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Allergic encephalomyelitis: Enzymatic degradation of the encephalitogenic basic protein from bovine spinal cord
ARCHIVES
OF
BIOCHEMISTRY
Allergic
AND
BIOPHYSICS
Encephalomyelitis:
Encephalitogenic GEORGE The Salk Institute Received
Basic
635-644 (1969)
129,
Enzymatic Protein
from
A. HASHIM for Biological
AND
Studies,
July 12, 1968: accepted
Degradation Bovine
of the
Spinal
Cord’
E. H. EYLAR San Diego, October
California
92112
22, 1968
The basic protein encephalitogen, wit,hnut Drier denaturation, was rapidly digested with such proteolytic enzymes as trypsin, pronase, chymotrypsin, and pepsin; from N-90% of the proteolysis occurred within 1 hr. The number of peptides released by these enzymes after 2 hr were about 28, 54, 20, and 14, respectively. Each proteolytic digest was chromatographed on Sepha,dax G-25 and G-75 in order to separate the peptides from large partially digested material. Only the peptide fractions obtained from the pepsin digestion possessed significant activity in inducing experimental allergic encephalomyelitis (EAE). One of the peptide fractions had higher encephalitogenic activity, on a weight basis, than either the original protein or the unfractionated digest. In contrast, the chymotryptic digest, while lacking encephalitogenic activity, possessed antibody-combining activity as shown by the Ouchterlony and passive hemagglutination-inhibition (PHI) test. The pepsin digest produced peptides which gave a negative Ouchterlony test. The tryptic peptides were negative in all tests-Ouchterlony, PHI, and EAE induction. These results suggest that the encephlitogenic and antibody-combining activities reside in separate or overlapping regions of the polypeptide chain. The retention of encephalitogenic activity in relatively small pepsin peptides further suggests that the EAE determinant may be defined by a region of the polypeptide chain lacking a folded tertiary structure.
About 30 % or more of the tot’al protein in in bovine myelin is an encephalitogenic basic protein that can be isolated in high yield and purity (1). When care is taken to prevent proteolysis in situ, the encephalitogen comprises more than 95% of t’he basic protein fraction. This protein, referred to as the Al protein, has been characterized in detail (2). It has a molecular weight of 16,400, corltains a high percent,age of lysine, arginine, and glycine and possesses a highly ext)ended conformation. Preparations of basic protein from brain or spinal cord have been variously reported to have molecular weights of from 4400 to 50,000 daltons (3-S). The widely differing result’s have been ascribed to 1 This work was supported by a grant from the National Foundation to Dr. Jonas Salk, in whose laboratory the work was carried out. 2Presently Postdoctoral Fellow, U. S. Public Health Service.
several factors: a nat~ural distribution of a family of proteins of varying size (9) ; a small peptide which binds t’o various macromolecules such as proteins or gangliosides (5) ; proteolysis during preparation at acidic pH (4); and aggregation-dissociation phenomena (8). JIoreover, it has been shown (1) that in situ degradation of t’he basic protein encephalitogen ma.v occur during lyophilization or incubation of the spinal cord at 4” or higher and perhaps for this reason mixtures of peptides which induce EAE are obtained from spinal cord (5, 9). The present study was initiated to examine the effect of knoll proteolytic enzymes in producing biologically active peptides. In the present study, t,he effect,s of trypsin, chymotrypsin, pepsin, and pronase on the Al prot’ein were determined. Differing results have been reported on the effect, of proteolytic enzymes in producing encephali635
636
HASHTM
AND
togenic peptides from basic proteins derived from the central nervous system; Carnegie et al. (10) reported that trypsin treatment for 3 hr resulted in highly encephalitogenic peptides; Caspary and Field (6) reported a partial loss of activity after 18 hr with trypsin; and Kies et al. (8) reported complete destruction of encephalitogenic activity under similar conditions. Our results are generally in agreement with those of Kies et al. (8) since trypsin treatment of the Al protein did not yield encephalitogenic peptides. However, highly encephalitogenic peptides were obtained from the pepsin digest of the Al protein. EXPERIMENTAL
PROCEDURE
Trypsin, specifically treated by the method of Kostka and Carpenter (11) to inhibit possible chymotrypsin, pronase, chymotryptic activity, and pepsin (Worthington Biochemical Corp.) were separately prepared in water and added to aliquots of the protein solution to provide an enzyme-toprotein ratio of 1:50. Incubation with gentle shaking was carried out in a 37” water bath for varying periods of time. A drop of toluene was added to the incubation mixture to prevent bacterial growth. The rate of proteolysis of the Al protein was followed periodically by analysis of small aliquots of the digestion mixtures using the ninhydrin technique (12). Hydrolysis with pepsin was carried out after adjusting the pH of the solution to 3.0 with acetic acid. Upon completion of the digestion, the digest was lyophilized. Fractionation of the enzymic digests. The peptide mixtures were applied to a column (4X100 cm) of Sephadex G-25 @O-cm layer) and G-75 (50-cm layer). The column was equilibrated for at least 24 hr with the eluting fluid (0.1 N acetic acid) after which the sample in a small volume of 0.1 N acetic acid was applied. Elution was carried out at room temperature; 10 ml per tube were collected. The elution pattern was followed by measuring the optical density of the tube contents at 280 rnp. The tubes related to each peak were combined; and then dried by lyophilization. Polyacrylamide gel electrophoresis. Electrophoresis of the Al protein and the enzymic digests, using the disc technique in polyacrylamide gel, was carried out at pH 4.2 according to the methods of Reisfeld et al. (13). Using 5(tlOO rg of the digestion mixtures, electrophoresis was run at 1 mA per tube for 15 min. This was followed by 7 mA per tube for 35 min. High-voltage electrophoresis. High-voltage electrophoresis was carried out with Whatman 3 MM
