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1.
Müthing J Meisen I Bulau P Langer M Witthohn K Lentzen H Neumann U Peter-Katalinić J 《Biochemistry》2004,43(11):2996-3007
Mistletoe lectin I (ML-I) is a type II ribosome-inactivating protein, which inhibits the protein biosynthesis at the ribosomal level. ML-I is composed of a catalytically active A-chain with rRNA N-glycosidase activity and a B-chain with carbohydrate binding specificities. Using comparative solid-phase binding assays along with electrospray ionization tandem mass spectrometry, ML-I was shown to preferentially bind to terminally alpha2-6-sialylated neolacto series gangliosides from human granulocytes. IV(6)Neu5Ac-nLc4Cer, VI(6)Neu5Ac-nLc6Cer, and VIII(6)Neu5Ac-nLc8Cer were identified as ML-I receptors, whereas the isomeric alpha2-3-sialylated neolacto series gangliosides were not recognized. Only marginal binding of ML-I to terminal galactose residues of neutral glycosphingolipids with a Galbeta1-4Glc or Galbeta1-4GlcNAc sequence was determined, whereas a distal Galalpha1-4Gal, GalNAcbeta1-3Gal, or GalNAcbeta1-4Gal disaccharide did not bind at all. Among the glycoproteins investigated in Western blot and microwell adsorption assays, only those carrying Neu5Acalpha2-6Galbeta1-4GlcNAc residues, exclusively, predominantly, or even as less abundant constituents in an assembly with Neu5Acalpha2-3Galbeta1-4GlcNAc-terminated glycans, displayed high ML-I binding capacity. From our data we conclude that (i) ML-I has to be considered as a sialic acid- and not a galactose-specific lectin and (ii) neolacto series gangliosides and sialoglycoproteins with type II glycans, which share the Neu5Acalpha2-6Galbeta1-4GlcNAc terminus, are true ML-I receptors. This strict preference might help to explain the immunostimulatory potential of ML-I toward certain leukocyte subpopulations and its therapeutic success as a cytotoxic anticancer drug. 相似文献
2.
Summary The acute toxicity of lectin ML I from the toxic drug, mistletoe, was demonstrated in previous experiments. Because the reason for this extremely high toxicity is not yet clear, mice were studied histochemically at different times after treatment with various doses of ML I, ML I A or ML I B chain separately, or recombinations of ML I A and ML I B. Various plasma membrane-associated hydrolases as well as Golgi apparatus-and endoplasmic reticulum-linked hydrolases, peroxisomal and extraperoxisomal oxidases, lysosomal hydrolases, mitochondrial dehydrogenases, the cytoskeletal proteins keratin and vimentin as well as iron, glycogen and lipids were analysed in all organs and tissues of female mice. Irrespective of the dose, a clear-cut response was only observed in the liver. After ML I treatment, glycogen disappeared completely from all hepatocytes, and this effect did not depend on the ML I-concentration and exposure time. The increase in activity of Golgiassociated thiamine pyrophosphatase in hepatocytes and of non-specific alkaline phosphatase in the sinusoidal endothelial cells depended on the applied ML I concentration and the time of treatment. Doses of 600 or 900 ng ML I/kg drastically increased the phosphatase activities. These clear-cut changes of glycogen and enzyme activities were not observed after administration of the ML I B chain alone, and less so when the mice were treated only with the ML I A chain, or were treated with a recombination of ML I A and ML I B even at concentrations higher than that of ML I. The glycogen as well as thiamine pyrophosphatase and non-specific alkaline phosphatase responses after ML I administration were highly reproducible. None of the other investigated cell constituents responded to treatment with ML I, irrespective of the organs, tissues and cells analysed. This was also true for mice treated with ML I concentrations higher than the LD50, after which the animals died with 2–3 days. In conclusion, application of ML I induces clear-cut liver cell changes, which are not observed after treatment with individual constituents of the ML I molecule either singly or in recombination. However, these changes do not explain the deaths of the animals.Supported by WTZ (Vereinbarung über wissenschaftlich-technische Zusammenarbeit: Projekt 13) 相似文献
3.
