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1.
Enteropathogenic transmissible gastroenteritis virus (TGEV), a porcine coronavirus, is able to agglutinate erythrocytes because of sialic acid binding activity. Competitive inhibitors that may mask the sialic acid binding activity can be inactivated by sialidase treatment of virions. Here, we show that TGEV virions with efficient hemagglutinating activity were also obtained when cells were treated with sialidase prior to infection. This method was used to analyze TGEV mutants for hemagglutinating activity. Recently, mutants with strongly reduced enteropathogenicity that have point mutations or a deletion of four amino acids within residues 145 to 155 of the S protein have been described. Here, we show that in addition to their reduced pathogenicity, these mutants also have lost hemagglutinating activity. These results connect sialic acid binding activity with the enteropathogenicity of TGEV.  相似文献   

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Sialic acids are prominent termini of mammalian glycoconjugates and are key binding determinants for cell-cell recog-nition lectins. Binding of the sialic acid-dependent lectin, myelin-associated glycoprotein (MAG), to nerve cells is implicated in the inhibition of nerve regeneration after injury. Therefore, blocking MAG binding to nerve cell sialoglycoconjugates might enhance nerve regeneration. Previously, we reported that certain sialoglycoconjugates bearing N-acetylneuraminic acid (NeuAc) but not N-glycolylneuraminic acid (NeuGc) support MAG binding (Collins et al., 1997a). We now report highly efficient conversion of sialic acids on living neural cells from exclusively NeuAc to predominantly NeuGc using a novel synthetic metabolic precursor, N-glycolylmannosamine pentaacetate (Man-NGc-PA). When NG108-15 neuroblastoma-glioma hybrid cells, which normally express only NeuAc (and bind to MAG), were cultured in the presence of 1 mM ManNGcPA, they expressed 80-90% of their sialic acid precursor pool as NeuGc within 24 h. Within 5 days, 80% of their ganglioside-associated sialic acids and 70% of their glycoprotein-associated sialic acids were converted to NeuGc. Consistent with this result, treatment of NG108-15 cells with ManNGcPA resulted in nearly complete abrogation of MAG binding. These results demonstrate that ManNGcPA treatment efficiently alters the sialic acid structures on living cells, with a commensurate change in recognition by a physiologically important lectin.  相似文献   

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Haemophilus ducreyi is a Gram-negative bacterium that causes chancroid, a sexually transmitted disease. Cell surface lipooligosaccharides (LOS) of H. ducreyi are thought to play important biological roles in host infection. The vast majority of H. ducreyi strains contain high levels of sialic acid (N-acetylneuraminic acid, NeuAc) in their LOS. Here we investigate the biosynthetic origin of H. ducreyi sialosides by metabolic incorporation studies using a panel of N-acylmannosamine and sialic acid analogues. Incorporation of sialosides into LOS was assessed by matrix-assisted laser desorption and electrospray ionization mass spectrometry. A Fourier transform ion cyclotron resonance mass spectrometer provided accurate mass measurements, and a quadrupole time-of-flight instrument was used to obtain characteristic fragment ions and partial carbohydrate sequences. Exogenously supplied N-acetylmannosamine analogues were not converted to LOS-associated sialosides at a detectable level. In contrast, exogenous (13)C-labeled N-acetylneuraminic acid ([(13)C]NeuAc) and N-glycolylneuraminic acid (NeuGc) were efficiently incorporated into LOS in a dose-dependent fashion. Moreover, approximately 1.3 microM total exogenous sialic acid was sufficient to obtain about 50% of the maximum production of sialic acid-containing glycoforms observed under in vitro growth conditions. Together, these data suggest that the expressed levels of sialylated LOS glycoforms observed in H. ducreyi are in large part controlled by the exogenous concentrations of sialic acid and at levels one might expect in vivo. Moreover, these studies show that to properly exploit the sialic acid biosynthetic pathway for metabolic oligosaccharide engineering in H. ducreyi and possibly other prokaryotes that share similar pathways, precursors based on sialic acid and not mannosamine must be used.  相似文献   

7.
Coronaviruses (CoV) mature by a budding process at intracellular membranes. Here we showed that the major surface protein S of a porcine CoV (transmissible gastroenteritis virus) is not transported to the cell surface but is retained intracellularly. Site-directed mutagenesis indicated that a tyrosine-dependent signal (YXXI) in the cytoplasmic tail is essential for intracellular localization of the S protein. Surface expression of mutant proteins was evident by immunofluorescence analysis and surface biotinylation. Intracellularly retained S proteins only contained endoglycosidase H-sensitive N-glycans, whereas mutant proteins that migrated to the plasma membrane acquired N-linked oligosaccharides of the complex type. Corresponding tyrosine residues are present in the cytoplasmic tails of the S proteins of other animal CoV but not in the tail portion of the S protein of severe acute respiratory syndrome (SARS)-CoV. Changing the SEPV tetrapeptide in the cytoplasmic tail to YEPI resulted in intracellular retention of the S protein of SARS-CoV. As the S proteins of CoV have receptor binding and fusion activities and are the main target of neutralizing antibodies, the differences in the transport behavior of the S proteins suggest different strategies in the virus host interactions between SARS-CoV and other coronaviruses.  相似文献   

