At least three distinct B cell epitopes reside in the C-terminal half of La protein, as determined by a recombinant DNA approach.

The La antigen is a nuclear protein that is one of the major target antigens of autoantibodies found in the sera of patients with primary Sjögren's syndrome. The formation of such autoantibodies is therefore likely to reflect the basic immunopathogenesis of this disorder. A recombinant DNA strategy has been used to examine the La protein for sequences that encode autoimmunizing B cell epitopes. We have isolated and characterized a 1.2-kb-long cDNA from a human liver cDNA library encoding a region of the La protein; this region contains 296 amino acids, including the C terminus. A sub-library of recombinant DNA in the expression vector pEX was made from portions of the La cDNA. Individual fusion proteins were tested by immunoblotting and enzyme-linked immunosorbent assay for their ability to react with anti-La autoantibodies contained in sera from patients with Sjögren's syndrome. In this way, we have identified at least three distinct epitopes in the C-terminal half of the La protein. Every anti-La serum tested contained antibodies against all three of the antigenic regions identified. Furthermore, most of the sera display similar ratios between the titers of antibodies with the three kinds of specificity. Our data suggest that the production of anti-La autoantibodies may be antigen driven.

Biochemical properties of the ras-related YPT protein in yeast: a mutational analysis.

Using site-directed mutagenesis, the ras-related and essential yeast YPT1 gene was changed to generate proteins with amino acid exchanges within conserved regions. Bacterially produced wild-type proteins were used for biochemical studies in vitro and were found to have properties very similar to mammalian ras proteins. Gene replacement allowed the study of physiological consequences of the mutations in yeast cells. Lys21----Met and Asn121----Ile substitutions rendered the protein incapable of binding GTP and caused lethality. Ser17----Gly and Ala65----Thr substitutions slightly changed the protein's apparent binding capacity for either GDP or GTP and altered its intrinsic GTPase activity. These mutations were without effect on cellular growth. The YPTgly17,thr65 mutant protein displayed a significantly altered relative capacity for guanine nucleotide binding but a GTPase activity comparable to the wild-type protein. In contrast to the Ala65----Thr substitution, the double mutant displayed a significantly reduced capacity for autophosphorylation and allowed cells to grow only poorly. Cellular growth was improved when this mutant protein was overproduced.