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1.
A lectin was purified from the bark of Robinia pseudoacaciaby sequential ion-exchange chromatography on DEAE-Sepharoseand CM-Toyopearl. The purified lectin was estimated to havea molecular weight of 106 kDa and to be a homotetramer of subunitswith a molecular weight of 29 kDa. Antibodies raised againstthe bark lectin cross-reacted with a 29-kDa polypeptide duringWestern blot analysis, showing that the antibodies are specificfor the bark lectin. The antibodies against the lectin fromRobinia bark cross-reacted with polypeptides in extracts ofthe seeds and bark of Sophora japonica, indicating that thelectin from Robinia bark is immunologically related to the lectinsof Sophora. However, the antibodies did not cross-react withproteins from Robinia seeds and leaves. The first twenty aminoacid residues from the N-terminus of the lectin from Robiniabark were determined and compared with those of the Sophoralectins. (Received July 13, 1991; Accepted December 12, 1991)  相似文献   

2.
A mannose-specific lectin was isolated from leaves of Neoregeliaflandria, an ornamental plant that belongs to Bromeliaceae,a family of monocotyledons. The amino acid composition and molecularmass of the lectin were similar to those of mannose-specificlectins from other monocotyledons. However, in a test to examinethe inhibition of hemagglutination, it became apparent thatthe isolated lectin recognized D-glucose and N-acetyl D-glucosaminein addition to D-mannose, unlike mannose-specific lectins fromthe monocotyledons that have been reported to date. (Received May 17, 1996; Accepted August 19, 1996)  相似文献   

3.
Purification and characterization of bovine mannan-binding protein   总被引:5,自引:0,他引:5  
Bovine mannan-binding protein (bMBP) was observed in serum byits Ca2+ -dependent binding to mannan and by an Mrof 28 kDaunder reducing conditions on sodium dodecyl sulphate—polyacrylamidegel electrophoresis (SDS—PAGE). The lectin was isolatedby precipitation with polyethyl-eneglycol (PEG), affinity chromatographyon mannan—Sepharose eluted with EDTA, and absorption onSepharose 4B rabbit anti-bovine Ig to remove anti-mannan antibodies.Fractions containing the lectin were reapplied to mannan—Sepharoseand eluted first with N-acetyl-D-glucosamine (GlcNAc) to removeconglutinin, and then with mannose to elute the 28 kDa lectin.Further purification was achieved by ion-exchange chromatographyon Mono-Q and by man-nose-gradient elution from a mannan-Sepharosecolumn. SDS—PAGE of the purified lectin showed three highmolecular weight bands under non-reducing conditions. The reducedprotein gave a single band of 28 kDa. On gel permeation chromatographyunder non-dissociating conditions, the protein emerged at avolume corresponding to Mr  相似文献   

4.
Lectins (agglutinins) are components of the immunobiologicalrecognition system of vertebrates and invertebrates. The presentstudy focused on the molecular properties of the agglutininfrom the albumen gland of Helix pomatia (HPA) and on the occurrenceof lectin-related molecules on the surface of H. pomatia haemocytes.According to the current model (Hammarström et al., 1972,Scandinavian Journal of Immunology, 1: 259–301), the hexamericHPA of about 79 kDa is composed of three non-covalently associateddimers (26 kDa), each consisting of two disulphide-bridged 13kDa monomers. However, on native-gradient polyacrylamide gelelectrophoresis (PAGE), we obtained high molecular weight bandsrepresenting lectin polymers. The stepwise dissociation of thesewas achieved by incubation with SDS at temperatures from 20to 40°C (1 h) and at 100°C (10 min). The results obtainedon SDS–PAGE included the occurrence of partially SDS-resistanthexamers of about 66 kDa, of two dimer bands of 22 and 19 kDa,and of two minor heteromonomer fractions. Complete dissociationinto heteromonomers of 13 and 11 kDa was achieved by boilingthe lectin (10 min) with SDS under reducing conditions. Fornative lectin molecules, both monomers occurred as disulphide-linkedhomodimers. Monomers or dimers electroeluted from an SDS–gel,reassociated to SDS-resistant oligomers upon re-electrophoresis.Finally, molecules antigenetically related to the lectin wereextracted from the membrane of H. pomatia haemocytes. Anti-HPAantibodies recognized peptides with an apparent molecular weightof about 30 and 56 kDa, which were shown to represent cell-surfacemolecules. (Received 4 March 2008; accepted 9 September 2008)  相似文献   

