232
Biochimica et Biophysica Acta, 532 (1978) 232--241 © Elsevier/North-Holland Biomedical Press
BBA 37833
PARTIAL CHARACTERIZATION OF THE HUMAN ANTI-ENCEPHALITOGENIC PROTEIN ISOLATION FROM SPINAL CORD AND SPINAL NERVES
KEVIN G. WEIR and CATHERINE F.C. MacPHERSON Departments of Biochemistry and Psychiatry, University of Western Ontario, London, Ontario (Canada)
(Received June 20th, 1977)
Summary 1. The human spinal cord protein (HSCP) is immunochemically closely related to the anti~encephalitogenic bovine spinal cord protein (BSCP) and immunoreactive HSCP is similarly distributed in brain, spinal cord and spinal nerve roots (a peripheral nervous tissue) in the proportions of 1 : 6 : 60. HSCP and protein immunochemically identical to HSCP (HSCP-PN), were purified from frozen spinal cords and frozen peripheral nervous tissue, respectively, by extraction with 0.15 M sodium chloride, carboxy-methyl cellulose (CM-cellulose) chromatography and gel filtration on Sephadex G-50. 2. Purified HSCP and HSCP-PN formed one band in sodium dodecyl sulfate polyacrylamide gel electrophoretograms and had estimated molecular sizes of 13 700 and 14 700, respectively. The amino acid compositions were similar except that HSCP had 16% glutamic acid and lacked half cystine while HSCPPN contained 11.9% of glutamic acid and 1.0% of half cystine. 3. Immunodiffusion analyses with anti-HSCP or anti-BSCP sera revealed that extracts of spinal cord and spinal nerves and purified HSCP and HSCP-PN are composed of immunogenically distinct major and minor forms. The major forms and the minor forms of HSCP and HSCP-PN are identical to each other but share different antigenic amino acid sequences with BSCP. Immunoelectrophoretic profiles of spinal cord and spinal nerve extracts indicated that the major and minor HSCP antigens also existed in two different molecular forms having the electrophoretic mobilities of a ~- or a y-serum globulin. The four dif-
Abbreviations: CM--cellulose, c a r b o x y m e t h y l cellulose; BSCP, bovine spinal cord protein; HSCP, h u m a n spinal cord protein; HSCP-PN, protein i m m u n o c h e m i c a l l y identical to HSCP, isolated from spinal nerves.
233 ferent molecular forms were present in purified HSCP-PN, but only the forms with 'l'-electrophoretic mobility were present in purified HSCP.
Introduction It was reported earlier [1,2] that guinea pigs pretreated with a bovine spinal cord protein [3,4] did not develop experimental allergic encephalomyelitis when subsequently immunized with disease-inducing doses of whole bovine spinal cord or purified myelin basic protein. More recently, MacPherson and Armstrong found that Lewis rats that were immunized with rat myelin basic protein in complete Freund's adjuvant were protected from disease [5] if they had been pretreated with the rat spinal cord protein [6] which shares some antigenic sequences with BSCP. Pretreatment with BSCP also protected the Lewis rats from disease, but the effective dose of BSCP was seven times larger than the protective doses of rat spinal cord protein [5]. Immunodiffusion studies demonstrated that human spinal cord and human spinal roots, a peripheral nervous tissue, contain, respectively, HSCP and HSCPPN, irnmunogenic proteins that are identical by criteria of immunodiffusion and that cross-react extensively with anti-BSCP sera [3,6] and are localized similaxly in the central and peripheral nervous systems. We considered it important to purify HSCP from central and peripheral nervous tissues to ascertain if the human proteins also had the capacity to prevent demyelinating diseases induced by immunization with myelin basic proteins. The purpose of this paper is to describe the methods of isolation and some of the chemical, physical and immunological properties of HSCP and HSCP-PN. Materials and Methods
Human spinal cords Human spinal cords were obtained within 12 h of death from patients who died from non-neurological causes. After the spinal nerve roots were removed from the spinal cords, the cords and nerves were prepared for extraction and stored in the same way as described earlier for bovine tissues [5]. Human brains were cut into 2-cm slices, wrapped in plastic film and stored at --20°C. Chemicals Cytochrome c (horse heart), ribonuclease (bovine pancreas), myoglobin, a-chymotrypsinogen (bovine pancreas), aprotinin (bovine lung), and Coomassie Brilliant Blue R were products of Sigma Chemical Co., St. Louis, Mo. Bovine serum albumin was obtained from Nutritional Biochemicals Corporation, Cleveland, Ohio. Sephadex G-50 superfine and Blue Dextran 2000 were supplied by A.B. Pharmacia, Uppsala, Sweden. Carboxymethyl cellulose was obtained from Whatman Biochemicals Ltd., Kent. Acrylamide (for electrophoresis), methylene bisacrylamide, N,N,N',N'-tetramethylethylene diamine and ~-mercaptoethanol were purchased from Eastman, Rochester, N.Y. Spectropor I was supplied by Spectrum Medical Industries Inc., Los Angeles, Calif. All other chemicals were reagent grade.
