Uncovering a Novel Molecular Mechanism for Scavenging Sialic Acids in Bacteria

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    hanscallanan
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    <br> Neu5Ac (blue), 2,7-anhydro-Neu5Ac (orange), or XY intermediate (gray). B and C, superposition of the 1H,13C HSQC reference spectra of Neu5Ac (blue) and 2,7-anhydro-Neu5Ac (orange) and 1H,13C HSQC reaction mixture at 30 min (where the XY peak is observed to peak, gray). The comparison of the 2D 1H,13C HSQC spectra of the substrate (2,7-anhydro-Neu5Ac), the product (Neu5Ac), and the reaction mixture at 30 min allowed the identification of an additional set of cross-peaks that did not belong to the substrate or to the product and were therefore assigned to the intermediate (Fig. 1, B and C). The product of the addition (2) is a 4-keto-Neu5Ac, in which the proton at C5 is now α to the keto and acidic. This acidic proton will exchange with solvent by the well-known keto enol tautomerization reaction, consistent with the NMR data (Fig. S1). If you have any concerns pertaining to where and the best ways to utilize sialic acid powder supplier, you could contact us at our own web page. This is consistent with the NMR observation that the axial, not equatorial proton, undergoes exchange (Fig. S1). 4-Keto-DANA exists as an equilibrium between two ring-flipped forms; however, the form with the equatorial glycerol and N-acetyl substituents would be expected to predominate. Based on this information, we proposed that in this species, the glycerol moiety is in the open form, indicating that the 2,7-anhydro bond is broken in the intermediate.<br>
    <br> The proton at C3 is now α to the keto group and thus more acidic and can be abstracted in an anti-periplanar elimination reaction in which the 2,7-anhydro bond is also broken. In the context of the two glycoproteins recognized in the array, AGP and apo-transferrin, the terminal α2,3/α2,6 trisaccharide motif is linked to several other sugars before they are N-linked to the protein, and thus the length of the chain is not likely to be limiting for binding. We used cell-based assays to show that α2,3 and α2,6 sialic acids that are present on N-linked glycoproteins facilitate cellular transduction by both AAV1 and AAV6 vectors. Here, using a combination of in silico, molecular, biochemical, and structural approaches, we elucidated the molecular mechanism of RgNanOx and showed that homologous enzymes are present across both Gram-positive and Gram-negative bacteria and are associated with different classes of predicted transporters. This analysis supported the earlier findings that YjhC could act on Neu5Ac (20) but also revealed that the enzyme was able to utilize 2,7-anhydro-Neu5Ac as a substrate in the same manner as RgNanOx.<br>
    <br> Analysis of the 3-4 ppm spectral region, where standard sugar-ring C-H signals typically show, suggests that the intermediate chemical shifts are closer to Neu5Ac than to 2,7-anhydro-Neu5Ac (Fig. S3). Analysis of the reaction curve, obtained by monitoring the signals from the methyl protons of the acetamide group at C5, revealed the presence of a new (third) molecule (XY). This molecule is formed with a very fast kinetics, reaching its highest concentration at very early time (Fig. 1A). The reaction mixture was analyzed by 2D NMR during the interval at which the intermediate was at its highest concentration, using a 600-MHz spectrometer equipped with a cryoprobe. Specifically, the presence of a heteronuclear cross-peak at 2.73 ppm/43.1 ppm (1H/13C) (signal 2 in Fig. 1B), characteristic of a proton in α to a keto group, strongly suggested the intermediate to be a keto-sugar. Signal 2, at 2.73 ppm, corresponds then to a proton neighboring a carbonyl on one side and at least two other protons on the other side with chemical shifts in the 3-4 ppm region.<br>
    <br> Following this conversion, Neu5Ac is then catabolized into N-acetylmannosamine (ManNAc) and pyruvate via the action of a Neu5Ac-specific aldolase (19). We confirmed the importance of this metabolic pathway in vivo by generating a R. gnavus nan cluster deletion mutant that lost the ability to grow on sialylated substrates. The human gut symbiont Ruminococcus gnavus scavenges host-derived N-acetylneuraminic acid (Neu5Ac) from mucins by converting it to 2,7-anhydro-Neu5Ac. We previously showed that 2,7-anhydro-Neu5Ac is transported into R. gnavus ATCC 29149 before being converted back to Neu5Ac for further metabolic processing. R. gnavus is a human gut symbiont that plays a major role in human health and disease. R. gnavus is widely distributed among individuals being represented in the most common 57 species present in ≥90% of individuals (6). Colonization by R. gnavus has been found in infants during the first days of life. The six cell lines utilized in the present study were obtained from the American Type Culture Collection (Manassas, VA) and maintained at 37°C with 5% CO2 in their respective media, supplemented with 10% fetal bovine serum and penicillin-streptomycin. 6 days in RPMI-1640 (Sigma, St Louis, MO) supplemented with 2 mm l-glutamine, 1% non-essential amino acids, 1% pyruvate, 100 μg/ml penicillin/streptomycin (Gibco, Grand Island, NY), 50 μm 2-mercaptoethanol, 10% fetal bovine serum (FBS) from Sigma and interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) from R&D Systems (Minneapolis, MN), to be differentiated into immature MDDCs, as described elsewhere.20,26 Whenever needed, mature MDDCs (mMDDCs) were induced at day 5 with 5 μg/ml lipopolysaccharide (LPS) (Sigma).<br>

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