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A solid-phase assay for the activity of CMPNeuAc:Gal beta 1-4GlcNAc-R alpha-2,6-sialyltransferase (2,6ST) has been developed. In the assay an acceptor glycoprotein is immobilized onto microtiter plate wells. The two glycoprotein acceptors used were asialofetuin (ASF), which contains oligosaccharides terminating in the sequence Gal beta 1-4GlcNAc-R, and a neoglycoprotein of bovine serum albumin containing covalently attached Gal beta 1-4GlcNAc-R units. Samples containing the donor CMPNeuAc and the 2,6ST were incubated with the immobilized acceptor to generate the product NeuAc alpha 2-6Gal beta 1-4GlcNAc-R. The product was detected by a biotin-streptavidin system using the biotinylated plant lectin Sambucus nigra agglutinin (SNA), which binds to sialic acid in alpha-2,6, but not in alpha-2,3, linkage. The biotinylated SNA bound to the product was then detected with streptavidin and biotinylated forms of either alkaline phosphatase or the recombinant bioluminescent protein aequorin. The assay was optimized with respect to the commercially available 2,6ST and shown to be dependent on the concentration of acceptor and CMPNeuAc and proportional to the 2,6ST activity in the range of 20 to 400 microU in a 1-h assay. The solid-phase assay also allows for the selective detection of 2,6ST activity in human and fetal bovine serum, where the activity was proportional in the range of 0.1 to 2 microliters of serum. 相似文献
3.
Y Abe C W Smith J P Katkin L M Thurmon X Xu L H Mendoza C M Ballantyne 《Journal of immunology (Baltimore, Md. : 1950)》1999,163(5):2867-2876
We have previously shown that costimulation of endothelial cells with IL-1 + IL-4 markedly inhibits VCAM-1-dependent adhesion under flow conditions. We hypothesized that sialic acids on the costimulated cell surfaces may contribute to the inhibition. Northern blot analyses showed that Gal beta 1-4GlcNAc alpha 2, 6-sialyltransferase (ST6N) mRNA was up-regulated in cultured HUVEC by IL-1 or IL-4 alone, but that the expression was enhanced by costimulation, whereas the level of Gal beta 1-4GlcNAc/Gal beta 1-3GalNAc alpha2,3-sialyltransferase (ST3ON) mRNA was unchanged. Removing both alpha 2,6- and alpha 2,3-linked sialic acids from IL-1 + IL-4-costimulated HUVEC by sialidase significantly increased VCAM-1-dependent adhesion, whereas removing alpha 2,3-linked sialic acid alone had no effect; adenovirus-mediated overexpression of ST6N with costimulation almost abolished the adhesion, which was reversible by sialidase. The same treatments of IL-1-stimulated HUVEC had no effect. Lectin blotting showed that VCAM-1 is decorated with alpha 2,6- but not alpha 2,3-linked sialic acids. However, overexpression of alpha 2,6-sialyltransferase did not increase alpha 2,6-linked sialic acid on VCAM-1 but did increase alpha 2,6-linked sialic acids on other proteins that remain to be identified. These results suggest that alpha 2,6-linked sialic acids on a molecule(s) inducible by costimulation with IL-1 + IL-4 but not IL-1 alone down-regulates VCAM-1-dependent adhesion under flow conditions. 相似文献
4.
Photobacterium damsela α2,6-sialyltransferase was cloned as N- and C- His-tagged fusion proteins with different lengths (16–497 aa or 113–497 aa). Expression and activity assays indicated that
the N-terminal 112 amino acid residues of the protein were not required for its α2,6-sialyltransferase activity. Among four truncated
forms tested, N-His-tagged Δ15Pd2,6ST(N) containing 16–497 amino acid residues had the highest expression level. Similar to the Δ15Pd2,6ST(N),
the shorter Δ112Pd2,6ST(N) was active in a wide pH range of 7.5–10.0. A divalent metal ion was not required for the sialyltransferase
activity, and the addition of EDTA and dithiothreitol did not affect the activity significantly.
Mingchi Sun and Yanhong Li contributed equally to this work. 相似文献
5.
Kitazume S Tachida Y Oka R Nakagawa K Takashima S Lee YC Hashimoto Y 《Glycoconjugate journal》2006,23(5-6):437-441
Deposition of amyloid β-peptide (Aβ) and neurofibrillary tangles in the brain are hallmarks of Alzheimer’s disease (AD) pathogenesis.
