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The appearance of a new antigenic determinant during the degradation of myelin basic protein
Journal o/Net~,oin, munolcgy, 2 (1982) 201-207 Elsevier Biomedi,al Press
201
The Appearance of a New Antigenic Determinant During the Degradation of Myelin Basic Protein John N. Whitaker Research o~,dNeurology Semices of tit,'. Memphis Veterans Administration Medical Center and of Neurology and Anatomy, University of Tennearee Center for the Health Sciences, Memphis, TIV 38104 (U.S.A.) (Received I0 August, 1981) (Revised, received 25 September, 1981) (Accepted i October, !)81)
Summary Studies were undertaken to determine if a previously unrecognized or inaccessible antigenic determinant might be exposed during the course of digestion of basic protein by a normal brain enzyme. As studied by double antibody radioimmunom~ say, exposure of boviite brain myelin basic protein to bovine brz~n cathepsin D led to the appearance of an antigenic de~.erminant recoLmiTed by an antibody reactive predominantly with the~.molecular region of BP encompassing residues 79-88. The 5 major microheterogeneous components of basic protein demonstrated this plmaomenon. These results indicate that a normally appearing enzyme in brain known to be present in a number of cell types including ofigodendrocytes can lead to the appearance of peptides of basic protein whose antigenic determinants may not be revealed in the intact mole¢ ude. This finding suggests that a number of basic protein pepfides may be released by a similar mechanism so that efforts made to detec~ and qoanfitate such peptides must be capable of recognizing their anique antigenic features. K e y w o r d s : Antigenic determinant -. Brain e n z y m e - M y e l i n basic p r m e i n - Peptides
Address tmmmponde~ce to Dr. Whitaker at: Neurology Service, Veterans Admini~tratiOQ IM[edi(~l
Center, 1030 Jeffe,soa Ave.aue, Mete,his, "IN 38104, U.S.A. This work was c~ducted trader the Vetermm Administration Research prcqpam stud warn aho tmpp0cted by Grant I 111.B-4 from the National Multiple ,%i~fosi~ Society. Dr. Whitak~ is a Medical Invettil~to¢ of the Veterans Administration. 0165-$728/82/0000--0000/$02.50 O 1982 Elsevier Biomedical press
202 Introduction Myelin basic protein (BP) has l~.¢n extensively investigated becaase of its disease, indu,:in$ property of producing experimental allergic encephalitis. The molecular regiohs responsible for disease induction vary, and a number of di fferent determinant,~ on the molecule have been shown to react with antibody a n d t o stiraulate cellular immunity (Day 1981). Whereas many of the initial serological studies of BP utilized antibodies to the intact molecule (Day et al. 1974; Driscoll et at. 1974; Whitaker et al. 1975; Whitaker and McFarlin 1977), other investigations have revealed that with the use of BP peptides (Barton et al. 19"12;Hung and Raa~h 1980) or with antibodies selectively directed toward BP peptides immunochemica! results may be different than findings with the intact molecule. For example, rabbit antibc~diesspecifically directed a~ peptide region 79-88 from the BP molecule of 169 residues fail to react with the intact BP moIecule'(Whitaker et al. 1977). Lewis rat antibodies to guinea pig BP pepride 43-88 also principally recognize the carboxyl end of the peptide (Fritz et al. ]978), and monoclonal rat antibodies to guinea pig BP peptide 68-88 may not react with intact BP (Fritz and McFadin 1981). BP is readily degraded by a variety of enzymes (Hashim and Eylar 1969). Peptides generated by naturally occurring enzymes in vivc may lead to the release of BP peptides which may carry uni~.ue ant.~genic d~terminants. In the present investigation it is demonstrated that bovine brain cathepsin D can digest bovine BP with the release and appearance of an antigenic deternfinant p~eviously undetected in the intact BP molecule. Materials a ~ Methods
Preparation of BP Bovine brain was c,btained from Pel[reez (Rogers, AR). BP was isolated from delipidated brain tissue which had been extracted at pH 3 and chromatographed over carboxymethylcellulose at pH 10.5 (Whitaker and Se~yer 1979b). The multiple peaks of BP eluting with the NaCI gradient were designated as microheterogeneous components 1-5 with component 1 the last to elute (Whitaker and Seyer 1979b). The separated 3P components were desalted on Sephadex G-25 ";n 0,5% acetic acid and lyophilized. Individual components were rechromatographed separately over ca~rboxymethylcellulose and desalted under identical conditions.,
Preparation of bovine brain cathepsin D Cathepsin D was purified from bovine brain using affinity chromatography with immobilized pepstatin as previously described (Whitaker and Seyer 1979a). The purified cathepsin D K/as dissolved in water, and its protein concentration was determined by the Fol~n phenol method (Lowry et al. 1951) with a standard of crystalline bovine serum albumin. The enzyme was stored at --90°C and thawed sh~:~rtlybefore use.
