the Encephalitogenic
June 6, 1969; accepted
August
19, 1969
Treatment of the bovine encephalitogen (Al protein) with cyanogen bromide resulted in complete cleavage of the two methionyl-alanine linkages present in the protein, giving rise to three peptides (CBl, CB2, and CB3). Although the reaction mixture retained full encephalitogenic activity, located exclusively in peptide CB2, the antibody-combining activity was lost. Neither peptide CBl nor CB2 was active in the passive hemagglutination inhibition test; and peptide CB2 was negative in the Ouchterlony test. It was concluded that encephalitogenic site of the Al protein does not coincide with the site involved in interaction with antibody. Peptide CBl contains N-acetylalanine, and thus occupies the N-terminal region of the Al protein. The amino acid sequence of peptide CBl was determined from tryptic, chymotryptic, and peptic peptides. The complete sequence is: N-Ac-Ala-Ser-Ala-Gln-Lys-Arg-Pro-Ser-Gln-ArgSer-Lys-Tyr-Leu-Ala-Ser-Ala-Ser-Thr-Hser-OH. Thus, the first methionine residue of the Al protein is located at position No. 26 from the N-terminal acetylalanine. The internal peptide (CBZ), containing approximately 141 amino acids, has an N-terminal alanine residue and contains the only tryptophan found in the original protein. The amino acidsequence of peptide CB3, H-AlaArg-Arg-OH, is identical with the C-terminal sequence obtained for the Al protein, and reveals the location of the second methionine residue at position No. 4 from the C-terminal arginine. The C-terminal region was further clarified by treating the Al protein and peptide CB2 with carboxypeptidase. The sequence was found to be: -His-Phe-Met-Ala-Arg-Arg-OH. The C-terminal sequence of -Met-Ala-Arg-Arg-OH was also established for encephalitogenic Al proteins derived from human, monkey, dog, and guinea pig brain.
The basic encephalitogenic protein from myelin of the central nervous system is of interest because it induces experimental allergic encephalomyelitis (EAE), an autoimmune demyelinating disease, and because of its role as a membrane protein (14). 1 Supported by U. S. Public Health Grant NB 0826801 and The National Foundation. 2 U. S. Public Health Postdoctoral Fellow. Present address: The Continental Research Institute, 25 Cedar St., New York, N. Y. 3 Recipient of a Research Career Development Award, U. S. Public Healt,h Service. 324
The isolated protein appears homogeneous by polyacrylamide gel and immunoelectrophoresis (4) and accounts for approximately 30 % of the total myelin protein (4,7). It has a molecular weight of approximately 16,400 daltons, behaves as a random coil, and is highly resistant to denaturation (5). The protein, however, is highly susceptible to proteolytic digestion (6). Previous studies (8, 9) have focused attention on the amino acid sequence around the tryptophan-containing region of 16 residues which appears to contain the major encephalitogenic de-
ENCEPHALITOGENIC
terminant. The present study, part of a program (10, 11) to elucidate the complete amino acid sequence of the protein, describes the terminal regions of the molecule where the two methionine residues are located. EX.PERIMENTAL
PROCEDURE
Al proiein encephalitogen. Procedures for the isolation and characterization of the Al protein from bovine spinal cord were described elsewhere (4, 5). For the studies reported here, the Al protein from human, monkey, bovine, dog, and guinea pig brain were purified by Cellex-P column chromatography (12), and appeared homogeneous by ultracentrifugation and polyacrylamide gel electrophoresis at pH 4.5. Treatment with cyanogen bromide. Oxidation and cleavage of the C-methionyl bond in the Al protein was performed in either 70yo formic acid or 0.15 N HCl at 25” water bath for 24 hr (13). Cyanogen bromide in either solvent was added to the protein solution (20 mg/ml) to obtain a 100-400 molar excess over methionine. Separation of peptides. After incubation with cyanogen bromide for 24 hr, the reaction mixture was dried by flash evaporation at room temperature and passed through a column (86 X 1.9 cm) containing equal portions of Sephadex G-25 and G-75. The peptides were eluted with 3Ooj, acetic acid; 7.0 ml per tube was collected. The elution of the peptides was monitored by measuring the absorption at 235 and 280 rnp. The symmetric peak eluting immediately aft’er