The acute toxicity of lectin ML I from the toxic drug, mistletoe, was demonstrated in previous experiments. Because the reason for this extremely high toxicity is not yet clear, mice were studied histochemically at different times after treatment with various doses of ML I, ML I A or ML I B chain separately, or recombinations of ML I A and ML I B. Various plasma membrane-associated hydrolases as well as Golgi apparatus-and endoplasmic reticulum-linked hydrolases, peroxisomal and extraperoxisomal oxidases, lysosomal hydrolases, mitochondrial dehydrogenases, the cytoskeletal proteins keratin and vimentin as well as iron, glycogen and lipids were analysed in all organs and tissues of female mice. Irrespective of the dose, a clear-cut response was only observed in the liver. After ML I treatment, glycogen disappeared completely from all hepatocytes, and this effect did not depend on the ML I-concentration and exposure time. The increase in activity of Golgi-associated thiamine pyrophosphatase in hepatocytes and of non-specific alkaline phosphatase in the sinusoidal endothelial cells depended on the applied ML I concentration and the time of treatment. Doses of 600 or 900 ng ML I/kg drastically increased the phosphatase activities. These clear-cut changes of glycogen and enzyme activities were not observed after administration of the ML I B chain alone, and less so when the mice were treated only with the ML I A chain, or were treated with a recombination of ML I A and ML I B even at concentrations higher than that of ML I.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
4.
Oda Y Senaha T Matsuno Y Nakajima K Naka R Kinoshita M Honda E Furuta I Kakehi K 《The Journal of biological chemistry》2003,278(34):32439-32447
In this report, we describe a new lectin from the fungus Rhizopus stolonifer that agglutinates rabbit red blood cells. Agglutinating activity was detected in the extract of mycelium-forming spores cultured on agar plates but not in the mycelium-forming no spores from liquid medium. This lectin, which we designated R. stolonifer lectin (RSL), was isolated by affinity chromatography with porcine stomach mucin-Sepharose. SDS-polyacrylamide gel electrophoresis and mass spectral analysis showed that RSL is approximately 4.5 kDa, whereas gel filtration indicated a mass of 28 kDa. This indicates that the lectin is a hexamer of noncovalently associated RSL monomers. RSL activity was very stable, since it was insensitive to heat treatment at 70 degrees C for 10 min. Analysis of RSL binding specificity by both microtiter plate and precipitation assays showed that N-glycans with l-fucose linked to the reducing terminal GlcNAc residues are the most potent inhibitors of RSL binding, whereas N-glycans without alpha(1-6)-linked fucose residues are approximately 100-fold weaker inhibitors of binding. Oligosaccharides with alpha(1-2, -3, and -4) linkages showed no inhibition of binding in these assays. In a mirror resonance biosensor assay, high affinity binding was observed between RSL and the glycopeptide of bovine gamma-globulin, which has N-glycans with alpha(1-6)-linked fucose residues. However, RSL showed only a weak interaction with the glycopeptide of quail ovomucoid, which lacks fucose residues. Finally, capillary affinity electrophoresis studies indicated that RSL binds strongly to N-glycans with alpha(1-6)-linked fucose residues. Together, these results show that RSL recognizes the core structure of N-glycans with alpha(1-6)-linked l-fucose residues. This specificity could make RSL a valuable tool for glycobiological studies. 相似文献
5.
The behaviour ofN-acetyllactosamine-type oligosaccharides and glycopeptides on a column of mistletoe lectin I (MLI) immobilized on Sepharose 4B was examined. The immobilized lectin does not show any affinity for asialo-N-glycosylpeptides and related oligosaccharides, which possess one to four unmaskedN-acetyllactosamine sequences. However, substitution of at least one of theN-acetyllactosamine sequences by sialic acid residues, either at O-3 or O-6 of galactose, slightly enhances the affinity of the lectin. Such sialylatedN-glycosylpeptides or oligosaccharides are eluted from the lectin column by the starting buffer as retarded fractions. Surprisingly, the affinity of the immobilized MLI is higher for P1 antigen-containing glycopeptide isolated from turtle-dove ovomucoid and for glycopeptides from bovine thyroglobulin containing terminal non-reducing Gal1–3Gal sequences. These structures are strongly bound on the lectin column and their elution is obtained with 0.15M galactose in the starting buffer.In memory of Hartmut Franz. 相似文献
6.