203
Preparation of bovine BP peptide 43-88 Bovin,.' BP peptide 43-88 was prepared :'tom bovine BP component one by digestion with brain cathepsin D ag previottsly described (Whitaker and S.-yer 1979b).
Antisera to BP peptide 43-88 The rabbit (R96) antiserum to bovine BP peptide 43-88 was the same as that previously produced (Whitaker et Id. 1977) b) injections with a mixture of bovine BP peptides 37-88 and 43-88 attach~l by carbodiimide to rabbit serum albumin. The characteristics of this antiserum, absorbed with bovine BP, have been described (Whitaker et al. 1977). It fails to react with intact bovine BP but reacts well with BP pepfides 43-88, 68-88 and 79-88 aad to a lesser extent with BP peptide 1-[8.
Assay Bovine BP components 115 w[~re incubated at 37~C in a volume of I ml of 0.05 molar citrate buffer, pH 3.5, with bovine bra/n cathep+in D at a protein-enzyme ratio of 1 : 100. The usual incubation assay contained 400 ttg of BP and 4/tg of brain cathepsin D. At periodic interval;;, an aliquot of the reaction mixture was removed and placed in a solution of Tris-acetate buffer, pH 7.2, containing 0.2~ methylated bovine serum albumin (TA-MBSA buffer). The diluted material was kept at 4oC for approximately I - 7 days prior to assay. The material was quantitated in a 3-day, equilibrium double antibody-r~dioimmunoassay using R96 antiserum as previously described (Wlrdtaker et al. 1977) except that bovine BP peptide 43-88 rather than an admixture of BP pepfides 37-88 and 43-88 was used as the rad..'oligand. The standard for the assay ,~as bovine BP peptide 43-88.
Polyacrylamide $ei electrophoresis Three different polyacrylan:tide gel systems were used for analysis of BP. Purity was assessed by electrophoresis at pH 8.8 (Benuck et al, 1975; Whitaker and Seyer 1979b) and at pH 2.5 in the presence of urea (Deibler et ad. 1972). The componen.s of BP were analyzed at pH 10.6 in t he presence of urea (Deibler et al. 1972; Shults et al. 1978).
Results
Characterization of BP components The 5 isolated components of bo~4ne BP migrated sim/~arly as single distinct bands when evaluated on gels at pH 8.8 or at pH 2.5 in the presence of urea. In both gel systems a +nore anodal minor band, interpreted to be a dimer of BP (Whitaker 1978b) alto occurred. At pH 10.6 ~n the presence of urea the microheterogeneity of BP was evident (Fig. 1). The BP in the pH 3 extract of bovine, brain appeared as major bands. The BP component represented in each of these band~ was enriched in the peaks obtained from the cation.exchange chromatography on carboxymethyl-cellulcse.
2O4
Fig. I. Polyac:rylamidedisc gel electrophoceticstady at pH 10.6in the presenceof urea of the pH .~extract of bovinebra.in (i) and microheterogene,:uscomponents five (2). four (3). three (4]. two (5) and one (6). 125/ag of material were applied to gel I and 25 pg to each of gels 2-6. Cathode at barton,.