the void volume of the column was divided into two fractions, tubes 10-25 and 26-40. Further fractionation of material from tubes 2640 was achieved by gel filtration on Sephadex G-10 and G-25 column (105 X 3.2 cm) in 0.1 M acetic acid. Amino acid analysis. The amino acid composition of the peptides was determined as described elsewhere (5). Tryptophan was estimated by the Spies and Chambers procedure (14) described previously (11). Gel electrophoresis. Polyacrylamide-gel electrophoresis (4) was performed at pH 4.5. Generally, 50 pg Al protein was applied; in the case of peptides 10&200 rg was used. Hi’gh voltage electrophoresis. Electrophoresis at pH 4.7 was carried out for 1 hr at 2500 V using Whatman 3MM filter paper. After electrophoresis and air drying, the paper was stained with 0.370 ninhydrin in acetone. For preparative highvoltage electrophoresis, the peptides were identified using guide strips and eluted from the paper with water. Paper chromatography. Chromatography for
BASIC
PROTEIN
325
16-24 hr in n-butanol:acetic acid:water (4:1:5 v/v/v). Hydrazinolysis. Peptide material was first dried in the desiccator at 60” using a vacuum pump; 0.1 ml of 97% anhydrous hydrazine was added and the solution was heated at 100” in Teflon-lined screw-capped test tube for 10 hr (15). Prior to amino acid analysis, the mixture was dried in vacuum, taken up in water, and washed two times with n-heptane and once with ethylacetate. Amino acid sequence studies. Both the direct Edman method (16) and the subtractive Edman combined with the dansylation techniques (17) were used. Enzyme digestion. Treatment with trypsin, chymotrypsin, and pronase was performed at 37” in 0.1 M triethylamine bicarbonate buffer at pH 8.0. An enzyme-to-protein weight ratio of 1:50 was used. For C-terminal sequence, carboxypeptidases A and B were used; generally, carboxypeptidase A was added first to a solution of peptide in 0.05 M KHC03 at pH 7.6; after 15 min at 25”, carboxypeptidase B was added. Samples were removed at timed intervals, lyophilized, and redissolved in acetate buffer pH 2.2 for amino acid analysis. Enzyme to protein weight ratio of 1:25 was used in the case of both enzymes. Identification of the N-terminal blocking group. After hydrazinolysis, the mixture was dried. The hydrazone derivative of the N-blocking group was identified by paper chromatography (18). Acetylalanine was identified directly by paper chromatography (19), and by gas chromatography of the silyl derivative of the peptide. Immunology and EAE assay. The peptide mixture and pure peptides from the cyanogen bromide-treated Al protein were mixed with complete Freund’s adjuvant and tested for encephalitogenic activity at l&100 pg in guinea pigs as described elsewhere (4). The reactivity of the peptides with the antibody, prepared in rabbits and against the Al protein, was measured by both the Ouchterlony 1 and the passive hemagglutination inhibition test (4). In the latter case, four hemagglutinating doses of rabbit antiserum were used along with tanned chicken erythrocytes to which Al protein was adsorbed. RESULTS
Treatment of the Al protein with cyanogen bromide in 70% formic acid or 0.15 N HCl for 24 hr resulted in complete oxidation and cleavage of the C-methionyl bonds. No trace of the original Al protein was detected by polyacrylamide-gel electrophoresis of the reaction mixture at pH 4.5. Instead, a single band was seen; it moved slightly but dis-
326
HASHIM
tinctly faster than the Al protein. Highvoltage electrophoresis, however, resolved the reaction mixture into three components which are referred to as peptides CBl, CB2, and CB3. All three peptides move toward the cathode on high-voltage electrophoresis at pH 4.7 (Fig. 1). Peptide CB3, the fastest moving peptide, migrates at the same rate as lysine. Purificatim of peptides CBl, CBS, and CBS. After gel filtration of the reaction mixture on a Sephadex G-25 and G-75 column, the peptides eluted in a symmetrical peak immediately after the void volume. The peak was divided into two fractions; tubes
AND
EYLAR 2.5
TUBE
NO.
FIG. 2. The gel filtration pattern of peptides CBl, CB2, and CB3 is shown using a Sephadex G-10 (lower half) and G-25 column (105 X 3.2 cm). The peptides were eluted with 30% acetic acid and 8 ml per tube was collected; optical density at 280 rnr (--a--); optical density at 235 rnp (+).
