Thermostable mannose-binding lectin from Dendrobium findleyanum with activities dependent on sulfhydryl content

A mannose-binding lectin was purified from Dendrobium (D.) findleyanum pseudobulb using mannan-agarose column chromatography. After heating in the presence of SDS with or without 2-mercaptoethanol on SDS-PAGE with a continuous gradient of 8%-20% acrylamide, the purified lectin showed only one protein band with a molecular mass of 14.5 kDa. Without heating, two bands were seen on the gel at the positions of 14.5 kDa and 53.7 kDa, but a higher amount of the 53.7 kDa protein was observed in the presence of 2-mercaptoethanol. Protein identification of both protein bands by liquid chromatography-tandem mass spectrometry showed three peptide fragments identical to parts of a lectin precursor from D. officinale; the lectin was named D. findleyanum agglutinin (DFA). Using various concentrations of native-PAGE and Ferguson plot, only one protein band revealed a molecular mass of 56.2 kDa, indicating four 14.5 kDa polypeptide subunits in the DFA. Isoelectric focusing revealed that the DFA had three conformational forms with an isoelectric point of 5.18, 4.87 and 4.72, whereas 2-mercaptoethanol-treated DFA showed only one band with an isoelectric point of 5.18. DFA exhibited specificity towards mannose using the solid-phase method. The binding activity, anti-fungal activity and hemagglutination activity of DFA were not affected by heat, but were increased by free sulfhydryl groups.     

Isolation and characterization of a lectin from Grifola frondosa fruiting bodies

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.     

Isolation and partial characterization of a novel lectin from Talisia esculenta seeds that interferes with fungal growth

A novel plant lectin has been isolated from the seeds of Talisia esculenta and partially characterized. The purified lectin showed two protein bands in SDS-PAGE (20,000 and 40,000 kDa) and agglutinated human and animal erythrocytes. Of the various sugars tested, the lectin was best inhibited by mannose. A search of sequence databases showed that the N-terminal sequence had no homology to any known protein. The lectin inhibited the growth of the fungi Fusarium oxysporum, Colletotrichum lindemuthianum and Saccharomyces cerevisiae.     

Isolation and Characterization of a Novel Chitosan-Binding Protein from Non-Heading Chinese Cabbage Leaves

To know the mechanism of ammonia assimilation in non-heading Chinese cabbage (Brassica campestris L. ssp. chinensis (L.) Makino) leaves regulated by chitosan (CTS), a CTS-binding protein was isolated from non-heading Chinese cabbage leaves using the chitosan affinity chromatography approach and this CTS-binding protein was partially characterized. The profile of the 53.1 kDa purified protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was compared with the native molecular weight of 106.5 kDa, which indicated that the purified protein was a dimer with identical subunits. After isoelectric focusing, a band was obtained at pH 8.25. The agglutination test and periodic acid-Schiff staining further revealed that the protein was a glycoprotein with lectin activity. Moreover, the purified protein contained 17.4% (w/w) neutral carbohydrate and 82.56% (w/w) protein. The comparison of this protein and the 67 kDa CTS-binding protein isolated previously from Rubus culture tissue exhibited some differences in characterization. According to results of peptide mass fingerprinting analysis, the protein purified in the present study does not show any similarity with any protein in the protein data bank. Thus, it was deduced that the protein purified in the present study is a novel CTS-binding protein.     

Isolation and Characterization of a Novel Chitosan-Binding Protein from Non-Heading Chinese Cabbage Leaves

To know the mechanism of ammonia assimilation in non-heading Chinese cabbage (Brassica campestris L. ssp. chinensis (L.) Makino) leaves regulated by chitosan (CTS), a CTS-binding protein was isolated from non-heading Chinese cabbage leaves using the chitosan affinity chromatography approach and this CTS-binding protein was partially characterized. The profile of the 53.1 kDa purified protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was compared with the native molecular weight of 106.5 kDa, which indicated that the purified protein was a dimer with identical subunits. After isoelectric focusing, a band was obtained at pH 8.25. The agglutination test and periodic acid-Schiff staining further revealed that the protein was a glycoprotein with lectin activity. Moreover, the purified protein contained 17.4 % (w/w) neutral carbohydrate and 82.56% (w/w) protein. The comparison of this protein and the 67 kDa CTS-binding protein isolated previously from Rubus culture tissue exhibited some differences in characterization. According to results of peptide mass fingerprinting analysis, the protein purified in the present study does not show any similarity with any protein in the protein data bank. Thus, it was deduced that the protein purified in the present study is a novel CTS-binding protein.     