Degradation of Bt' On exposure of bovine BP to cathepsin D an antigen~.c determinant, which had previously been detected only ~.t low levels or been undetcctable, appeared (Fig. 2). A 2. fold increase in the antigen measured was noted w,.'t.hin 15 min of incubation a~ 37°C, and values continued to increase for approximately 2h, at which time litde, additional increa.-,e occurred. There was no significant difference in the extent of reactivity of the 5 microheterogeneous components of intacg BP with this antiserum. This indicated that among the microheterogeneou:; components of bovino BP this antiseru:n and assay detected no structural or conformatior~al differences. Although there were minor differences in the rates of appearance of the new determinant during the degradation of the different components, many of the values overlapped. If cathepsin I-} was omitted or if pepstatin, 1 l,~g/ml, was included, no incre~¢ in irnmunoreactive material occurred. Discussion
BP (Cohen et al. 19":6) or its peptides (Whitaker 1977) enter cerebrospina~ fluid. and probably other body fluids, immediately after injury to cenu'al nervous s.',stem
205
°-'6
~b
d~
do
',h""-,s,,
,~o
~,
MltttJTEI Of: INCUI~TIOtl
Fig. 2. ,~,ppearance of antigenic d~'.terminaql reactive with antiserum to BP region containing residue, 79-8L lgwin© BP components I-5 were incubated ia :: volume of ! ml ~vit~hbovine brain cathepsin D at a prot,:in t~) enzyme ratio of 100: ! at pH 3.5 and 37°C. At intervals, 5 91 of sample was withdrawn from file reaction mixture and mixed with 2 ml of TA-MBSA buffer. Value~ shown in thL; figure are from one reprc~ntative assay and were obtained by multiplying tl~¢ numerical r~'~dt from the ~adioinmmnoassay to accoant for the dilution factor.
myelin. Although BP (Whitaker 1978a; Itoyama et al. 1980) and the myelin-associated glycoprot¢in (ltoyama et al. 1980) disappear as the process of demydination evolves in multiple sclerosis (MS), the mechanisms for bo'~h removal ~md degradation are poorly understood. A number of prot-.inases increase: in the tissue lesions of MS (HLtsoh 1981). One of the proteinases wi~ch show elevated activity in the lt:sions of MS is cathepsin D (Einstein et al. 1972). Macrophages and microglia are the major phagocytic cells in the demyelinating lesioas ef MS (Pr~neas and Wright 1978). and it is usually assumed that these, two cell types ~a,e the cellalar sources ~forcathepsin D and possibly other proteinases and lipases. Re~:ent immunocytochemi,:al studies have revealed that in the rat, the ol/godendrocyte also conta/ns cathepsin D (Whitaker et al. 1981). Whatever the source, this naturally occt~rring acidic prc,teinase readily degrades BP with the two phenylalanyl-phenylalan/ne bonds at residues 42-43 and 88-89 very susceptible to cleavage (Whitaker ~ d Seye~" 19791)). Results of the present investigation "indicate that a potential ant/genic determimint, previously inaccessible or in a different conformafon, becomes ,~xposed daring the degradation of bovine BP by ~tthepsin D. No~e of the 5 microheterogeneous c.mnponents of BP was reactive with antiserum directed at the carbc,xyl end of BP peptide 43-88 (Whitaker et al. 1.977) and all 5 displayeJ a similar susceptibility to degradation. This suggests that the ::ontrol of the processes of selective deamidation and pho6phorylation of BP, which account for its microheterogeneity (Chou et al. 1976), is not associated with a chang." in the conformation of the BP molecule in the region of peptide 79-88.