FIG. 1. The high-voltage electrophoresis pattern at pH 4.7 of peptides from the Al protein treated with cyanogen bromide: (1) the reaction mixture; (2) peptide CB2; (3) peptide CBl; (4) peptide CB3; (5) lysine.
10-25, containing only peptide CB2 as shown by high-voltage electrophoresis, and a second fraction from tubes 2640 which contained a mixture of all three peptides as revealed by high-voltage electrophoresis. Further fractionation of the peptide mixture (tubes 2640) by gel filtration on Sephadex G-10 and G-25 (Fig. 2) produced peptide CB2 in tubes 3047, peptide CBl along with traces of CB2 in tubes 48-57, and peptide CB3 along with traces of CBl in tubes 58-75. It can be seen that this latter fraction does not have a significant optical density at 280 rnp. Further purification of peptides CBl and CB3 from tubes 48-57 was achieved by preparative high-voltage electrophoresis using the system shown in Fig. 1. Recovery and purity of isolated peptides.
ENCEPHALITOGENIC
THE
Amino
acid
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methioninec Isoleucine Leucine Tyrosine Phenylalanine Trypt0pha.n Total
BASIC PROTEIN
TABLE I Aailr~o ACID COMPOSITIONS Peptide
Alb
Protein 13 10 17 10 9 19 10 12 24 14 4 2 3 10 3 8 1 169
327
CBl
CB2
CB3
2 0 2 0 1 5 2 1 0 4
11 9 13 10 6 14 10 12 23 9
0 1 1 0 0 20
(t) 2 8 2 6 1 141
0 0 2 0 0 0 0 0 0 1 0 0 0 0 0 0 0 3
Pl
2 0 2 0 0 3 2 1 0 2 0 0 0 0 1 0 0 13
Tl
1 0 0 0 0 1 1 0 0 2 0 0 0 0 0 0 0 5
T2
T3
0 0 2 0 0 1 1 1 0 0 0 0 0 0 0 0 0 5
1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 2
T4
0 0 0 0 1 2 0 0 0 2 0 1 0 1 1 0 0 8
Cl
1 0 0 1 0 1 0 0 0 0 0 0 0 0 1 0 0 4
C2
1 0 1 0 0 1 1 1 0 0 0 0 0 0 0 0 0 5
Ccc
1 0 1 0 0 1 0 0 0 0 0 0 0 0 1 0 0 4
QThe number of amino acid residues per molecule is computed assuming one leucine or tyrosine residue for peptide CBl; two tyrosine or isoleucine residues for peptide CB2; one alanine residue for peptide CB3; one tyrosine or two alanine residues for peptide Pl; one lysine or one glutamic acid residue for peptide Tl; one proline or one glutamie acid residue for peptide T2; one Ieucine or one tyrosine for peptide T4; one lysine or one tyrosine for peptides Cl and C3; one lysine or one proline residue for peptide C2. b The number of amino acid residues per Al protein molecule was calculated from data assuming three tyrosine and one tryptophan residue, and molecular weight values near 18,OOOdaltons (12). This value is higher than previous estimates of 16,400 daltons and 142 residues (5). c Methionine coIltent of peptide CBl was determined as homoserine. Traces of homoserine and homoserine lactone were detected in peptide CB2.
Peptides CBl, CB2, and CB3 appeared homogeneous by high-voltage eleetrophoresis (Fig. 1) and end-group analysis. Peptide CB2 also appeared as a single band on polyacrylamide gel electrophoresis. Based on dry weight, 42 mg of peptide CBl, 238 mg of peptide CB2 and 2 mg of CB3 were obtained. Based on the original protein used, these values represent 11, 63, and 0.5% respectively, or a total recovery of approximately 7.5%. Size and composition of peptides CBI , CBZ, and CBS. Peptide CBl contains 20 amino acid residues, assuming 1.0 mole of leucine or proline per mole of peptide (Table I), with a calculated molecular weight of 2168. Peptide CBl contains 1 tyrosine, 2 glutamic acid residues, and a lysine to arginine ratio
of 1.O. In contrast to our earlier findings (lo), peptide CBl does not contain histidine or glycine. Peptide CB2 contains a representative of all of the amino acids, including the only tryptophan residue found in the Al protein. Based on the best fit to the amino acid analysis, it was calculated that peptide CB2 contains approximately 141 amino acid residues. As seen in Table I, good agreement exists between the sum of the amino acid residues of peptides CBl, CB2, and CB3 with the analysis of the Al protein. The composition of the Al protein shown in Table I represents a revision from a previous analysis (12). Peptide CB3, the smallest of the three peptides, contains 1 alanine and 2 arginine residues.
