Purification, characterization, and cDNA cloning of alpha-N-acetylgalactosamine-specific lectin from starfish, Asterina pectinifera

We report here the purification, characterization, and cDNA cloning of a novel N-acetylgalactosamine-specific lectin from starfish, Asterina pectinifera. The purified lectin showed 19-kDa, 41-kDa, and 60-kDa protein bands on SDS-PAGE, possibly corresponding to a monomer, homodimer, and homotrimer. Interestingly, on 4-20% native PAGE the lectin showed at least nine protein bands, among which oligomers containing six to nine subunits had potent hemagglutination activity for sheep erythrocytes. The hemagglutination activity of the lectin was specifically inhibited by N-acetylgalactosamine, Tn antigen, and blood group A trisaccharide, but not by N-acetylglucosamine, galactose, galactosamine, or blood group B trisaccharide. The specificity of the lectin was further examined using various glycosphingolipids and biotin-labeled lectin. The lectin was found to bind to Gb5Cer, but not Gb4Cer, Gb3Cer, GM1a, GM2, or asialo-GM2, indicating that the lectin specifically binds to the terminal alpha-GalNAc at the nonreducing end. The hemagglutination activity of the lectin was completely abolished by chelation with EDTA or EGTA and completely restored by the addition of CaCl(2). cDNA cloning of the lectin showed that the protein is composed of 168 amino acids, including a signal sequence of 18 residues, and possesses the typical C-type lectin motif. These findings indicate that the protein is a C-type lectin. The recombinant lectin, produced in a soluble form by Escherichia coli, showed binding activity for asialomucin in the presence of Ca(2+) but no hemagglutination.     

Purification and characterization of a D‐mannose specific lectin from the green marine alga,Bryopsis plumosa

A d ‐mannose specific lectin was purified from the green marine alga, Bryopsis plumosa (Huds.) Ag. The lectin agglutinated horse and sheep erythrocytes. Matrix assisted laser desorption/ionization time of flight mass spectrometry, size exclusion chromatography, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and two dimensional gel electrophoresis (2DE) results showed that the lectin was a monomer with molecular weight of 17 kDa and pI 7.3. The agglutinating activity was inhibited by d ‐mannose (1 mM), α‐methyl‐D‐mannose (4 mM) and l ‐fucose (8 mM). d ‐glucose (125 mM) showed weak inhibition. The lectin did not need divalent cations for agglutinating activity. N‐terminal amino acid sequence of the lectin was analyzed. As the lectin was novel, we named it BPL‐2 (Bryopsis plumosa lectin 2). Full cDNA sequence of BPL‐2 was obtained using cDNA library. It was comprised of 624 bp of open reading frame and 167 bp/57 bp of 3′/5′ untranslated regions as well as N‐terminal signal peptide. No antimicrobial activity of BPL‐2 was observed in four bacteria strains tested.     

Purification, characterization, cDNA cloning and expression of a novel ketoreductase from Zygosaccharomyces rouxii.

A novel ketoreductase isolated from Zygosaccharomyces rouxii catalyzes the asymmetric reduction of selected ketone substrates of commercial importance. The 37.8-kDa ketoreductase was purified more than 300-fold to > 95% homogeneity from whole cells with a 30% activity yield. The ketoreductase functions as a monomer with an apparent Km for 3,4-methylenedioxyphenyl acetone of 2.9 mM and a Km for NADPH of 23.5 microM. The enzyme is able to effectively reduce alpha-ketolactones, alpha-ketolactams, and diketones. Inhibition is observed in the presence of diethyl pyrocarbonate, suggesting that a histidine is crucial for catalysis. The 1.0-kb ketoreductase gene was cloned and sequenced from a Z. rouxii cDNA library using a degenerate primer to the N-terminal sequence of the purified protein. Furthermore, it was expressed in both Escherichia coli and Pichia pastoris and shown to be active. Substrate specificity, lack of a catalytic metal, and extent of protein sequence identity to known reductases suggests that the enzyme falls into the carbonyl reductase enzyme class.     

A fibrinolytic enzyme from a marine green alga, Codium latum

A fibrinolytic enzyme was isolated from a marine green alga, Codium latum, and designated C. latum protease (CLP). It also had fibrinogenolytic activity, hydrolyzing A alpha, B beta and gamma chains with preference in this order. As CLP hydrolyzed oxidized insulin B chain at position Arg22-Gly23, and the peptide map of lysozyme digested with CLP was similar to that with trypsin, CLP would be expected to have a high substrate specificity, similar to that of trypsin. Protease activity peaked at pH 10, and was completely inhibited by diisopropyl fluorophosphate (DFP). Therefore, we conclude that CLP is a trypsin-like serine protease.     