EYLAR
filter paper for 1 hr at 2506 V in pyridine:acetic acid:n-butanol:water (1:1:2:36 v/v/v/v) at pH 4.7, The chromatogram was air-dried and stained with 0.3yo ninhydrin in acetone. Peptide mapping. A tryptic digest (0.25 mg) was applied 2 in. from the edge of a Whatman 3 MM filter paper (18 X 24 in.) and chromatographed for 18 hr in n-butanol:acetic acid: water (4: 1:5 v/v/v upper phase) and air-dried. This was followed by high-voltage electrophoresis at right angles to the chromatography direction at 2500 V for 60 min at pH 4.7 in pyridine:acetic acid:n-butanol:water (1:1:2:36 v/v/v/v). The chromatogram was then air-dried and stained with 0.3% ninhydrin in acetone. Amino acid analysis. The amino acid composition was determined as described previously (2). Immunologic analysis. Aliquots of the enzymic digests and peptide fractions were prepared in 0.15 M NaCl. These solutions were applied to Ouchterlony plate(s) at original, l:l, 1:2, and 1:4 dilutions according to procedures described elsewhere (1). The antiserum used for this test was prepared from rabbits as described previously (1). The passive hemagglutination test (1) was performed using tannic acid-treated chicken erythrocytes, and rabbit antisera prepared against the purified Al protein or an acid extract of bovine cord as described previously (1). Encephalitogenic activity. The enzymic digests and fractions therefrom were mixed with complete Freund’s adjuvant (CFA) and tested for encephalitogenic activity at 100 and 10 pg in guinea pigs as described previously (I). RESULTS
Proteolysis of the Al protein was found to proceed rapidly in the case of each enzyme tested; at 50 min, digestion is 80-90 % complete (Fig. 1). The rate of digestion is particularly fast for trypsin and is more than 55% complete after 15 min. The maximum number of peptides generated by the proteolysis, expressed as leucine equivalents, occurred with pronase followed in order by trypsin, chymotrypsin, and pepsin. An approximation of the number of peptides produced by the proteolysis can be made from the leucine equivalents based on the ninhydrin values; for pronase, trypsin, chymotrypsin, and pepsin, respectively; the results after
3 hr were 33.6, 17.4, 13.6, and 6.7 moles of peptide per mole protein. These figures represent a minimum that peptides and
value some
since it is known amino acids give
E?;CEPHALITOGE?JIC
PEPTIDES
637
MINUTES
FIQ. 1. The time course is shown of the digestion of the Al protein with proteolytic enzymes pronase (-a-), trypsin (-0-), chymotrypsin (-A-), and pepsin (-A-). The degree of hydrolysis, expressed as pmoles leucine, was determined from ninhydrin values measured at various times and compared with ninhydrin values produced by n-leucine standards. Generally, 10 mg Al protein per ml was incubated with 0.2 mg of enzyme at 37“. A 0.1 M triethylamine-bicarbonate buffer, pH 8, was used for all enzymes except pepsin.
i
FIG. 2. The polyacrylamide gel electrophoretic patterns of the proteolytic digests of the Al protein after 1 and 4 hr at pH 4.2 are shown. Electrophoresis was performed for 50 min in order to give optimal resolution of the peptides. The gels are: (1) Al protein; trypsin digest, 1 hr (2) and 4 hr (3); chymotrypsin digest, 1 hr (4) and 4 hr (5); pepsin digest, 1 hr (6) and 4 hr (7); pronase digest, 1 hr (8).
lower ninhydrin values in general than leutine (14). It is apparent that pronase and trypsin digestion is more extensive than that due to pepsin. When the enzymic digests were examined by electrophoresis on polyacrylamide gel
(Fig. 2), it was seen that the band corresponding to the Al protein was not detectable after 1 hr at the concentrations used. The rapid digestion noted above with pronase and trypsin was apparent from the gel patt’erns; t,he only distinct bands arose from
638
HASHIM
AND EYLAR
small fast-moving peptides. Two diffuse bands were also seen in the tryptic digest. Moreover, with t’he tryptic and pronase digests, no change was observed when compared aft,er 1 hr and 4 hr of incubation. However, the chymotryptic and pepsin digests yielded several peptides of various sizes and charge as shown by the electrophoretic patterns. In most cases they migrat,ed more slowly than the fast band noted with the pronase and tryptic digests. The 4-hr peptide pattern of the chymotryptic digest showed two additional fast-moving bands not present in the I-hr sample. It should be noted that the bulk of the peptides from the pepsin digest moved less rapidly than the peptides from the other digests, which is indicative of larger size or less charge. Generally, a lOO-pg peptide material was applied to the gels in order to detect as many peptides as possible; for the Al protein, 25 pg was routinely used. Only the larger 5( )-
2.4 -
z* -
1.6 -
: 22
1.2 -
d 0.8 -
0.4 -
5.0 -
4.5 -
1
4.0 -
(3a.l
3.5 4c )-
3.0 2 c?g 6
3.c )-
2.0 -
c
d d
2.5 -
1.5 -
1.0 -
20
1.0 TUBE
i 40
60
80
100
I20
TUBE
140
160
180
NO.
3. The elution patterns of the enzyme digests of the Al protein using trypsin (a), chymotrypsin (b), and pepsin (c) are shown using columns of Sephadex G-25 and G-75 as described in the Experimental section. The elution of peptides was followed by optical density measurement at 280 mp. Ten-milliliter fractions were collected. FIG.
No
peptides appeared after electrophoresis since the smaller peptides would either migrate out of the gel or would be eluted from the gel during the staining and washing procedures. Preparation of peptide families from the enzyme digests. The enzymic digests of the Al protein derived from pepsin, trypsin, and chymotrypsin treatment were applied to a column containing Sephadex G-75 and
ENCEPHALITOGENIC
FIG. 4. The gel electrophoresis patterns at pH 4.2 are shown for the small peak preceding the major peptide peak (Fig. 3) obtained by gel filtration of digests of the Al protein using trypsin (2 hr), chymotrypsin (1.5 hr), and pepsin (2 hr). The gels are: (1) 81 prot)ein; (2) tryptic digest; (3) chymotryptic digest.