8.
Sialic acid (Sia) is a family of acidic nine-carbon sugars that occupies the nonreducing terminus of glycan chains. Diversity of Sia is achieved by variation in the linkage to the underlying sugar and modification of the Sia molecule. Here we identified Sia-dependent epitope specificity for GL7, a rat monoclonal antibody, to probe germinal centers upon T cell-dependent immunity. GL7 recognizes sialylated glycan(s), the alpha2,6-linked N-acetylneuraminic acid (Neu5Ac) on a lactosamine glycan chain(s), in both Sia modification- and Sia linkage-dependent manners. In mouse germinal center B cells, the expression of the GL7 epitope was upregulated due to the in situ repression of CMP-Neu5Ac hydroxylase (Cmah), the enzyme responsible for Sia modification of Neu5Ac to Neu5Gc. Such Cmah repression caused activation-dependent dynamic reduction of CD22 ligand expression without losing alpha2,6-linked sialylation in germinal centers. The in vivo function of Cmah was analyzed using gene-disrupted mice. Phenotypic analyses showed that Neu5Gc glycan functions as a negative regulator for B-cell activation in assays of T-cell-independent immunization response and splenic B-cell proliferation. Thus, Neu5Gc is required for optimal negative regulation, and the reaction is specifically suppressed in activated B cells, i.e., germinal center B cells.  相似文献   

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We purified a serum calcium-decreasing factor, which showed chymotrypsin-like protease activity, from porcine pancreas to homogeneity. The factor administered to mice intravenously at a dose of 20 micrograms/kg b.w. decreased serum calcium by 15%. Treatment of the factor with the serine protease inhibitor, PMSF, caused a leftward shift in the dose-response curve, showing strengthened activity. It also caused a decrease in serum calcium and hydroxyproline levels in rats. At a dose of 10 ng/ml, the factor inhibited 45Ca release from cultured fetal long bone stimulated by parathyroid hormone (PTH) and PTH-related protein, but not by interleukin-1 alpha, prostaglandin E1 and 1,25-dihydroxy vitamin D3. No other well-known pancreatic proteases had these effects. In view of the results of experiments using protease inhibitor and pancreatic proteases, and in view of the specificity of this factor in vitro, we propose that the factor exerts its serum calcium-decreasing activity most probably not through proteolytic degradation of PTH, but through an inhibition of PTH action on bones by a yet undefined mechanism.  相似文献   

10.
Elafin (elastase-specific inhibitor) is a low molecular weight inhibitor of neutrophil elastase which is secreted in the lung. Using synthetic peptides corresponding to full-length elafin (H2N-1AVT.....95Q-OH), the NH2-terminal domain (H2N-1AVT.....50K-OH) and the COOH-terminal domain (H2N-51PGS.....95Q-OH), we demonstrate that elafin's anti-elastase activity resides exclusively in the COOH-terminus. Several characteristics of elafin suggest potential anti-microbial activity. The anti-microbial activity of elafin, and of its two structural domains, was tested against the respiratory pathogens Pseudomonas aeruginosa and Staphylococcus aureus. Elafin killed both bacteria efficiently, with 93% killing of P. aeruginosa by 2.5 microM elafin and 48% killing of S. aureus by 25 microM elafin. For both organisms, full-length elafin was required to optimise bacterial killing. These findings represent the first demonstration of co-existent anti-proteolytic and anti-microbial functions for elafin.  相似文献   

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A sialic acid binding lectin, AchatininH, was purified in single step from the hemolymph of the land snail, Achatina fulica, by the affinity chromatography on sheep submaxillary mucin coupled to Sepharose 4B. The yield of the lectin was found to be 3 mg from 100 ml of hemolymph. The homogeneity of the lectin was established by alkaline gel electrophoresis, immunodiffusion, immunoelectrophoresis and analytical isoelectrophoresis. The molecular weight of the native protein was 242000, having identical subunits of Mr 15000. The lectin agglutinated rabbit erythrocytes in the presence of Ca2–. The inhibition study clearly suggests that the binding site of the lectin recognizes sialic acid as the immunodominant sugar. This was further confirmed by the observation that there was a marked decrease of agglutinating activity of the lectin with neuraminidase treated rabbit erythrocytes and asialofetuin was unable to inhibit the activity of AchatininH. Among the inhibitors used the glycoconjugate containing 2-6 linkages of N-acetylneuraminic acid with subterminal galactopyranose or 2-acetamido-2-deoxy-galactopyranose residue was found to be better inhibitor than that containing 23 linkages of N-acetyl neuraminic acid. Besides that sialoglycoprotein containing both N and O type of glycosidic linkages plays an important role in binding with the lectin. Fetuin was found to be the best inhibitor.  相似文献   

