Millie Thompson

Allergic encephalomyelitis: the physico-chemical properties of the basic protein encephalitogen from bovine spinal cord

Abstract A protein encephalitogen, isolated in a homogeneous state from bovine spinal cord, was found to have unusual properties. It was highly basic, containing 38 moles/ mole of basic amino acids (arginine, lysine, histidine) and only 7 moles/mole of acidic amino acids (glutamic and aspartic acid); the calculated isoionic point exceeded 12. Only one tryptophan (determined by three independent methods) and two methionine residues were present; no carbohydrate or lipid was associated with the isolated protein. Although similar to certain histone fractions, it differed from these proteins in its much higher content of histidine, glycine, and serine, and its lower content of alanine. The N-terminal position is blocked. The molecular weight determined by sedimentation equilibrium techniques was 16,400 daltons; by sedimentation-viscosity, 16,200; and by UV absorption studies based on the tyrosine-tryptophan content, 16,200. Estimates of molecular weight based on the amino acid analysis gave values of 15,500–18,200 daltons. An intrinsic viscosity of 9.27 ml/g and viscosity increment of 12.9 were found. From these data an axial ratio (a/b) of approximately 10:1 (assuming no hydration and a prolate ellipsoid) was calculated, revealing that the molecule is highly unfolded and approximates a rod-like shape. This property accounts for the unexpected behavior of this basic protein on gel nitration which has led to erroneously high molecular weight values. A small degree of aggregation was observed as indicated by the high molecular weight estimate of 22,300 found near the cell bottom using a long-column sedimentation equilibrium technique. No evidence of aggregation was observed at pH 2.6, 7.0, or 9.5 during sedimentation velocity studies. An S 0 20 , w value of 1.72S was found at pH 2.6, ionic strength 0.25. Considerable variation of the S 20, w was found with protein concentration, but not with pH. The highly extended conformation of the Al molecule, found from viscosity studies, is compatible with its resistance to denaturation; no loss in encephalitogenic activity was found following heating at 100 ° for 1 hr, treatment with 8 m urea for 8 hr, or incubation at pH 10.0 for 8 hr. Moreover, these procedures do not diminish interaction with antibody measured by the Ouchterlony and passive hemagglutination inhibition tests. Polyacrylamide gel electrophoresis also revealed no drastic changes in conformation arising from these procedures. It was concluded from these data that hydrophobic and hydrogen bonding play a minor role in the overall conformation of the A1 protein molecule. This property is of interest in considering the role of this protein as a membrane component.

The Encephautogenic Dose Response in Guinea Pigs of the Tryptophan Region of Myelin Basic Protein

The encephalitogenic dose response for the synthetic tryptophan peptide from the basic protein of myelin is given. The best dose in guinea pigs for disease induction using the peptide is between 1 and 3ug. Using doses higher or lower induces EAE in fewer animals.

An encephalitogenic region for rabbits

Abstract A synthetic peptide containing residues 154–162 within the rabbit myelin basic protein has been found to be encephalitogenic in rabbits. Synthetic analogs of this region have also been tested for their disease-producing ability.

Encephalitogenic Regions for the Lewis Rat within the Myelin Basic Protein

The sequence, phe lys asn ile val thr pro arg thr pro pro pro ser gln gly lys gly arg gly leu ser ser arg phe ser trp gly ala glu gly gln isolated from the peptic digestion of guinea pig myelin basicprotein is able to produce EAE in Lewis rats. The synthetic peptide phe lys phe gly gly arg asp ser arg, an analog of residues 154-162, is encephalitogenic in Lewis rats when B. pertussis is used as the adjuvant.

Further Definition of the Encephalitogenic Region for Guinea Pigs

The effect on encephalitogenicity of using a serinyl substitution for the glutaminyl group in the peptide ser arg phe ser trp gly ala glu gly gln arg is reported. We conclude from these results that the function of the glutaminyl residue is assisting this peptide to produce allergic encephalomyelitis in guinea pigs is as a hydrogen donor-receptor.

The proposed sequence of the encephalitogenic protein from chimpanzee brain

Abstract A proposed sequence of the chimpanzee encephalitogenic protein from the central nervous system myelin is reported. When compared to the human sequence, it contains one addition, no deletions and one substitution. In the case of the chimpanzee, at residue 144 there is an alanine instead of a valine and the adjacent residue is a glutamine. This latter change occurs also in the porcine basic protein.

The Effects of Imidates on the Encephalitogenicity of Myelin Basic Protein

Because the encephalitogenic tryptophan region within the porcine basic protein contains a lysine essential for disease induction, modification of the residue with imidates should further elucidate its contribution to basic protein encephalitogenicity. The modification of the encephalitogenic basic protein from myelin does not eliminate encephalitogenicity irrespective of the size of the imidate employed. This result suggests that the positive group in the tryptophan region functions only to help maintain a necessary overall conformation of the region.

Induction of Allergic Encephalomyelitis Using Hydrosoluble Adjuvant and the Tryptophan Region of Myelin Basic Protein

Experimental allergic encephalomyelitis has been induced in guinea pigs using the encephalitogenic tryptophan peptide as antigen and a hydrosoluble adjuvant extracted from Mycobacterium tuberculosis, var. hominis, strain H37Ra. The maximum response was observed using 100mug of adjuvant per animal. This is a quantity of adjuvant substantially higher than was necessary to induce disease utilizing the whole myelin basic protein as antigen.