I ‘-
1
2
3
4
5
PEPTIDES
639
Sephadex G-25. The elution patterns, as recorded by optical density at 280 mc(, were basically similar (Fig. 3a, b, c). In each case the enzyme emerged first, near t’he void volume of the column (430 ml), followed by a small intermediat,e peak and then bv the bulk of the peptide material. It is evident that the small intermediate peak contains large peptides in addition to material which migrates on electrophoresis as did the original Al protein (lcig. 4). This lat’ter material may represent slightly degraded Al protein rather than the intact molecule. Selected tubes from each peptide peak were examined for peptides by high-voltage elect,rophoresis at pH 4.7 as shown in Figs. 5-7. This technique provides a clear indication of the distribution of t’he individual peptides over the elution curve and is particularly well illustrated by the tryptic peptides shown in E’ig. 5. From these patterns the total number of peptides can be closely approximat’ed since they are separated according t’o bot#h size and charge. The appearance or disappearance of any given peptide can
6
FIG. 5. High-voltage electrophoresis on paper at pH 1.7 of selected tubes from the major peak obtained by gel filtration of the 2.hr trypsirr digest showu in Fig. 3a. The 0.25.mg sample was applied at the origin (0). Electrophoresis was performed at 2500 V for GOmin. Other details are given in the test. The peptides are from: (1) tube 111, (2) tube 114, (3) tube 118, (4) tube 121, (5) tube 123, (6) tube 126, (7) tube 130, (8) tube 134, (9) tube 139, (10) tube 142, (11) tltbe 148, (12) trypsin digest.
640
HASHIM
AND
EYLAR
FIG. 6. High-voltage electrophoresis on paper at pH 4.7 of selected tubes from the major peak obtained by gel filtration of the 1.5-hr chymotrypsin digest shown in Fig. 3b. Other details are given in Fig. 5 and in the text. The peptides are from (1) chymotrypsin digest, (2) tube 75, (3) tube 112, (4) tube 118, (5) tube 122, (6) tube 126, (7) tube 130, (8) tube 151.
easily be followed by its relative intensity in the electrophoretic pattern. For trypsin, 28 peptides were identified in this way as shown in Fig. 5; for chymotrypsin and pepsin (Figs. 6 and 7) 20 and 14 peptides, respectively, were counted. These numbers are appreciably higher than estimated from the ninhydrin values (Fig. 1). This technique, therefore, provides an alternate method of peptide mapping to the electrophoresis-chromatographic procedure generally used. A peptide map of the trypsin digest, shown in Fig. 8 for comparison, reveals 29 peptides, and is in agreement with the results obtained by gel filtration and electrophoresis. Immunologic activity 0f the peptide frac-
tions. Pronase, trypsin, and chymotrypsin digests of the Al protein formed distinct precipitating bands when the digests were tested by immunodiffusion against rabbit antiserum prepared against the purified protein (Table I). A positive test was obtained, whether the enzyme treatment was carried out for 1 hour or for 24 hr. By contrast, pepsin treatment of the Al protein for as little as 30 min destroys the reactivity in the immunodiff usion test (Table I). It is seen (Table II) that material in the small peak representing partially digested protein and large peptides forms a precipitin band in the immunodiff usion test in the case of the trypsin (tubes 50-60) and chymo-
1
2
3
4
5
FIG. 7. High-voltage electrophoresis on paper at pH 4.7 of major peak obtained by gel filtration of the 2-hr pepsin digest Fig. 3c. Other details are given in Fig. 5 and in the text. The treated Al protein, (2) chymotrypsin digest, (3) tubes 106-114, 119-125, (6) tubes 126-138.
a c 0 w A W
6 selected fractions from the of the Al protein shown in peptides are from: (1) un(4) tubes l&118, (5) tubes
‘J _-... t-.-J --0-
..-.p
: .__’ r
CHROMATOGRAPHY
-
FIG. 8. The mapping of peptides obtained by tryptic digestion of the Al protein. Chromatography was performed for 18 hr in n-butanol:acetic acid:water (4:1:5 v/v/v, upper phase), and high-voltage elect,rophoresis u-as run at right angles moving toward the cathode. Other details are given in the text.
642
HASHIM TABLE
AND
I
THE
ANTIBODY-COMBINING ACTIVITY OF ENZYMIC DIGESTS OF THE Al PROTEIN as MEASURED BY THE OCCHTERLONY TECHNIQUE: Digestion time
ElK.YlIle
Concentrationa
(mg/ml)
5.0
2.5
1.25
0.6
+ + + + -
+ + + + -
+ tr + + -
+
+ + +
+ + +
+ +
(hr)
Trypsin Chymotrypsin Pepsin Pronase Untreated protein
Al
1 21 1 24 0.5 1 1 24
EYLAR
tein (Table II). Only the peptide fractions from the pepsin digest showed significant encephalitogenic activity; the peptide fraction represented by tubes 119-125 appeared to be at least as active on a weight basis as the untreated Al protein. By contrast, the tryptic and chymotryptic peptides showed negligible encephalitogenic activity. DISCUSSION
+ tr + +
a Each sample was tested using the concentrations of digested protein shown. (+) refers to a strong band generally developed in the first 3 days; (-) refers to the absence of a band, and (tr) refers to a faint trace of a band. Observations were made for a minimum of 28 days. Control sera, obtained either from animals prior to sensitization, or from animals injected with Freund’s adjuvant alone, was negative.
