Michael J. Weise

Basic proteins of rodent peripheral nerve myelin: immunochemical identification of the 21.5K, 18.5K, 17K, 14K, and P2 proteins.

Abstract: Proteins in peripheral nervous system and central nervous system myelin and homogenates of sciatic nerve and brain from young and adult mice and rats were characterized with affinity‐purified anti‐P2 and anti‐myelin basic protein sera after electrophoretic transfer from sodium dodecyl sulfate‐polyacrylamide gels to nitrocellulose sheets. Using this method we have identified a component of rodent peripheral nervous system myelin as P2 protein. Peripheral nervous system myelin also showed the presence of four basic proteins in addition to P2 protein. These were found to be analogous to the 14, 17, 18.5, and 21.5K species found in the central nervous system myelin. A number of high‐molecular‐weight proteins were also detected with anti‐myelin basic protein serum in peripheral nervous system, as well as central nervous system myelin. In addition, we report the presence of a high‐molecular‐weight P2 cross‐reactive protein in rodent brain stem homogenates, but not in central nervous system myelin.

Experimental allergic neuritis. I. Rat strain differences in the response to bovine myelin antigens.

Strain differences among rats to the induction and severity of experimental allergic neuritis (EAN) in response to whole PNS myelin were observed. Lewis rats were highly susceptible and developed severe EAN without central nervous system lesions (EAE), while Brown Norway rats were most resistant. Wistar, Sprague-Dawley, and Buffalo rats were susceptible but developed less severe disease than Lewis rats. Only Lewis rats consistantly developed EAN in response to isolated P2 protein. The severity of EAN was enhanced by treatment of the P2 with mercaptoethanol prior to injection. None of the strains developed EAN in response to galactocerebroside and none developed the lesions of EAE in response to any of the bovine myelin antigens tested. Myelin protein profiles from these rat strains were similar which suggests that factors other than target tissue differences, such as genetically determined immune responses to bovine myelin antigens, must be involved in these differing responses.

A T cell epitope for experimental allergic neuritis is an amphipathic α-helical structure

Abstract The bovine P2 protein is known to produce experimental allergic neuritis (EAN) in Lewis rats. The longest region of amphiphatic α-helix within the bovine P2 protein was identified as residues 61–72. A synthetic peptide representing this sequence was synthesized and shown to have the ability to (1) produce EAN in Lewis rats by direct sensitization, (2) produce a lymphoproliferative response in a P2-specific T cell line, and (3) stimulate a P2 protein-specific T cell line sufficiently to transfer EAN to naive recipient Lewis rats. Residues 61–72 is the shortest sequence thus far identified that will produce EAN in the Lewis rat and supports the contention that T cell determinants tend to be amphipathic α-helices.

Bovine peripheral nervous system myelin P2 protein: chemical and immunological characterization of the cyanogen bromide peptides.

Cleavage of bovine P2 protein by cyanogen bromide (CNBr) produced peptide fractions CN1, CN2, and CN3 which were isolated by gel filtration chromatography. CN2 was found to contain two NH2‐terminals (lysine and valine) and accounted for both of the cysteine residues of P2. When reduced carboxymethylated P2 (RCM‐P2) was digested with CNBr, peptides CN1 and CN3 were obtained as were (1) a peptide with NH2‐terminal lysine (Lys) that contained no homoserine and only one cysteine residue and (2) a peptide with NH‐2‐terminal valine (Val) that was co‐eluted with CN3. These data and the chemical characterization of all the CNBr peptides obtained from P2 and RCM‐P2 suggest that isolated P2 protein has a structure composed of the CNBr peptides in the order CN3‐CN1‐CN2(Val)‐CN2(Lys) with an intrachain disulfide bond between the cysteine residues located in the two constituent peptides of CN2, CN2(Lys) and CN2(Val), To locate the neuritogenic region(s) within the P2 protein structure, CN1, CN2, and CN3 were tested for the ability to induce experimental allergic neuritis (EAN) in Lewis rats. The disease‐inducing sites of P2 protein were found only in CN1; neither CN2 nor CN3 produced disease. EAN induced by CN1 was comparable to that induced with P2 protein as determined by disease onset, clinical symptoms, and histologic lesions.

An approach to searching protein sequences for superfamily relationships or chance similarities relevant to the molecular mimicry hypothesis: application to the basic proteins of myelin.

