Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.1.53 (sialidase)
 2,694 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Porcine interferon-gamma (SfPoIFN-gamma) was expressed with high efficiency in Spodoptera frugiperda (Sf9) cells by means of the baculovirus expression system. Up to 10(5) U/ml of antivirally active SfPoIFN-gamma could be tracked down in the culture medium at 64 h postinfection. Three proteins (17, 19, and 21 kDa), which under nondenaturing conditions primarily exist as mutual-dimeric combinations, were purified by immunoaffinity chromatography. Carbohydrate labeling and kinetic deglycosylation studies suggested that the 19- and 21-kDa proteins are N-glycosylated variants of a single 17-kDa protein carrying no N-linked sugars, in which one respectively two N-glycosylation sequons are occupied by glycans of 2 kDa. Both the quantitative recovery of SfPoIFN-gamma from a Con A column at 0.2 M methyl-alpha-mannopyranoside and the results of lectin blots, revealing strong affinity of the 19- and 21-kDa species for Galanthus nivalis agglutinin, support the presence of N-glycosidically linked high mannose-type chains in the carbohydrate moiety of SfPoIFN-gamma. Intriguingly, both 19- and 21-kDa glycoforms, but not their sialidase-treated derivatives, showed clear reactivity with the Sambucus nigra and Maackia amurensis agglutinins. These agglutinins specifically recognize sialic acid linked alpha(2-6) and alpha(2-3), respectively, to penultimate galactose residues. Their affinity for the larger glycoforms of PoIFN-gamma suggests that the biosynthetic pathways in Sf9 cells are able to modify oligomannose structures to complex or hybrid glycans.
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PMID:Glycoform heterogeneity of porcine interferon-gamma expressed in Sf9 cells. 799 24

An analytical system is presented for rapid assessment of site-specific microheterogeneity of the two potential N-linked glycosylation sites of recombinant human interferon-gamma (IFN-gamma) derived from Chinese hamster ovary cell culture. The target protein is first purified from culture supernatant by immunoaffinity chromatography, and the acidic eluent is neutralized via an in-line mixing tee. On-line proteolysis is rapidly performed by an immobilized trypsin cartridge, and reversed-phase chromatography isolates the two pools of glycopeptides representing the potential glycosylation sites. Following off-line analysis by matrix-assisted laser-desorption ionization/time-of-flight (MALDI/TOF) mass spectrometry, observed mass shifts of glycopeptides relative to the known masses of their amino acid portions are correlated to site-specific oligosaccharide structures. Desialylation of glycopeptides by sialidase treatment on the MALDI sample plate allows for quantitative estimations of asialoglycan structures by MALDI/TOF. This methodology permits glycoprotein microheterogeneity to be evaluated in a time frame of approximately 2 h, utilizing as little as 0.5 microgram (25 pmol) of product. Results of monitoring a batch culture are presented as well as analysis of a culture containing deoxymannojirimycin, an inhibitor of glycoprotein processing.
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PMID:Rapid monitoring of site-specific glycosylation microheterogeneity of recombinant human interferon-gamma. 881 42

To understand the modulation and the behavior of glycocalyx elements during adhesion, we explored one of its components, the CD43 molecule, on human monocytic THP-1 cells exposed to cytokine stimulation and its redistribution during heterotypic adhesion to opsonized erythrocytes. First we demonstrated by immunofluorescence and immunoprecipitation that CD43 is dys-sialylated in monocytic THP-1 cells stimulated by interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) and stimulation increased correlated to heterotypic adhesion. CD43 anti-adhesive effect seemed to be related to sialic acid moeties because an increase in adhesion was also induced by sialidase treatment and by monoclonal antibodies recognizing sialic acid-dependent epitopes on CD43. Second, a redistribution of CD43 molecules was observed after adhesion, resulting in the exclusion of CD43 molecules from contact areas as demonstrated by immunofluorescence and by ultrastructural immunogold localization. We therefore demonstrated in monocytic THP-1 cells that some glycocalyx molecules can be modulated by cytokines and redistributed during adhesion. These results support the concept that CD43 can regulate cell interactions.
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PMID:Leukosialin (CD43) behavior during adhesion of human monocytic THP-1 cells to red blood cells. 912 10

Trypanosoma cruzi. the protozoan parasite that causes Chagas' disease, does not synthesize sialic acid, but expresses a trans-sialidase (TS) that catalyzes the transfer of sialic acid from host glycoconjugates to the parasite surface. Here, we review studies that characterize the immune response to the catalytic domain of the enzyme in humans during Chagas' disease or in mice following immunization with the TS gene. In both cases, there are antibodies that strongly inhibit the enzymatic activity and generation of interferon-gamma-producing T cells.
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PMID:Trans-sialidase delivered as a naked DNA vaccine elicits an immunological response similar to a Trypanosoma cruzi infection. 1034 60