trypsin (tubes 60-90) digests. The only peptide fraction giving a positive precipitin band is found in tubes 105-114 from the chymotryptic digest. This chymotryptic fraction, when examined by high-voltage electrophoresis as shown in Fig. 6, reveals several peptides which move toward t,he cathode. After pepsin digestion, none of the fractions gave a precipitin band on immunodiffusion. Many of the peptide fractions from the pepsin and chymotryptic digests retained antibody-combining activity as demonstrated by the passive hemagglutinationinhibition (PHI) t’est. For pepsin peptides, the activities ranged from $4 to 364 that of the intact Al protein; for the chymotryptic peptides, the activities ranged from go to x4. In contrast, none of the fractions obtained from tryptic digests were positive in the PHI test even at 1000~pg levels (Table II), except the fraction representing the undigested or “core” material. Induction of EAE. In general, the unfractionated proteolytic digests appeared less encephalitogenic than the original Al pro-
With the isolation of a highly purified encephalitogen (Al protein), it was of interest to investigate the effect of specific prot’eolytic enzymes on immunologic react,ivity in order to explore the relationship between chemical structure and biological function. The results clearly show that of the four proteolytic enzymes tested, only pepsin resulted in the formation of pept’ides that were encephalitogenic. Past studies on the proteolysis of basic proteins from bovine spinal cord are difficult to interpret because the original material was not precisely characterized in many cases (6, 8). Furthermore, comparison with other data is difficult since slight changes in the incubation conditions could modify the number and activity of the peptides produced; there may also be a question of enzyme purity when working w&h trypsin and chymotrypsin. Although we found t,hat pepsin digestion produced highly encephalitogenic peptides, ot’hers have reported (15) that TCA-soluble peptides, derived from pepsin treatment of encephalitogenic protein, were inactive. It was reported (S) also that papain digestion liberated TCA-soluble encephalitogenic peptides; however, since the original material itself contained TCA-soluble encephalitogenie peptides, this conclusion is open to question. Our results are in accord with those of Kies et al. (8) regarding the destructive effect of trypsin on encephalitogenic activity; in contrast, Carnegie et al. (10) reported the finding of highly encephalitogenic activity after trypsin treatment of encephalitogenic protein and peptides from bovine spinal cord. Although the explanation for these differences is not apparent, they might arise because of only partial digestion of the original materials. In this regard, it was desirable to remove the incompletely
ENCEPHALITOGENIC TABLE IMMUNOLOGIC
REACTIVITY
613
PEPTIDES
II
OF FRACTIONS OBTMNED BY GEL DIGESTS OF THE Al PROTEIX
FILTRSTIOS
OF ENZYMIC
Tests Tube ~os.~
Enzyme
EAE induction"
Ouchterlonyb 10.00
5 .oo
2.50
1.25
PHIC
(m/ml )
Trypsin
(2 hr)
Chymotrypsin
Pepsin
(1.5 hr)
(2 hr)
PIYJll:iS;e
Untreated
Al protein
Digest
-
-
-
-
lo&1 14 115-118 IlO& 12G138 Digest
-
-
-
-
li320 l/500
+ +
+ +
+ +
+ +
1.0
Digest 50~GO 1088117 118~124 1255127 138-150 Digest GO-00 105511-l 11512ti 127-150
+ + -
+ + -
+ + -
-
+ + + -
+ + tr -
+ + -
+ + -
100 M
10 CF
l/5 o/5 1,/s 0;5 1,/5 1,‘5 2;‘5 0,/5 OS’5 o/5 l/5 l/4 2 ‘5 4/5 5!5 215 1,/5 4:5
l/5 l/5 O/5 l/5 l/5 l/5 O/5 l/5 O/5 O/5 01’5 I;‘-1 2/‘5 O/3 3/4 3/5 I,/5 l/4
u Tttbe ntrmbers refer to their position on gel filtration shown in Fig. 3a, b, and c. b For details see Foot,not,e a in Table I, and in the text. c The PHI activity of digests or fract,ions are given relative to the untreated Al protein (expressed as 1.0) which inhibits aggltttination due to antibody at 0.5.pg levels. In the passive hemagglutination test, the control sera obtained from rabbits prior to sensitizat,ion or from rabbits injected with Freund’s adjuvant, alone were negative. d The EAE assay is expressed as the ratio of the number of guinea pigs with disease (clinical basis) to the number tested (1) at the levels given.
digested “core” material before definitive conclusions concerning biological activities could be drawn. After removal of the “core” material by gel filtration, our results reveal that’ the peptides from the kypsin and chymotrypsin digests are devoid of significant encephalitogenic activit,y; treatment with pepsin, however, produces highly active encephalitogenic peptides. It was found that the Al protein was readily hydrolyzed by proteolytic enzymes. The rate of proteolysis by each of t’he enzymes tested reached a plat,eau in approximately l-l.5 hr. Trypsin hydrolysis gave rise to 25 peptides shown by gel filtrat,ion and high-volt’age electrophoresis. This technique provides a useful alternative to peptide mapping procedures. The peptide map of the tryptic digest revealed 29 pep-
tides corresponding to that expected based on the number of lysine and arginine residues, 13 and 16 respectively. It should be emphasized that prior denaturation is not required for rapid proteolysis of the Al protein. This observation is in keeping with t’he earlier proposal of a highly unfolded molecule having the extended conformation of a random coil (2). By contrast, most globular proteins are relatively resistant t(o proteolysis unless first denatured (16). The other aspect, of this study, the antibody-combining activity of the peptides, revealed that pept.ide fractions from both the pepsin and chymotryptic digests retained some of the activity of t’he original Al protein as shown by the PHI test. The only peptide fraction, however, revealing antibody-combining activit~y by the Ouchterlony
644
HASHIM
test was one of the fractions from t,he chymotryptic digests, the largest peptide material as shown by its early elution upon gel filtration. The tryptic peptides contained no antibody-combining activity as demonstrated by either test. It should be noted that it was essential to remove the “core” material containing undigested protein and large partially digested peptides, which was positive by the Ouchterlony test, before the peptides could be tested. These result’s demonstrate that the antibody-combining site of the Al protein, like the EAE determinant, is at least partially retained in peptide fragments. ACKNOWLEDGMENTS We are grateful for the interest and encouragement of Dr. Jonas Salk throughout the course of this work. We wish to acknowledge the able assistance of Miss Millie Thompson and Mr. John Griesgraber in many aspects of this work, and we are grateful to Mr. D. K. Miller for the photography. REFERENCES 1. EYLAR, E. H., SALK, J. E., BEVERIDGE, G., BROWN, L. V., AND THOMPSON, M., Arch. Biochem.. Biophys., in press.