5.
A novel lectin has been identified in rainbow trout serum and plasma. The lectin binds to Sepharose (an agarose polymer) in a calcium-dependent manner. Glucose, N-acetyl-glucosamine, mannose, N-acetyl-mannosamine, l-fucose, maltose and α-methyl-mannoside are good inhibitors of this binding, whereas glucosamine and d-fucose inhibits to a lesser degree and mannosamine and galactose do not inhibit the binding to Sepharose. When analysed by SDS-PAGE under non-reducing conditions, the lectin appears as a characteristic ladder of bands with approximately 16 kDa between consecutive bands. Upon reduction, the lectin appears as a 16-kDa band. On size-exclusion chromatography of trout serum and plasma, the protein emerges over a broad range corresponding to sizes from about 2000 kDa to less than 200 kDa. The NH2-terminal sequence (AAENRNQXPPG) shows no significant homology with known proteins. Because of the characteristic appearance in non-reducing SDS-PAGE and the lectin activity, we propose to name the protein “ladderlectin.”  相似文献   

6.
N-Glycoloylneuraminic acid (Neu5Gc) is synthesized as its CMP-giycosideby the action of CMPN-acetylneuramlnic acid (CMP-Neu5Ac) hydroxylase.This enzyme is a soluble cytochrome bs-dependent monooxygenaseand has been purified to apparent homogeneity from pig submandibularglands by precipitation with N-cetyN,N,N-trimethylam-moniumbromide and fractionation on Q-Sepharose, Cibacron Blue 3GA-Agarose,Reactive Brown 10-Agarose, Hexyl-Agarose and Superose S.12.This procedure resulted in an 8960-fold purification of thehydroxylase with a recovery of 0.8%. The molecular mass of thisprotein was shown to be 65 kDa on SDS-PAGE and 60 kDa as determinedby gel filtration on Superose S.12, which suggests that theenzyme is a monomer. The purified CMP-Neu5Ac hydroxylase isactivated by FeSO4 and inhibited by iron-binding reagents suchas o-phenanthroline, KCN, Tiron and ferro-zine. An apparentKm of 11 µM was determined for the substrate CMP-Neu5Acusing purified hydroxylase in the presence of Triton X-100-solubilizedmicrosomes. In a reconstituted system consisting of purifiedhydroxylase, cytochrome b5, cytochrome b5 reductase and catalase,an apparent Km of 3 µM was measured. The apparent Kmforcytochrome b5 in this system was 0.24 µM. Immunizationof a rabbit with enriched and purified hydroxylase led to anantiserum that inhibited CMP-Neu5Ac hydroxylase activity andreacted with the purified 65 kDa protein on a Western blot afterSDS-PAGE. Antibodies specific for this 65 kDa protein were isolatedand showed a strong reaction with the purified CMP-Neu5Ac hydroxylasefrom mouse liver after immunoblotting. Initial experiments withthis monospecific antibody suggest that the activity of thehydroxylase in a particular tissue correlates with the amountof immuno-reactive protein. cytochrome b5 N-glcoloylneuraminic acid hydroxylase pig submandibular gland mucin sialic acid  相似文献   

7.
An N-acetylgalactosamine-specific lectin (GFL) was isolated from Grifola frondosa fruiting bodies by affinity chromatographies on acid-treated Sepharose CL-4B and then GalNAc-Toyopearl. The isolated lectin agglutinated all types of erythrocytes equally. Molecular masses estimated by gel filtration under various buffers and matrices varied from 30 to 52 kDa. On the other hand, SDS-PAGE in the presence or absence of 2-mercaptoethanol showed three major bands of 33, 66 and 100 kDa and a faint band of 65 kDa. This lectin exhibited GalNAc-specificity. The protein was a glycoprotein containing 3.3% total sugar, and the amino acid analysis revealed a high content of acidic and hydroxy amino acids and a low content of methionine and histidine. GFL was cytotoxic against HeLa cells. The toxicity did not appear after preincubating the lectin with the haptenic sugar N-acetylgalactosamine.  相似文献   