Benjamin B. Gengenbach Linda L. Keil Patrick Opdensteinen Catherine R. Müschen Georg Melmer Hans Lentzen Jens Bührmann Johannes F. Buyel 《Biotechnology and bioengineering》2019,116(9):2236-2249
Cancer is the leading cause of death in industrialized countries. Cancer therapy often involves monoclonal antibodies or small-molecule drugs, but carbohydrate-binding lectins such as mistletoe (Viscum album) viscumin offer a potential alternative treatment strategy. Viscumin is toxic in mammalian cells, ruling them out as an efficient production system, and it forms inclusion bodies in Escherichia coli such that purification requires complex and lengthy refolding steps. We therefore investigated the transient expression of viscumin in intact Nicotiana benthamiana plants and Nicotiana tabacum Bright Yellow 2 plant-cell packs (PCPs), comparing a full-length viscumin gene construct to separate constructs for the A and B chains. As determined by capillary electrophoresis the maximum yield of purified heterodimeric viscumin in N. benthamiana was ~7 mg/kg fresh biomass with the full-length construct. The yield was about 50% higher in PCPs but reduced 10-fold when coexpressing A and B chains as individual polypeptides. Using a single-step lactosyl-Sepharose affinity resin, we purified viscumin to ~54%. The absence of refolding steps resulted in estimated cost savings of more than 80% when transient expression in tobacco was compared with E. coli. Furthermore, the plant-derived product was ~3-fold more toxic than the bacterially produced counterpart. We conclude that plants offer a suitable alternative for the production of complex biopharmaceutical proteins that are toxic to mammalian cells and that form inclusion bodies in bacteria. 相似文献
7.
The elderberry (Sambucus nigra L.) bark lectin recognizes the Neu5Ac(alpha 2-6)Gal/GalNAc sequence 总被引:17,自引:0,他引:17
N Shibuya I J Goldstein W F Broekaert M Nsimba-Lubaki B Peeters W J Peumans 《The Journal of biological chemistry》1987,262(4):1596-1601
Carbohydrate binding properties of a new plant lectin isolated from elderberry (Sambucus nigra L.) (SNA) bark were studied using the techniques of quantitative precipitation, hapten inhibition, and equilibrium dialysis. Purified SNA precipitates highly sialylated glycoproteins such as fetuin, orosomucoid, and ovine submaxillary mucin, but not their asialo derivatives. Hapten inhibition experiments showed that both D-Gal and D-GalNAc are weak inhibitors of SNA-glycophorin precipitation, but neither New5Ac nor Neu5Gc is an inhibitor. A series of oligosaccharides which contain the terminal Neu5Ac(alpha 2-6)Gal sequence showed an extremely high inhibitory potency (1,600-10,000 times more inhibitory than Gal). On the other hand, oligosaccharides with the Neu5Ac(alpha 2-3)Gal linkage were only 30-80 times more inhibitory than Gal, thus showing a marked preference for the 2,6-linked isomer. Hapten inhibition with Gal and its epimers suggested that the equatorial OH at C-3 and the axial OH at C-4 of the D-pyranose ring are strict requirements for binding. Conversion of the Neu5Ac residue to its 7-carbon analogue by selective periodate oxidation of its glyceryl side chain, followed by NaBH4 reduction, completely destroyed the ability of fetuin and orosomucoid to precipitate with SNA. Moreover, the same treatment of Neu5Ac(alpha 2-3)lactitol also abolished its ability to inhibit the precipitation reaction, suggesting that the glyceryl side chain of NBu5Ac (especially the C-8 and/or C-9 portion) is an important determinant for SNA. The increased inhibitory potency of various glycosides with beta-linked nonpolar aglycons suggested the presence of a hydrophibic interacting region adjacent to the carbohydrate binding site. The results of equilibrium dialysis using [3H] Neu5Ac(alpha 2-6)lactitol as ligand showed the presence of two equivalent, noninteracting carbohydrate binding sites in this tetrameric glycoprotein lectin (Ka = 3.9 X 10(5) M-1). 相似文献
8.