206 It has become evident that there are antigenic determinants (Whitaker et al. 1977: Day et al. 1981; Fritz and McFarfin 1981) f ~ in the c~arboxyl portion of BF peptide 43-8~° which have limited accessibility in the intact BP molecule. This may also be the case with the encephafitogerde determinant of BP for the Lewis :rat (Hashim et al. 1979). For serological studies of BP peptides, the detection of the peptides depends on the availability of selective antisera, and the detection of antibody requires that the correct BP pepfide be used. Other antigenic determinants may appear from other molecular re~,ions of the BP molecule when degradation occurs and fragments of varying sizes and conformation are released. T h e size and nature of the BP peptides formed will be determined by the specif'wity of the proteinase or combination of proteinases. Immunochemical detection of such newly appearing deI:erminants will require that the appropriate antisera and assays are utilized.
Acknowledgemews Mr. Jeff Goode, Ms. Laura Muehl and Mrs. 0live Smith p r o d d e d excellent technical assistal~ce. The aumoJ ~.hanks Mrs. Sara Brewer and Mrs. Ann Marie Bobrowski for secretarial assistance in ~,e preparation of the matms~ript.
References Bareon, M.A., T.A. McPherson, R.U. LemJeux and O.O. Bain, Experimentalallergic eneephalomyelith; (E.A.E.)--Synthesis of the 7.11, 11-20, and 12-20 peptides of human encephalitoi~nk:protein and inhibition of histologicalE.A.E. by the }-~) peptide, Canad. J. Biochem.,50 (1972) 689-696. Benuck, M., N. Marks and G.A. Hashim, Metabolicinstability of myelin proteins ~ Breakdownof bask protein induced by brain cathepsin D, Europ. J. Biochem.,52 (1975) 615-621. Cho,,. F.C.-H., C.-H.J. Cliou, Re Shapira and R.F. FAbler, Ba~ 04' mk:rohetewf,eneieyof myelin basic protein, J. Biol. Chem., 251 (1976) 2671-2679. Cohen, S.R., R.M. Herndon and G.M. McKhann, Radiohnmunonssayfor myelin basic pt,otein in q,imd fluid--An index of active demyelination,N. F.nO. J. Med,, 295 (1976) 145~-!457. Day, ED., Myelin basic protein. In: F.P. inman and W.J. Mandy (Eds.), Contt~nporary Topics in Mol~ular Immunology,Vol. 8, Plenum, New Yolk, NY, 1981, pp. !-39. Day, E.D. and O.M. Pitts, Radioimmunoassayof myelin basic protein in r,odJum sulfate, Imm,Jnochem., 11 (1974) 651-659. Day, E.D., G.A. Hashim, V.A. Varitek and P.Y. Paterson, Immuno~nieity of synthetic peptide tequen¢~ $81 and $82 (residues 68-83 and 65-83) of bovine myelin I)asic protein, j. Neuroimmmml., I (1981) 205-216. Deibler, G.E., R.E. Ma~'tensonand M.W. Kies, Large scale preparation of myehn balk protein from central nervous tissue of severalmammalian specks, PFep. Biochcm.,2 (1972) 139-165. Driscoll, B.F., A.I. Kfamer and M.W. Kies, Myelin bathe protein~Locatim of multiple independent antigenic regions, Science, 18,~(1974) 73-75. Einstein, E.R., J. Csejtey, K.B. Dalai, C.W.M. Adams, O.B. Bayliu and J.IL Hallpik¢. Proteoly~ activity and basic protein loss in and around multiple ~lerosht plaques -- Combintd biochemical at~d histochemicalobservations, J. Neurochem., 19 (1972) 653-662. Fritz, R.B., C.-H.J. Chou, D.H. Randolph, A.F. De~al.'dinsand R.F, Kibler, Sl~'if'tcity of I m ~ from Lewis rats immunized with encephalitoge~¢ fraiFr~©nt43.88 of IWine-tpig myelin basic isro~in. ; lmmunol., 121 (1978) 1865-1869.