Mitochondrial phosphate transport. Large scale isolation and characterization of the phosphate transport protein from beef heart mitochondria

The phosphate transport protein from beef heart mitochondria has been purified on a large scale by hydroxylapatite chromatography in the presence of sodium dodecyl sulfate and urea. As shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (silver stain), the pure phosphate transport protein preparation consists of two protein bands (alpha and beta, ratio 1:1) with similar mobilities (34 kDa) which display identical peptide maps if fragmented with either CNBr or HCl/dimethyl sulfoxide/HBr. The complete amino acid composition of phosphate transport protein is presented. Quantitative determination of N-terminal amino acids underlines the purity of the preparation and shows for alpha and beta the identical amino-terminals H2N-Ala-Val-Glu-Glu-Glx-Tyr-. Qualitative digestion shows that carboxypeptidase A is able to release at least three amino acids from the C termini of the alpha as well as the beta band of phosphate transport protein. The nature of these two protein bands is discussed. The sum of phosphate transport protein (alpha + beta) per total mitochondrial protein amounts to 2.3% or 1.4 nmol of phosphate transport protein (34 kDa) per nmol of cytochrome b.     

Mannan-Binding Lectin of the Sea Urchin Strongylocentrotus nudus

A novel lectin specific to low-branched mannans (MBL-SN) was isolated from coelomic plasma of the sea urchin Strongylocentrotus nudus by combining anion-exchange liquid chromatography on DEAE Toyopearl 650 M, affinity chromatography on mannan-Sepharose and gel filtration on the Sephacryl S-200. The molecular mass of MBL-SN was estimated by sodium dodecyl sulphate polyacrylamide gel electrophoresis under non-reducing conditions to be about 34 kDa. MBL-SN was shown to be a dimer with two identical subunits of about 17 kDa. The native MBL-SN exists as a tetramer. The physico-chemical properties of MBL-SN indicate that it belongs to C-type mannan-binding lectins. The cDNA encoding MBL-SN was cloned from the total cDNA of S. nudus coelomocytes and encodes a 17-kDa protein of 144 amino acid residues that contains a single carbohydrate-recognition domain of C-type lectins. Prediction of the MBL-SN tertiary structure using comparative modelling revealed that MBL-SN is an α/β-protein with eight β-strands and two α-helices. Comparison of the MBL-SN model with available three-dimensional structures of C-type lectins revealed that they share a common fold pattern.     

Purification and Properties of a Lectin from the Fruitbodies of Flammulina velutipes

A lectin was purified to homogeneity from the fruitbodies of Flammulina velutipes by conventional purification procedures. The purified lectin was demonstrated to be a dimeric protein consisting of two identical subunits with an apparent molecular mass of 11 kDa. The lectin was an acidic protein with a pI value of 5.4, and devoid of cysteine, methionine, and histidine as amino acid constituents. Its hemagglutinating activity was totally unaffected by mono- and oligosaccharides and glycosides, but inhibited by some desialylated glycoproteins. Immunological assays revealed that no protein cross-reacting with rabbit anti-i⁷. velutipes lectin antibody was apparently present in vegetatively growing mycelia but was distributed throughout the fruitbody at different concentrations.     

Isolation and characterization of a thermolabile beta-2 macroglycoprotein ('thermolabile substance' or 'Hakata antigen') detected by precipitating (auto) antibody in sera of patients with systemic lupus erythematosus.

A novel thermolabile beta-2 macroglycoprotein ('thermolabile substance' (TLS) or 'Hakata antigen' (HA], which was detected by the precipitating (auto) antibodies of patients with systemic lupus erythematosus, was isolated and characterized. The purification procedure entailed the following steps: isoelectric precipitation in the range between pH 5.2-6.1, hydroxyapatite absorption chromatography, 35% saturated ammonium sulfate precipitation, Sephadex G-200 gel filtration, Pevikon block electrophoresis, lentil lectin affinity chromatography and immobilized rabbit anti-human whole serum IgG column chromatography. Utilizing these procedures, 0.1 mg of HA was purified from 3 1 of pooled human serum. The molecular mass of HA was determined as 650 kDa by Sepharose 4B gel filtration. On SDS-PAGE analysis, HA showed a single band at 35 kDa under reduced conditions and numerous ladder bands between 35 kDa to more than 300 kDa under nonreduced conditions. On analytical ultracentrifugation, HA gave a molecular mass of 520 kDa with a single meniscus and a sedimentation constant of 12.0. The amino acid and carbohydrate analysis of reduced and S-pyridylethylated HA revealed that it contained five residues of hydroxyproline and an N-linked type sugar chain.     