Adam EC Holgate ST Lackie PM 《American journal of physiology. Lung cellular and molecular physiology》2007,292(2):L462-L468
The effective repair of damage to the airway epithelium is essential to maintain the ability to exclude airborne particulates and protect against potential pathogens. Carbohydrates on the cell surface have an important role in cell-cell and cell substrate interactions. Using a model of repair with airway epithelial-derived cells of the 16HBE 14o(-) cell line, we have examined the effect of the Aleuria aurantia lectin (AAL), which binds very selectively to alpha(1,6)-linked fucose residues. Addition of unconjugated or FITC-labeled AAL reduced the rate of epithelial repair to approximately one-third of control values as measured by image analysis while cell viability was maintained. Pulse labeling with AAL-FITC for 30 min followed by incubation in AAL-free medium caused similar inhibition of repair but could be reversed by addition of fucose up to 7 h after AAL removal. By confocal microscopy, AAL binding was found to be on the apical, but not basolateral, surfaces of cells, and internalization of the labeled lectin was seen. Preincubation of the lectin with fucose prevented this effect. Ulex europeaus I lectin, which is also fucose specific, resulted in similar binding to the cells and internalization, but it did not affect the speed of the repair process. We conclude that alpha(1,6)-fucose binding sites play an important role in epithelial repair. Better understanding of this process will provide a deeper insight into the crucial mechanisms of epithelial repair. 相似文献
9.
A Domeyne C Pinset D Montarras V Garandel R G Rosenfeld B Barenton 《European journal of biochemistry》1992,208(2):273-279
We have studied insulin-like-growth-factor (IGF) binding in two subclones of the C2 myogenic cell line. In the permissive parental subclone, myoblasts differentiate spontaneously into myotubes in medium supplemented with fetal calf serum. Unlike permissive myoblasts, inducible myoblasts require high concentrations of insulin (1.6 microM) or lower concentrations of IGF-I (25 nM) to differentiate, and expression of MyoD1 is not constitutive. IGF receptors were studied in microsomal membranes of proliferating and quiescent myoblasts and myotubes. IGF-II binding was also studied in inducible myoblasts transfected with the MyoD1 cDNA (clone EP5). Both inducible and permissive cells exhibited a single class of binding sites with similar affinity for IGF-I (Kd 0.8-1.2 nM). Affinity cross-linking of [125I]IGF-I to microsomal membranes, under reducing conditions, revealed a binding moiety with an apparent molecular mass of 130 kDa in permissive cells and 140 kDa in inducible cells, which corresponded to the alpha subunit of the IGF-I receptor. In permissive quiescent myoblasts, linear Scatchard plots suggested that [125I]IGF-II bound to a single class of binding sites (Kd 0.6 nM) compatible with binding to the IGF-II/M6P receptor. This was confirmed by affinity cross-linking experiments showing a labeled complex with an apparent molecular mass of 260 kDa and 220 kDa when studied under reducing and non-reducing conditions, respectively. In contrast, competitive inhibition of [125I]IGF-II binding to inducible quiescent myoblasts generated curvilinear Scatchard plots which could be resolved into two single classes of binding sites. One of them corresponded to the IGF-II/M6P receptor (Kd 0.2 nM) as evidenced by cross-linking experiments. The second was the binding site of highest affinity (Kd 0.04 nM) which was less inhibited by IGF-I than by IGF-II and was not inhibited by insulin. It migrated in SDS/PAGE at a position equivalent a molecular mass of 140 kDa, under reducing conditions, and at approximately 300 kDa, under non-reducing conditions. The labeling of this atypical binding moiety was not inhibited by anti(IGF-II/M6P-receptor) immunoglobulin. It was also observed in permissive and inducible myoblasts at proliferating stage. It was absent for permissive quiescent myoblasts and from permissive and inducible myotubes. Forced expression of MyoD1 in inducible cells (EP5 cells) dramatically reduced [125I]IGF-II binding to this atypical receptor. It emerges from these experiments that C2 cells express a putative alpha 2 beta 2 IGF-II receptor structurally related to the insulin/IGF-I receptor family. It is present in myoblasts but not in myotubes.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
10.