AND
EYLAR
2. EYLSR, E. H., AND THOI\IPSON, M., Arch. Biochem. Biophys. 129, 468 (1969). 3. KIES, M. W., MURPHY, J. B., .IXD ALVORD, E. C., Federation Proc. 19, 207 (19GO). 4. Na~
OF
BIOCHEMISTRY
Allergic
AND
BIOPHYSICS
Encephalomyelitis:
Encephalitogenic GEORGE The Salk Institute Received
Basic
635-644 (1969)
129,
Enzymatic Protein
from
A. HASHIM for Biological
AND
Studies,
July 12, 1968: accepted
Degradation Bovine
of the
Spinal
Cord’
E. H. EYLAR San Diego, October
California
92112
22, 1968
The basic protein encephalitogen, wit,hnut Drier denaturation, was rapidly digested with such proteolytic enzymes as trypsin, pronase, chymotrypsin, and pepsin; from N-90% of the proteolysis occurred within 1 hr. The number of peptides released by these enzymes after 2 hr were about 28, 54, 20, and 14, respectively. Each proteolytic digest was chromatographed on Sepha,dax G-25 and G-75 in order to separate the peptides from large partially digested material. Only the peptide fractions obtained from the pepsin digestion possessed significant activity in inducing experimental allergic encephalomyelitis (EAE). One of the peptide fractions had higher encephalitogenic activity, on a weight basis, than either the original protein or the unfractionated digest. In contrast, the chymotryptic digest, while lacking encephalitogenic activity, possessed antibody-combining activity as shown by the Ouchterlony and passive hemagglutination-inhibition (PHI) test. The pepsin digest produced peptides which gave a negative Ouchterlony test. The tryptic peptides were negative in all tests-Ouchterlony, PHI, and EAE induction. These results suggest that the encephlitogenic and antibody-combining activities reside in separate or overlapping regions of the polypeptide chain. The retention of encephalitogenic activity in relatively small pepsin peptides further suggests that the EAE determinant may be defined by a region of the polypeptide chain lacking a folded tertiary structure.
About 30 % or more of the tot’al protein in in bovine myelin is an encephalitogenic basic protein that can be isolated in high yield and purity (1). When care is taken to prevent proteolysis in situ, the encephalitogen comprises more than 95% of t’he basic protein fraction. This protein, referred to as the Al protein, has been characterized in detail (2). It has a molecular weight of 16,400, corltains a high percent,age of lysine, arginine, and glycine and possesses a highly ext)ended conformation. Preparations of basic protein from brain or spinal cord have been variously reported to have molecular weights of from 4400 to 50,000 daltons (3-S). The widely differing result’s have been ascribed to 1 This work was supported by a grant from the National Foundation to Dr. Jonas Salk, in whose laboratory the work was carried out. 2Presently Postdoctoral Fellow, U. S. Public Health Service.
several factors: a nat~ural distribution of a family of proteins of varying size (9) ; a small peptide which binds t’o various macromolecules such as proteins or gangliosides (5) ; proteolysis during preparation at acidic pH (4); and aggregation-dissociation phenomena (8). JIoreover, it has been shown (1) that in situ degradation of t’he basic protein encephalitogen ma.v occur during lyophilization or incubation of the spinal cord at 4” or higher and perhaps for this reason mixtures of peptides which induce EAE are obtained from spinal cord (5, 9). The present study was initiated to examine the effect of knoll proteolytic enzymes in producing biologically active peptides. In the present study, t,he effect,s of trypsin, chymotrypsin, pepsin, and pronase on the Al prot’ein were determined. Differing results have been reported on the effect, of proteolytic enzymes in producing encephali635
636
HASHTM
AND
togenic peptides from basic proteins derived from the central nervous system; Carnegie et al. (10) reported that trypsin treatment for 3 hr resulted in highly encephalitogenic peptides; Caspary and Field (6) reported a partial loss of activity after 18 hr with trypsin; and Kies et al. (8) reported complete destruction of encephalitogenic activity under similar conditions. Our results are generally in agreement with those of Kies et al. (8) since trypsin treatment of the Al protein did not yield encephalitogenic peptides. However, highly encephalitogenic peptides were obtained from the pepsin digest of the Al protein. EXPERIMENTAL
PROCEDURE
Trypsin, specifically treated by the method of Kostka and Carpenter (11) to inhibit possible chymotrypsin, pronase, chymotryptic activity, and pepsin (Worthington Biochemical Corp.) were separately prepared in water and added to aliquots of the protein solution to provide an enzyme-toprotein ratio of 1:50. Incubation with gentle shaking was carried out in a 37” water bath for varying periods of time. A drop of toluene was added to the incubation mixture to prevent bacterial growth. The rate of proteolysis of the Al protein was followed periodically by analysis of small aliquots of the digestion mixtures using the ninhydrin technique (12). Hydrolysis with pepsin was carried out after adjusting the pH of the solution to 3.0 with acetic acid. Upon completion of the digestion, the digest was lyophilized. Fractionation of the enzymic digests. The peptide mixtures were applied to a column (4X100 cm) of Sephadex G-25 @O-cm layer) and G-75 (50-cm layer). The column was equilibrated for at least 24 hr with the eluting fluid (0.1 N acetic acid) after which the sample in a small volume of 0.1 N acetic acid was applied. Elution was carried out at room temperature; 10 ml per tube were collected. The elution pattern was followed by measuring the optical density of the tube contents at 280 rnp. The tubes related to each peak were combined; and then dried by lyophilization. Polyacrylamide gel electrophoresis. Electrophoresis of the Al protein and the enzymic digests, using the disc technique in polyacrylamide gel, was carried out at pH 4.2 according to the methods of Reisfeld et al. (13). Using 5(tlOO rg of the digestion mixtures, electrophoresis was run at 1 mA per tube for 15 min. This was followed by 7 mA per tube for 35 min. High-voltage electrophoresis. High-voltage electrophoresis was carried out with Whatman 3 MM