13.
N-Glycolylneuraminic acid (Neu5Gc), precious sialic acid which could not be synthesized by a chemical method, occurrs in the body of holothuroidea, Gumi Cucumaria echinata. Gumi contains 85% of total sialic acid, as Neu5Gc, in the body. Neu5Gc was purified from dry powder of the body using Dowex 1-x8 (HCOO* form) anion exchange chromatography after mild acid hydrolysis with 0.1 N trifluoroacetic acid. Using GC-MS and 1H-NMR spectroscopy, the purified Neu5Gc was correctly identified to be Neu5Gc. The purity of Neu5Gc was more than 99%. This is the first report of purification and identification of Neu5Gc from holothuroidea by using anion exchange chromatography, GC-MS, and 1H-NMR.  相似文献   

14.
Pulmonary surfactant is a complex mixture of lipids and proteins, of which surfactant protein A (SP-A) is the most abundant glycoprotein. The SP-A molecule has several distinct structural features that include a lectin-like domain, sharing structural features with other mammalian lectins. We have tested the hypothesis that lectin activity of the SP-A molecule is required for the binding to its receptor on the surface of alveolar Type II cells. By using colloidal gold immunocytochemistry in conjunction with electron microscopy, we evaluated the ability of mannosylated proteins to inhibit canine SP-A binding to rat Type II cells in vitro. After preincubation of SP-A with the mannosylated protein horse-radish peroxidase (HRP), SP-A was incubated with isolated filter-grown Type II cells. HRP did not alter the binding of SP-A to the Type II cell surface. Evidence that SP-A did bind to HRP was shown by coincident observation of gold-labeled SP-A and HRP precipitates. These results provide visual evidence that the lectin activity associated with SP-A is not required for its binding to receptor on rat alveolar Type II epithelial cells.  相似文献   

15.
Pyrazinamide (PZA), an essential component of short-course anti-tuberculosis chemotherapy, was shown by Saturation Transfer Difference (STD) NMR methods to act as a competitive inhibitor of NADPH binding to purified Mycobacterium tuberculosis fatty acid synthase I (FAS I). Both PZA and pyrazinoic acid (POA) reversibly bind to FAS I but at different binding sites. The competitive binding of PZA and NADPH suggests potential FAS I binding sites. POA was not previously known to have any specific binding interactions. The STD NMR of NADPH bound to the mycobacterial FAS I was consistent with the orientation reported in published single crystal X-ray diffraction studies of fungal FAS I. Overall the differences in binding between PZA and POA are consistent with previous recognition of the importance of intracellular accumulation of POA for anti-mycobacterial activity.  相似文献   

16.
R Kaplan  S S Li  J M Kehoe 《Biochemistry》1977,16(19):4297-4303
The sialic acid binding lectin, limulin, was isolated by gel filtration and ion-exchange chromatography from the hemolymph of Limulus polyphemus. When the purified protein was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of beta-mercaptoethanol, two major protein bands were observed. These two bands, subsequently found to contain carbohydrate as well, corresponded to molecular weights of 25 000 and 27 000. Amino acid sequence analyses were performed on both the intact protein and isolated cyanogen bromide fragments. The following primary structural features were noted in the amino-terminal region of limulin: (1) the absence of histidine and alanine from the NH2-terminal 50 residues; (2) the presence of five of the total eight prolines of the molecule between positions 13 and 30; and (3) a possible carbohydrate attachment site consisting of only the amino acids proline and serine between residues 13 and 19. The resultsof cyanogen bromide cleavage studies confirmed the presence of 2 methionine residues per subunit, at positions 25 and 58 respectively. No sequence heterogeneity was observed in this study. While it is quite possible that limulin plays some role in the defense mechanisms of the horseshoe crab, there is no obvious sequence homology between this invertebrate lectin and vertebrate immunoglobulins.  相似文献   