Purification and characterization of a fibrinolytic enzyme and identification of fibrinogen clotting enzyme in a marine green alga, Codium divaricatum

A fibrinolytic enzyme was isolated from a marine green alga, Codium divaricatum, and designated C. divaricatum protease (CDP). This protease effectively hydrolyzed fibrinogen A alpha chain, while it had very low hydrolyzing efficiency for B beta and gamma chains. This property was similar to that of alpha-fibrinogenase isolated from snake venom. Protease activity peaked at pH 9, and was completely inhibited by diisopropyl fluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF), identifying it as a serine protease. Its molecular form was single polypeptide structure and molecular weight was estimated as 31,000 by SDS-PAGE. Fibrinogen clotting enzyme was also identified in a fraction by ion-exchange chromatography. Analysis of clots formed by the enzyme and by thrombin by SDS-PAGE showed that the fibrinogen clotting enzyme would act like thrombin and have high substrate specificity.     

Purification, characterization, and cDNA cloning of a novel acidic endoglycoceramidase from the jellyfish, Cyanea nozakii

Endoglycoceramidase (EC ) is an enzyme capable of cleaving the glycosidic linkage between oligosaccharides and ceramides in various glycosphingolipids. We report here the purification, characterization, and cDNA cloning of a novel endoglycoceramidase from the jellyfish, Cyanea nozakii. The purified enzyme showed a single protein band estimated to be 51 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme showed a pH optimum of 3.0 and was activated by Triton X-100 and Lubrol PX but not by sodium taurodeoxycholate. This enzyme preferentially hydrolyzed gangliosides, especially GT1b and GQ1b, whereas neutral glycosphingolipids were somewhat resistant to hydrolysis by the enzyme. A full-length cDNA encoding the enzyme was cloned by 5'- and 3'-rapid amplification of cDNA ends using a partial amino acid sequence of the purified enzyme. The open reading frame of 1509 nucleotides encoded a polypeptide of 503 amino acids including a signal sequence of 25 residues and six potential N-glycosylation sites. Interestingly, the Asn-Glu-Pro sequence, which is the putative active site of Rhodococcus endoglycoceramidase, was conserved in the deduced amino acid sequences. This is the first report of the cloning of an endoglycoceramidase from a eukaryote.     

Purification, characterization, and cDNA cloning of a novel metallothionein-like, cadmium-binding protein from Caenorhabditis elegans

Caenorhabditis elegans adapted for survival in high concentrations of Cd(II) express a heavy metal binding protein designated C. elegans metallothionein-like protein or MT-Ce. This protein was purified to homogeneity and characterized. MT-Ce binds 6 mol of Cd(II)/mol protein. The sequence of 39 amino-terminal residues in MT-Ce was determined. A radiolabeled 41-mer oligonucleotide, designed from the partial MT-Ce sequence, was used in conjunction with sucrose gradient centrifugation to obtain size-fractionated poly(A+) RNA enriched in MT-Ce sequences. Subsequently, cloned cDNAs, corresponding to MT-Ce mRNA sequences, were isolated from a lambda ZapII cDNA library prepared from the enriched template mRNA. cDNA and protein sequence analysis revealed that MT-Ce comprises 62 amino acid residues and has a predicted Mr of 6462. Seventeen of the 18 Cys residues in the nematode cadmium-binding protein are included in Cys-X-Cys and X-Cys-Cys-X motifs that are characteristic of mammalian metallothioneins (MTs). However, the resemblance of MT-Ce to mammalian MTs is superficial. The amino acid sequence of MT-Ce is unique, and neither its putative alpha and beta domains nor its Cys residues can be readily aligned with the corresponding regions of other eukaryotic MTs. This suggests that MT-Ce is an example of convergent evolution. The MT-Ce mRNA level in nematodes that were selected and grown with Cd(II) concentrations that are lethal for wild-type worms, was 55-fold higher than the level of MT-Ce mRNA in wild-type C. elegans. Comparison of the sequences of MT-Ce cDNAs revealed the occurrence of two types of MT-Ce mRNA. Each contains an identical coding region, but the cDNAs diverge markedly in their 5'-untranslated regions. This suggests the possibilities of regulation by alternative splicing and/or the presence of multiple MT-Ce genes encoding a single protein, but controlled by different regulatory elements.     