234
Extracts o f spinal cords and spinal nerves Approximately 125 g of frozen tissue were cut into 2-mm shavings and homogenized at 0°C in sufficient 0.15 M sodium chloride to make a 10% homogenate (w/v). A Tri.R blender at speed No. 7 for 2 min and a Sorval Omni-Mixer at speed No. 10 for 3 min were used to homogenize spinal cord and spinal nerves, respectively. The homogenate was centrifuged at 0°C at 14 000 ×g for 30 min, the supernatant removed by suction and the precipitate rehomogenized in one-third the original volume of 0.15 M sodium chloride and centrifuged as above. The supernatants were combined, adjusted to pH 4.5 with 5 M acetic acid, and centrifuged to remove the insoluble proteins. The supernatant was concentrated 3-fold by flash evaporation and dialyzed overnight against several changes of 0.05 M sodium acetate buffer, pH 4.5, until the conductivity of the dialysand was about 3.6 ms. Batch absorption o f extracts on CM-52 cellulose Sufficient preswollen CM-52 cellulose (equal to 50 times the weight of total protein of the extract) equilibrated with 0.05 M sodium acetate buffer, pH 4.5, was added to the tissue extracts and stirred for 2 h at 4°C. The CM-52 cellulose was collected by vacuum filtration on a Buchner funnel, transferred to a 4.5 × 60 cm chromatography column and washed sequentially with (1) 0.05 M sodium acetate buffer, pH 4.5, (2) 0.05 M sodium acetate buffer, pH 5.5, and (3) 0.05 M sodium acetate buffer, pH 5.8, containing 0.05 M sodium chloride. Each wash buffer was passed through the CM-52 cellulose column until the A at 280 nm was approximately 0.09 or less for at least 100 ml of eluant. Elution o f HSCP from CM-52 cellulose HSCP or HSCP-PN was eluted with 0.05 M sodium acetate buffers, pH 5.8, containing increasing concentrations of sodium chloride. The areas of the chromatogram where HSCP, other nervous system antigens and human serum proteins were eluted were determined by semi-quantitative immunodiffusion analyses using rabbit anti-BSCP sera, an anti-human brain extract serum and an anti-human serum serum. Gel filtration chromatography Fractions that contained HSCP were pooled, concentrated by flash evaporation and dialyzed in Spectropor No. 1 against distilled water. The purity of each fraction was checked by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis [7]. Fractions that contained similar kinds of impurities were pooled and chromatographed on Sephadex G-50 superfine. A n tisera The anti-BSCP serum was the same serum used in an earlier study on bovine spinal cord protein [5]. Anti-sera were prepared against a sodium chloride extract of human brain or normal human serum by immunizing rabbits with 2.0 ml of inoculums prepared by emulsifying 1.0 ml of human brain extract or normal human serum (each containing about 30 mg protein per ml) with 1.0 ml of complete Freund's adjuvant. About 0.3 ml of inoculum was injected intradermally into toe pads in each hind foot and into two sites in each thigh.
