Localization of Antigenic Determinants on Bovine Encephalitogenic Protein

Abstract

By peptic or tryptic proteolysis of different fragments of bovine encephalitogenic protein, a set of small fragments covering the following regions of the native protein have been obtained: 44-68, 44-89, 75-91, 93-116, 114-130, 134-150, and 154-170 (the amino acid residues are numbered according to [l]). These fragments were used in the macrophage migration inhibition technique in guinea pigs (an assay which is vividly considered as a correlate in vitro to delayed type hypersensitivity) to study the localization of antigenic determinants in the protein.The results indicate the presence of a t least one determinant in each of regions 44-68, 134-140, and 154-170. Furthermore, the rather strong determinant, which has previously been localized around the single tryptophan residue in the protein does not seem to be contained within peptide 114-130, as this peptide is considered unreactive in the assay. On the other hand, this peptide shows full encephalitogenic activity.Experimental allergic encephalomyelitis is an experimentally induced disease in animals, which is considered to be predominantly due to a cell-mediated autoimmunity against central nervous system myelin [2]. The disease-inducing component of the myelin is a strongly basic protein which has been well characterized [l -61. This encephalitogenic protein (from bovine tissue) has been shown to have a t least four different sites capable of inducing the disease in rabbits [7-91. The activities of these sites seem to vary considerably. Guinea pigs, on the other hand, respond predominantly to only one of these regions, whereas the others have marginal, if any, activity [9-111. Investigations in this laboratory have been concerned with the relation between disease-inducing sites and antigenic determinants on the protein. I n a recent report [12] we described the isolation and partial characterization of a set of different proteolytically and chemically derived fragments of bovine encephalitogenic protein. These fragments have been used to study antigenic determinants on the protein capable of reacting in the macrophage migration inhibition test, which is a well-documented correlate in vitro to delayed type hypersensitivity.There could be demonstrated 1131 a t least four different reactive regions in the protein with the apparently most reactive sequence containing the single tryptophan residue in position 116.I n the present communication, attempts a t a narrower localization of the determinants are described, using a new set of fragments and the macrophage migration inhibition assay in guinea pigs. MATERIALS AND METHODSPepsin (P-6875 Lot 7OC-0280) and trypsin (T-1005 Lot 7OC-8070) were both obtained from Sigma Chemical Company. Fig.1 shows the schematical description of the preparation of the different fragments. Preparation of the Different PolypeptidesThe isolation and characterization of fragments 44-116, 90-170, and 117-170, which provided starting material for the preparation of new fragments, has previously been described in detail [12].Fragments 44-89 ...

Large peptides of bovine and guinea pig myelin basic proteins produced by limited peptic hydrolysis

“…These cleavages have been shown previously to occur at relatively high pepsin/substrate ratios (Carnegie, 1971;Eylar et al, 1971;Bergstrand, 1972).…”

Evolutionary Divergence in the Structure of Myelin Basic Protein: Comparison of Chondrichthye Basic Proteins with Those from Higher Vertebrates

“…Guinea pigs and Lewis rats immunized with shark MBP , and guinea pigs immunized with dogfish MBP (Agrawal et al, 1971), have previously been reported to show no evidence of EAE. Since MBP has several antigenic determinants (Hruby et al, 1985) it is possible that the observed cellular immunity is directed toward a part of the antigen that is not involved in the induction of EAE in guinea pigs (Bergstrand, 1972;Spitler et al, 1972;Bergstrand and Kallen, 1973).…”

Antigenic determinants on bovine encephalitogenic protein. Localization of regions that induce transformation of lymph node cells from immunized rabbits

“…Fragment 93-170 was isolated directly from the acid extract of defatted bovine brain tissue (Bergstrand, submitted, c.f. [ 15,211) and an aliquot of the purified peptide was reacted with HNB-Br according to the method of Barman and Koshland [22] to give fragment HNB-93-170. The degree of substitution was determined to 1.2 HNB-residues/tryptophan residue.…”

“…The results of these examinations, except for those concerning peptides 154-168 and 154-1 69, have been given in appropriate reports. In previous publications [12][13][14][15]19, 2 11 the nomenclature of some of the peptides differs from that presently used. Previously, peptides 44-89,tyr, 44-68, tyr, 44-11 6,tyr, 1 17-170,tyr, and 134-150,tyr were referred to as solely 44-89,44-68,44-116, 117-170 and 134-150, respectively.…”

“…Rabbits respond with EAE at several different sites, which have been localized t o the following peptides: 1-20 and 65-74 [9],44-68 [lo], 114-122 [ l l ] , 134-150and 154-170 [12, 131. The properties of the BEP molecule have also been studied with reference to cell-mediated immunity. Specific determinants capable of reacting in the macrophage migration inhibition assay using guinea pigs were examined at our laboratory ( [14][15][16] In a previous study [ 191 we investigated a set of polypeptide fragments of BEP for their ability to cause lymphocyte transformation with the blood leukocyte test. A crude localization was obtained showing activity within regions 1-43, 44-1 16 and 1 17-170, but no activity was found in the region around the trytophan residue (residue 116).…”