An anticoagulant proteoglycan from the marine green alga, Codium pugniformis

An anticoagulant isolated from the marine green alga Codium pugniformis was composed mainly of glucose with minor amounts of arabinose and galactose. It was highly sulfated (326 μg mg^-1 polysaccharide) and contained protein(52 μg mg^-1 polysaccharide) and was thus a proteoglycan. The anticoagulant properties of the purified proteoglycan were compared with those of heparin by studying the activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time(TT) using normal human plasma. The proteoglycan showed similar activities to heparin, but was weaker than heparin. On the other hand, the proteoglycan did not affect PT even at the concentration at which APTT and TT were prolonged. The anticoagulation mechanism of this proteoglycan was due to the direct inhibition of thrombin and the potentiation of antithrombin III. This revised version was published online in August 2006 with corrections to the Cover Date.     

Purification and characterization of a new lectin from the red marine alga Hypnea musciformis

A lectin from the red marine alga Hypnea musciformis (HML) was purified by extraction with 20 mM PBS, precipitation with 70% saturated ammonium sulphate, ion-exchange DEAE-Cellulose chromatography and RP-HPLC. The 9.3 kDa polypeptide agglutinates erythrocytes from various sources and shows oligomerization tendencies under certain MALDI-TOF/MS conditions. Preliminary N-terminal sequencing and biological assays strongly suggest that the HML may belong to a new class of algae lectins.     

Isolation, cloning, and characterization of a novel phosphomannan-binding lectin from porcine serum

Mannan-binding protein (MBP) is a C-type serum lectin that is an important constituent of the innate immune defense because it activates the complement system via the lectin pathway. While the pig has been proposed to be an attractive source of xenotransplantable tissues and organs, little is known about porcine MBP. In our previous studies, phosphomannan, but not mannan, was found to be an effective inhibitor of the C1q-independent bactericidal activity of newborn piglet serum against some rough strains of Gram-negative bacteria. In contrast, the inhibitory activities of phosphomannan and mannan were very similar in the case of MBP-dependent bactericidal activity against rough strains of Escherichia coli K-12 and S-16. Based on these findings, we inferred that an MBP-like lectin with slightly or completely different carbohydrate binding specificity might exist in newborn piglet serum and be responsible for the C1q-independent bactericidal activity. Herein we report that a novel phosphomannan-binding lectin (PMBL) of 33 kDa under reducing conditions was isolated from both newborn and adult porcine serum and characterized. Porcine PMBL functionally activated the complement system via the lectin pathway triggered by binding with both phosphomannan (P-mannan) and mannan, which, unlike MBP, was effectively inhibited by mannose 6-phosphate- or galatose-containing oligosaccharides. Our observations suggest that porcine PMBL plays a critical role in the innate immune defense from the newborn stage to adult-hood, and the establishment of a newborn piglet experimental model for the innate immune system studies is a valuable step toward elucidation of the physiological function and molecular mechanism of lectin pathway.     

Purification,cDNA cloning,and characterization of plant chitinase with a novel domain combination from lycophyte Selaginella doederleinii

Chitinase-A from a lycophyte Selaginella doederleinii (SdChiA), having molecular mass of 53 kDa, was purified to homogeneity by column chromatography. The cDNA encoding SdChiA was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1477 nucleotides and its open reading frame encoded a polypeptide of 467 amino acid residues. The deduced amino acid sequence indicated that SdChiA consisted of two N-terminal chitin-binding domains and a C-terminal plant class V chitinase catalytic domain, belonging to the carbohydrate-binding module family 18 (CBM18) and glycoside hydrolase family 18 (GH18), respectively. SdChiA had chitin-binding ability. The time-dependent cleavage pattern of (GlcNAc)₄ by SdChiA showed that SdChiA specifically recognizes the β-anomer in the + 2 subsite of the substrate (GlcNAc)₄ and cleaves the glycoside bond at the center of the substrate. This is the first report of the occurrence of a family 18 chitinase containing CBM18 chitin-binding domains.

Abbreviations: AtChiC: Arabidopsis thaliana class V chitinase; CBB: Coomassie brilliant blue R250; CBM: carbohydrate binding module family; CrChi-A: Cycas revolute chitinase-A; EaChiA: Equisetum arvense chitinase-A; GH: glycoside hydrolase family, GlxChi-B: gazyumaru latex chitinase-B; GlcNAc: N-acetylglucosamine; HPLC: high performance liquid chromatography; LysM; lysin motif; MtNFH1: Medicago truncatula ecotypes R108-1 chitinase; NCBI: national center for biotechnology information; NF: nodulation factor; NtChiV: Nicotiana tabacum class V chitinase; PCR: polymerase chain reaction; PrChi-A: Pteris ryukyuensis chitinase-A; RACE: rapid amplification of cDNA ends; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SdChiA: Selaginella doederleinii chitinase-A.     

