Crystal structures of antigenic peptides bound to class I MHC proteins suggest that chemical modifications of the central part of the bound peptide should not alter binding affinity to the MHC restriction protein but could perturb the T-cell response to the parent epitope. In our effort in designing nonpeptidic high-affinity ligands for class I MHC proteins, oligomers of (R)-3-hydroxybutanoate and(or) beta-homoalanine have been substituted for the central part of a HLA-B27-restricted T-cell epitope of viral origin. The affinity of six modified peptides to the B2705 allele was determined by an in vitro stabilization assay. Four out of the six designed analogues presented an affinity similar to that of the parent peptide. Two compounds, sharing the same stereochemistry (R,R,S,S) at the four stereogenic centers of the nonpeptidic spacer, bound to B2705 with a 5-6-fold decreased affinity. Although the chiral spacers do not strongly interact with the protein active site, there are configurations which are not accepted by the MHC binding groove, probably because of improper orientation of some lateral substituents in the bound state and different conformational behavior in the free state. However we demonstrate that beta-amino acids can be incorporated in the sequence of viral T-cell epitopes without impairing MHC binding. The presented structure-activity relationships open the door to the rational design of peptide-based vaccines and of nonnatural T-cell receptor antagonists aimed at blocking peptide-specific T-cell responses in MHC-associated autoimmune diseases.
Total protein variation as revealed by two-dimensional electrophoresis (2D-E) was studied in 18 isolates from populations of Meloidogyne arenaria (six isolates), Meloidogyne incognita (10 isolates), and Meloidogyne javanica (one isolate) plus an unclassified isolate. Gels (80 x 60 x 0.75 mm) were silverstained and digitized in order to compare their protein patterns. Optical density and position of protein patterns were measured using statistical cluster analysis and computer-assisted image analysis software. Only those protein stains or positions that were clearly defined (i.e., without background) were considered. The number of positions in gels ranged from 86 to 203. Each of these positions had 95 clearly expressed proteins that were present in at least two replicates for each isolate. Spot position was considered a taxonomical character with two different states: presence (1) and absence (0). Accordingly, genetic distance was estimated among isolates and species, and a phylogenetic tree was constructed following the cladistic approach based on maximum parsimony analysis. Isolates of M. arenaria--M. javanica--Meloidogyne sp. and of M. incognita formed two separate monophyletic groups. Both groups were clearly defined on the basis of two sets of protein positions that can be considered as diagnostic characters. An attempt to identify these proteins by mass spectrometry was made. Group diagnostic proteins for M. incognita and M. arenaria (and for other proteins common to all isolates) were distinguished by protonated mass signals in the MALDI fingerprinting spectrum.
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