Fritz, R.B. and D.E, McFarlin, Monoclonal ra~ anti1:.~lies against enccphatitogenic pcptide 68-8g o¢ guinea pig myelin basic protein, Fed, Proc., 4~} (19~ 1) 996. Hashim, G.A. and E,H. Eylar, Allergic encephah~myel tis -- Enzymatic de;gradation of Ihe ¢aceph~togenie basic protein from bovine spinal cord, Arch. i!]iorhem, Biophys., 129 (1969) 635-644. Hashim, G.A., R.D. Sharpe and E.F. Carvalho, Exi~imental allergic encepbalomyelitis--SequesteRd encepl~litogenic determinant in the bovine myelin 13asieprotein, J. N©urochem., 32 (1979) 73-77. Hirsch, H.F_..,Proteinases and demyelination, J. Histoc~enl. Cytochem., 29, 14umber 3A (1981) 425-430. Hung, T.C. and H.C. Rauch, Antibody response to syn~her ¢ encephalitogenic oeptide, MoL lmmunoL. IV (19~0) 527-53 I. Itoyama, Y., N.H. Sternberser, H. de F. Webster, I;t.H. Quades~ S.R Cohen ~Lnd E.P. Rkhardsom, lmmunocytochemical observations on the distribution of myelin-associated glycoprotein and myelin basic pro:ein in multiple sclerosis lesions, Ann. Ne~srol.,7 (1980) 167-177. Lowry, O.H., N.Y. RcL~ebrough,A.L. Farr and R.J. Re ~dall, Proteit~ mea.~urementwith the Folin phenol reagent, J. I~ioi. Chem., I93 (1951) 265-27S. ..~rine,as, J.W. and R,G. Wright, Macropha~es, lymlr~hOcytes, and plasma cells in d¢ ~qeriuascular compartment in c]u'onic multiple sclerosis, Lab. lax ~t., 38 (1978) 4U9-421. Shults, C.W., J.N. Whi.~akerand J.G. Wood, Myelin b~sic protein microheterogeneity in subfractions of rat brain myelin, J. Neurochem., .~0 (1978) 1543-1551. Whitaker, J.N., The di:~tribution of myelin basic protein in central nervous system lesions of multiple sclerosis and acute t:xperimental allergic el~cephalon~yelifis,Ann. Neurol., 3 (1978a) 291-298. Whitaker, J.N., Immunochemical comparisons among l~yelin basic proteins, Comic. Bicchem. Physiot., 59B (1978b) 299-306. Whitaker, J.N. and J.M. Seyer, Isolation and characterizalion ot bovine brain cathep:;in D, J. Neurochem., 32 (1979a) 325-333. Whitaker, J.N. and J.M. Seyer, The sequential fimited degradation of bovine myelin basic protein by bovine brain cathepsinD, J. Biol. Chem., 254 (1979b) 6956-6963. Whitaker, J.N., C.-HJ. Chou, F.C.-H. Chou and R.F. Kibler, Molecular intentali~atio~ of a t~gioa o~,' myelin basic protein, J. Exp, Med., 146 (1977) 317-331. Whitaker, J.N., L.C. Terry and W.D. Whetseli, lmmunocytochemical localization of cathepsln D in rat neural tissue, Bra,n Res., 216 (1981) 109-124.