Anticoagulant properties of a sulfated galactan preparation from a marine green alga, Codium cylindricum

An anticoagulant was isolated from a marine green alga, Codium cylindricum. The anticoagulant was composed mainly of galactose with a small amount of glucose, and was highly sulfated (13.1% as SO3Na). The anticoagulant properties of the purified anticoagulant were compared with that of heparin by assays of activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) using normal human plasma. The anticoagulant showed similar activities with heparin, however, weaker than heparin. On the other hand, the anticoagulant did not affect PT even at the concentration at which APTT and TT were strongly prolonged. The anticoagulant did not potentiate antithrombin III (AT III) and heparin cofactor II (HC II), thus the anticoagulant mechanism would be different from that of other anticoagulants isolated so far from the genus Codium.     

Purification and cDNA cloning of a lectin and a lectin-like protein from Apios americana Medikus tubers

An Apios americana lectin (AAL) and a lectin-like protein (AALP) were purified from tubers by chromatography on Butyl-Cellulofine, ovomucoid-Cellulofine, and DEAE-Cellulofine columns. AAL showed strong hemagglutinating activity toward chicken and goose erythrocytes, but AALP showed no such activity toward any of the erythrocytes tested. The hemagglutinating activity of AAL was not inhibited by mono- or disaccharides, but was inhibited by glycoproteins, such as asialofetuin and ovomucoid, suggesting that AAL is an oligosaccharide-specific lectin. The cDNAs of AAL and AALP consist of 1,093 and 1,104 nucleotides and encode proteins of 302 and 274 amino acid residues, respectively. Both amino acid sequences showed high similarity to known legume lectins, and those of their amino acids involved in carbohydrate and metal binding were conserved.     

Purification and characterization of a novel lectin from a freshwater cyanobacterium, Oscillatoria agardhii

In the survey of 14 species of laboratory-cultured cyanobacteria for hemagglutinins, we newly detected the activity in two species, Oscillatoria agardhii, strain NIES-204, and Phormidium foveolarum, strain NIES-503. From the extract of O. agardhii, which showed the highest activity with trypsin-treated erythrocytes of rabbit, a lectin was purified to homogeneity by the combination of precipitation with (NH4)2SO4, gel filtration, hydrophobic chromatography and reverse phase chromatography. The purified lectin, designated OAA, was a monomeric protein with an apparent molecular weight of 13,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 16,000 on gel filtration. The amino acid composition was rich in glycine and acidic amino acids. The hemagglutination activity was inhibited by glycoproteins such as yeast mannan, but not by any of the monosaccharides tested. The activity was stable over a wide range of pH (4-11) and at a high temperature of 80 degrees C, and independent on the presence of divalent cations. The features of OAA resembled those of many of lectins from marine macroalgae. The sequence of amino-terminal residues of OAA was determined as ALYNVENQWGGSSAPWNEGG, which was highly homologous to those of lectins from macroalgae of the genus Eucheuma and that of a myxobacterium Myxococcus xanthus hemagglutinin.     

Antiangiogenic properties of a sulfated galactan isolated from a marine green alga, Codium cylindricum

An anticoagulant sulfated galactan isolated from the marine green alga, Codium cylindricum, was shown to have antiangiogeinic activity. This galactan suppressed microvessel formation in an ex vivo serum-free matrix culture model using rat aortic ring. It also inhibited human umbilical vein endothelial cells (HUVEC) tube formation on reconstituted basement membrane gel. These results show the value of algal sulfated galactans in the design of antiangiogenic agents.     

Purification, characterization, and cDNA cloning of lipoate-activating enzyme from bovine liver

In mammals, lipoate-activating enzyme (LAE) catalyzes the activation of lipoate to lipoyl-nucleoside monophosphate. The lipoyl moiety is then transferred to the specific lysine residue of lipoate-dependent enzymes by the action of lipoyltransferase. We purified LAE from bovine liver mitochondria to apparent homogeneity. LAE activated lipoate with GTP at a 1000-fold higher rate than with ATP. The reaction absolutely required lipoate, GTP, and Mg(2+) ion, and the reaction product was lipoyl-GMP. LAE activated both (R)- and (S)-lipoate to the respective lipoyl-GMP, although a preference for (R)-lipoate was observed. Similarly, lipoyltransferase equally transferred both the (R)- and (S)-lipoyl moieties from the respectively activated lipoates to apoH-protein. Interestingly, however, only H-protein carrying (R)-lipoate was active in the glycine cleavage reaction. cDNA clones encoding a precursor LAE with a mitochondrial presequence were isolated. The predicted amino acid sequence of LAE is identical with that of xenobiotic-metabolizing/medium-chain fatty acid:CoA ligase-III, but an amino acid substitution due to a single nucleotide polymorphism was found. These results indicate that the medium-chain acyl-CoA synthetase in mitochondria has a novel function, the activation of lipoate with GTP.     

