The high affinity immunoglobulin E (IgE) receptor is an alpha beta gamma 2 tetrameric complex. The truncated extracellular segment (alpha t) of the heavily glycosylated alpha chain is sufficient for high affinity binding of IgE. Here we have expressed various alpha t mutants in eukaryotic and prokaryotic cells to analyze the role of glycosylation in the folding, stability, and secretion of alpha t. All seven N-linked glycosylation sites in alpha t are glycosylated and their mutations have an additive effect on the folding and secretion of alpha t. Mutation of the seven N-glycosylation sites (delta 1-7 alpha t) induces misfolding and retention of alpha t in the endoplasmic reticulum. Similarly, tunicamycin treatment reduces substantially the folding efficiency of wild-type alpha t. In contrast, no difference in folding efficiency is detected between wild-type alpha t and delta 1-7 alpha t expressed in Escherichia coli. In addition, maturation of N-linked oligosaccharides and addition of O-linked carbohydrates are not required for either the transport or the IgE-binding function of alpha t. Furthermore, complete enzymatic deglycosylation does not affect the stability and the IgE-binding capacity of alpha t. Therefore, glycosylation is not intrinsically necessary for proper folding of alpha t but is required for folding in the endoplasmic reticulum. Our data are compatible with the concept that specific interactions between N-linked oligosaccharides and the folding machinery of the endoplasmic reticulum are necessary for efficient folding of alpha t in eukaryotic cells.
Objective. To assay antifilaggrin autoantibodies, we developed an enzyme-linked immunosorbent assay (ELISA) using a "citrullinated" recombinant rat filaggrin. Our objectives were to assess its value for diagnosing rheumatoid arthritis (RA) and to compare the results with those obtained using 4 other reference methods for detection of antifilaggrin autoantibodies, including the commercially available ELISA that uses a modified "citrullinated" synthetic peptide derived from the sequence of human filaggrin (CCP-ELISA).Methods. We analyzed 711 sera from patients with well-characterized rheumatic diseases, including 240 patients with RA. Antifilaggrin autoantibodies were detected by an ELISA using a recombinant rat filaggrin deiminated in vitro as immunosorbent (ArFA-ELISA). The results considered were the differences between the optical densities obtained on deiminated and nondeiminated proteins. Antibodies to rat esophagus epithelium were detected by indirect immunofluorescence, while antibodies to human filaggrin were detected by immunoblotting and by a recently described ELISA using a deiminated recombinant human filaggrin. Finally, CCP-ELISA was performed according to the manufacturer's recommendations.Results. At the titer thresholds allowing diagnostic specificities of 0.95, 0.985, and 0.99 to be reached, the diagnostic sensitivities of the ArFA-ELISA were 0.76, 0.67, and 0.65, respectively. At these 3 thresholds, the sensitivities were significantly higher than those of the 4 other tests. Despite incomplete overlapping of the 5 tests, the high diagnostic performance of the ArFA-ELISA allows us to propose this test to replace all the other methods for antifilaggrin autoantibody detection.
Conclusion.ArFA-ELISA appears to be the most efficient test among those available for the detection of antifilaggrin autoantibodies, in terms of diagnostic accuracy for RA. Its diagnostic performance in early RA and its prognostic value are currently under evaluation.
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