BACE1, a membrane-bound aspartic protease that cleaves amyloid precursor protein (APP) to produce Aβ, has been implicated
in triggering the pathogenesis of the disease. We previously reported that BACE1 also cleaved α2,6-sialyltransferase (ST6Gal
I) in the Golgi apparatus and induced its secretion from the cell. Since most glycosyltransferases show Golgi localization
and many of these are cleaved and secreted from the cell, we hypothesized that other glycosyltransferases may also be BACE1
substrates. Here, we focused on a series of sialyltransferases as candidates for BACE1 substrates. We found that BACE1 cleaved
polysialyltransferase ST8Sia IV (PST) in vitro. We further found that BACE1 overexpression in COS cells enhanced the secretion of ST3Gal I, II, III and IV, although these
sialyltransferases were not cleaved by BACE1 in vitro. These results suggest that BACE1 expression affects glycosylation not only by directly cleaving glycosyltransferases but
also by modifying the secretion of glycosyltransferases via some other mechanisms. 相似文献
6.
Terminal sialylation of therapeutic glycoprotein is important for biological activity and in vivo stability. The enzyme α2,3-sialyltransferase is the key enzyme that links sialic acids to the termini of glycans in the Chinese
hamster ovary (CHO) cell line. Terminal sialylation is affected by numerous factors, but the elements that regulate α2,3-sialyltransferase
are not known. We investigated the relationship between α2,3-sialyltransferase activity, ammonium concentration, and cell
attachment area-based cell concentration in a recombinant human erythropoietin (rhEPO)-producing CHO cell line. We found that
ammonium in the culture medium had almost no effect on α2,3-sialyltransferase activity, but that the activity was affected
by cell attachment area-based cell concentration; α2,3-sialyltransferase activity and terminal sialylation of rhEPO decreased
with increasing the cell concentration. These results demonstrate that the cell attachment area-based cell concentration is
an important factor that affects 2,3-sialyltransferase activity and terminal sialylation of CHO cells. 相似文献
7.
Marie-Ange Krzewinski-Recchi Sylvain Julien Sylvie Juliant Mélanie Teintenier-Lelièvre Bénédicte Samyn-Petit Maria-Dolores Montiel Anne-Marie Mir Martine Cerutti Anne Harduin-Lepers Philippe Delannoy 《European journal of biochemistry》2003,270(5):950-961
BLAST analysis of the human and mouse genome sequence databases using the sequence of the human CMP-sialic acid:beta-galactoside alpha-2,6-sialyltransferase cDNA (hST6Gal I, EC2.4.99.1) as a probe allowed us to identify a putative sialyltransferase gene on chromosome 2. The sequence of the corresponding cDNA was also found as an expressed sequence tag of human brain. This gene contained a 1590 bp open reading frame divided in five exons and the deduced amino-acid sequence didn't correspond to any sialyltransferase already known in other species. Multiple sequence alignment and subsequent phylogenic analysis showed that this new enzyme belonged to the ST6Gal subfamily and shared 48% identity with hST6Gal-I. Consequently, we named this new sialyltransferase ST6Gal II. A construction in pFlag vector transfected in COS-7 cells gave raise to a soluble active form of ST6Gal II. Enzymatic assays indicate that the best acceptor substrate of ST6Gal II was the free disaccharide Galbeta1-4GlcNAc structure whereas ST6Gal I preferred Galbeta1-4GlcNAc-R disaccharide sequence linked to a protein. The alpha2,6-linkage was confirmed by the increase of Sambucus nigra agglutinin-lectin binding to the cell surface of CHO transfected with the cDNA encoding ST6Gal II and by specific sialidases treatment. In addition, the ST6Gal II gene showed a very tissue specific pattern of expression because it was found essentially in brain whereas ST6Gal I gene is ubiquitously expressed. 相似文献
8.