Purification and partial characterization of thiol protease inhibitors from embryos of the brine shrimp Artemia.

Thiol protease inhibitor (TPI) proteins in embryos of the brine shrimp Artemia were purified to apparent homogeneity and several of their properties were studied. Four protein fractions containing thiol protease inhibitor activity were obtained by high performance liquid chromatography of Artemia embryo proteins on a C-18 reverse-phase column and these were designated as TPI-1a, -1b, -2, and -3. Acrylamide gel electrophoresis showed that TPI-1a and TPI-1b each consisted of two bands of 11.8 and 13.6 kilodaltons (kDa), while TPI-2 and TPI-3 consisted of only one band of 12.5 kDa. Isoelectric focusing experiments demonstrated that TPI-3 contained one band at pH 5.3, while both TPI-1b and TPI-2 yielded bands at pH 5.2 and 5.3. TPI-1a did not yield any major bands. Amino acid composition analyses of the Artemia TPI proteins showed them to be remarkably similar to one another. All were rich in valine and aspartic and glutamic acids, and devoid of cysteine. Partial trypsin digestion of TPI-1b, TPI-2, and TPI-3 yielded several peptides with identical mobilities on a reverse-phase column and several other peptides with different mobilities, suggesting that the multiple forms of Artemia TPIs may have originated from the same parental protein. N-terminal amino acid sequence analyses of TPI-3 suggest that Artemia TPI proteins are members of the type I cystatin family of protease inhibitors.     

Production and purification of a recombinant human 14 kDa β-galactoside-binding lectin

The cDNA for a 14 kDa human β-galactoside-binding lectin was inserted into a plasmid carrying a taq promoter, and the lectin protein was expressed in E. coli cells. The recombinant lectin was extracted from the cells and purified to apparent homogeneity by a single-step chromatography on an asialofetuin-agarose column. Subunit molecular mass (14 kDa), hemagglutinating activity and antigenicity were indistinguishable from those of the human placental lectin. Though the N-terminal of the placental lectin is blocked with an acetyl group, the recombinant lectin was found to have a free amino group. However, the N-terminal amino acid sequences were identical. The recombinant lectin was considered to have the same three-dimensional structure as the placental lectin.     

A Novel Core Fucose-specific Lectin from the Mushroom Pholiota squarrosa

Fucα1–6 oligosaccharide has a variety of biological functions and serves as a biomarker for hepatocellular carcinoma because of the elevated presence of fucosylated α-fetoprotein (AFP) in this type of cancer. In this study we purified a novel Fucα1–6-specific lectin from the mushroom Pholiota squarrosa by ion-exchange chromatography and affinity chromatography on thyroglobulin-agarose. The purified lectin was designated as PhoSL (P. squarrosa lectin). SDS-PAGE, MALDI-TOF mass spectrometry, and N-terminal amino acid sequencing indicate that PhoSL has a molecular mass of 4.5 kDa and consists of 40 amino acids (NH₂-APVPVTKLVCDGDTYKCTAYLDFGDGRWVAQWDTNVFHTG-OH). Isoelectric focusing of the lectin showed bands near pI 4.0. The lectin activity was stable between pH 2.0 and 11.0 and at temperatures ranging from 0 to 100 °C for incubation times of 30 min. When PhoSL was investigated with frontal affinity chromatography using 132 pyridylaminated oligosaccharides, it was found that the lectin binds only to core α1–6-fucosylated N-glycans and not to other types of fucosylated oligosaccharides, such as α1–2-, α1–3-, and α1–4-fucosylated glycans. Furthermore, PhoSL bound to α1–6-fucosylated AFP but not to non-fucosylated AFP. In addition, PhoSL was able to demonstrate the differential expression of α1–6 fucosylation between primary and metastatic colon cancer tissues. Thus, PhoSL will be a promising tool for analyzing the biological functions of α1–6 fucosylation and evaluating Fucα1–6 oligosaccharides as cancer biomarkers.     