HASHIM
-(Ol-
3
4
5
0
AND
6
-
0°0
0 0
0
0
FIG. 3. The high-voltage electrophoresis at pH 4.7 and chromatography of peptides derived from peptide CBl after trypsin treatment: (1) lysine; (2) trypsin digest of peptide CBl; (3) chromatography of the slower migrating spot from electrophoresis showing peptides Tl (upper) and T4 (lower) ; (4) serine-lysine dipeptide; (5) chromatography of the faster migrating spot from electrophoresis showing peptides T2 (upper) and T3 (lower) ; (6) lysine.
Peptide C’Bl. Peptide CBl has an Nterminal block as demonstrated by the direct Edman and the dansylation procedures. Treatment with carboxypeptidase A for 2 hr released 0.7 moles homoserine along with 0.6 moles threonine, 0.5 moles serine, and 0.35 moles alanine per mole of peptide; the following sequence is suggested for the C-terminal region of this peptide: -AlaSer-Thr-Hser-OH. High-voltage electrophoresis of peptide CBl treated with trypsin revealed only two peptide bands (Fig. 3). These two bands were eluted and subjected to paper chromatography; each band from electrophoresis contained two peptides which were clearly separated by chromatography and are referred to as peptides Tl, T2, T3, and T4 (Fig. 3). Based on the amino acid analysis,
EYLAR
which is shown in Table I, these peptides appeared to be highly purified; the amino acid molar ratios varied within f5 % and no trace of other amino acids was seen. Three chymotryptic peptides were derived from peptide CBl and designated Cl, C2, and C3. These peptides were purified by electrophoresis alone. When examined by chromatography, they appeared homogeneous. Peptide Pl. The peptide was obtained from the pepsin digest of the Al protein obtained as described previously using Cellex-P column chromatography (8), and subsequently purified by paper chromatography. The N-terminal amino acid of peptide Pl was not detected by either the direct Edman or the dansylation technique. Carboxypeptidase A and B released 0.9 moles tyrosine, 0.8 moles lysine, 0.4 moles serine, and 0.2 moles arginine per mole of peptide Pl. These data suggest the following Cterminal sequence for peptide Pl: -ArgSer-Lys-Tyr-OH. Peptide T3. Amino acid analysis of this peptide revealed only two amino acids, serine and lysine. Based on the known specificity of trypsin, the sequence is SerLys-OH. Peptide T4. This peptide contains eight amino acid residues. Edman degradation gave the sequence H-Tyr-Leu-Ala-Ser-Ala-. Carboxypeptidase A released 0.9 moles homoserine, 0.7 moles threonine, 0.5 moles serine, and 0.5 moles alanine. Thus, the sequence of the final three residues is: (Ala, Ser)-Thr-Hser-OH. This sequence corresponds with the sequence of the C-terminal region of peptide CBl and thus positions peptide T4. Peptide Tl . Peptide Tl has an N-terminal block; no reaction was indicated by either the direct Edman or the dansylation procedure. It was concluded that this peptide is derived from the N-terminal region of peptide CBl. Treatment of peptide Tl with carboxypeptidase A and B liberated 1 mole of lysine per mole of peptide in the first 5 min. This was followed by 1 mole of glutamine and 0.5 moles alanine. Treatment with pronase released 0.9 mole each of lysine, glutamine, and alanine and 0.3 moles of serine per mole of peptide in addition t,o
ENCEPHALITOGENIC
peptide ‘1’1-1 which did not stain with ninhydrin. Peptide Tl-1 was obtained by eluting the anodic region of the electrophoretogram after high-voltage electrophoresis of the pronase digest. Hydrazinolysis of peptide Tl-1 gave alanine and serine in approximately equal molar ratios, suggesting the presence of equivalent amounts of two peptides. This was confirmed by paper chromatography of peptide Tl-1 which revealed two peptides after staining for monosubstituted amides (19). One of the spots was identical to Nacetylalanine with an R, value of 0.7. Further, gas chromatography of the silyl derivative of peptide Tl-1 gave two peaks, one corresponding to N-acetylalanine. The presence of acetate as the N-terminal block was further confirmed by identifying acetylhydrazide by paper chromatography