Koma M Miyagawa S Honke K Ikeda Y Koyota S Miyoshi S Matsuda H Tsuji S Shirakura R Taniguchi N 《Glycobiology》2000,10(7):745-751
The effect of the various glycosyltransferases on glycosphingolipids was examined, using transfected swine endothelial cell (SEC) lines. The reactivity of parental SEC to normal human serum (NHS) and Griffonia simplicifolia IB(4) (GSIB4) lectin, which binds to the Gal alpha1-3 Gal beta 1-4 GlcNAc-R (alpha-galactosyl epitope), was reduced by approximately 20% by the treatment with D-PDMP (D-threo-1-phenyl-2-decan- oylamino-3-morpholino-1-propanol), suggesting that glycosphingolipids contained by SEC have a considerable amount of the alpha-galactosyl epitope. The overexpression of two different types of glycosyltransferase, N-acetylglucosaminyl transferase III (GnT-III), as well as alpha2, 6-sialyltransferase (ST6Gal I), alpha2,3-sialyltransferase (ST3Gal III), and alpha1,2-fucosyltransferase (alpha1,2FT), suppresses the total antigenicity of SEC significantly. However, the reduction in reactivities toward NHS and GSIB4 lectin in the case of GnT-III transfectants was milder than those in other transfectants. Western blot analysis indicated that the glycoproteins in all transfectants had diminished reactivity to NHS and GSIB4 lectin to approximately the same extent. Therefore, the neutral glycosphingolipids of these transfectants were separated by thin layer chromatography, followed by immunostaining with NHS and GSIB4 lectin. The levels of the alpha-galactosyl epitope in glycosphingolipids were not decreased in the GnT-III transfectants but were in the ST6Gal I, ST3Gal III, and alpha1,2FT transfectants. These data indicate that ST6Gal I, ST3Gal III, and alpha1,2FT reduced the alpha-galactosyl epitope in both glycoproteins and glycosphingolipids, while GnT-III reduced them only in glycoproteins. 相似文献
9.
Influenza A neuraminidase (NA) is a target for anti-influenza drugs. The function of this enzyme is to cleave a glycosidic
linkage of a host cell receptor that links sialic acid (Sia) to galactose (Gal), to allow the virus to leave an infected cell
and propagate. The receptor is an oligosaccharide on the host cell surface. There are two types of oligosaccharide receptor;
the first, which is found mainly on avian epithelial cell surfaces, links Sia with Gal by an α2,3 glycosidic linkage; in the
second, found mainly on human epithelial cell surfaces, linkage is via an α2,6 linkage. Some researchers believe that NAs
from different viruses show selectivity for each type of linkage, but there is limited information available to confirm this
hypothesis. To see if the linkage type is more specific to any particular NA, a number of NA-receptor complexes of human influenza
A H1N1 (1918), avian influenza A H5N1 (2004), and a pandemic strain of H1N1 (2009) were constructed using homology modeling
and molecular dynamics simulation. The results show that the two types of receptor analogues bound to NAs use different mechanisms.
Moreover, it was found that a residue unique to avian virus NA is responsible for the recognition of the Siaα2,3Gal receptor,
and a residue unique to human virus NA is responsible for the recognition of Siaα2,6Gal. We believe that this finding could
explain how NAs of different virus origins always possess some unique residues. 相似文献
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Chang KH Lee JM Hwang-Bo J Yoo KH Sohn BH Yang JM Chung IS 《Biotechnology letters》2007,29(12):1803-1809
We examined the expression of human cyclooxygenase-1 (COX-1) in Drososphila melanogaster S2 (S2) cells transformed with cDNAs encoding β1,4-galactosyltransferase (GalT) and Galβ1,4-GlcNAc α2,6-sialyltransferase
(ST). Southern blot analysis indicated that multiple copies of the glycosyltransferases genes were integrated into the S2
cell genome. A lectin blot analysis also indicated that recombinant COX-1 from S2COX-1/GalT-ST cells contained the glycan
residues of β1,4-linked galactose and α2,6-linked sialic acid. The specific peroxidase activity of recombinant sialylated
COX-1 from S2COX-1/GalT-ST cells was 41,250 U mg−1, indicating an increase of approximately 22% compared with a non-sialylated control (33,850 U mg−1) from S2COX-1 cells.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
12.
