By comparing the amino acid sequences of 11 mammalian and 1 avian prion proteins (PrP), structural analyses predicted four a-helical regions. Peptides corresponding to these regions of Syrian hamster PrP were synthesized, and, contrary to predictions, three ofthe four spontaneously formed amyloids as shown by electron microscopy and Congo red staining. By IR spectroscopy, these amyloid peptides exhibited secondary structures composed largely of 13-sheets. The first of the predicted helices is the 14-amino acid peptide corresponding to residues 109-122; this peptide and the overlapping 15-residue sequence 113-127 both form amyloid. The most highly amyloidogenic peptide is AGAAAAGA, which corresponds to Syrian hamster PrP residues 113-120 and is conserved across all species for which the PrP sequence has been determined. Two other predicted a-helices corresponding to residues 178-191 and 202-218 form amyloids and exhibit considerable 13-sheet structure when synthesized as peptides. These findings suggest the possibility that the conversion of the cellular isoform of PrP to the scrapie isoform of PrP Involves the transition of one or more putative PrP a-helices into 13-sheets and that prion diseases are disorders of protein conformation. 27-30 (19, 20).In the study reported here, we synthesized peptides homologous to portions of the Syrian hamster (SHa) PrP sequence and examined their physical characteristics by attenuated total reflection (ATR) Fourier transform IR spectroscopy (FTIR) (21). The particular peptides were chosen based on structural predictions where the PrP amino acid sequences from 11 mammalian and 1 avian sources were compared (22). Those analyses suggested the possibility that PrP might fold into a monomeric molecule with four a-helices (ref. 23; J.-M.G., F.C., R.F., and S.B.P., unpublished results). Unexpectedly, synthetic peptides corresponding to three of the four putative a-helices exhibit extensive 13-sheet structure as well as the ultrastructural and tinctorial properties of amyloid.MATERIALS AND METHODS All PrP peptides (Table 1) were assembled by using the 9-fluorenylmethoxycarbonyl variant of the Merrifield solidphase method on an Applied Biosystems model 432A Synergy peptide synthesizer. Peptides with side-chain protection were cleaved from the resins by using thioanisole/ethanedithiol/ trifluoroacetic acid (TFA) and precipitated with tert-butyl methyl ether. They were pelleted by centrifugation, suspended in ether, washed three times, repelleted, dissolved in water/ isopropanol, and filtered. Peptides without side-chain protection were cleaved with 90%o (vol/vol) TFA. The solutions were lyophilized, and the peptides were analyzed by reversed-phase HPLC and plasma desorption mass spectrometry. The majority ofpeptides were >80%o pure and were used without further purification. The initial product for peptide H4 was =20%6 pure, and it was purified by preparative HPLC and reanalyzed. The synthesis of SHaPrP-(113-120) gave 40%o Ala6Gly2 (the desired product) and 60% Ala5Gly2, which ...

Prion diseases are a group of neurodegenerative disorders in human and animals that seem to result from a conformatlonal change in the prion protein (PrP). Utilizing data obtained by circular dichroism and in spectroscopy, computational studies prdIe the tedi nal s ture of the cellular form of PrP (PrPC). A heuristic approach consistng of the prediction of so structures and of an evalnation of the pig of s y elements was used to search for plausible teriay strucures. After a series of experimental and theoretical ants were applied, four structural models of four-helix bundles emerged. A group of amino acids within the four predicted helices were Identified as important for tertiary interactions between helices. These amino acids could be essential for ma ining a stable tertiary suture of PrPC. Among four plausible s al models for PrPC, the X-bundle model seemed to correlate best with 5 of 11 known point mutations that segregate with the inherited prion diseases. These 5 mutions cluster around a central hydrophobic core in the X-bundle structure. Furthermore, these mutations occur at or near those amino acds which are predicted to e important for helix-helix interactions. The three-dimensonal structure of

Fractions enriched for acetylcholine receptor-inducing activity from chicken brain were found to contain a protein that was -30% homologous with mmalian prion proteins [Harris, D.

We report here on a patient with anti-myelin-associated glycoprotein (MAG) neuropathy in whom examination of a sural nerve biopsy by multichannel confocal microscopy showed a partly overlapping distribution of MAG and IgM deposits in myelinated fibers. Our data demonstrate that MAG in Schmidt-Lanterman incisures and paranodal loops, as well as some additional HNK-1-positive components of the basal lamina, are the major targets of the anti-MAG monoclonal IgM autoantibodies in this neuropathy in vivo. Perforation of the basal lamina can allow the penetration and binding of anti-MAG IgM inside myelinated fibers. Our results support and extend the notion that the production of monoclonal anti-MAG IgM may be antigenically driven by MAG molecules and that this process may occur in the immunologically privileged environment of the nerve prior to the appearance of a genuine gammopathy in serum.

The IgM monoclonal autoantibodies of patients with demyelinating paraproteinaemic polyneuropathy recognize a carbohydrate structure present on both myelin-associated glycoprotein (MAG) and protein zero (P0). These autoantibodies are sufficient to cause the disease but the mechanism of demyelination remains unclear. We have analysed nerve biopsies from eight patients with polyneuropathy and anti-MAG antibodies by quantitative immunohistochemistry and find a concordant pattern of reduced expression of myelin markers with the loss of myelinated fibres. We report here novel features of this disease, in particular a selective lack of detectable MAG in a large proportion of myelinated-fibres containing P0, myelin basic protein (MBP) and periaxin. There is also an inverse correlation of the distribution of MAG in peripheral nerve myelin with the serum anti-MAG antibody titres but no correlation of these titres with the loss of myelinated fibres. Double immunofluorescence staining of paraproteinaemic polyneuropathy (PPN) nerves shows anti-MAG IgM deposited on the periphery of myelinated fibres associated with or lacking MAG staining. These data suggest that the binding of anti-MAG antibodies to MAG and/or other myelin component(s) results in MAG downregulation and may have an essential role in the molecular mechanisms leading to demyelination and partial regeneration in this disease.