after hydrazinolysis of peptide Tl-1. Moreover, the presence of an N-acetyl blocking group in peptides Tl, CBl, and PI, as well as the Al protein, was revealed by the identification of acetylhydrazide after hydrazinolysis. From these results, the following structure for peptide Tl is: N-Ac-Ala-Ser-Ala-GlnLys. Peptide T2. This peptide contains five amino acid residues. The Edman degradation procedure gave the sequence Arg-Prcof Ser. . . . From the known specificity trypsin and the amino acid analysis, the sequence of this peptide is H-Arg-Pro-SerGln-Arg-OH. Peptide Cl. Edman degradation of peptide Cl gave a Leu-Ala-Ser-Ala . . . sequence. Carboxypeptidase released 0.8 moles homoserine, 0.7 moles threonine, 0.5 moles serine, and 0.3 moles alanine per mole peptide giving the following sequence for peptide Cl, H-Leu-Ala-Ser-Ala-Ser-Thr-Hser-OH. Peptide C2. The direct Edman and dansylation procedures gave an H-Lys-ArgPro sequence; hydrazinolysis gave a Cterminal glutamic acid. Carboxypeptidase A and B treatment gave 0.7 moles glutamine and 0.2 moles of serine. The sequence of this peptide is H-Lys-Arg-Pro-Ser-Gln-OH. Peptide CS. An N-terminal Arg-Ser sequence was established by the dansylation procedure. Further, a C-terminal tyrosine was found by hydrazinolysis. Thus, the
BASIC
PROTEIN
329
sequence of this peptide is H-Arg-Ser-LysTyr-OH. The structure of peptide CBl. In common with the Al protein, peptide CBl has an N-terminal block; thus this peptide occupies the N-terminal region in the parent molecule. Since peptide CBl contains 20 residues, the methionine residue must be at position No. 20 from the S-terminal end of the Al molecule. The amino acid sequence of peptide CBl, shown in Table II, is constructed from the sequences of the peptides derived from trypsin, chymotrypsin, and pepsin treatment. The C-terminal region of the Al protein. Carboxypeptidases A and B were used to investigate the C-terminal region. When the Al protein was treated with these enzymes, as shown in Fig. 4, approximately 2 moles of arginine and 1 mole alanine/mole protein were rapidly released. Lesser amounts (0.5, 0.3, 0.2 moles) of methionine, phenylalanine, and histidine were subsequently released; less than 0.1 mole of valine, leucine, and isoleucine were observed. These data suggest a His-Phe-Met-Ala-Arg-Arg-OH sequence for the C-terminal region of the Al protein. Further clarification of this region of the Al protein comes from peptides CB2 and CB3. The peptide CB3, which gave alanine by the Edman degradation, has an Ala-ArgArg sequence which is identical with the last three residues of the Al protein and firmly establishes the structure of this region. The C-terminal region of peptide CB2, derived from the carboxypeptidase data shown in Fig. 4, has the sequence (Ileu, Val, Leu) -Arg-His-Phe. Thus, the region of the Al protein surrounding the second methionine residue appears to have a sequence of -Arg-His-Phe-Met-Ala-Arg-ArgOH. The absence of homoserine from peptide CB2 suggests that this amino acid is cleaved off during or after the reaction with cyanogen bromide. Using several preparations of peptide CB2, the amino acid analysis of the carboxypeptidase-treated material gave approximately 10 % of the expected homoserine or homoserine lactone. This result was veiified by hydrazinolysis of peptide CB2 which gave phenylalanine in high yield, approximately 10 times that of the homoserine. In order to evaluate the phylogenetic
HASHIM
330
AND EYLAR
TABLE THE AMINO Peptide
ACID
II
SEQUENCE
h-0. of residues
OF PEPTIDE
CBl
Sequence determineda
CBl 20 T4 8 Cl 7 Pl 13 c3 4 T3 2 T2 5 Tl 5 c2 5 Tl-1 1, 2 CBl (complete)
-Ala-Ser.Thr-Hser-OH H-Tyr-LetI-Ala-Ser-Ala-Ser-Thr-Hser-OH H-Leu-Ala-Ser.Ala-Ser-Thr-Haer-OH -Arg-Ser-Lys-Tyr-OH H-Arg-Ser.Lys-Tyr-OH H-&I-Lys-OH H-Am-Pro-Ser.Gin-Arp-OH N-4c-.ila-Ser-Ala-Gin-Lys-OH H-Lys-hrg-Pro-Ser.Gin-OH N-AC-Ala, N-AC-Ala-&r-OH N-Ac-Alrt-Ser-Ala-GIn-Lys-4rg-Pro-Ser-Gln-Ar~-Se~-Lys-Tyr-~u-Ala-Ser-Ala-Ser-Thr-Hser-OH
a The amino acid sequences shown represent that portion of the peptide for which the sequence w&s determined.