Natural human interferon- (hIFN-) contains mainly biantennary complex-type sugar chains. We previously remodeled the branch structures of N-glycans on hIFN- in Chinese hamster ovary (CHO) cells by overexpressing UDP-N-acetylglucosamine: 1,6-D-mannoside 1,6-N-acetylglucosaminyltransferase (GnT-V). Normal CHO cells primarily produced hIFN- having biantennary sugar chains, whereas a CHO clone, designated IM4/Vh, transfected with GnT-V, primarily produced hIFN- having GlcNAc1-6 branched triantennary sugar chains when sialylation was incomplete and an increase in poly-N-acetyllactosamine (Gal1-4GlcNAc1-3)n was observed. In the present study, we introduced mouse Gal1-3/4GlcNAc-R 2,3-sialyltransferase (ST3Gal IV) and/or rat Gal1-4GlcNAc-R 2,6-sialyltransferase (ST6Gal I) cDNAs into the IM4/Vh cells to increase the extent of sialylation and to examine the effect of sialyltransferase (ST) type on the linkage of sialic acid. Furthermore, we speculated that sialylation extent might affect the level of poly-N-acetyllactosamine. We isolated four clones expressing different levels of 2,3-ST and/or 2,6-ST. The extent of sialylation of hIFN- from the IM4/Vh clone was 61.2%, which increased to about 80% in every ST transfectant. The increase occurred regardless of the type of overexpressed ST, and the proportion of 2,3- and 2,6-sialic acid corresponded to the activity ratio of 2,3-ST to 2,6-ST. Furthermore, the proportion of N-glycans containing poly-N-acetyllactosamine was significantly reduced (less than 10%) in the ST transfectants compared with the parental IM4/Vh clone (22.9%). These results indicated that genetic engineering of STs is highly effective for regulating the terminal structures of sugar chains on recombinant proteins in CHO cells. 相似文献
13.
4-Methylumbelliferyl N-acetyllactosaminide and 4-methylumbelliferyl sialyl N-acetyllactosaminides, which are used for the assay of sialytransferase, neuraminidase and fucosyltransferase, were synthesized,
respectively, by the β-D-galactosidase from Bacillus circulans and by a recombinant rat α2,3-(N)-sialyltransferase or rat liver α2,6-(N)-sialyltransferase with CMP-N-acetylneuraminic acid as donor. 相似文献
14.
Alzheimer's beta-secretase (BACE1) is a membrane-bound protease that cleaves the amyloid precursor protein (APP) in the trans-Golgi
network, an initial step in the pathogenesis of Alzheimer's disease. Although BACE1 is distributed among various tissues including
brain, its physiological substrate other than APP have not been identified. We have recently found that when BACE1 was overexpressed
in COS cells together with α2,6-sialyltransferase (ST6Gal I), the secretion of ST6Gal I markedly increased, suggesting that
BACE1 cleaves ST6Gal I as a physiological substrate. Thus BACE1 is the first identified protease that is responsible for the
cleavage and secretion of glycosyltransferases. Published in 2004.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
15.
Saito S Aoki H Ito A Ueno S Wada T Mitsuzuka K Satoh M Arai Y Miyagi T 《The Journal of biological chemistry》2003,278(29):26474-26479
Monosialosyl globopentaosylceramide (MSGb5), originally described as stage-specific embryonic antigen-4, is expressed in testicular germ cell tumors and in aggressive cases of human renal cell carcinoma (RCC). Clarification of the molecular mechanisms regulating synthesis of MSGb5 is very important to understand testicular carcinogenesis and the malignant progression of human RCC. For this purpose, we have investigated alpha2,3-sialyltransferase involved in the synthesis of MSGb5. We used the method of expression cloning combined with polymerase chain reaction targeted to sialylmotif to isolate a cDNA clone from RCC cell line ACHN library. The cloned cDNA was found to be identical to the previously cloned ST3Gal II in sequence. A soluble recombinant form of the protein in COS-1 cells showed an enzyme activity of alpha2,3-sialyltransferase toward globopentaosylceramide (Gb5) in addition to asialo-GM1 and GM1a. Transient transfection of COS-7 and ACHN cells with this cDNA induced an increase of MSGb5, whereas stable transfection of antisense ST3Gal II cDNA suppressed expression of MSGb5 in ACHN cells. The ST3Gal II mRNA level was increased in 7 of 8 RCC cell lines and in all six RCC tissues surgically obtained, although it was not necessarily consistent with the MSGb5 level in RCC cell lines. This study indicates that ST3Gal II is a MSGb5 (stage-specific embryonic antigen-4) synthase and that its increased expression level is closely related to renal carcinogenesis. 相似文献
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17.
