The aim of this work was to identify Lactobacillus johnsonii NCC533 (La1) surface molecules mediating attachment to intestinal epithelial cells and mucins. Incubation of Caco-2 intestinal epithelial cells with an L. johnsonii La1 cell wall extract led to the recognition of elongation factor Tu (EF-Tu) as a novel La1 adhesin-like factor. The presence of EF-Tu at the surface of La1 was confirmed by analysis of purified outer surface protein extract by immunoblotting experiments, by electron microscopy, and by enzyme-linked immunosorbent assays of live bacteria. Furthermore, tandem mass spectrometry analysis proved that EF-TU was expressed at the La1 surface as an intact molecule. Using recombinant La1 EF-Tu protein, we were able to determine that its binding to intestinal cells and to mucins is pH dependent. Competition experiments suggested that EF-Tu has an important role in La1 mucin binding capacity. In addition, immunomodulation studies performed on HT29 cells showed that EF-Tu recombinant protein can induce a proinflammatory response in the presence of soluble CD14. Our in vitro results indicate that EF-Tu, through its binding to the intestinal mucosa, might participate in gut homeostasis.Probiotic bacteria, mainly lactic acid bacteria and bifidobacteria, have been shown to have beneficial effects on the immune defenses and to alleviate or prevent diverse intestinal disorders (3,4,16,25,27,30,39,42,47). Several in vitro studies have shown that one of them, Lactobacillus johnsonii NCC533 (La1), is able to bind to epithelial cell lines (5,8,9,21) and can induce the secretion of different cytokines in coculture systems (9, 24). Furthermore, human and animal studies have demonstrated that La1 has immune adjuvant effects (40, 44) and can also act as a modulator of nonspecific immune responses (6,14,29,48,53,62).The mechanisms underlying these beneficial effects are not completely understood, but it is believed that the maximum probiotic effects can be achieved if the organisms adhere to mucus and/or intestinal epithelial cells (31, 62). It has recently been shown that lipoteichoic acid (LTA), a molecule associated with the surface of La1 bacteria, participates in their adhesion to intestinal cells (21) and has an immunomodulatory effect on gut homeostasis (64). However, competition experiments indicated that LTA is not the only surface molecule mediating La1 binding to epithelial cells (21). Indeed, it had already been suggested by Bernet et al. (5) that proteinaceous compounds are involved in the attachment of bacteria to these cells. This observation is in accordance with recent studies showing that surface proteins of other lactobacilli participate in adhesion to epithelial cell lines, gastrointestinal mucins, or extracellular matrix proteins (1,26,58,60).In this work, therefore, we have investigated the ability of La1 surface proteins to attach to intestinal epithelial cells and mucoproteins. We have identified the elongation factor Tu as a novel surface protein possessing the characteristics of an adhesion ...
We recently demonstrated the presence of a new asparaginelinked complex glycan on plant glycoproteins that harbors the Lewis a (Le a ), or Gal(1-3)[Fuc␣(1-4)]GlcNAc, epitope, which in mammalian cells plays an important role in cell-to-cell recognition. Here we show that the monoclonal antibody JIM 84, which is widely used as a Golgi marker in light and electron microscopy of plant cells, is specific for the Le a antigen. This antigen is present on glycoproteins of a number of flowering and non-flowering plants, but is less apparent in the Cruciferae, the family that includes Arabidopsis. Le a -containing oligosaccharides are found in the Golgi apparatus, and our immunocytochemical experiments suggest that it is synthesized in the trans-most part of the Golgi apparatus. Le a epitopes are abundantly present on extracellular glycoproteins, either soluble or membrane bound, but are never observed on vacuolar glycoproteins. Double-labeling experiments suggest that vacuolar glycoproteins do not bypass the late Golgi compartments where Le a is built, and that the absence of the Le a epitope from vacuolar glycoproteins is probably the result of its degradation by glycosidases en route to or after arrival in the vacuole.
Objective. To identify a new autoantigen/ autoantibody population in rheumatoid arthritis (RA) sera.Methods. Following a population-based recruitment effort, 255 patients with very early arthritis (median disease duration 4 months) were studied using different clinical, biologic, and radiologic assessments. After a followup period of 1 year, patients were classified as having RA (n ؍ 145), non-RA rheumatic diseases (n ؍ 70), and undifferentiated arthritis (n ؍ 40). Patients' sera were analyzed by one-dimensional (1D) and 2D Western blotting. The recognized 50-kd protein was analyzed by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS). RA serum reactivities were evaluated against the recombinant protein synthesized by an in vitro coupled transcription-translation system.Results. On 1D Western blots, 36 of the 145 RA sera bound to a 50-kd polypeptide. On 2D Western blots, anti-50-kd؉ RA sera recognized a triplet of isoelectric point 6.5-7.0 and a molecular mass of 50 kd. The 3 spots of the triplet were analyzed by MALDI-TOF MS and were shown to correspond to human ␣-enolase. A goat anti-enolase antiserum, which recognized a band comigrating with the 50-kd antigen on 1D Western blots, gave a labeling pattern on 2D Western blots similar to that observed with anti-50-kd؉ RA sera. Among the 36 RA sera that identified ␣-enolase in protein maps, only 8 recognized the recombinant (unmodified) ␣-enolase. The specificity of anti-␣-enolase antibodies for RA was 97.1%. Half of the anti-␣-enolase-positive RA patients were negative for both rheumatoid factor and antifilaggrin antibodies. The presence of anti-␣-enolase antibodies was the greatest predictive factor of radiologic progression in the first 66 RA patients included.Conclusion. Autoantibodies to ␣-enolase, an enzyme of the glycolytic pathway, are present in the sera of patients with very early RA and have potential diagnostic and prognostic value for RA.
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