1.0
2 r” $ 0.5 P
15
30
45
60
75
90
MINUTES
0.0
15
30
45
60
MINUTES
FIG. 4. The moles of amino acid per mole protein released from the Al protein (left), and peptide CB2 (right) after treatment with carboxypeptidase at 25 and 37”, respectively. Samples were removed at indicated times and analyzed for amino acids.
of the sequence around the methionine in the C-terminal region, the Al proteins, isolated from several species (12), were chosen for study. The results shown in Table III reveal an identical sequence of -Phe-Met-Ala-Arg-Arg-OH for human, monkey, dog, and guinea pig, w well as the bovine material. Immunologic studies. The biological properties of peptides CBl and CB2 are compared to the Al protein in Table IV. In order to measure interaction with antibody, prepared against the Al protein, the inhibition of hemagglutination (PHI) and the
variation
immunodiffusion tests were used. The data reveal the absence of antibody-combining activity of both peptides CBl and CB2. However, the encephalitogenic activity of the Al protein appears to be fully retained in peptide CB2. In contrast, peptide CBl did not induce EAE when tested at 10-100 pg doses. Pyrrolidonecarboxylic acid. In order to evaluate the possible presence of pyrrolidonecarboxylic acid (PCA) at the N-terminal position of the Al protein, peptide CBl (2 pmoles), peptide Pl (2 pmoles) and the Al protein (0.5 pmole) were each incu-
ENCEPHALITOGENIC TABLE
III
THE -MOLES AMINO ACID/MOLE PROTEIN CARBOXYPEPTIDASE RELEASED BY TREATMENT OF ENCEPHALITOGENIC Al PROTEIN” Amino acid
Arginine Alanine Methionine Phenylalanine
Human
Monkey
Dog
Guinea pig
1.53 1.00 0.15 0.11
1.94 0.67 0.20 0.13
1.33 0.88 0.12 0.10
1.44 0.79 0.16 0.11
a Carboxypeptidases A and B were added together. The samples were removed for amino acid analysis after incubation at 37” for 1 hr. Other details are given in the text. TABLE
IV
THE IMMUNOLOGIC REACTIVITY OF THE PEPTIDES OBTAINED FROM CYANOGEN BROMIDE TREATMENT
Material
Pept,ide mixture Peptide Cl31 Peptide Cl32 Al protein
EAE activitp
Ouchterlonyb
17/20 o/10 9/10 18/20
Negative Negative Negative Positive
Passive hemagglutin&ion inhibitionC
1000 1000 1000 1.0
a The EAE activity was evaluated from both clinical and histologic criteria. The materials were tested at doses from 10-100 pg per guinea pig as described elsewhere (4). The figures give the ratio of guinea pigs with disease over those tested. b The Ouchterlony test (4) was performed using peptide concentrations from 0.5-10 mg/ml and observed 2 weeks. c The PHI test refers to the amount of material relative to the Al protein which inhibits hemagglutination by antiserum. The Al protein inhibits four hemagglutinating doses of antiserum at a level of 0.03-0.1 pg.