201
The Appearance of a New Antigenic Determinant During the Degradation of Myelin Basic Protein John N. Whitaker Research o~,dNeurology Semices of tit,'. Memphis Veterans Administration Medical Center and of Neurology and Anatomy, University of Tennearee Center for the Health Sciences, Memphis, TIV 38104 (U.S.A.) (Received I0 August, 1981) (Revised, received 25 September, 1981) (Accepted i October, !)81)
Summary Studies were undertaken to determine if a previously unrecognized or inaccessible antigenic determinant might be exposed during the course of digestion of basic protein by a normal brain enzyme. As studied by double antibody radioimmunom~ say, exposure of boviite brain myelin basic protein to bovine brz~n cathepsin D led to the appearance of an antigenic de~.erminant recoLmiTed by an antibody reactive predominantly with the~.molecular region of BP encompassing residues 79-88. The 5 major microheterogeneous components of basic protein demonstrated this plmaomenon. These results indicate that a normally appearing enzyme in brain known to be present in a number of cell types including ofigodendrocytes can lead to the appearance of peptides of basic protein whose antigenic determinants may not be revealed in the intact mole¢ ude. This finding suggests that a number of basic protein pepfides may be released by a similar mechanism so that efforts made to detec~ and qoanfitate such peptides must be capable of recognizing their anique antigenic features. K e y w o r d s : Antigenic determinant -. Brain e n z y m e - M y e l i n basic p r m e i n - Peptides
Address tmmmponde~ce to Dr. Whitaker at: Neurology Service, Veterans Admini~tratiOQ IM[edi(~l
Center, 1030 Jeffe,soa Ave.aue, Mete,his, "IN 38104, U.S.A. This work was c~ducted trader the Vetermm Administration Research prcqpam stud warn aho tmpp0cted by Grant I 111.B-4 from the National Multiple ,%i~fosi~ Society. Dr. Whitak~ is a Medical Invettil~to¢ of the Veterans Administration. 0165-$728/82/0000--0000/$02.50 O 1982 Elsevier Biomedical press
202 Introduction Myelin basic protein (BP) has l~.¢n extensively investigated becaase of its disease, indu,:in$ property of producing experimental allergic encephalitis. The molecular regiohs responsible for disease induction vary, and a number of di fferent determinant,~ on the molecule have been shown to react with antibody a n d t o stiraulate cellular immunity (Day 1981). Whereas many of the initial serological studies of BP utilized antibodies to the intact molecule (Day et al. 1974; Driscoll et at. 1974; Whitaker et al. 1975; Whitaker and McFarlin 1977), other investigations have revealed that with the use of BP peptides (Barton et al. 19"12;Hung and Raa~h 1980) or with antibodies selectively directed toward BP peptides immunochemica! results may be different than findings with the intact molecule. For example, rabbit antibc~diesspecifically directed a~ peptide region 79-88 from the BP molecule of 169 residues fail to react with the intact BP moIecule'(Whitaker et al. 1977). Lewis rat antibodies to guinea pig BP pepride 43-88 also principally recognize the carboxyl end of the peptide (Fritz et al. ]978), and monoclonal rat antibodies to guinea pig BP peptide 68-88 may not react with intact BP (Fritz and McFadin 1981). BP is readily degraded by a variety of enzymes (Hashim and Eylar 1969). Peptides generated by naturally occurring enzymes in vivc may lead to the release of BP peptides which may carry uni~.ue ant.~genic d~terminants. In the present investigation it is demonstrated that bovine brain cathepsin D can digest bovine BP with the release and appearance of an antigenic deternfinant p~eviously undetected in the intact BP molecule. Materials a ~ Methods
Preparation of BP Bovine brain was c,btained from Pel[reez (Rogers, AR). BP was isolated from delipidated brain tissue which had been extracted at pH 3 and chromatographed over carboxymethylcellulose at pH 10.5 (Whitaker and Se~yer 1979b). The multiple peaks of BP eluting with the NaCI gradient were designated as microheterogeneous components 1-5 with component 1 the last to elute (Whitaker and Seyer 1979b). The separated 3P components were desalted on Sephadex G-25 ";n 0,5% acetic acid and lyophilized. Individual components were rechromatographed separately over ca~rboxymethylcellulose and desalted under identical conditions.,
Preparation of bovine brain cathepsin D Cathepsin D was purified from bovine brain using affinity chromatography with immobilized pepstatin as previously described (Whitaker and Seyer 1979a). The purified cathepsin D K/as dissolved in water, and its protein concentration was determined by the Fol~n phenol method (Lowry et al. 1951) with a standard of crystalline bovine serum albumin. The enzyme was stored at --90°C and thawed sh~:~rtlybefore use.
