SUMMARYThe 52 000 MW Ro/SS-A (Ro52) protein is a major target of autoantibodies in autoimmune conditions such as systemic lupus erythematosus and Sjö gren's syndrome. Recent genomic and bioinformatic studies have shown that Ro52 belongs to a large family of related RING/ Bbox/coiled-coil (RBCC) tripartite motif proteins sharing overall domain structure and 40-50% identity at the amino acid level. Ro52 also has a B30.2 domain at the C-terminus. Using the human genome draft sequence, the genomic organization of the Ro52 gene on human chromosome 11p15.5 has been deduced and related to the protein domain structure. We show that the steady-state levels of Ro52 mRNA are normally very low but are induced by cell activation with interferon-c. In transient transfection of HeLa cells, epitope-tagged Ro52 protein was localized to unidentified membrane proximal rod-like structures. Using in vitro coupled transcription/translation followed by immunoprecipitation, the autoimmune response to Ro52 protein was investigated and two distinct interactions were resolved. The Ro52 C-terminal B30.2 domain interacts with human immunoglobulin independently of antibody specificities. Sera derived from patients with Sjö gren's syndrome and systemic lupus erythematosus, in addition, contained specific autoantibodies directed towards the rest of the Ro52 molecule. The majority of these autoimmune sera also immunoprecipitated the Ro52-related molecule RNF15. A possible role for Ro52 protein in alterations of plasma membranes during cellular activation or apoptosis is discussed.
SunllTlaryHuman histocompatibility leukocyte antigen (HLA)-DM is a facilitator of antigen presentation via major hlstocompatibility complex (MHC) class II molecules. In the absence of HLA-DM, MHC class II molecules do not present natural peptides, but tend to remain associated with class II-associated invariant chain peptides (CLIP). Recently, DM was shown to catalyze the release of CLIP from HLA-DR. We have investigated which peptides bound to HLA-DR are vulnerable to release upon encountering DM. By directed substitution of allele-specific anchor residues between CLIP and DR3-cognate peptldes and the application of recombinant DM we show that DM catalyzes the release of those peptides bound to HLA-DR3 that do not have appropriate anchor residues and, hence, no optimal ligand binding motif. Thus, HLA-DM acts as a peptide editor, facilitating selection of peptides that stably bind to class II molecules for eventual presentation to the immune system from the pool of available peptides.
The peptide motifs of two HLA molecules, B8 and DR3(17), which are associated with autoimmune diseases including myasthenia gravis, were determined from natural peptide pools using Edman degradation. The majority of HLA-B8 ligands are nonamers preferentially terminated by leucine. As a characteristic feature of the HLA-B8 motif, there is a high degree of conservation of positively charged amino acids at position 3 and 5, exclusively lysine at position 3, and lysine or arginine at position 5. Additional evidence for this allele-specific motif is the presence of these features in several viral peptides recognized by HLA-B8 restricted T cells. The DR3(17) motif is characterized by four conserved anchor-like positions ordered in an almost symmetrical arrangement, as has been found for DR1 and DR5 motifs. A first hydrophobic/aromatic anchor three to four residues apart from the N-terminus (at relative position 1) appears to be a common feature of DR ligands. The second anchor is an aspartate at relative position 4, which is likely to be the DR3(17)-specific contact site in the groove. Two additional conserved positions closer to the C-terminus are occupied by charged amino acids at relative position 6 and by hydrophobic/aromatic residues at positions 8, 9, or 10. Eight individual naturally processed DR17 ligands were sequenced and were found to be derived from exogenous proteins and cytoplasmic membrane receptors. These natural peptides conform well to the determined motif. A single exchange of the anchor-like positions in a model peptide abrogated binding to DR17+ cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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