17.
Four new porcine respiratory coronavirus (PRCV) isolates were genetically characterized. Subgenomic mRNA patterns and the nucleotide sequences of the 5' ends of the S genes, the open reading frame (ORF) 3/3a genes, and the ORF 3-1/3b genes of these PRCV isolates were determined and compared with those of other PRCV and transmissible gastroenteritis virus (TGEV) isolates. The S, ORF 3/3a, and ORF 3-1/3b genes are under intense study because of their possible roles in determining tissue tropism and virulence. Northern (RNA) blot analysis of subgenomic mRNAs revealed that mRNA 2, which encodes for the S gene, of the PRCV isolates migrated faster than the mRNA 2 of TGEV. The PRCV isolates AR310 and LEPP produced eight subgenomic mRNA species, the same number as produced by the virulent Miller strain of TGEV. However, the PRCV isolates IA1894 and ISU-1 produced only seven subgenomic mRNA species. All four of the PRCV isolates were found to have a large in-frame deletion in the 5' end of the S gene; however, the size and location of the deletion varied. Analysis of the ORF 3/3a gene nucleotide sequences from the four PRCV isolates also showed a high degree of variability in this area. The ORF 3 gene of the PRCV isolates AR310 and LEPP was preceded by a CTAAAC leader RNA-binding site, and the ORF 3 gene was predicted to yield a protein of 72 amino acids, the same size as that of the virulent Miller strain of TGEV. The PRCV isolates AR310 and LEPP are the first PRCV isolates found to have an intact ORF 3 gene. The ORF 3a gene of the PRCV isolate IA1894 was preceded by a CTAAAC leader RNA-binding site and was predicted to yield a truncated protein of 54 amino acids due to a 23-nucleotide deletion. The CTAAAC leader RNA-binding site and ATG start codon of ORF 3 gene of the PRCV isolate ISU-1 were removed because of a 168-nucleotide deletion. Analysis of the ORF 3-1/3b gene nucleotide sequences from the four PRCV nucleotides isolates also showed variability.  相似文献   

18.
DNA damage promotes the activation of a signal transduction cascade referred to as the DNA damage checkpoint. This pathway initiates with the Mec1/ATR kinase, which then phosphorylates the Rad53/Chk2 kinase. Mec1 phosphorylation of Rad53 is then thought to promote Rad53 autophosphorylation, ultimately leading to a fully active Rad53 molecule that can go on to phosphorylate substrates important for DNA damage resistance. In the absence of DNA repair, this checkpoint is eventually downregulated in a Cdc5-dependent process referred to as checkpoint adaptation. Recently, we showed that overexpression of Cdc5 leads to checkpoint inactivation and loss of the strong electrophoretic shift associated with Rad53 inactivation. Interestingly, this same overexpression did not strongly inhibit Rad53 autophosphorylation activity as measured by the in situ assay (ISA). The ISA involves incubating the re-natured Rad53 protein with γ 32P labeled ATP after electrophoresis and western blotting. Using a newly identified Rad53 target, we show that despite strong ISA activity, Rad53 does not maintain phosphorylation of this substrate. We hypothesize that, during adaptation, Rad53 may be in a unique state in which it maintains some Mec1 phosphorylation but does not have the auto-phosphorylations required for full activity towards exogenous substrates.Key words: DNA damage, checkpoint, adaptation, CDC5, RAD53, ISA  相似文献   

19.
D M Gibbs  W Vale  J Rivier  S S Yen 《Life sciences》1984,34(23):2245-2249
The effects of CRF(41), oxytocin (OT), and arginine vasopressin (AVP) on ACTH secretion were studied alone and in combination in an in vitro system of superfused rat hemipituitaries. CRF(41) (10(-9)M) and AVP (10(-8)M) alone produced a significant increase in ACTH secretion while OT (10(-8)M) alone had no effect. However the same concentration of OT markedly potentiated the ACTH response to CRF(41) while having no effect on the ACTH response to AVP. The data support a physiologic role for OT in the regulation of ACTH secretion.  相似文献   

20.
The echinoderm microtubule-associated protein (EMAP) is a 75-kDa, WD-repeat protein associated with the mitotic spindle apparatus. To understand EMAP's biological role, it is important to determine its affinity for microtubules (MTs) and other cytoskeletal components. To accomplish this goal, we utilized a low-cost, bubble-column bioreactor to express EMAP as a hexahistidine fusion (6his) protein in baculovirus-infected insect cells. After optimizing cell growth conditions, up to 30 mg of EMAP was obtained in the soluble cell lysate from a 1-liter culture. EMAP was purified to homogeneity in a two-step process that included immobilized metal-affinity chromatography (IMAC) and anion-exchange chromatography. In vitro binding studies on cytoskeletal components were performed with the 6his-EMAP. EMAP bound to MTs, but not actin or vimentin filaments, with an intrinsic dissociation constant of 0.18 microM and binding stoichiometry of 0.7 mol EMAP per mol tubulin heterodimer. In addition, we show that a strong MT binding domain resides in the 137 amino acid, NH(2)-terminus of EMAP and a weaker binding site in the WD-domain. Previous work has shown that the EMAP concentration in the sea urchin egg is over 4 microM. Together, these results show that there is sufficient EMAP in the egg to regulate the assembly of a large pool of maternally stored tubulin.  相似文献   

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