8.
The hamster gene for uridine diphosphate N-acetyl-D-glucosamine:dolichyl phosphate N-acetylglucosaminyl phosphoryl transferase(L-G1PT) was found to extend over 6.5 kb and to contain nineexons. The exons ranged in size from 63 to 214 bp, encodingthe 408 amino acid protein. The introns ranged from 85 bp to1.4 kb. Upstream 5' sequences included two possible TATA boxes,one possible CCAAT box and at least two potential GC boxes.Heterologous expression was successful in Schizosaccharomycespombe, and resulted in cells that were tunicamycin resistantand had 12-fold more L-G1PT activity than wild-type cells. Antiserumprepared to a hydrophilic peptide (residues 300–320) ofthe L-G1PT protein reacted with a 35–36 kDa protein inmembrane samples from Chinese hamster ovary (CHO) cells andS.pombe cells that had increased levels of L-G1PT activity.In both cases, antigenic peptide competed with the 35–36kDa protein detected by the antiserum. N-acetylglucosarmine 1-phosphate transferase dolichol glycosylation  相似文献   

9.
A divalent cation-independent 16 kDa d-galactose binding lectin (AKL-2) was isolated from eggs of sea hare, Aplysia kurodai. The lectin recognized d-galactose and d-galacturonic acid and had a 32 kDa dimer consisting of two disulfide-bonded 16 kDa subunits. Eighteen N-terminus amino acids were identified by Edman degradation, having unique primary structure. Lectin blotting analysis with horseradish peroxidase-conjugated lectins has shown that AKL-2 was a glycoprotein with complex type oligosaccharides with N-acetyl d-glucosamine and mannose at non-reducing terminal. Two protein bands with 38 and 36 kDa in the crude extract of sea hare eggs after purification of the lectin was isolated by AKL-2-conjugated Sepharose column and elution with 0.1 M lactose containing buffer. It suggested that the lectin binds with an endogenous ligand in the eggs. AKL-2 kept extreme stability on haemagglutination activity if it was treated at pH 3 and 70 °C for 1 h. Glycan binding profile of AKL-2 by frontal affinity chromatography technology using 15 pyridylamine labeled oligosaccharides has been appeared that the lectin uniquely recognized globotriose (Galα1-4Galβ1-4Glc; Gb3) in addition to bi-antennary complex type N-linked oligosaccharides with N-acetyllactosamine. Surface plasmon resonance analysis of AKL-2 against a neo-glycoprotein, Gb3-human serum albumin showed the k ass and k diss values are 2.4 × 103 M?1 s?1 and 3.8 × 10?3 s?1, respectively. AKL-2 appeared cytotoxicity against both Burkitt’s lymphoma Raji cell and erythroleukemia K562. The activity to Raji by the lectin was preferably cancelled by the co-presence of melibiose mimicing Gb3. On the other hand, K562 was cancelled effectively by lactose than melibiose. It elucidated that AKL-2 had cytotoxic ability mediated glycans structure to cultured cells.  相似文献   

10.
The Saccharomyces cerevisiae mnn10 mutant is defective in thesynthesis of N-linked oligosaccharides (Ballou et al., 1989).This mutation has no effect on O-linked sugars, but resultsin the accumulation of glycoproteins that contain severely truncatedN-linked outer-chain oligosaccharides. We have cloned the MNN10gene by complementation of the hygromycin B sensitivity conferredby the mutant phenotype. Sequence analysis predicts that Mnn10pis a 46.7 kDa type II membrane protein with structural featurescharacteristic of a glycosyltransferase. Subcellular fractionationdata indicate that most of the Mnn10 protein cofractionateswith Golgi markers and away from markers for the endoplasmicreticulum (ER), suggesting Mnn10p is localized to the Golgicomplex. A comparison of the Mnn10 protein sequence to proteinsin the two different databases identified five proteins thatare homologous to Mnn10p, including a well characterized Schizosaccharomycespombe  相似文献   

11.
Chigira Y  Oka T  Okajima T  Jigami Y 《Glycobiology》2008,18(4):303-314
Development of a heterologous system for the production of homogeneoussugar structures has the potential to elucidate structure–functionrelationships of glycoproteins. In the current study, we usedan artificial O-glycosylation pathway to produce an O-fucosylatedepidermal growth factor (EGF) domain in Saccharomyces cerevisiae.The in vivo O-fucosylation system was constructed via expressionof genes that encode protein O-fucosyltransferase 1 and theEGF domain, along with genes whose protein products convertcytoplasmic GDP-mannose to GDP-fucose. This system allowed identificationof an endogenous ability of S. cerevisiae to transport GDP-fucose.Moreover, expression of EGF domain mutants in this system revealedthe different contribution of three disulfide bonds to in vivoO-fucosylation. In addition, lectin blotting revealed differencesin the ability of fucose-specific lectin to bind the O-fucosylatedstructure of EGF domains from human factors VII and IX. Furtherintroduction of the human fringe gene into yeast equipped withthe in vivo O-fucosylation system facilitated the addition ofN-acetylglucosamine to the EGF domain from factor IX but notfrom factor VII. The results suggest that engineering of anO-fucosylation system in yeast provides a powerful tool forproducing proteins with homogenous carbohydrate chains. Suchproteins can be used for the analysis of substrate specificityand the production of antibodies that recognize O-glycosylatedEGF domains.  相似文献   