EYLAR
filter paper for 1 hr at 2506 V in pyridine:acetic acid:n-butanol:water (1:1:2:36 v/v/v/v) at pH 4.7, The chromatogram was air-dried and stained with 0.3yo ninhydrin in acetone. Peptide mapping. A tryptic digest (0.25 mg) was applied 2 in. from the edge of a Whatman 3 MM filter paper (18 X 24 in.) and chromatographed for 18 hr in n-butanol:acetic acid: water (4: 1:5 v/v/v upper phase) and air-dried. This was followed by high-voltage electrophoresis at right angles to the chromatography direction at 2500 V for 60 min at pH 4.7 in pyridine:acetic acid:n-butanol:water (1:1:2:36 v/v/v/v). The chromatogram was then air-dried and stained with 0.3% ninhydrin in acetone. Amino acid analysis. The amino acid composition was determined as described previously (2). Immunologic analysis. Aliquots of the enzymic digests and peptide fractions were prepared in 0.15 M NaCl. These solutions were applied to Ouchterlony plate(s) at original, l:l, 1:2, and 1:4 dilutions according to procedures described elsewhere (1). The antiserum used for this test was prepared from rabbits as described previously (1). The passive hemagglutination test (1) was performed using tannic acid-treated chicken erythrocytes, and rabbit antisera prepared against the purified Al protein or an acid extract of bovine cord as described previously (1). Encephalitogenic activity. The enzymic digests and fractions therefrom were mixed with complete Freund’s adjuvant (CFA) and tested for encephalitogenic activity at 100 and 10 pg in guinea pigs as described previously (I). RESULTS
Proteolysis of the Al protein was found to proceed rapidly in the case of each enzyme tested; at 50 min, digestion is 80-90 % complete (Fig. 1). The rate of digestion is particularly fast for trypsin and is more than 55% complete after 15 min. The maximum number of peptides generated by the proteolysis, expressed as leucine equivalents, occurred with pronase followed in order by trypsin, chymotrypsin, and pepsin. An approximation of the number of peptides produced by the proteolysis can be made from the leucine equivalents based on the ninhydrin values; for pronase, trypsin, chymotrypsin, and pepsin, respectively; the results after
3 hr were 33.6, 17.4, 13.6, and 6.7 moles of peptide per mole protein. These figures represent a minimum that peptides and
value some
since it is known amino acids give
E?;CEPHALITOGE?JIC
PEPTIDES
637
MINUTES
FIQ. 1. The time course is shown of the digestion of the Al protein with proteolytic enzymes pronase (-a-), trypsin (-0-), chymotrypsin (-A-), and pepsin (-A-). The degree of hydrolysis, expressed as pmoles leucine, was determined from ninhydrin values measured at various times and compared with ninhydrin values produced by n-leucine standards. Generally, 10 mg Al protein per ml was incubated with 0.2 mg of enzyme at 37“. A 0.1 M triethylamine-bicarbonate buffer, pH 8, was used for all enzymes except pepsin.
i
FIG. 2. The polyacrylamide gel electrophoretic patterns of the proteolytic digests of the Al protein after 1 and 4 hr at pH 4.2 are shown. Electrophoresis was performed for 50 min in order to give optimal resolution of the peptides. The gels are: (1) Al protein; trypsin digest, 1 hr (2) and 4 hr (3); chymotrypsin digest, 1 hr (4) and 4 hr (5); pepsin digest, 1 hr (6) and 4 hr (7); pronase digest, 1 hr (8).
lower ninhydrin values in general than leutine (14). It is apparent that pronase and trypsin digestion is more extensive than that due to pepsin. When the enzymic digests were examined by electrophoresis on polyacrylamide gel
(Fig. 2), it was seen that the band corresponding to the Al protein was not detectable after 1 hr at the concentrations used. The rapid digestion noted above with pronase and trypsin was apparent from the gel patt’erns; t,he only distinct bands arose from
638
HASHIM
AND EYLAR
small fast-moving peptides. Two diffuse bands were also seen in the tryptic digest. Moreover, with t’he tryptic and pronase digests, no change was observed when compared aft,er 1 hr and 4 hr of incubation. However, the chymotryptic and pepsin digests yielded several peptides of various sizes and charge as shown by the electrophoretic patterns. In most cases they migrat,ed more slowly than the fast band noted with the pronase and tryptic digests. The 4-hr peptide pattern of the chymotryptic digest showed two additional fast-moving bands not present in the I-hr sample. It should be noted that the bulk of the peptides from the pepsin digest moved less rapidly than the peptides from the other digests, which is indicative of larger size or less charge. Generally, a lOO-pg peptide material was applied to the gels in order to detect as many peptides as possible; for the Al protein, 25 pg was routinely used. Only the larger 5( )-
2.4 -
z* -
1.6 -
: 22
1.2 -
d 0.8 -
0.4 -
5.0 -
4.5 -
1
4.0 -
(3a.l
3.5 4c )-
3.0 2 c?g 6
3.c )-
2.0 -
c
d d
2.5 -
1.5 -
1.0 -
20
1.0 TUBE
i 40
60
80
100
I20
TUBE
140
160
180
NO.
3. The elution patterns of the enzyme digests of the Al protein using trypsin (a), chymotrypsin (b), and pepsin (c) are shown using columns of Sephadex G-25 and G-75 as described in the Experimental section. The elution of peptides was followed by optical density measurement at 280 mp. Ten-milliliter fractions were collected. FIG.
No
peptides appeared after electrophoresis since the smaller peptides would either migrate out of the gel or would be eluted from the gel during the staining and washing procedures. Preparation of peptide families from the enzyme digests. The enzymic digests of the Al protein derived from pepsin, trypsin, and chymotrypsin treatment were applied to a column containing Sephadex G-75 and
ENCEPHALITOGENIC
FIG. 4. The gel electrophoresis patterns at pH 4.2 are shown for the small peak preceding the major peptide peak (Fig. 3) obtained by gel filtration of digests of the Al protein using trypsin (2 hr), chymotrypsin (1.5 hr), and pepsin (2 hr). The gels are: (1) 81 prot)ein; (2) tryptic digest; (3) chymotryptic digest.