203
Preparation of bovine BP peptide 43-88 Bovin,.' BP peptide 43-88 was prepared :'tom bovine BP component one by digestion with brain cathepsin D ag previottsly described (Whitaker and S.-yer 1979b).
Antisera to BP peptide 43-88 The rabbit (R96) antiserum to bovine BP peptide 43-88 was the same as that previously produced (Whitaker et Id. 1977) b) injections with a mixture of bovine BP peptides 37-88 and 43-88 attach~l by carbodiimide to rabbit serum albumin. The characteristics of this antiserum, absorbed with bovine BP, have been described (Whitaker et al. 1977). It fails to react with intact bovine BP but reacts well with BP pepfides 43-88, 68-88 and 79-88 aad to a lesser extent with BP peptide 1-[8.
Assay Bovine BP components 115 w[~re incubated at 37~C in a volume of I ml of 0.05 molar citrate buffer, pH 3.5, with bovine bra/n cathep+in D at a protein-enzyme ratio of 1 : 100. The usual incubation assay contained 400 ttg of BP and 4/tg of brain cathepsin D. At periodic interval;;, an aliquot of the reaction mixture was removed and placed in a solution of Tris-acetate buffer, pH 7.2, containing 0.2~ methylated bovine serum albumin (TA-MBSA buffer). The diluted material was kept at 4oC for approximately I - 7 days prior to assay. The material was quantitated in a 3-day, equilibrium double antibody-r~dioimmunoassay using R96 antiserum as previously described (Wlrdtaker et al. 1977) except that bovine BP peptide 43-88 rather than an admixture of BP pepfides 37-88 and 43-88 was used as the rad..'oligand. The standard for the assay ,~as bovine BP peptide 43-88.
Polyacrylamide $ei electrophoresis Three different polyacrylan:tide gel systems were used for analysis of BP. Purity was assessed by electrophoresis at pH 8.8 (Benuck et al, 1975; Whitaker and Seyer 1979b) and at pH 2.5 in the presence of urea (Deibler et ad. 1972). The componen.s of BP were analyzed at pH 10.6 in t he presence of urea (Deibler et al. 1972; Shults et al. 1978).
Results
Characterization of BP components The 5 isolated components of bo~4ne BP migrated sim/~arly as single distinct bands when evaluated on gels at pH 8.8 or at pH 2.5 in the presence of urea. In both gel systems a +nore anodal minor band, interpreted to be a dimer of BP (Whitaker 1978b) alto occurred. At pH 10.6 ~n the presence of urea the microheterogeneity of BP was evident (Fig. 1). The BP in the pH 3 extract of bovine, brain appeared as major bands. The BP component represented in each of these band~ was enriched in the peaks obtained from the cation.exchange chromatography on carboxymethyl-cellulcse.
2O4
Fig. I. Polyac:rylamidedisc gel electrophoceticstady at pH 10.6in the presenceof urea of the pH .~extract of bovinebra.in (i) and microheterogene,:uscomponents five (2). four (3). three (4]. two (5) and one (6). 125/ag of material were applied to gel I and 25 pg to each of gels 2-6. Cathode at barton,.