BALB/c or C57Bl/6 mice immunized with plasmids containing Trypanosoma cruzi genes developed specific immune responses and protective immunity against lethal parasitic infection. In contrast, in the highly susceptible mouse strain A/Sn, DNA vaccination reduced the peak parasitemia but promoted limited mouse survival after challenge. In the present study, we tested whether the immunogenicity and protective efficacy of vaccination could be improved by combining DNA and recombinant protein immunization regimens. A/Sn mice immunized with plasmid p154/13 which harbours the gene encoding Trypanosoma cruzi trans-sialidase developed a predominant type 1 immune response. In contrast, immunization with the recombinant Trypanosoma cruzi trans-sialidase protein adsorbed to alum generated a typical type 2 immune response. Simultaneous administration of both p154/13 and recombinant Trypanosoma cruzi trans-sialidase protein also led to a predominant type 2 immune response. Sequential immunization consisting of two priming doses of p154/13 followed by booster injections with recombinant Trypanosoma cruzi trans-sialidase protein significantly improved specific type 1 immune response, as revealed by a drastic reduction of the serum IgG1/IgG2a ratio and by an increase in the in vitro interferon-gamma secretion by CD4 T cells. Our observations confirm and extend previous data showing that a DNA-priming protein-boosting regimen might be a general strategy to enhance type 1 immune response to DNA vaccines. Upon challenge with Trypanosoma cruzi, no improvement in protective immunity was observed in mice immunized with the DNA-priming protein-boosting regimen when compared to animals that received DNA only. Therefore, our results suggest that in this experimental model there is no correlation between the magnitude of type 1 immune response and protective immunity against Trypanosoma cruzi infection.
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PMID:A DNA-priming protein-boosting regimen significantly improves type 1 immune response but not protective immunity to Trypanosoma cruzi infection in a highly susceptible mouse strain. 1263 Dec 35

Among the three isoenzymes of neuraminidase (Neu) or sialidase, Neu-1 has been suggested to be induced by cell activation and to be involved in IL (interleukin)-4 biosynthesis in murine T-cells. In the present study, we found that antigen-induced airway eosinophilia, a typical response dependent on Th2 (T-helper cell type 2) cytokines, as well as mRNA expression of Th2 cytokines, including IL-4, are suppressed in Neu-1-deficient mice, thereby demonstrating the in vivo role of murine Neu-1 in regulation of Th2 cytokines. To elucidate the roles of various sialidases in human T-cell activation, we investigated their tissue distribution, gene induction and function. Neu-1 is the predominant isoenzyme at the mRNA level in most tissues and cells in both mice and humans, including T-cells. T-cells also have significant levels of Neu-3 mRNAs, albeit much lower than those of Neu-1, whereas the levels of Neu-2 mRNAs are minimal. In human T-cells, both Neu-1 and Neu-3 mRNAs are significantly induced by T-cell-receptor stimulation, as is sialidase activity against 4-methylumbelliferyl- N -acetylneuramic acid (a substrate for both Neu-1 and Neu-3) and the ganglioside G(D1a) [NeuAcalpha2-3Galbeta1-3GalNAcbeta1-4(NeuAcalpha2-3)Galbeta1-4Glcbeta1-cer] (a substrate for Neu-3, but not for Neu-1). The expression of the two sialidase genes may be under differential regulation. Western blot analysis and enzymic comparison with recombinant sialidases have revealed that Neu-3 is induced as a major isoform in activated cells. The induction of Neu-1 and Neu-3 in T-cells is unique. In human monocytes and neutrophils stimulated with various agents, the only observation of sialidase induction has been by IL-1 in neutrophils. Functionally, a major difference has been observed in Jurkat T-cell lines over-expressing Neu-1- and Neu-3. Upon T-cell receptor stimulation, IL-2, interferon-gamma, IL-4 and IL-13 are induced in the Neu-1 line, whereas in the Neu-3 line the same cytokines are induced, with the exception of IL-4. Taken together, these results suggest an important immunoregulatory role for both Neu-1 and Neu-3 in humans.
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PMID:Induction of lysosomal and plasma membrane-bound sialidases in human T-cells via T-cell receptor. 1499 89

