The orthologue of the "acatalytic" mammalian ART4 in chicken is an arginine-specific mono-ADP-ribosyltransferase

<p>Abstract</p> <p>Background</p> <p>Human ART4, carrier of the GPI-(glycosyl-phosphatidylinositol) anchored Dombrock blood group antigens, is an apparently inactive member of the mammalian mono-ADP-ribosyltransferase (ART) family named after the enzymatic transfer of a single ADP-ribose moiety from NAD⁺to arginine residues of extracellular target proteins. All known mammalian ART4 orthologues are predicted to lack ART activity because of one or more changes in essential active site residues that make up the R-S-EXE motif. So far, no other function has been detected.</p> <p>Results</p> <p>Here we report the identification and characterisation of ART4 in chicken, which to our knowledge is the first true non-mammalian orthologue of a mammalian ART family member. The chicken <it>ART4 </it>gene has the same physical structure as its mammalian counterparts (three coding exons separated by two introns in phase 0 and phase 1, respectively) and maps to a region of conserved linkage synteny on chromosome 1. Its mRNA encodes a 289 amino acid protein with predicted N-terminal signal peptide and C-terminal GPI-anchor sequences and 47% sequence identity to human ART4. However, in striking contrast to its mammalian orthologues, the chicken protein contains an intact R-S-EXE motif. Upon ectopic expression in C-33A cells, recombinant chicken ART4 localized at the cell surface as a GPI-anchored, highly glycosylated protein, which displayed arginine-specific ART activity (apparent K_mof the recombinant protein for etheno-NAD⁺1.0 ± 0.18 μM).</p> <p>Conclusion</p> <p>The avian orthologue of the "acatalytic" mammalian ART4 is a mono-ADP-ribosyltransferase with enzymatic activity comparable to that of other, catalytically active and GPI-anchored members of the mammalian ART family.</p

IL-4 receptor-alpha-dependent control of Cryptococcus neoformans in the early phase of pulmonary infection

Cryptococcus neoformans is an opportunistic fungal pathogen that causes lung inflammation and meningoencephalitis in immunocompromised people. Previously we showed that mice succumb to intranasal infection by induction of pulmonary interleukin (IL)-4Rα-dependent type 2 immune responses, whereas IL-12-dependent type 1 responses confer resistance. In the experiments presented here, IL-4Rα −/− mice unexpectedly show decreased fungal control early upon infection with C. neoformans , whereas wild-type mice are able to control fungal growth accompanied by enhanced macrophage and dendritic cell recruitment to the site of infection. Lower pulmonary recruitment of macrophages and dendritic cells in IL-4Rα −/− mice is associated with reduced pulmonary expression of CCL2 and CCL20 chemokines. Moreover, IFN-γ and nitric oxide production are diminished in IL-4Rα −/− mice compared to wild-type mice. To directly study the potential mechanism(s) responsible for reduced production of IFN-γ, conventional dendritic cells were stimulated with C. neoformans in the presence of IL-4 which results in increased IL-12 production and reduced IL-10 production. Together, a beneficial role of early IL-4Rα signaling is demonstrated in pulmonary cryptococcosis, which contrasts with the well-known IL-4Rα-mediated detrimental effects in the late phase

In the presence of IL-4Rα elevated pulmonary chemokine expression, IFN-γ mRNA expression and NO production.

<p>Following infection, WT (open circle) and IL-4Rα^−/− (gray circle) mice were sacrificed at the time points indicated (A, C) or at 14 days after infection (B). RT-qPCR analyses were done to determine the expression of mRNAs as indicated. Data are derived from one (A, C, 0 and 7 dpi) or one representative out of two (A, 14 dpi) or three (C, 14 dpi) experiments (n = 6–7 mice per genotype and experiment). Statistical analysis was done using the unpaired Student's t-test (ns, not significant; *P<0.05; **P<0.01; ***P<0.001 (A, C)). The concentration of nitric oxide (NO) in cell culture supernatants was determined using the Griess reaction. Pooled data from two different experiments are shown. Dotted line represents detection limit. Statistical analysis was done using the Mann-Whitney test. **P<0.01 (B).</p

Stronger pulmonary inflammation, eosinophilia, and mucus production in WT as compared with IL-4Rα^−/− mice.

<p>Lung slices from WT and IL-4Rα^−/− mice infected for 14 days were stained with H&E (A-F) and periodic acid Schiff reagent (G, H). Leukocyte infiltration and fungal load are depicted in panels A, C and B, D. Sites of inflammation contain eosinophils (arrowheads) and large, multinucleated macrophages (E) or lymphocytes (F). Mucus production by bronchial epithelial cells is depicted in G and H. One representative experiment out of three with n = 6–7 animals per group is shown.</p

Early fungal growth control in pulmonary infection with <i>C. neoformans</i> in the presence of IL-4Rα signaling.

<p>Wild-type (WT, open circle) and IL-4Rα^−/− (gray circle) mice on C57BL/6J background were infected intranasally with <i>C. neoformans</i>. Analysis of fungal burdens in the lung was done at different days <i>post infectionem</i> (dpi) as indicated. Shown is data from n = 7 mice per group from one representative of three independent experiments (14 dpi) or from two independent experiments (7; 21 and 42 dpi). Statistical analysis was done using the unpaired Student's t-test (7 dpi) or Mann-Whitney test. *P<0.05; **P<0.01.</p