Characterization and cDNA cloning of monomeric lectins that correspond to the B-Chain of a type 2 ribosome-inactivating protein from the bark of Japanese elderberry (Sambucus sieboldiana)

Two monomeric lectins, SSA-b-3 and SSA-b-4, were purified from the bark tissue of Japanese elderberry, Sambucus sieboldiana. SDS-PAGE of the purified lectins showed the presence of single bands of 35 and 33 kDa for SSA-b-3 and SSA-b-4, respectively, irrespective of the presence of reducing agent. MS analysis as well as gel filtration of these lectins indicated that they exist mostly as monomeric lectins. Analysis of the N-terminal amino acid sequences of SSA-b-3 and SSA-b-4 yielded an identical sequence, indicating their close structural relationship. Four cDNA clones with extensive homology were obtained from the bark cDNA library and indicated to encode SSA-b-3 or SSA-b-4 from the comparison with the N-terminal sequences of these lectins. These clones were classified into two groups, three for SSA-b-3 and one for SSA-b-4, based on the predicted isoelectric points. The amino acid sequences of the encoded polypeptides were almost identical with the B-chain of a type 2 ribosome-inactivating protein from the same bark tissue, sieboldin-b, except for the absence of a small peptide containing a cystein residue, which is critical for the heteromeric dimerization with an A-subunit. Carbohydrate binding specificity and biological activity of these lectins are also reported.     

Comparison of sucrase-free isomaltase with sucrase-isomaltase purified from the house musk shrew Suncus murinus

We purified sucrase-isomaltase and sucrase-free isomaltase from a normal and a sucrase-deficient line, respectively, of the house musk shrew Suncus murinus and examined the effects of mutation on enzyme structure and activities. Recent cDNA cloning studies have predicted that sucrase-free mutant isomaltase lacks the C-terminal 69 amino acids of normal isomaltase, as well as the entire sucrase. On SDS-polyacrylamide gel electrophoresis purified sucrase-free isomaltase gave a single protein band of 103 kDa, while sucrase-isomaltase gave two major protein bands of 106 and 115 kDa. The 115, but not 106, kDa band was quite similar to the 103 kDa band on Western blotting with Aleuria aurantia lectin and antibody against shrew sucrase-isomaltase, suggesting that the 115 and 103 kDa bands are due to normal and mutant isomaltases, respectively, in accordance with the above prediction. Purified isomaltase and sucrase-isomaltase were similar in Km and Vmax (based on isomaltase mass) values for isomaltose hydrolysis and in inhibition of isomaltase activity by antibody against rabbit sucrase-isomaltase, suggesting that the enzymatic properties of isomaltase are mostly unaffected by mutation.     

Molecular cloning of the lectin and a lectin-related protein from common Solomon's seal (Polygonatum multiflorum)

The most prominent protein ofPolygonatum multiflorum (common Solomon's seal) rhizomes has been identified as a mannose-binding lectin. Analysis of the purified lectin demonstrated that it is a tetramer of four identical subunits of 14 kDa. Molecular cloning further revealed that the lectin from this typical Liliaceae species belongs to the superfamily of monocot mannose-binding proteins. Screening of cDNA libraries constructed with RNA isolated from buds, leaves and flowers ofP. multiflorum also yielded cDNA clones encoding a protein, which contains two tandemly arranged domains with an obvious sequence homology to the mannose-binding lectins. Molecular modelling of thePolygonatum lectin and lectin-related protein indicated that the three-dimensional structure of both proteins strongly resembles that of the snowdrop lectin. In addition, this approach suggested that the presumed carbohydrate-binding sites of the lectin can accommodate a mannose residue whereas most of the carbohydratebinding sites of the lectin-related protein cannot.Abbreviations GNA Galanthus nivalis agglutinin - HCA hydrophobic cluster analysis - LECPMA cDNA clone encoding PMA - PM30 30 kDa protein isolated fromPolygonatum multiforum - PMA Polygonatum multiflorum agglutinin - PMLRP Polygonatum multiflorum lectin-related protein     

Vitellogenin-derived yolk proteins in a hybrid sturgeon, bester (Huso huso x Acipencer ruthenus): identification, characterization and course of proteolysis during embryogenesis

Vitellogenin (Vg) and its corresponding yolk protein (YP) products, YP1, YP2 and YP3, were isolated from serum of estrogen-treated hybrid sturgeon (bester; Huso huso X Acipencer ruthenus) and eggs from untreated fish, respectively. Vitellogenin had an apparent molecular mass of 580 kDa and appeared as two major bands corresponding to 180 kDa and 120 kDa after SDS-PAGE. Apparent molecular weights of YP1, YP2 and YP3 were 370 kDa, 88 kDa and 19 kDa, respectively. After SDS-PAGE, YP1 appeared as a main band of 110 kDa, while YP2 was resolved as a single band of 94 kDa and 29 kDa band under non-reducing and reducing conditions, respectively. Yolk protein 3 appeared as a diffuse band corresponding to 16 kDa and two faint bands below 14.4 kDa after SDS-PAGE. However, the 16 kDa band alone was observed after dephosphorylation with alkaline phosphatase. The course of cleavage of yolk proteins in bester embryos and alevins was observed by SDS-PAGE and Western blotting from fertilization onward. After hatching, the main 110 kDa band of YP1 was degraded into smaller peptides during development, while YP2 hardly showed any such structural changes. The amino acid compositions of purified yolk proteins indicated that YP1, YP2 and YP3 were bester lipovitellin, beta-component, and phosvitin, respectively.     