Cloning of the T-cell receptor genes is a critical step when generating T-cell receptor transgenic mice. Because T-cell receptor molecules are clonotypical, isolation of their genes requires reverse transcriptase-assisted PCR using primers specific for each different Valpha or Vbeta genes or by the screening of cDNA libraries generated from RNA obtained from each individual T-cell clone. Although feasible, these approaches are laborious and costly. The aim of the present study was to test the application of the non-palindromic adaptor-PCR method as an alternative to isolate the genes encoding the T-cell receptor of an antigen-specific T-cell hybridoma. For this purpose, we established hybridomas specific for trans-sialidase, an immunodominant Trypanosoma cruzi antigen. These T-cell hybridomas were characterized with regard to their ability to secrete interferon-gamma, IL-4, and IL-10 after stimulation with the antigen. A CD3+, CD4+, CD8- interferon-gamma-producing hybridoma was selected for the identification of the variable regions of the T-cell receptor by the non-palindromic adaptor-PCR method. Using this methodology, we were able to rapidly and efficiently determine the variable regions of both T-cell receptor chains. The results obtained by the non-palindromic adaptor-PCR method were confirmed by the isolation and sequencing of the complete cDNA genes and by the recognition with a specific antibody against the T-cell receptor variable beta chain. We conclude that the non-palindromic adaptor-PCR method can be a valuable tool for the identification of the T-cell receptor transcripts of T-cell hybridomas and may facilitate the generation of T-cell receptor transgenic mice.
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PMID:The non-palindromic adaptor-PCR method for the identification of the T-cell receptor genes of an interferon-gamma-secreting T-cell hybridomaspecific for trans-sialidase, an immunodominant Trypanosoma cruzi antigen. 1650 14

Previous work from our laboratory has shown that modifying cell surface glycosylation with either a Clostridium perfringens-derived sialidase (CP-Siase), or an O-linked glycoprotein endopeptidase (OSGE) can enhance the function of CD4 T cells from both young and old mice at multiple levels. Here we have re-assessed the effect of age on CD8 T-cell function, and examined the outcome of enzymatic treatment with CP-Siase and OSGE on its different aspects. Pre-treatment of CD8 T cells with either CP-Siase or OSGE led to a significant increase in anti-CD3-mediated Ca2+ response in both young and old mice. Pre-treated CD8 T cells from both age groups also displayed a significant increase in activation-induced CD69 and CD25 expression, and produced significantly higher amounts of interleukin-2 and interferon-gamma in comparison to their untreated counterparts. Furthermore, pretreatment with either enzyme enhanced granzyme B expression in CD8 T cells, and increased their cytolytic activity in vitro. These data support the notion that glycosylated surface proteins hinder CD8 T-cell activation and function in both young and old mice, and raise the possibility of significantly improving CD8 T cell function in older individuals through enzymatic alteration of surface glycoproteins.
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PMID:Enhancement of CD8 T-cell function through modifying surface glycoproteins in young and old mice. 1680 88

Since sialic acid content is known to be a critical determinant of the biological properties of glycoproteins, it is essential to characterize and monitor sialylation patterns of recombinant glycoproteins intended for therapeutic use. This study reports site- and branch-specific differences in sialylation of human interferon-gamma (IFN-gamma) derived from Chinese hamster ovary (CHO) cell culture. Sialylation profiles were quantitated by reversed-phase HPLC separations of the site-specific pools of tryptic glycopeptides representing IFN-gamma's two potential N-linked glycosylation sites (i.e., Asn(25) and Asn(97)). Although sialylation at each glycosylation site was found to be incomplete, glycans of Asn(25) were more heavily sialylated than those of Asn(97). Furthermore, Man(alpha1-3) arms of the predominant complex biantennary structures were more favorably sialylated than Man(alpha1-6) branches at each glycosylation site. When the sialylation profile was analyzed throughout a suspension batch culture, sialic acid content at each site and branch was found to be relatively constant until a steady decrease in sialylation was observed coincident with loss of cell viability. The introduction of a competitive inhibitor of sialidase into the culture supernatant prevented the loss of sialic acid after the onset of cell death but did not affect sialylation prior to cell death. This finding indicated that incomplete sialylation prior to loss of cell viability could be attributed to incomplete intracellular sialylation while the reduction in sialylation following loss of cell viability was due to extracellular sialidase activity resulting from cell lysis. Thus, both intracellular and extracellular processes defined the sialic acid content of the final product. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 390-398, 1977.
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PMID:Site- and branch-specific sialylation of recombinant human interferon-gamma in Chinese hamster ovary cell culture. 1863 97