bated with a purified pyrrolidonyl peptidase preparation from Pseutiomonas jluorescens (20). After incubation at 28” for 16 hr, under conditions which release PCA from fibrinopeptides, the mixture was examined for end groups by the Edman method and for PCA by low-voltage electrophoresis (pH 4.1). No trace of PCA or an uncovered N-terminal amino acid was found. DISCUSSION
The present study confirms the presence of two methionine residues as previously
BASIC
PROTEIN
331
reported for the bovine Al protein (5) ; cleavage of the C-methionyl bonds with cyanogen bromide gave three peptides. These results are at variance with Carnegie (21) who reported only 1 methionine residue for a human encephalitogenic protein, and only 2 peptides after cyanogen bromide treatment. The action of carboxypeptidase reported here demonstrates the presence of a second methionine residue in the C-terminal region in both bovine and human Al proteins. The strategic location of the methionine residues facilitated the determination of the amino acid sequence in the Nand C-terminal regions of the Al molecule. One methionine residue is present at position No. 20 from the N-terminal end and the other at position No. 4 from the C-terminal end. Previous studies revealed a dislocation between the antibody-combining sites of the Al protein and the encephalitogenic determinant. Pepsin treatment (6), for example, rapidly destroys the antibody-combining activity (using the Ouchterlony test) whereas the encephalitogenic determinant was maintained in a peptide (peptide E) whose sequence has been determined (8, 9). The influence of the cyanogen bromide treatment on the immunologic properties of the Al protein, therefore, is of particular interest. It is clear that the encephalitogenic activity is unaffected; the reaction mixture is as potent as the untreated Al protein. Of the three peptides produced, only peptide CB2 induces EAE; full activity appears to be retained in this portion of the molecule. Although the claim has been made that peptide CBl induces histologic lesions characteristic of EAE at high relative doses (21), we found no evidence that peptide CBl was encephalitogenic either by clinical or histologic criteria. By contrast, peptide CB2, which contains the inner 141 residues, appears as active as the Al protein. These data are consistent with a previous finding of a major encephalitogenic determinant located in a small region of 16 residues containing the single tryptophan residue. This region is located within peptide CB2; it would be anticipated, therefore, that this large peptide would be highly encephalitogenie in view of the absence of appreciable
332
HASHIM
AND
secondary or tertiary structure in the Al molecule. In contrast to the full encephalitogenic activity of peptide CB2, the antibody-combining activity as measured by the PHI and Ouchterlony tests is lost. It is apparent that cleavage of the C-methionyl bonds disrupts one or more of the sites involved in interserum. Beaction with hyperimmune cause of the random, unfolded nature of the polypeptide chain, it is likely that one or more of the combining sites of the Al molecule is in the proximity of one (or both) of the methionine residues, and is directly affected by the cyanogen bromide cleavage. It is possible, however, that this treatment, indirectly influences the antigen-antibody interaction by inducing conformational changes in the remaining large peptide CB2 which partially masks the antigenic site(s). Peptide CBl, derived from the N-terminal region of the Al protein, is a basic peptide with four positive charges (two- lysine and two arginine) and only one negative charge (C-terminal carboxyl). This region also contains one of the three tyrosine residues present in the Al protein. It is noteworthy that this peptide has N-acetylalanine at the terminal position; the unreactivity of the N-terminal amino group found previously in the Al protein is thereby resolved. No evidence was found for the presence of pyrrolidonecarboxylic acid in the