Human milk oligosaccharides differentially support gut barrier integrity and enhance Th1 and Th17 cell effector responses in vitro.

Human milk oligosaccharides (HMOs) can modulate the intestinal barrier and regulate immune cells to favor the maturation of the infant intestinal tract and immune system, but the precise functions of individual HMOs are unclear. To determine the structure-dependent effects of individual HMOs (representing different structural classes) on the intestinal epithelium as well as innate and adaptive immune cells, we assessed fucosylated (2'FL and 3FL), sialylated (3'SL and 6'SL) and neutral non-fucosylated (LNT and LNT2) HMOs for their ability to support intestinal barrier integrity, to stimulate the secretion of chemokines from intestinal epithelial cells, and to modulate cytokine release from LPS-activated dendritic cells (DCs), M1 macrophages (MØs), and co-cultures with naïve CD4+ T cells. The fucosylated and neutral non-fucosylated HMOs increased barrier integrity and protected the barrier following an inflammatory insult but exerted minimal immunomodulatory activity. The sialylated HMOs enhanced the secretion of CXCL10, CCL20 and CXCL8 from intestinal epithelial cells, promoted the secretion of several cytokines (including IL-10, IL-12p70 and IL-23) from LPS-activated DCs and M1 MØs, and increased the secretion of IFN-γ and IL-17A from CD4+ T cells primed by LPS-activated DCs and MØs while reducing the secretion of IL-13. Thus, 3'SL and 6'SL supported Th1 and Th17 responses while reducing Th2 responses. Collectively, our data show that HMOs exert structure-dependent effects on the intestinal epithelium and possess immunomodulatory properties that confer benefits to infants and possibly also later in life.

Spectroscopic and biochemical studies on protein variants of quinaldine 4-oxidase: Role of E736 in catalysis and effects of serine ligands on the FeSI and FeSII clusters.

Quinaldine 4-oxidase (Qox), which catalyzes the hydroxylation of quinaldine to 1H-4-oxoquinaldine, is a heterotrimeric (LMS)2 molybdo-iron/sulfur flavoprotein belonging to the xanthine oxidase family. Variants of Qox were generated by site-directed mutagenesis. Replacement in the large subunit at E736, which is presumed to be located close to the molybdenum, by aspartate (QoxLE736D) resulted in a marked decrease in kcat app for quinaldine, while Km app was largely unaffected. Although a minor reduction of the glutamine substituted variant QoxLE736Q by quinaldine occurred, its activity was below detection, indicating that the carboxylate group of E736 is crucial for catalysis. Replacement of cysteine ligands C40, C45, or C60 (FeSII) and of the C120 or C154 ligands to FeSI in the small subunit of Qox by serine led to decreased iron contents of the protein preparations. Substitutions C40S and C45S (Fe1 of FeSII) suppressed the characteristic FeSII EPR signals and significantly reduced catalytic activity. In QoxSC154S (Fe1 of FeSI), the g-factor components of FeSI were drastically changed. In contrast, Qox proteins with substitutions of C48 and C60 (Fe2 of FeSII), and of the C120 ligand at Fe2 of FeSI, retained considerable activity and showed less pronounced changes in their EPR parameters. Taken together, the properties of the Qox variants suggest that Fe1 of both FeSI and FeSII are the reducible iron sites, whereas the Fe2 ions remain in the ferric state. The location of the reducible iron sites of FeSI and FeSII appears to be conserved in enzymes of the xanthine oxidase family.

N-acetylanthranilate amidase from Arthrobacter nitroguajacolicus Rü61a, an alpha/beta-hydrolase-fold protein active towards aryl-acylamides and -esters, and properties of its cysteine-deficient variant.

N-acetylanthranilate amidase (Amq), a 32.8-kDa monomeric amide hydrolase, is involved in quinaldine degradation by Arthrobacter nitroguajacolicus Rü61a. Sequence analysis and secondary structure predictions indicated that Amq is related to carboxylesterases and belongs to the alpha/beta-hydrolase-fold superfamily of enzymes; inactivation of (His(6)-tagged) Amq by phenylmethanesulfonyl fluoride and diethyl pyrocarbonate and replacement of conserved residues suggested a catalytic triad consisting of S155, E235, and H266. Amq is most active towards aryl-acetylamides and aryl-acetylesters. Remarkably, its preference for ring-substituted analogues was different for amides and esters. Among the esters tested, phenylacetate was hydrolyzed with highest catalytic efficiency (k(cat)/K(m) = 208 mM(-1) s(-1)), while among the aryl-acetylamides, o-carboxy- or o-nitro-substituted analogues were preferred over p-substituted or unsubstituted compounds. Hydrolysis by His(6)Amq of primary amides, lactams, N-acetylated amino acids, azocoll, tributyrin, and the acylanilide and urethane pesticides propachlor, propham, carbaryl, and isocarb was not observed; propanil was hydrolyzed with 1% N-acetylanthranilate amidase activity. The catalytic properties of the cysteine-deficient variant His(6)AmqC22A/C63A markedly differed from those of His(6)Amq. The replacements effected some changes in K(m)s of the enzyme and increased k(cat)s for most aryl-acetylesters and some aryl-acetylamides by factors of about three to eight while decreasing k(cat) for the formyl analogue N-formylanthranilate by several orders of magnitude. Circular dichroism studies indicated that the cysteine-to-alanine replacements resulted in significant change of the overall fold, especially an increase in alpha-helicity of the cysteine-deficient protein. The conformational changes may also affect the active site and may account for the observed changes in kinetic properties.