Kazuya I.P.J. Hidari Kin-ichi Oyama Miho Nakayama Shiho Goto Kei-ichi Watanabe Takumi Furuta Toshiyuki Kan 《Biochemical and biophysical research communications》2009,382(3):609-613
Sialyltransferases biosynthesize sialyl-glycoconjugates involved in many biological and pathological processes. We investigated and characterized synthetic flavonoid derivatives as sialyltransferase inhibitors. We first examined 54 compounds by solid-phase enzyme assay using β-galactoside α2,6-sialyltransferase 1 (ST6Gal I) and β-galactoside α2,3-sialyltransferase. Several compounds inhibited sialyltransferase enzyme activity regardless of sialyl-linkage reactions. Among them, two compounds showed inhibitory activity against ST6Gal I with IC50 values less than 10 μM. Three characteristic features of flavonoids were determined by structure-inhibitory activity relationships. First, a double bond between C2-C3 linkages is required for the activity. Second, increasing hydrophilic properties on the B-ring markedly augmented the inhibitory effect. Third, a hydrophobic functional group introduced on the hydroxyl groups of the A-ring enhanced the inhibitory activity. Kinetic analysis using human ST6Gal I indicated a mixed inhibition mechanism of the compounds. In conclusion, the flavonoids identified could be applied for control of cellular expression of sialic acid. 相似文献
18.
Suppression of a sialyltransferase by antisense DNA reduces invasiveness of human colon cancer cells in vitro 总被引:5,自引:0,他引:5
Zhu Y Srivatana U Ullah A Gagneja H Berenson CS Lance P 《Biochimica et biophysica acta》2001,1536(2-3):148-160
Transfer of terminal alpha 2,6-linked sialic acids to N-glycans is catalyzed by beta-galactoside alpha 2,6-sialyltransferase (ST6Gal I). Expression of ST6Gal I and its products is reportedly increased in colon cancers. To investigate directly the functional effects of ST6Gal I expression, human colon cancer (HT29) cells were transfected with specific antisense DNA. ST6Gal I mRNA and protein were virtually undetectable in six strains of transfected HT29 cells. ST6Gal activity was reduced to 14% of control (P<0.005) in transfected cells. Expression of terminal alpha 2,6- and alpha 2,3-linked sialic acids, and unmasked N-acetyllactosamine oligosaccharides, respectively, was assessed using flow cytometry and fluoresceinated Sambucus nigra, Maackia amurensis and Erythrina cristagalli lectins. Results indicated a major reduction in expression of alpha 2,6-linked sialic acids and counterbalancing increase in unmasked N-acetyllactosamines in antisense DNA-transfected cells, without altered expression of alpha 2,3-linked sialic acids or ganglioside profiles. The ability of transfected cells to form colonies in soft agar and to invade extracellular matrix material (Matrigel), respectively, in vitro was reduced by approx. 98% (P<0.0001) and more than 3-fold (P<0.005) compared to parental HT29 cells. These results indicate that N-glycans bearing terminal alpha 2,6-linked sialic acids may enhance the invasive potential of colon cancer cells. 相似文献
19.
Peter R. Andreana Przemyslaw Kowal Adam J. Janczuk Peng George Wang 《Glycoconjugate journal》2003,20(2):107-118
Galactose oxidase (EC 1.1.3.9, GAO) was used to convert the C-6′ OH of Galβ(1 → 4)Glcβ–OBn (5) to the corresponding hydrated
aldehyde (7). Chemical modification, through dehydratative coupling and reductive amination, gave rise to a small library
of Galβ(1 → 4)Glcβ–OBn analogues (9a–f, 10, 11). UDP-[6-3H]Gal studies indicated that α1,3-galactosyltransferase recognized the C-6′ modified Galβ(1 → 4)Glcβ–OBn analogues (9a–f,
10, 11). Preparative scale reactions ensued, utilizing a single enzyme UDP-Gal conversion as well as a dual enzymatic system
(GalE and α1,3GalT), taking full advantage of the more economical UDP-Glc, giving rise to compounds 6, 15–22. Galα(1 → 3)Galβ(1
→ 4)Glcβ–OBn trisaccharide (6) was produced on a large scale (2 g) and subjected to the same chemoenzymatic modification as
stated above to produce C-6″ modified derivatives (23–30). An ELISA bioassay was performed utilizing human anti-αGal antibodies
to study the binding affinity of the derivatized epitopes (6, 15–30). Modifications made at the C-6′ position did not alter
the IgG antibody's ability to recognize the unnatural epitopes. Modifications made at the C-6″ position resulted in significant
or complete abrogation of recognition. The results indicate that the C-6′ OH of the αGal trisaccharide epitope is not mandatory
for antibody recognition. Published in 2004.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献