235 Rabbits were test-bled at the end of the first month and reinjected as before if their sera did not contain adequate levels of antibody for immunodiffusion studies. Rabbits that were producing suitable amounts of antibodies were bled at monthly intervals and reinjected when necessary to maintain the serum antibody concentrations at the desired levels. Anti-HSCP sera was raised in rabbits as described [5], using purified HSCP and HSCP-PN. All sera were stored at --20 ° C. The anti-HSCP sera were tested for the presence of antibodies against antigenic contaminants by immunodiffusion analyses using various dilution of human brain extracts and human serum.
Amino acid analyses The amino acid analyses were performed in the laboratory of Dr. D.B. Smith of the Department of Biochemistry, University of Western Ontario, in a Beckman 120C automatic amino acid analyzer. The samples were hydrolyzed under vacuum with constant boiling HC1 at l l 0 ° C for 24 h. Other methods The HSCP preparations were examined for purity by SDS-polyacrylamide gel electrophoresis using 10% gels according to Weber and Osborn [7], but without prior incubation with ~-mercaptoethanol. When the molecular weights of HSCP and HSCP-PN were determined [7], the preliminary incubation with ~-mercaptoethanol was carried out. Proteins used as standards for the molecular weight determination were bovine serum albumin, a-chymotrypsinogen, myoglobin, ribonuclease and cytochrome c. Molecular sizes were also estimated by gel-filtration chromatography according to Andrews [8]. Qualitative immunodiffusion analyses were performed by the method of Ouchterlony [9], in 1.2% agarose containing 0.05 M barbital buffer, pH 8.6, and either 10% aprotinin or 0.10 M sodium chloride. Immunoelectrophoretic analyses were run according to the method of Scheidegger [10] on microscope slides coated with 1.3% agar containing 0.05 M barbital buffer, pH 8.6, at a constant current of 15 mA per slide for 45 min. Protein concentration was determined by the method of Lowry et al. [11]. Results
H S C P was more difficult to isolate than HSCP-PN because the extracts of spinal cord contained significantly larger amounts of contaminating proteins and roughly one-tenth the immunoreactive H S C P of extracts of spinal nerves. A large proportion of the impurities were removed by "washing" the CM-52 cellulose column sequentially with 0.05 M sodium acetate buffers at p H values of 4.5, 5.5 and 5.8. The elution profile of H S C P from CM-52 celluloseis shown in Fig. 1. El and E2 were combined for gel filtrationchromatography because the protein compositions of these eluates were similar. Usually, the fraction containing H S C P had to be recl~omatographed to remove all impurities. The elution pattern of HSCP-PN from CM-52 cellulose differed from that of H S C P as peak El contained more contaminants and significantlylessimmunoreactive H S C P than peak E2. Thus, fractions El and E2 were chromatographed separately. Pure H S C P or HSCP-PN was collected between fractions 62 and 75
236 0.7 • 0.6
c~
2
a5
El
0.4,
E2
8
0.3
Z
~0.2
020
0.1 0.0
0.10
100
200
300
400
500
600
700
0.00
Elution volume ( ml ) Fig. 1. C M - 5 2 c e l l u l o s e c h r o m a t o g r a p h y o f b a t c h - a b s o r b e d H S C P . S t e p w i s e e l u t i o n o f H S C P w a s c a r r i e d o u t w i t h 0 . 0 5 M s o d i u m a c e t a t e b u f f e r s , p H 5 . 8 , c o n t a i n i n g i n c r e a s i n g c o n c e n t r a t i o n s o f NaCI: E l , 0 . 1 0 M NaCI a n d E 2 , 0 . 2 0 M N a C I . F r a c t i o n s o f 5 m l w e r e c o l l e c t e d a t a f l o w r a t e o f 9 0 m l / h a t 2 0 ° C . T h e shaded areas indicate the eluates that contained HSCP.
as shown in Fig. 2 for the gel-filtration o f HSCP-PN. As shown in Fig. 3 purified HSCP's formed one band on SDS gel electrophoresis whether or not the proteins had been previously exposed to ~-mercaptoethanol before electrophoresis. The molecular weights of HSCP and HSCP-PN estimated by gel electrophoresis were found to be 13 700 -+ 1370 and 14 700 + 1470, respectively. The molecular weight of 15 000 was estimated for HSCP-PN by gel-exclusion chromatography.