Abstract: A rapid method for similarity searches (FASTP program) was used to identify similarities between a protein database and the human basic proteins from myelin [P2 protein and 17.2K, 18.5K, and 21.5K variants of myelin basic protein (MBP)]. From similarity scores, we concluded that none of the presently known proteins are in a family containing the MBPs. No new members were found for the lipid‐binding family of which P2 is a member. Sequence similarities deemed relevant to the molecular mimicry hypothesis for virus‐induced autoimmunity were identified in FASTP data with the aid of microcomputer programs. Several MBP/viral protein similarities were found that have not been reported previously. Of note because of their association with demyelinating conditions were proteins from visna and vaccinia. Similarity with visna was specific to the 21.5K and 20.2K MBPs. The most interesting new possibility for mimicry involving the P2 protein was between the influenza A NS2 protein and a sequence region of P2 thought to be neuritogenic in animals and mitogenic for lymphocytes from some patients with Guillain‐Barre syndrome (GBS). This may have relevance for some cases of GBS associated with the 1976 U.S.A. swine flu vaccination program. Because FASTP reports only the best similarities between proteins, searches with FASTP may not have detected all the examples of mimicry present in the database. Searches might also be more effective if similarities could be scored on immunological rather than structural relatedness.

AN IMMUNOLOGICAL CHARACTERIZATION OF THE BASIC PROTEINS OF RODENT SCIATIC NERVE MYELIN

Radioimmunoassays (RIA) for the myelin basic protein (MBP) and P2 protein together with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to establish the identities of and relationships between the basic proteins (BP) of rodent peripheral nervous system (PNS) myelin. The PNS myelin proteins studied, in order of increasing mobility of SDS-PAGE, are P1, PR (R = rodent) and PB (B = breakdown). The majority of the acid extractable proteins of rodent PNS myelin are MBP related as shown by MBP-RIA. When tested individually, rodents P1, PR and PB were each found to cross-react in the RIA for MBP but not that for P2. The acid extracts of rodent PNS myelin were found to contain P2, although in minute quantities. P2 accounts for approximately 0.05-1.0% of the acid extractable protein in rodent PNS myelin.

Bovine P2 myelin basic protein crystallizes in three different forms

Abstract: P2 protein is a minor component of the myelin membrane. We have crystallized this protein for high‐resolution crystallographic study. Three crystal morphologies are available. Two of them are from ammonium sulfate, and one is from polyethyleneglycol (PEG). The unit cell of the most suitable crystals from PEG 4000 has the dimensions a= 91.3 Å, b= 99.8 Å, c= 56.0 Å; is of space group P212121; and contains up to four molecules per asymmetric unit. The limit of resolution is 2.7 Å.

Protein Composition of PNS Myelin: Developmental Comparison of Control and Quaking Mice

Abstract: Protein compositions were determined for sciatic nerve myelin isolated from young and adult control and quaking (Qk) mice. Age‐related changes in the relative amounts of large (P1) and small (Pr) basic proteins were found. In control animals, the ratio Pr/P1 increased with age, a change similar to that observed for the large (B1) and small (Bs) CNS myelin basic proteins of adult mice. Pr/P1 also increased with age in the Qk mouse sciatic nerve, but only to the point that the value in the adult Qk mouse was similar to that observed for young control animals, a situation reminiscent of the effect of the Qk mutation on CNS basic proteins. Thus, our data suggest that the Qk mutation has a similar effect on peripheral nervous system (PNS) and CNS basic proteins. Our findings are consistent with recent electrophoretic and immunochemical data showing that PNS and CNS myelin basic proteins in rodents are analogous, and they suggest that the genetic program controlling basic protein expression is common to oligodendroglia and Schwann cells.

Conformation of Bovine Nerve Root P2 Protein: Characteristics of the Molecule from Circular Dichroism Spectra

Abstract: Circular dichroism (CD) was used to study the conformations of bovine nerve root P2 basic protein, its reduced and carboxymethylated derivative (RCM‐P2), and its large cyanogen bromide fragment (CN1). Data in the far UV show that both the parent protein and RCM‐P2 have conformations dominated by a large amount of β structure. However, the CN1 peptide appears to exist in a largely unordered conformation. Since CN1 lacks short (20 residue) amino‐ and carboxy‐terminal segments of the P2 protein, the spectral data suggest that these regions are important for determining and/or maintaining folding of the P2 protein in aqueous solutions. The P2 protein was found to have a distinctive CD spectrum in the near UV. The characteristics of molar ellipticities indicate that the spectrum contains significant contributions from tyrosine residues, and that these contributions suggest different environments for the two tyrosines in P2 protein. Both environments depend on protein conformation, since CD side chain absorptions are lost when P2 protein is denatured with 5 M urea.

Research Methods in Studies with the P2 Basic Protein

Purification of proteins from PNS myelin began in the early 1970s. Isolation procedures were developed to obtain the basic proteins (Uyemura et al., 1970; London, 1971; Brostoff et al., 1972), and an early study (Brostoff et al., 1972) suggested that one of them was able to induce experimental allergic neuritis (EAN).