12.
A lectin was isolated from hemolymph of the Southeast Asian horseshoe crab Tachypleus gigas by using glycophorin HA affinity chromatography and Sephacryl S-300 gel filtration. The purified lectin had a molecular mass of approximately 396 kDa and was composed of 13 identical subunits with molecular masses of 31 kDa. The serological specificity of the purified lectin was specifically inhibited by sialic acids sialoglycoproteins, but not by neutral sugars, hexosamines, N-acetylhexosamines, or asialoglycoproteins. Although the N-terminal amino acid sequence of the lectin from T. gigas was identical to that from American horseshoe crab (liphemin) by the same purification method and cross reacted with the anti-liphemin serum, the calcium concentration of hemagglutinating activity of the purified lectin showed a smaller optimal concentration than that of liphemin.  相似文献   

13.
Bryohealin is a lectin involved in the wound-healing process of the marine green alga Bryopsis plumosa. In the previous purification study, it has been shown that lectin was composed of two identical subunits of 27 kDa, cross-linked by disulfide bond, and showed binding specificity to N-acetyl-d-glucosamine and N-acetyl-d-galactosamine (GlcNAc and GalNAc, respectively). To determine if the lectin recognize the two different sugars at the same binding domain, the carbohydrate binding sites of Bryohealin was analyzed using chromatography and chemical modification methods. Results showed that the same binding site of the lectin was responsible for the recognition of two sugars, GalNAc as well as GlcNAc. Chemical modification studies showed that hemagglutinating activities of Bryohealin were not affected by modification of histidine, tryptophan, aspartic acid, and glutamic acid. When arginine residues were modified with 1,2-cyclohexanedione, the activity of Bryohealin rapidly decreased. The sugar binding sites remained intact when the lectin was treated with inhibitory sugars (0.2 M GalNAc and/or GlcNAc) prior to 1,2-cyclohexanedione treatment. The sugar binding domain of Bryohealin was predicted from the MALDI-TOF analysis and the full cDNA sequence of the lectin gene.  相似文献   

14.
A lectin, with a specificity for N-acetylgalactosamine and N-acetylglucosamine and a molecular weight of 43 kDa, was isolated from fruiting bodies of the edible shiitake mushroom Lentinus edodes. The purification procedure entailed extraction with aqueous buffer, ammonium sulfate precipitation, gel filtration on Sephadex G-100 and affinity chromatography on N-acetylgalactosamine-agarose. The lectin was unique in that it was tenaciously bound on anion exchangers including DEAE-cellulose, DEAE-Sepharose, DEAE-Sephadex, Q-Sepharose, Dowex and PEI-cellulose and also on hydroxyapatite and phenyl Sepharose. It was largely unadsorbed on cation exchangers including CM-cellulose, CM-Sepharose, SP-Sepharose and Amberlite, and also on protein G-Sepharose, Red Sepharose and Affi-gel Blue gel, wheat germ lectin-Sepharose and p-aminophenyl-d-glucopyranoside agarose.  相似文献   

15.
Phytohaemagglutinin (PHA) is the major lectin present in theseeds of the common bean, Phaseolus vulgaris, and PHA-L is theleucocyte-agglutinating form of this lectin. This tetramericglycoprotein accumulates in the vacuoles of storage parenchymacells. Based on amino acid sequence comparisons of legume lectinsand the three-dimensional structure of lectin-carbohydrate complexes,Asn128 can be identified as a likely candidate for site-directedmutagenesis to create a mutant PHA-L that does not bind carbohydrate.PHA-L N128  相似文献   