14 12
0.8 0~6
0-4
0
Biochimica et Biophysica Acta, 532 (1978) 232--241 © Elsevier/North-Holland Biomedical Press
BBA 37833
PARTIAL CHARACTERIZATION OF THE HUMAN ANTI-ENCEPHALITOGENIC PROTEIN ISOLATION FROM SPINAL CORD AND SPINAL NERVES
KEVIN G. WEIR and CATHERINE F.C. MacPHERSON Departments of Biochemistry and Psychiatry, University of Western Ontario, London, Ontario (Canada)
(Received June 20th, 1977)
Summary 1. The human spinal cord protein (HSCP) is immunochemically closely related to the anti~encephalitogenic bovine spinal cord protein (BSCP) and immunoreactive HSCP is similarly distributed in brain, spinal cord and spinal nerve roots (a peripheral nervous tissue) in the proportions of 1 : 6 : 60. HSCP and protein immunochemically identical to HSCP (HSCP-PN), were purified from frozen spinal cords and frozen peripheral nervous tissue, respectively, by extraction with 0.15 M sodium chloride, carboxy-methyl cellulose (CM-cellulose) chromatography and gel filtration on Sephadex G-50. 2. Purified HSCP and HSCP-PN formed one band in sodium dodecyl sulfate polyacrylamide gel electrophoretograms and had estimated molecular sizes of 13 700 and 14 700, respectively. The amino acid compositions were similar except that HSCP had 16% glutamic acid and lacked half cystine while HSCPPN contained 11.9% of glutamic acid and 1.0% of half cystine. 3. Immunodiffusion analyses with anti-HSCP or anti-BSCP sera revealed that extracts of spinal cord and spinal nerves and purified HSCP and HSCP-PN are composed of immunogenically distinct major and minor forms. The major forms and the minor forms of HSCP and HSCP-PN are identical to each other but share different antigenic amino acid sequences with BSCP. Immunoelectrophoretic profiles of spinal cord and spinal nerve extracts indicated that the major and minor HSCP antigens also existed in two different molecular forms having the electrophoretic mobilities of a ~- or a y-serum globulin. The four dif-
Abbreviations: CM--cellulose, c a r b o x y m e t h y l cellulose; BSCP, bovine spinal cord protein; HSCP, h u m a n spinal cord protein; HSCP-PN, protein i m m u n o c h e m i c a l l y identical to HSCP, isolated from spinal nerves.
233 ferent molecular forms were present in purified HSCP-PN, but only the forms with 'l'-electrophoretic mobility were present in purified HSCP.
Introduction It was reported earlier [1,2] that guinea pigs pretreated with a bovine spinal cord protein [3,4] did not develop experimental allergic encephalomyelitis when subsequently immunized with disease-inducing doses of whole bovine spinal cord or purified myelin basic protein. More recently, MacPherson and Armstrong found that Lewis rats that were immunized with rat myelin basic protein in complete Freund's adjuvant were protected from disease [5] if they had been pretreated with the rat spinal cord protein [6] which shares some antigenic sequences with BSCP. Pretreatment with BSCP also protected the Lewis rats from disease, but the effective dose of BSCP was seven times larger than the protective doses of rat spinal cord protein [5]. Immunodiffusion studies demonstrated that human spinal cord and human spinal roots, a peripheral nervous tissue, contain, respectively, HSCP and HSCPPN, irnmunogenic proteins that are identical by criteria of immunodiffusion and that cross-react extensively with anti-BSCP sera [3,6] and are localized similaxly in the central and peripheral nervous systems. We considered it important to purify HSCP from central and peripheral nervous tissues to ascertain if the human proteins also had the capacity to prevent demyelinating diseases induced by immunization with myelin basic proteins. The purpose of this paper is to describe the methods of isolation and some of the chemical, physical and immunological properties of HSCP and HSCP-PN. Materials and Methods
Human spinal cords Human spinal cords were obtained within 12 h of death from patients who died from non-neurological causes. After the spinal nerve roots were removed from the spinal cords, the cords and nerves were prepared for extraction and stored in the same way as described earlier for bovine tissues [5]. Human brains were cut into 2-cm slices, wrapped in plastic film and stored at --20°C. Chemicals Cytochrome c (horse heart), ribonuclease (bovine pancreas), myoglobin, a-chymotrypsinogen (bovine pancreas), aprotinin (bovine lung), and Coomassie Brilliant Blue R were products of Sigma Chemical Co., St. Louis, Mo. Bovine serum albumin was obtained from Nutritional Biochemicals Corporation, Cleveland, Ohio. Sephadex G-50 superfine and Blue Dextran 2000 were supplied by A.B. Pharmacia, Uppsala, Sweden. Carboxymethyl cellulose was obtained from Whatman Biochemicals Ltd., Kent. Acrylamide (for electrophoresis), methylene bisacrylamide, N,N,N',N'-tetramethylethylene diamine and ~-mercaptoethanol were purchased from Eastman, Rochester, N.Y. Spectropor I was supplied by Spectrum Medical Industries Inc., Los Angeles, Calif. All other chemicals were reagent grade.