N-terminal position. The presence of N-acetylalanine as the terminal residue of the Al protein is of interest in comparison with calf thymus histones. It appears that the “arginine-rich” histone (F3) also contains a terminal Nacetylalanine residue (22). Other fractions, however, such as the “slightly lysine-rich” histone, contain N-acetylproline (F2b) and N-acetylserine (F2al and F2a2) at the Nterminal position (23). It should be noted that the sequence of peptide CBl, derived from bovine Al protein, differs in several respects from that obtained by cyanogen bromide treatment from a human basic protein reported by Carnegie (21). The His-Gly sequence, reported for residues 11-12 (between the Arg-Ser linkage) of the human protein is deleted in the bovine protein. The bovine protein has a N-AcAla-Ser-Ala-Gln sequence, whereas the hu-
EYLAR
man protein was reported to have an N-AcAla-Ser-Gln sequence. Thus, it appears in the latter case that an alanine residue is deleted. Finally, the bovine protein has a Leu-Ala-Ser-Ala sequence compared to a Leu-Ala-Thr-Ala sequence in the human protein. Thus, a serine residue is replaced by a threonine residue. Aside from the three aforementioned differences, the human and bovine proteins appear identical. A considerable portion of the amino acid sequence in the C-terminal region was determined using carboxypeptidase on the Al protein and peptide CB2, giving: -(Leu, Val, Ileu)-Arg-His-Phe-Met-Ala-Arg-Arg-OH. The tripeptide CB3, Ala-Arg-Arg-OH, was split from this region by the cyanogen bromide treatment. No phylogenetic variation in the -Phe-Met-Ala-Arg-Arg-OH sequence was found in the species examined, which include human, monkey, bovine, dog, and guinea pig. ACKNOWLEDGMENTS We are very grateful to Dr. R. Doolittle, University of California, San Diego, for the pyrrolidonyl peptidase studies. We thank Mr. John Griesgraber and Mrs. Barbara Thompson for technical assistance, Dr. R. Sanchez for the gas chromatography, Mrs. Ruth Goldman for the amino acid analyses, Mr. D. K. Miller for the photography, and Mrs. Bessie Wood for typing the manuscript. REFERENCES 1. ALVORD, E. C., JR., in “The Central Nervous System,” Intern. Acad. Pathol. Monograph No. 9 Williams & Wilkins, Baltimore (1968). 2. NAKAO, A., DAVIS, W., AND RABOZ-EINSTEIN, E., Biochim. Biophys. Acta 130, 163 (1966). 3. KIES,M., Ann. iv. Y. Acad. Sci. l22,161 (1965). 4. EYLAR, E. H., SALK, J., BEVERIDGE, G., AND BROWN, L., Arch. Biochem. Biophys. 130, 34 (1969). 5. EYLAR, E. H., AND THOMPSON, M., Arch. Biochem. Biophys. 129, 468 (1969). 6. HASHIM, G. A., AND EYLAR, E. H., Arch. Biochem. Biophys. 129, 635 (1969). 7. ENG, L. F., CHAO, F. C., GERSTL, B., PRATT, D., AND TAWASTSTJERNA, M. G., Biochemistry 7, 4455 (1968). 8. HASHIM, G. A., AND EYLAR, E. H., Arch. Biochem. Biophys. 129, 645 (1969).
ENCEPHALITOGENIC 9. EYLAK, E. H., AND HASHIM, G. A., Proc. N&Z. Aca.d. Sci. U. S. 61, 644 (1968). 10. HASHIM, G. A., AND EYLAR, E. H., Biochem. Biophys. Res. Commun. 34, 770 (1969). 11. EYLAR, E. H., AND HASHIM, G. A., Arch. Biochem. Biophys. 130, 215 (1969). 12. Os~rno, Y., AND EYLAR, E. H., Arch. Biochem. Biophys. Submitted. 13. GROSS, E., in “Methods in Enzymology” (Cl. H. W. Him, ed.), Vol. 11, p. 238. Academic Press, New York (1967). 14. SPIES, J., AND CHAMBERS, O., Anal. Chem. 21, 1249 (1949). 15. AKAB~RI, S., OHNO, K., AND NARITA, K., Bull. Chem. Sot. Japan 26, 214 (1952). 16. BLOMBACK, B., BLOMBACK, M., EDMAN, P.
BASIC
17.
18. 19.
20. 21. 22. 23.
PROTEIN
333
AND HESSEL, B., Biochim. Biophys. Acta 116, 371 (1966). GRAY, W. R., in “Methods in Enzymology” (C. H. W. Him, ed.), Vol. 11, p. 469. Academic Press, New York (1967). ANDREAE, W. A., Can. J. Biochem. Physiol. 36, 71 (1958). MACEK, K., in “Paper Chromatography” (I. M. Hais and K. Macek, eds.), p. 784. Academic Press, New York (1963). DOOLITTLE, R. F., AND ARMENTROUT, R. W., Biochemistry 7, 516 (1968). CARNEGIE, P. R., Biochem. J. 111, 240 (1969). PHILLIPS, D. M. P., Biochem. J. 87,268 (1963). PHILLIPS, D. M. P., Biochem. J. 107, 135 (1968).