Sialic acids are essential components of cell surface receptors used by influenza viruses. To determine the molecular mechanisms of viral recognition of two major species of sialic acids,
...N-acetylneuraminic acid (Neu5Ac) and
N-glycolylneuraminic acid (Neu5Gc), we tested the binding reactivity of nine human H3 influenza A viruses to sialylglycolipids containing type II sugar chain and different molecular species of terminal sialic acids. All human H3 viruses tested except A/Memphis/1/71 bound both Neu5Ac and Neu5Gc. Nucleotide sequence analysis suggests that amino acids at 143, 155, and 158 are linked to the viral recognition of Neu5Gc.
We have recently shown that binding of Helicobacter pylori to sialylated carbohydrates is dependent on the presence of the carboxyl group and the glycerol chain of Neu5Ac. In this work we ...investigated the importance of GlcNAc in the binding trisaccharide Neu5Acα3Galβ4GlcNAc and the role of the N-acetamido groups of both Neu5Ac and GlcNAc. An important part of the project was epitope dissection, that is chemical derivatizations of the active carbohydrate followed by binding studies. In addition we used a panel of various unmodified carbohydrate structures in the form of free oligosaccharides or glycolipids. These were tested for binding by hemagglutination inhibition assay, TLC overlay tests, and a new quantitative approach using radiolabeled neoglycoproteins. The studies showed that the N-acetamido group of Neu5Ac is important for binding by H. pylori, whereas the same group of GlcNAc is not. In addition, Fuc attached to GlcNAc, as tested with sialyl-Lewis x, did not affect the binding. Free Neu5Ac was inactive as inhibitor, and Neu5Acα3Gal turned out to be active. The binding preference for neolacto structures was confirmed, although one strain also was inhibited by lacto chains. The combined results revealed that an intact Neu5Ac is crucial for the interactions with H. pylori. Parts of Gal also seem to be necessary, whereas the role of the GlcNAc is secondary. GlcNAc does influence binding, however, primarily serving as a guiding carrier for the binding epitope rather than being a part of the binding structure.
Our previous studies have shown extensively diverse structures in oligo/polymers of sialic acid (oligo/polySia) that are expressed often in developmentally regulated manner on animal glycoconjugates. ...The aim of this study was to establish highly sensitive and specific methods that can be used to identify diverse types of oligo/polySia and thus can be applied to studies of biological phenomena associated with the differential expression of oligo/polySia chains with different degree of polymerization (DP). As model compounds, we analyzed five different homologous series of oligo/polySia, (→8Neu5Acα2→)n, (→9Neu 5Acα2→)n, (→8Neu5Gcα2→)n, (→5-Oglycolyl-Neu5Gcα2→)n, and Neu5Gc9SO4 α2→(→5-Oglycolyl-Neu5Gcα2→)n, expressed in various biopolymers. The latter two structures have recently been identified in sea urchin egg receptor for sperm. First we examined application of high-performance anion-exchange chromatography (HPAEC) on a CarboPac PA-100 column with pulsed electrochemical detection (PED) to new types of oligo/polySia compounds and confirmed that resolution of high polymers (DP > 70) of sialic acids was remarkable as reported previously. However, there are limitations in sensitivity and selectivity in PED that become significant when material is available only in a minute amount or material contained a large proportion of protein. These limitations can be circumvented by fluorometric detection of oligo/polySia tagged with 1,2-diamino-4,5-methyl-enedioxybenzene (DMB) at the reducing terminal residues after separation on a MonoQ HR5/5 column. The latter method can be applied to any type of oligo/polySia we examined if we choose the derivatization conditions and is more sensitive and specific than the method with PED for analysis of oligo/polySia with DP up to 25.
HPLC analysis of sialic acid released from recombinant variants of human tissue plasminogen activator, human chimeric plasminogen activator, human erythropoietin, and human follitropin, expressed in ...Chinese hamster ovary cells, demonstrates for each glycoprotein the presence of
N-acetylneuraminic and
N-glycolylneuraminic acid in a ratio of 97:3. Structural analysis by 500 MHz
1H-NMR spectroscopy, of the enzymatically released N-linked carbohydrate chains of chimeric plasminogen activator and of erythropoietin, showed that α2-3 linked
N-glycolylneuraminic acid can occur in different
N-acetyllactosamine type antennary structures.
Structural analysis by ID and 2D
1H NMR spectroscopy of three acidic O-linked oligosaccharide alditols, released from porcine zona pellucida glycoproteins by alkaline borohydride treatment, afforded ...the following structures: Galβl-4(6SO
4-)GlcNAcβl-3Galβl-4GlcNAcβ1-3Galβ1-3GalNAc-ol Neu5Gcα2-3Gal/91 -4(6SO
4-)GlcNAqβl -3Ga1βl -4GlcNAcβl -3Galβl -3GalNAc-ol Neu5Acα2-3Galβl -4(6SO
4-)GlcNAcβl -3Galβl -4GlcNAcβl -3Galβl-3GalNAc-ol These oligosaccharides are the smallest compounds that contain the structural elements which are present in the acidic, high-molecular mass O-linked carbohydrate chains of porcine zona pellucida glycoproteins.
CMP-Neu5Gc has been shown to be transported into mouse liver Golgi vesicles by a specific carrier the characteristics of which were investigated in detail. In the system employed, CMP-Neu5Gc enters ...the Golgi vesicles within 2 min; transport was saturable with high concentrations of the sugar-nucleotide and was dependent on temperature. A kinetic analysis gave an apparent
K
m of 1.3 μM and a maximal transport velocity of 335 pmol/mg protein per min. Almost identical values were obtained with CMP-Neu5Ac, under the same incubation conditions. Furthermore, the uptake of CMP-Neu5Gc was inhibited by CMP-Neu5Ac, a substrate analogue. Conversely, the uptake of CMP-Neu5Ac was inhibited by CMP-Neu5Gc to the same extent, leading to the conclusion that the transport of CMP-Neu5Ac and CMP-Neu5Gc is mediated by the same carrier molecule. This transport system for CMP-Neu5Gc involves both CMP and CMP-Neu5Gc since intravesicular CMP induced the entry of external CMP-Neu5Gc.
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