16.
17.
The galectin family of β-galactoside binding lectins isinvolved in normal and pathological processes. Altered expressionof galectin-3 has been described in many cancers, and studiesof cancer cell lines have implicated this lectin in variousaspects of the tumorigenic cascade. The goal of this reportwas to directly assess the importance of galectin-3 in tumorbiology by introducing the galectin-3 null mutation (galectin-3–/–)into mouse lines genetically programmed to develop cancers.We used two mouse models of human intestinal cancer, the ApcMinand Apc1638N lines, to study tumor initiation and tumor progression.We also crossed the galectin-3–/– mice with PyMTtransgenic animals, a model in which primary mammary gland tumorsgive rise to lung metastases at high frequency. Unexpectedly,we show that the absence of galectin-3 does not affect the evolutionof the disease in any of these three situations.  相似文献   

18.
Cytolytic activity against invading microorganisms is one of the innate forms of immunity in invertebrates. A serine protease-associated sialic acid-specific cytolytic lectin was purified using glutaraldehyde-fixed ox erythrocytes from the larval extract of blowfly (Chrysomya megacephala). The purified lectin lysed vertebrate erythrocytes with effective haemolysis of ox red blood cells (RBCs) in an isotonic medium. The degree of haemolytic (HL) activity of the purified cytolytic lectin depended on its concentration, pH, temperature, and calcium ions. It was sensitive to ethylenediaminetetraacetic acid. The native molecular mass of the C-type lectin was 260 ± 26 kDa, comprising four different polypeptide subunits of 75 kDa (pI ~8), 69 kDa (pI ~7.0), 61 kDa (pI ~5.3), and 55 kDa (pI ~4.6). The association between the C-type lectin and serine protease was confirmed by MALDI-TOF-MS analysis that revealed its homology in the same spectral peak as well as the proteases and phenylmethylsulphonyl fluoride inhibition of HL activity. Haemolysis inhibition by N-acetylneuraminic acid and other sugars revealed the properties of the lectin. The purified lectin distorted the integrity of ox RBCs and Paenalcaligenes hermetiae. This in vitro study documents the presence of a cytolytic system in blowfly (C. megacephala) larvae for the clearance of invading microbial pathogens in their feeding niche.  相似文献   

19.
A novel lectin was isolated and characterized from Bryopsis plumosa (Hudson) Agardh and named BPL-3. This lectin showed specificity to N-acetyl-d-galactosamine as well as N-acetyl-d-glucosamine and agglutinated human erythrocytes of all blood types, showing slight preference to the type A. SDS-PAGE and MALDI-TOF MS data showed that BPL-3 was a monomeric protein with molecular weight of 11.5 kDa. BPL-3 was a non-glycoprotein with pI value of ∼7.0. It was stable in high temperatures up to 70°C and exhibited optimum activity in pH 5.5–10. The N-terminal and internal amino acid sequences of the lectin were determined by Edman degradation and enzymatic digestion, which showed no sequence homology to any other reported proteins. The full sequence of the cDNA encoding this lectin was obtained from PCR using cDNA library, and the degenerate primers were designed from the N-terminal amino acid sequence. The size of the cDNA was 622 bp containing single ORF encoding the lectin precursor. This lectin showed the same sugar specificity to previously reported lectin, Bryohealin, involved in protoplast regeneration of B. plumosa. However, the amino acid sequences of the two lectins were completely different. The homology analysis of the full cDNA sequence of BPL-3 showed that it might belong to H lectin group, which was originally isolated from Roman snails.  相似文献   

20.
We purified and characterized a lectin from the corn coleoptyle (Zea mays). The lectin (CCL) was purified by affinity chromatography on a Lactosyl–Sepharose 4B column. It is a glycoprotein of 88.7 kDa, composed mainly by glutamic, aspartic, glycine, and Ser residues; in a minor proportion, it contained methionine and cysteine residues. Carbohydrates that constituted 12% of the total weight comprised galactose, mannose, and N-acetyl-D-glucosamine. The lectin contained the blocked amino-terminus. Analysis of the lectin, determined from peptides obtained after trypsin digestion by MALDI-TOF (matrix-assisted laser desorption ionization-time of flight), indicated that CCL has 18% homology with a putative calcium-dependent Ser/Thr protein kinase, from Arabidopsis thaliana, and 39% homology with a NADPH-dependent reductase from Z. mays. The lectin showed hemagglutinating activity toward several erythrocytes, including human A, B, and O. Hapten inhibition assays indicated that the lectin interacts specifically with the OH on C4 from galactose residues. OH- on C1 plays a relevant role in the interaction with CCL, since β-galactose residues are better recognized than those from the anomeric α-galactose. Lack of lectin activity was observed in corn extracts; the highest specific activity was obtained from coleoptyle obtained at the 7th day after seeding.  相似文献   

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