Affinity purification, physicochemical and immunological characterization of a galactose-specific lectin from the seeds of Dolichos lablab (Indian lablab beans)

A galactose-specific lectin has earlier been isolated from the seeds of Dolichos lablab in our laboratory by conventional protein purification methods. We now established conditions to bind the lectin on Sepharose-galactose gel in the presence of 1.5 M ammonium sulfate in Tris-buffered saline, pH 7.4. It can be specifically eluted with 0.3 M galactose. The purified lectin is a glycoprotein, binds to Con A, agglutinates erythrocytes, and has an apparent native molecular weight of 120 +/- 5 kDa. In SDS-PAGE under reducing conditions, it dissociates into two subunits of molecular mass (Mr) 31 and 29 kDa. Among a number of sugars tested for inhibitory activity of the lectin, galactose was found to be a potent inhibitor. Rabbit polyclonal antibody to the purified lectin specifically reacted with the lectin subunits in Western blot analysis and additionally, an antibody raised to the isolated 31 kDa subunit show reactivity with both the subunits. Amino terminal sequences of both the subunits are identical. The purified lectin is stable up to 40 degrees C with a pH optimum of 7.4. The lectin has a high content of acidic amino acids and lacks sulfur-containing amino acids. Chemical modification of the lectin with group-specific reagents indicates the possible role of histidine, lysine, and tyrosine residues in lectin activity.     

A Rainbow Trout Lectin with Multimeric Structure

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.”     

Establishment of a heterologous system for the expression of Canavalia brasiliensis lectin: a model for the study of protein splicing

During its biosynthesis in developing Canavalia brasiliensis seeds, the lectin ConBr undergoes a form of protein splicing in which the order of the N- and C-domains of the protein is reversed. To investigate whether these events can occur in other eukaryotic organisms, an expression system based on Pichia pastoris cells was established. A DNA fragment encoding prepro-ConBr was cloned into the vector pPICZB, and the recombinant plasmid was transformed in P. pastoris strain GS115. Ten clones were screened for effective recombinant protein production. Based on Western blot analysis of the two clones with the highest level of protein expression: 1) diffuse high-molecular mass immunoreactive bands were produced as early as 24 h after induction; 2) a single-, high-molecular mass protein was secreted into the medium, and 3) a significant fraction of the recombinant polypeptides that cross-reacted with anti-ConBr antibodies comprised a band of approximately 34.5 kDa. Diffuse protein bands with high molecular masses are attributed to hyperglycosylation at the single potential N-glycosylation site located in the linker peptide of prepro-ConBr. In contrast, native ConBr is made up of three polypeptides, the intact alpha chain (aa 1-237) and the fragments beta (aa 1-118) and gamma (aa 119-237), which have apparent molecular masses of 30, 16 and 12 kDa, respectively. Apparently, the yeast P. pastoris is not able to carry out all the complex post-translational proteolytic processing necessary for the biosynthesis of ConBr.     

Purification and characterization of a T-antigen specific lectin from the coelomic fluid of a marine invertebrate, sea cucumber (Holothuria scabra)

A novel lectin was purified from the coelomic fluid of the sea cucumber Holothuria scabra (HSL), subjected to bacterial challenge. HSL is a monomeric glycoprotein of molecular mass 182 kDa. The lectin is highly thermostable as it retains full activity for 1 h at 80 degrees C. Further, the hemagglutination activity of HSL is unaffected by pH in the range 2-11. Unlike other lectins purified from marine invertebrates, the hemagglutination activity of HSL does not require any divalent metal ions. The affinity profile of HSL was studied by a combination of hemagglutination inhibition and fluorescence spectroscopy. HSL binds to desialylated glycoproteins, MealphaGal, T-antigen and T (alpha-ser)-antigen with a distinction between beta1-4 and beta1-3 linkages. Mealpha-T-antigen was a potent ligand having highest affinity (Ka 8.32 x 10(7)M(-1)). Monosaccharide binding is enthalphically driven while disaccharide binding involves both entropic and enthalpic contributions.