I ‘-
1
2
3
4
5
PEPTIDES
639
Sephadex G-25. The elution patterns, as recorded by optical density at 280 mc(, were basically similar (Fig. 3a, b, c). In each case the enzyme emerged first, near t’he void volume of the column (430 ml), followed by a small intermediat,e peak and then bv the bulk of the peptide material. It is evident that the small intermediate peak contains large peptides in addition to material which migrates on electrophoresis as did the original Al protein (lcig. 4). This lat’ter material may represent slightly degraded Al protein rather than the intact molecule. Selected tubes from each peptide peak were examined for peptides by high-voltage elect,rophoresis at pH 4.7 as shown in Figs. 5-7. This technique provides a clear indication of the distribution of t’he individual peptides over the elution curve and is particularly well illustrated by the tryptic peptides shown in E’ig. 5. From these patterns the total number of peptides can be closely approximat’ed since they are separated according t’o bot#h size and charge. The appearance or disappearance of any given peptide can
6
FIG. 5. High-voltage electrophoresis on paper at pH 1.7 of selected tubes from the major peak obtained by gel filtration of the 2.hr trypsirr digest showu in Fig. 3a. The 0.25.mg sample was applied at the origin (0). Electrophoresis was performed at 2500 V for GOmin. Other details are given in the test. The peptides are from: (1) tube 111, (2) tube 114, (3) tube 118, (4) tube 121, (5) tube 123, (6) tube 126, (7) tube 130, (8) tube 134, (9) tube 139, (10) tube 142, (11) tltbe 148, (12) trypsin digest.
640
HASHIM
AND
EYLAR
FIG. 6. High-voltage electrophoresis on paper at pH 4.7 of selected tubes from the major peak obtained by gel filtration of the 1.5-hr chymotrypsin digest shown in Fig. 3b. Other details are given in Fig. 5 and in the text. The peptides are from (1) chymotrypsin digest, (2) tube 75, (3) tube 112, (4) tube 118, (5) tube 122, (6) tube 126, (7) tube 130, (8) tube 151.
easily be followed by its relative intensity in the electrophoretic pattern. For trypsin, 28 peptides were identified in this way as shown in Fig. 5; for chymotrypsin and pepsin (Figs. 6 and 7) 20 and 14 peptides, respectively, were counted. These numbers are appreciably higher than estimated from the ninhydrin values (Fig. 1). This technique, therefore, provides an alternate method of peptide mapping to the electrophoresis-chromatographic procedure generally used. A peptide map of the trypsin digest, shown in Fig. 8 for comparison, reveals 29 peptides, and is in agreement with the results obtained by gel filtration and electrophoresis. Immunologic activity 0f the peptide frac-
tions. Pronase, trypsin, and chymotrypsin digests of the Al protein formed distinct precipitating bands when the digests were tested by immunodiffusion against rabbit antiserum prepared against the purified protein (Table I). A positive test was obtained, whether the enzyme treatment was carried out for 1 hour or for 24 hr. By contrast, pepsin treatment of the Al protein for as little as 30 min destroys the reactivity in the immunodiff usion test (Table I). It is seen (Table II) that material in the small peak representing partially digested protein and large peptides forms a precipitin band in the immunodiff usion test in the case of the trypsin (tubes 50-60) and chymo-
1
2
3
4
5
FIG. 7. High-voltage electrophoresis on paper at pH 4.7 of major peak obtained by gel filtration of the 2-hr pepsin digest Fig. 3c. Other details are given in Fig. 5 and in the text. The treated Al protein, (2) chymotrypsin digest, (3) tubes 106-114, 119-125, (6) tubes 126-138.
a c 0 w A W
6 selected fractions from the of the Al protein shown in peptides are from: (1) un(4) tubes l&118, (5) tubes
‘J _-... t-.-J --0-
..-.p
: .__’ r
CHROMATOGRAPHY
-
FIG. 8. The mapping of peptides obtained by tryptic digestion of the Al protein. Chromatography was performed for 18 hr in n-butanol:acetic acid:water (4:1:5 v/v/v, upper phase), and high-voltage elect,rophoresis u-as run at right angles moving toward the cathode. Other details are given in the text.
642
HASHIM TABLE
AND
I
THE
ANTIBODY-COMBINING ACTIVITY OF ENZYMIC DIGESTS OF THE Al PROTEIN as MEASURED BY THE OCCHTERLONY TECHNIQUE: Digestion time
ElK.YlIle
Concentrationa
(mg/ml)
5.0
2.5
1.25
0.6
+ + + + -
+ + + + -
+ tr + + -
+
+ + +
+ + +
+ +
(hr)
Trypsin Chymotrypsin Pepsin Pronase Untreated protein
Al
1 21 1 24 0.5 1 1 24
EYLAR
tein (Table II). Only the peptide fractions from the pepsin digest showed significant encephalitogenic activity; the peptide fraction represented by tubes 119-125 appeared to be at least as active on a weight basis as the untreated Al protein. By contrast, the tryptic and chymotryptic peptides showed negligible encephalitogenic activity. DISCUSSION
+ tr + +
a Each sample was tested using the concentrations of digested protein shown. (+) refers to a strong band generally developed in the first 3 days; (-) refers to the absence of a band, and (tr) refers to a faint trace of a band. Observations were made for a minimum of 28 days. Control sera, obtained either from animals prior to sensitization, or from animals injected with Freund’s adjuvant alone, was negative.