Degradation of Bt' On exposure of bovine BP to cathepsin D an antigen~.c determinant, which had previously been detected only ~.t low levels or been undetcctable, appeared (Fig. 2). A 2. fold increase in the antigen measured was noted w,.'t.hin 15 min of incubation a~ 37°C, and values continued to increase for approximately 2h, at which time litde, additional increa.-,e occurred. There was no significant difference in the extent of reactivity of the 5 microheterogeneous components of intacg BP with this antiserum. This indicated that among the microheterogeneou:; components of bovino BP this antiseru:n and assay detected no structural or conformatior~al differences. Although there were minor differences in the rates of appearance of the new determinant during the degradation of the different components, many of the values overlapped. If cathepsin I-} was omitted or if pepstatin, 1 l,~g/ml, was included, no incre~¢ in irnmunoreactive material occurred. Discussion
BP (Cohen et al. 19":6) or its peptides (Whitaker 1977) enter cerebrospina~ fluid. and probably other body fluids, immediately after injury to cenu'al nervous s.',stem
205
°-'6
~b
d~
do
',h""-,s,,
,~o
~,
MltttJTEI Of: INCUI~TIOtl
Fig. 2. ,~,ppearance of antigenic d~'.terminaql reactive with antiserum to BP region containing residue, 79-8L lgwin© BP components I-5 were incubated ia :: volume of ! ml ~vit~hbovine brain cathepsin D at a prot,:in t~) enzyme ratio of 100: ! at pH 3.5 and 37°C. At intervals, 5 91 of sample was withdrawn from file reaction mixture and mixed with 2 ml of TA-MBSA buffer. Value~ shown in thL; figure are from one reprc~ntative assay and were obtained by multiplying tl~¢ numerical r~'~dt from the ~adioinmmnoassay to accoant for the dilution factor.
myelin. Although BP (Whitaker 1978a; Itoyama et al. 1980) and the myelin-associated glycoprot¢in (ltoyama et al. 1980) disappear as the process of demydination evolves in multiple sclerosis (MS), the mechanisms for bo'~h removal ~md degradation are poorly understood. A number of prot-.inases increase: in the tissue lesions of MS (HLtsoh 1981). One of the proteinases wi~ch show elevated activity in the lt:sions of MS is cathepsin D (Einstein et al. 1972). Macrophages and microglia are the major phagocytic cells in the demyelinating lesioas ef MS (Pr~neas and Wright 1978). and it is usually assumed that these, two cell types ~a,e the cellalar sources ~forcathepsin D and possibly other proteinases and lipases. Re~:ent immunocytochemi,:al studies have revealed that in the rat, the ol/godendrocyte also conta/ns cathepsin D (Whitaker et al. 1981). Whatever the source, this naturally occt~rring acidic prc,teinase readily degrades BP with the two phenylalanyl-phenylalan/ne bonds at residues 42-43 and 88-89 very susceptible to cleavage (Whitaker ~ d Seye~" 19791)). Results of the present investigation "indicate that a potential ant/genic determimint, previously inaccessible or in a different conformafon, becomes ,~xposed daring the degradation of bovine BP by ~tthepsin D. No~e of the 5 microheterogeneous c.mnponents of BP was reactive with antiserum directed at the carbc,xyl end of BP peptide 43-88 (Whitaker et al. 1.977) and all 5 displayeJ a similar susceptibility to degradation. This suggests that the ::ontrol of the processes of selective deamidation and pho6phorylation of BP, which account for its microheterogeneity (Chou et al. 1976), is not associated with a chang." in the conformation of the BP molecule in the region of peptide 79-88.
206 It has become evident that there are antigenic determinants (Whitaker et al. 1977: Day et al. 1981; Fritz and McFarfin 1981) f ~ in the c~arboxyl portion of BF peptide 43-8~° which have limited accessibility in the intact BP molecule. This may also be the case with the encephafitogerde determinant of BP for the Lewis :rat (Hashim et al. 1979). For serological studies of BP peptides, the detection of the peptides depends on the availability of selective antisera, and the detection of antibody requires that the correct BP pepfide be used. Other antigenic determinants may appear from other molecular re~,ions of the BP molecule when degradation occurs and fragments of varying sizes and conformation are released. T h e size and nature of the BP peptides formed will be determined by the specif'wity of the proteinase or combination of proteinases. Immunochemical detection of such newly appearing deI:erminants will require that the appropriate antisera and assays are utilized.
Acknowledgemews Mr. Jeff Goode, Ms. Laura Muehl and Mrs. 0live Smith p r o d d e d excellent technical assistal~ce. The aumoJ ~.hanks Mrs. Sara Brewer and Mrs. Ann Marie Bobrowski for secretarial assistance in ~,e preparation of the matms~ript.
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