Combined N-glycome and N-glycoproteome analysis of the lotus japonicus seed globulin fraction shows conservation of protein structure and glycosylation in legumes

Legume food allergy, such as allergy toward peanuts and soybeans, is a health issue predicted to worsen as dietary advice recommends higher intake of legume-based foods. Lotus japonicus (Lotus) is an established legume plant model system for studies of symbiotic and pathogenic microbial interactions and, due to its well characterized genotype/phenotype and easily manipulated genome, may also be suitable for studies of legume food allergy. Here we present a comprehensive study of the Lotus N-glycoproteome. The global and site-specific N-glycan structures of Lotus seed globulins were analyzed using mass spectrometry-based glycomics and glycoproteomics techniques. In total, 19 N-glycan structures comprising high mannose (∼20%), pauci-mannosidic (∼40%), and complex forms (∼40%) were determined. The pauci-mannosidic and complex N-glycans contained high amounts of the typical plant determinants β-1,2-xylose and α-1,3-fucose. Two abundant Lotus seed N-glycoproteins were site-specifically profiled; a predicted lectin containing two fully occupied N-glycosylation sites carried predominantly pauci-mannosidic structures in different distributions. In contrast, Lotus convicilin storage protein 2 (LCP2) carried exclusively high mannose N-glycans similar to its homologue, Ara h 1, which is the major allergen in peanut. In silico investigation confirmed that peanut Ara h 1 and Lotus LCP2 are highly similar at the primary and higher protein structure levels. Hence, we suggest that Lotus has the potential to serve as a model system for studying the role of seed proteins and their glycosylation in food allergy.10 page(s

Tir Is Essential for the Recruitment of Tks5 to Enteropathogenic Escherichia coli Pedestals.

Enteropathogenic Escherichia coli (EPEC) is a bacterial pathogen that infects the epithelial lining of the small intestine and causes diarrhea. Upon attachment to the intestinal epithelium, EPEC uses a Type III Secretion System to inject its own high affinity receptor Translocated intimin receptor (Tir) into the host cell. Tir facilitates tight adhesion and recruitment of actin-regulating proteins leading to formation of an actin pedestal beneath the infecting bacterium. The pedestal has several similarities with podosomes, which are basolateral actin-rich extensions found in some migrating animal cells. Formation of podosomes is dependent upon the early podosome-specific scavenger protein Tks5, which is involved in actin recruitment. Although Tks5 is expressed in epithelial cells, and podosomes and EPEC pedestals share many components in their structure and mechanism of formation, the potential role of Tks5 in EPEC infections has not been studied. The aim of this study was to determine the subcellular localization of Tks5 in epithelial cells and to investigate if Tks5 is recruited to the EPEC pedestal. In an epithelial MDCK cell line stably expressing Tks5-EGFP, Tks5 localized to actin bundles. Upon infection, EPEC recruited Tks5-EGFP. Tir, but not Tir phosphorylation was essential for the recruitment. Time-lapse microscopy revealed that Tks5-EGFP was recruited instantly upon EPEC attachment to host cells, simultaneously with actin and N-WASp. EPEC infection of cells expressing a ΔPX-Tks5 deletion version of Tks5 showed that EPEC was able to both infect and form pedestals when the PX domain was deleted from Tks5. Future investigations will clarify the role of Tks5 in EPEC infection and pedestal formation

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Tks5 localized with bundled actin in MDCK cells.

<p>A: Western blot of lysates of Tks5-EGFP-MDCK cells and untransfected MDCK cells probed with anti-Tks5 (top panels) and α-tubulin (bottom panels) antibodies. Arrows indicate bands of Tks5-EGFP and Tks5 isoforms. α-tubulin was included as loading control. One splice variant of Tks5 was observed in WT MDCK cells. Tks5-EGFP-MDCK cells expressed various splice variants of endogenous Tks5 and/or Tks5-EGFP (125–140 kDa); Tks5-EGFP (150–175 kDa) was expressed at a higher level than endogenous Tks5 in the transfected cell line. B and C: Tks5-EGFP-MDCK cells were stained with Hoechst to visualize nuclei (blue) and phalloidin-rhodamine to visualize actin or α-tubulin antibody (red). Images are maximum projections of Z-stacks of 20 or 17 slices, respectively. White arrows in B indicate a site of colocalization of Tks5-EGFP and actin, whereas the yellow arrows in C point to Tks5-EGFP not co-localizing with α-tubulin, and the pink arrows point to α-tubulin. D: Tks5-EGFP-MDCK cells were transiently transfected with LifeAct-mRuby and treated with cytochalasin D to disrupt actin fibers and imaged live with fluorescence microscopy. Timepoints after cytochalasin D addition are indicated. Arrows indicate aggregates of actin, to which Tks5-EGFP also localized. Scale bars correspond to 10 μm.</p

Tks5 was recruited simultaneously with actin and N-WASp to the site of EPEC infection and they colocalized in the pedestal.

<p>A: Tks5-EGFP-MDCK cells were infected with WT EPEC for four hours. Cells and bacteria were then fixed and stained with phalloidin to visualize actin (red), Hoechst to visualize DNA and an antibody against lipid A in the bacteria membrane (both in blue). Tks5-EGFP co-localized with actin in the pedestal. B: Tks5-EGFP-MDCK cells transiently transfected with N-WASp-mCherry were infected with WT EPEC for four hours, then fixed and stained with Hoechst and lipid A antibody. Tks5-EGFP co-localized with N-WASp-mCherry at the infection site. Cells and bacteria in A and B were imaged on a confocal microscope, and the shown slices were selected to both show EPEC bacteria as well as protein localization. Arrows point to positions of individual bacteria. Scale bars correspond to 5 μm. C and D: Tks5-EGFP-MDCK cells transiently transfected with LifeAct-mRuby (C) or N-WASp-mCherry (D) were infected with low numbers of WT EPEC bacteria and imaged with widefield microscopy. Tks5-EGFP and LifeAct-mRuby or N-WASp-mCherry are shown as inverted contrast to ease the interpretation. A flash of Tks5-EGFP and LifeAct-mRuby or Tks5-EGFP and N-WASp were observed instantly upon adhesion (exemplified with Tks5-EGFP and LifeAct-mRuby in C). Upon adhesion, Tks5-EGFP was rearranged in the membrane simultaneously with LifeAct-mRuby or N-WASP-mCherry (exemplified with N-WASp-mCherry in D). Arrows point to positions of the initial rearrangements of Tks5 and LifeAct-mRuby or N-WASp-mCherry. Scale bars correspond to 3 μm.</p

Tks5 localized to EPEC infection site and Tir is essential for the localization.

<p>Tks5-EGFP-MDCK cells were infected with WT, Δ<i>tir</i>, Δ<i>escN</i>, JPN15, or Tir_Y474F EPEC for four hours, as indicated. Cells and bacteria were fixed and stained with phalloidin to visualize actin (red) and Hoechst and an antibody directed against lipid A in the bacterial membrane to visualize nuclei and EPEC bacteria (both shown in blue). Z-stacks were acquired on a confocal microscope, and the shown slices were selected to both show EPEC bacteria as well as Tks5-EGFP and actin localization. Tks5-EGFP and actin were recruited to the infection site of WT, Tir_Y474F, and JPN15 EPEC, where they accumulated around the infecting bacteria. No recruitment was observed for Δ<i>tir</i> and Δ<i>escN</i> EPEC. Arrows point to the positions of individual bacteria. Scale bars correspond to 5 μm.</p