Three-dimensional structures of the complexes of concanavalin A (ConA) with alpha(1-2) linked mannobiose, triose and tetraose have been generated with the X-ray crystal structure data on native ConA using the CCEM (contact criteria and energy minimization) method. All the constituting mannose residues of the oligosaccharide can reach the primary binding site of ConA (where methyl-alpha-D-mannopyranose binds). However, in all the energetically favoured complexes, either the non-reducing end or middle mannose residues of the oligosaccharide occupy the primary binding site. The middle mannose residues have marginally higher preference over the non-reducing end residue. The sugar binding site of ConA is extended and accommodates at least three alpha(1-2) linked mannose residues. Based on the present calculations two mechanisms have been proposed for the binding of alpha(1-2) linked mannotriose and tetraose to ConA. 相似文献
11.
H Shimizu H Tsuchie H Honma K Yoshida T Tsuruoka H Ushijima T Kitamura 《Japanese journal of medical science & biology》1990,43(3):75-87
The effect of N-(3-phenyl-2-propenyl)-1-deoxynojirimycin (ppDNM) on the lectin binding to HIV-1 glycoprotein was analyzed by using biotinylated lectins of various sugar specificities as probes. ppDNM potentially inhibited HIV-1-induced syncytium formation and viral infectivity of HIV-1 without cytotoxicity. The lectin binding assay showed that ppDNM treatment reduced Con A binding to gp120 of HIV-1. 相似文献
12.
Preferential binding of collagenase to alpha 2-macroglobulin in the presence of the tissue inhibitor of metalloproteinases 总被引:5,自引:0,他引:5
The binding of collagenase to both alpha 2-macroglobulin and the tissue inhibitor of metalloproteinases was studied using purified materials. Collagenase bound preferentially to alpha 2-macroglobulin although no transfer of collagenase to alpha 2-macroglobulin occurred if the enzyme was first mixed with the tissue inhibitor of metalloproteinases. The sequences of amino acids in both inhibitors likely to be responsible for the binding of collagenase are discussed and compared to the cleavage site in the collagen molecule. 相似文献
13.
The specificity of viral and bacterial sialidases for alpha(2-3)- and alpha(2-6)-linked sialic acids in glycoproteins 总被引:5,自引:0,他引:5
The anomeric specificity of six sialidases (Vibrio cholerae, Arthrobacter ureafaciens, Clostridium perfringens, Newcastle disease virus, fowl plague virus and influenza A2 virus sialidases) was assessed with sialylated antifreeze glycoprotein, ovine submandibular gland glycoprotein and alpha 1-acid glycoprotein, resialylated specifically in alpha(2-3) or alpha(2-6) linkage with N-acetylneuraminic acid or N-glycolylneuraminic acid using highly purified sialyltransferases. The rate of release of sialic acid from these substrates was found to correlate well with the specificity observed earlier with the same sialidases using small oligosaccharide substrates, i.e., alpha(2-3) glycosidic linkages are hydrolyzed faster than alpha(2-6) linkages, with the exception of the enzyme from A. ureafaciens. Sialidase activity was higher with N-acetylneuraminic acid when compared with N-glycolylneuraminic acid. The studies also showed that the core oligosaccharide and protein structure in glycoproteins may influence the rate of release for different glycosidic linkages. 相似文献
14.
We previously reported the chemical synthesis of oligonucleotides containing thymine glycol, a major form of oxidative DNA damage. In the preparation of the phosphoramidite building block, the predominant product of the osmium tetroxide oxidation of protected thymidine was (5R,6S)-thymidine glycol. To obtain the building block of the other isomer, (5S,6R)-thymidine glycol, in an amount sufficient for oligonucleotide synthesis, the Sharpless asymmetric dihydroxylation (AD) reaction was examined. Although the reaction was very slow, (5S,6R)-thymidine glycol was obtained in preference to the (5R,6S) isomer. The ratio of (5S,6R)- and (5R,6S)-thymidine glycols was 2:1, and a trans isomer was also formed. When an ionic liquid, 1-butyl-3-methylimidazolium hexafluorophosphate, was used as a co-solvent, the reaction became faster, and the yield was improved without changing the preference. The phosphoramidite building block of (5S,6R)-thymidine glycol was prepared, and oligonucleotides containing 5S-thymine glycol were synthesized. One of the oligonucleotides was used to analyze the binding of distamycin A to thymine glycol-containing DNA by Circular dichroism (CD) spectroscopy and surface plasmon resonance (SPR) measurements. Distamycin A bound to a duplex containing either isomer of thymine glycol within the AATT target site, and its binding was observed even when the thymine glycol was placed opposite cytosine. 相似文献
15.