trypsin (tubes 60-90) digests. The only peptide fraction giving a positive precipitin band is found in tubes 105-114 from the chymotryptic digest. This chymotryptic fraction, when examined by high-voltage electrophoresis as shown in Fig. 6, reveals several peptides which move toward t,he cathode. After pepsin digestion, none of the fractions gave a precipitin band on immunodiffusion. Many of the peptide fractions from the pepsin and chymotryptic digests retained antibody-combining activity as demonstrated by the passive hemagglutinationinhibition (PHI) t’est. For pepsin peptides, the activities ranged from $4 to 364 that of the intact Al protein; for the chymotryptic peptides, the activities ranged from go to x4. In contrast, none of the fractions obtained from tryptic digests were positive in the PHI test even at 1000~pg levels (Table II), except the fraction representing the undigested or “core” material. Induction of EAE. In general, the unfractionated proteolytic digests appeared less encephalitogenic than the original Al pro-
With the isolation of a highly purified encephalitogen (Al protein), it was of interest to investigate the effect of specific prot’eolytic enzymes on immunologic react,ivity in order to explore the relationship between chemical structure and biological function. The results clearly show that of the four proteolytic enzymes tested, only pepsin resulted in the formation of pept’ides that were encephalitogenic. Past studies on the proteolysis of basic proteins from bovine spinal cord are difficult to interpret because the original material was not precisely characterized in many cases (6, 8). Furthermore, comparison with other data is difficult since slight changes in the incubation conditions could modify the number and activity of the peptides produced; there may also be a question of enzyme purity when working w&h trypsin and chymotrypsin. Although we found t,hat pepsin digestion produced highly encephalitogenic peptides, ot’hers have reported (15) that TCA-soluble peptides, derived from pepsin treatment of encephalitogenic protein, were inactive. It was reported (S) also that papain digestion liberated TCA-soluble encephalitogenic peptides; however, since the original material itself contained TCA-soluble encephalitogenie peptides, this conclusion is open to question. Our results are in accord with those of Kies et al. (8) regarding the destructive effect of trypsin on encephalitogenic activity; in contrast, Carnegie et al. (10) reported the finding of highly encephalitogenic activity after trypsin treatment of encephalitogenic protein and peptides from bovine spinal cord. Although the explanation for these differences is not apparent, they might arise because of only partial digestion of the original materials. In this regard, it was desirable to remove the incompletely
ENCEPHALITOGENIC TABLE IMMUNOLOGIC
REACTIVITY
613
PEPTIDES
II
OF FRACTIONS OBTMNED BY GEL DIGESTS OF THE Al PROTEIX
FILTRSTIOS
OF ENZYMIC
Tests Tube ~os.~
Enzyme
EAE induction"
Ouchterlonyb 10.00
5 .oo
2.50
1.25
PHIC
(m/ml )
Trypsin
(2 hr)
Chymotrypsin
Pepsin
(1.5 hr)
(2 hr)
PIYJll:iS;e
Untreated
Al protein
Digest
-
-
-
-
lo&1 14 115-118 IlO& 12G138 Digest
-
-
-
-
li320 l/500
+ +
+ +
+ +
+ +
1.0
Digest 50~GO 1088117 118~124 1255127 138-150 Digest GO-00 105511-l 11512ti 127-150
+ + -
+ + -
+ + -
-
+ + + -
+ + tr -
+ + -
+ + -
100 M
10 CF
l/5 o/5 1,/s 0;5 1,/5 1,‘5 2;‘5 0,/5 OS’5 o/5 l/5 l/4 2 ‘5 4/5 5!5 215 1,/5 4:5
l/5 l/5 O/5 l/5 l/5 l/5 O/5 l/5 O/5 O/5 01’5 I;‘-1 2/‘5 O/3 3/4 3/5 I,/5 l/4
u Tttbe ntrmbers refer to their position on gel filtration shown in Fig. 3a, b, and c. b For details see Foot,not,e a in Table I, and in the text. c The PHI activity of digests or fract,ions are given relative to the untreated Al protein (expressed as 1.0) which inhibits aggltttination due to antibody at 0.5.pg levels. In the passive hemagglutination test, the control sera obtained from rabbits prior to sensitizat,ion or from rabbits injected with Freund’s adjuvant, alone were negative. d The EAE assay is expressed as the ratio of the number of guinea pigs with disease (clinical basis) to the number tested (1) at the levels given.
digested “core” material before definitive conclusions concerning biological activities could be drawn. After removal of the “core” material by gel filtration, our results reveal that’ the peptides from the kypsin and chymotrypsin digests are devoid of significant encephalitogenic activit,y; treatment with pepsin, however, produces highly active encephalitogenic peptides. It was found that the Al protein was readily hydrolyzed by proteolytic enzymes. The rate of proteolysis by each of t’he enzymes tested reached a plat,eau in approximately l-l.5 hr. Trypsin hydrolysis gave rise to 25 peptides shown by gel filtrat,ion and high-volt’age electrophoresis. This technique provides a useful alternative to peptide mapping procedures. The peptide map of the tryptic digest revealed 29 pep-
tides corresponding to that expected based on the number of lysine and arginine residues, 13 and 16 respectively. It should be emphasized that prior denaturation is not required for rapid proteolysis of the Al protein. This observation is in keeping with t’he earlier proposal of a highly unfolded molecule having the extended conformation of a random coil (2). By contrast, most globular proteins are relatively resistant t(o proteolysis unless first denatured (16). The other aspect, of this study, the antibody-combining activity of the peptides, revealed that pept.ide fractions from both the pepsin and chymotryptic digests retained some of the activity of t’he original Al protein as shown by the PHI test. The only peptide fraction, however, revealing antibody-combining activit~y by the Ouchterlony
644
HASHIM
test was one of the fractions from t,he chymotryptic digests, the largest peptide material as shown by its early elution upon gel filtration. The tryptic peptides contained no antibody-combining activity as demonstrated by either test. It should be noted that it was essential to remove the “core” material containing undigested protein and large partially digested peptides, which was positive by the Ouchterlony test, before the peptides could be tested. These result’s demonstrate that the antibody-combining site of the Al protein, like the EAE determinant, is at least partially retained in peptide fragments. ACKNOWLEDGMENTS We are grateful for the interest and encouragement of Dr. Jonas Salk throughout the course of this work. We wish to acknowledge the able assistance of Miss Millie Thompson and Mr. John Griesgraber in many aspects of this work, and we are grateful to Mr. D. K. Miller for the photography. REFERENCES 1. EYLAR, E. H., SALK, J. E., BEVERIDGE, G., BROWN, L. V., AND THOMPSON, M., Arch. Biochem.. Biophys., in press.
AND
EYLAR
2. EYLSR, E. H., AND THOI\IPSON, M., Arch. Biochem. Biophys. 129, 468 (1969). 3. KIES, M. W., MURPHY, J. B., .IXD ALVORD, E. C., Federation Proc. 19, 207 (19GO). 4. Na~