234
Extracts o f spinal cords and spinal nerves Approximately 125 g of frozen tissue were cut into 2-mm shavings and homogenized at 0°C in sufficient 0.15 M sodium chloride to make a 10% homogenate (w/v). A Tri.R blender at speed No. 7 for 2 min and a Sorval Omni-Mixer at speed No. 10 for 3 min were used to homogenize spinal cord and spinal nerves, respectively. The homogenate was centrifuged at 0°C at 14 000 ×g for 30 min, the supernatant removed by suction and the precipitate rehomogenized in one-third the original volume of 0.15 M sodium chloride and centrifuged as above. The supernatants were combined, adjusted to pH 4.5 with 5 M acetic acid, and centrifuged to remove the insoluble proteins. The supernatant was concentrated 3-fold by flash evaporation and dialyzed overnight against several changes of 0.05 M sodium acetate buffer, pH 4.5, until the conductivity of the dialysand was about 3.6 ms. Batch absorption o f extracts on CM-52 cellulose Sufficient preswollen CM-52 cellulose (equal to 50 times the weight of total protein of the extract) equilibrated with 0.05 M sodium acetate buffer, pH 4.5, was added to the tissue extracts and stirred for 2 h at 4°C. The CM-52 cellulose was collected by vacuum filtration on a Buchner funnel, transferred to a 4.5 × 60 cm chromatography column and washed sequentially with (1) 0.05 M sodium acetate buffer, pH 4.5, (2) 0.05 M sodium acetate buffer, pH 5.5, and (3) 0.05 M sodium acetate buffer, pH 5.8, containing 0.05 M sodium chloride. Each wash buffer was passed through the CM-52 cellulose column until the A at 280 nm was approximately 0.09 or less for at least 100 ml of eluant. Elution o f HSCP from CM-52 cellulose HSCP or HSCP-PN was eluted with 0.05 M sodium acetate buffers, pH 5.8, containing increasing concentrations of sodium chloride. The areas of the chromatogram where HSCP, other nervous system antigens and human serum proteins were eluted were determined by semi-quantitative immunodiffusion analyses using rabbit anti-BSCP sera, an anti-human brain extract serum and an anti-human serum serum. Gel filtration chromatography Fractions that contained HSCP were pooled, concentrated by flash evaporation and dialyzed in Spectropor No. 1 against distilled water. The purity of each fraction was checked by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis [7]. Fractions that contained similar kinds of impurities were pooled and chromatographed on Sephadex G-50 superfine. A n tisera The anti-BSCP serum was the same serum used in an earlier study on bovine spinal cord protein [5]. Anti-sera were prepared against a sodium chloride extract of human brain or normal human serum by immunizing rabbits with 2.0 ml of inoculums prepared by emulsifying 1.0 ml of human brain extract or normal human serum (each containing about 30 mg protein per ml) with 1.0 ml of complete Freund's adjuvant. About 0.3 ml of inoculum was injected intradermally into toe pads in each hind foot and into two sites in each thigh.