16.
Differential binding of a CCAAT DNA binding factor to the promoters of the mouse alpha 2(I) and alpha 1(III) collagen genes 总被引:11,自引:0,他引:11
A Hatamochi B Paterson B de Crombrugghe 《The Journal of biological chemistry》1986,261(24):11310-11314
An exonuclease III assay (Wu, C. (1985) Nature 317, 84-87) was used to identify in nuclear extracts of NIH 3T3 cells a factor which binds to the CCAAT segment of the alpha 2(I) collagen promoter between -80 and -84. This sequence is located on the coding strand in the alpha 2(I) collagen promoter. Binding is specific since only promoter fragments which contain the CCAAT box sequences on one or the other DNA strand inhibit binding to the alpha 2(I) collagen CCAAT box. The CCAAT binding factor protects approximately 26 base pairs of the alpha 2(I) collagen promoter from exonuclease III digestion. Binding to the alpha 2(I) collagen promoter CCAAT box is not inhibited by a fragment of the alpha 1(III) collagen promoter (from -396 to +16), which does not contain a CCAAT sequence on either one or the other strand. Our data suggest that two genes such as the alpha 2(I) and alpha 1(III) collagen genes, which are coordinately expressed in many tissues, are not necessarily regulated by the same trans-acting DNA binding proteins. 相似文献
17.
18.
The asymmetric synthesis of novel, potent phosphoramidate alpha(2-6)sialyltransferase transition-state analogue inhibitors such as (R)-9 (K(i)=68 microM) is described, via condensation of cytidine phosphitamide 6 with key chiral, non-racemic alpha-aminophosphonates, prepared in >98% ee by Mitsunobu azidation followed by Staudinger reduction of the corresponding chiral, non-racemic alpha-hydroxyphosphonates. 相似文献
19.
An NMR investigation of the binding of the anticancer drug actinomycin D to oligodeoxyribonucleotides with isolated 5'd(GC)3' binding sites 总被引:1,自引:0,他引:1
Imino proton and 31P NMR studies were conducted on the binding of actinomycin D (ActD) to self-complementary oligodeoxyribonucleotides with one GC binding site [d(ATATGCATAT) (1), d-(ATACGCGTAT) (2), and d(ATATACGCGTATAT) (3)] and with two GC sites [d(ATGCATGCAT) (4)]. At R = 1 (molar ratio of ActD to oligomer duplex) ActD caused a doubling of the number of imino proton signals at, and adjacent to, the GC binding site of 1. One of the G.C base pair signals shifted upfield while the other shifted downfield. Both of the signals for the A.T base pairs adjacent to the binding site shifted downfield. All imino proton signals of 2 and the longer sequence, 3, shifted upfield on binding of ActD to the GC site, indicating a sequence-dependent change in base stacking on complex formation. For both 1 and 2 addition of ActD resulted in a similar pattern of three downfield 31P NMR signals. The two most downfield signals have chemical shift and temperature dependence which are characteristic of phosphate groups at isolated intercalation sites. At R = 1 the ActD complex with 4 has very complex spectra with both upfield and downfield A.T and G.C imino signals. All these data were consistent with two 1:1 complexes with the unsymmetrical phenoxazone ring adopting both of the two possible orientations.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
20.
S Hakomori E Nudelman S B Levery C M Patterson 《Biochemical and biophysical research communications》1983,113(3):791-798
A new monoclonal antibody (IB9) was prepared by hybridoma technique directed specifically to sialosyl alpha 2 leads to 6 galactosyl residue. With this reagent, accumulation of two major gangliosides in human colonic and liver adenocarcinoma has been detected, and these gangliosides were isolated and characterized as structures A and B (below). Another ganglioside with a ceramide nonasaccharide structure, which reacted to anti-X-hapten antibody after desialylation, was also isolated and partially characterized. (formula; see text) These gangliosides were absent or present in very small quantity in normal tissue and may represent human cancer-associated markers. 相似文献