Purification, characterization, and cDNA cloning of profilin from Phaseolus vulgaris.

Profilin from common bean (Phaseolus vulgaris L.) was purified to homogeneity by poly-L-Pro affinity chromatography and gel filtration. The hypocotyl and symbiotic root nodule protein was detected as a single isoform with a 14.4-kD molecular mass and an isoelectric point of 5.3. Partial amino acid and DNA sequencing of a full-length cDNA clone confirmed its identity as profilin. An antibody generated against the purified protein binds to a protein with the same molecular mass in leaves and nodules. Immunolocalization of the protein showed a diffuse distribution in the cytoplasm of hypocotyls and nodules but enhanced staining at the vascular bundles. The strong identity of the sequence among the profilins of birch, maize, and bean suggests that it may play an important role in the signal transduction mechanism of plant cells and plant-bacterial symbioses.     

Phytochrome from the green alga Mesotaenium caldariorum. Purification and preliminary characterization

A method to purify the phytochrome photoreceptor from the unicellular green alga Mesotaenium caldariorum is presented. Preparative scale formation of algal protoplasts and controlled osmotic cell lysis have permitted separation of intact organelles from the phytochrome-enriched soluble protein fraction. We have utilized the observation that red light-absorbing (Pr) and far-red light-absorbing (Pfr) forms of phytochrome are differentially retained on an anion exchange matrix to purify M. caldariorum phytochrome to apparent homogeneity. M. caldariorum phytochrome preparations with A650/A280 ratios greater than 0.78 exhibit a single 120-kDa band on silver-stained sodium dodecyl sulfate-polyacrylamide gels. Immunoblot analyses using a cross-reactive pea phytochrome monoclonal antibody reveal that 1) the 120-kDa band represents the full-length polypeptide, 2) phytochrome is predominantly localized in the algal cytoplasm, and 3) there are 150,000-250,000 phytochrome molecules/cell. Steric exclusion high pressure liquid chromatography analysis under nondenaturing conditions indicates that M. caldariorum phytochrome has an apparent mass of 355 kDa. The absorption maxima for Pr and Pfr are 650 and 722 nm, respectively. Both are blue-shifted compared with those of phytochromes from dark-grown angiosperm tissue. The molar absorption coefficient for Pr at 650 nm is 86,800 +/- 2800 liter mol-1 cm-1, which is lower than that of higher plant phytochromes. The significance of the similarities and differences of the molecular properties of phytochromes from M. caldariorum and higher plant sources is discussed.     

A preponderantly 4-sulfated, 3-linked galactan from the green alga Codium isthmocladum

The green algae of the genus Codium have recently been demonstrated to be an important source of sulfated galactans from the marine environment. Here, a sulfated galactan was isolated from the species Codium isthmocladum and its structure was studied by a combination of chemical analyses and NMR spectroscopy. Two fractions (SG 1, approximately 14 kDa, and SG 2, approximately 20 kDa) were derived from this highly polydisperse and heterogeneous polysaccharide. Both exhibited similar structures in (1)H 1D NMR spectra. The structural features of SG 2 and its desulfated derivative were analyzed by COSY, TOCSY, DEPT-HSQC, HSQC, and HMBC. This sulfated galactan is composed preponderantly of 4-sulfated, 3-linked beta-D-galactopyranosyl units. In minor amounts, it is sulfated and glycosylated at C-6. Pyruvate groups are also found, forming five-membered cyclic ketals as 3,4-O-(1'carboxy)-ethylidene-beta-D-galactose residues. A comparison of sulfated galactans from different marine taxonomic groups revealed similar backbones of 3-beta-D-Galp-1.     

Molecular cloning and characterization of a novel mannose-binding lectin gene from Amorphophallus konjac

A new lectin gene was cloned from Amorphophallus konjac. The full-length cDNA of Amorphophallus konjac agglutinin (aka) was 736 bp and contained a 474 bp open reading frame encoding a 158 amino acid protein. Homology analysis revealed that the lectin from this Araceae species belonged to the superfamily of monocot mannose-binding proteins. Molecular modeling of AKA indicated that the three-dimensional structure of AKA strongly resembles that of the snowdrop lectin. Southern blot analysis of the genomic DNA revealed that aka belonged to a low-copy gene family. Northern blot analysis demonstrated that aka expression was tissue-specific with the strongest expression being found in root.