235 Rabbits were test-bled at the end of the first month and reinjected as before if their sera did not contain adequate levels of antibody for immunodiffusion studies. Rabbits that were producing suitable amounts of antibodies were bled at monthly intervals and reinjected when necessary to maintain the serum antibody concentrations at the desired levels. Anti-HSCP sera was raised in rabbits as described [5], using purified HSCP and HSCP-PN. All sera were stored at --20 ° C. The anti-HSCP sera were tested for the presence of antibodies against antigenic contaminants by immunodiffusion analyses using various dilution of human brain extracts and human serum.
Amino acid analyses The amino acid analyses were performed in the laboratory of Dr. D.B. Smith of the Department of Biochemistry, University of Western Ontario, in a Beckman 120C automatic amino acid analyzer. The samples were hydrolyzed under vacuum with constant boiling HC1 at l l 0 ° C for 24 h. Other methods The HSCP preparations were examined for purity by SDS-polyacrylamide gel electrophoresis using 10% gels according to Weber and Osborn [7], but without prior incubation with ~-mercaptoethanol. When the molecular weights of HSCP and HSCP-PN were determined [7], the preliminary incubation with ~-mercaptoethanol was carried out. Proteins used as standards for the molecular weight determination were bovine serum albumin, a-chymotrypsinogen, myoglobin, ribonuclease and cytochrome c. Molecular sizes were also estimated by gel-filtration chromatography according to Andrews [8]. Qualitative immunodiffusion analyses were performed by the method of Ouchterlony [9], in 1.2% agarose containing 0.05 M barbital buffer, pH 8.6, and either 10% aprotinin or 0.10 M sodium chloride. Immunoelectrophoretic analyses were run according to the method of Scheidegger [10] on microscope slides coated with 1.3% agar containing 0.05 M barbital buffer, pH 8.6, at a constant current of 15 mA per slide for 45 min. Protein concentration was determined by the method of Lowry et al. [11]. Results
H S C P was more difficult to isolate than HSCP-PN because the extracts of spinal cord contained significantly larger amounts of contaminating proteins and roughly one-tenth the immunoreactive H S C P of extracts of spinal nerves. A large proportion of the impurities were removed by "washing" the CM-52 cellulose column sequentially with 0.05 M sodium acetate buffers at p H values of 4.5, 5.5 and 5.8. The elution profile of H S C P from CM-52 celluloseis shown in Fig. 1. El and E2 were combined for gel filtrationchromatography because the protein compositions of these eluates were similar. Usually, the fraction containing H S C P had to be recl~omatographed to remove all impurities. The elution pattern of HSCP-PN from CM-52 cellulose differed from that of H S C P as peak El contained more contaminants and significantlylessimmunoreactive H S C P than peak E2. Thus, fractions El and E2 were chromatographed separately. Pure H S C P or HSCP-PN was collected between fractions 62 and 75
236 0.7 • 0.6
c~
2
a5
El
0.4,
E2
8
0.3
Z
~0.2
020
0.1 0.0
0.10
100
200
300
400
500
600
700
0.00
Elution volume ( ml ) Fig. 1. C M - 5 2 c e l l u l o s e c h r o m a t o g r a p h y o f b a t c h - a b s o r b e d H S C P . S t e p w i s e e l u t i o n o f H S C P w a s c a r r i e d o u t w i t h 0 . 0 5 M s o d i u m a c e t a t e b u f f e r s , p H 5 . 8 , c o n t a i n i n g i n c r e a s i n g c o n c e n t r a t i o n s o f NaCI: E l , 0 . 1 0 M NaCI a n d E 2 , 0 . 2 0 M N a C I . F r a c t i o n s o f 5 m l w e r e c o l l e c t e d a t a f l o w r a t e o f 9 0 m l / h a t 2 0 ° C . T h e shaded areas indicate the eluates that contained HSCP.
as shown in Fig. 2 for the gel-filtration o f HSCP-PN. As shown in Fig. 3 purified HSCP's formed one band on SDS gel electrophoresis whether or not the proteins had been previously exposed to ~-mercaptoethanol before electrophoresis. The molecular weights of HSCP and HSCP-PN estimated by gel electrophoresis were found to be 13 700 -+ 1370 and 14 700 + 1470, respectively. The molecular weight of 15 000 was estimated for HSCP-PN by gel-exclusion chromatography.
14 12
0.8 0~6
0-4
0
