Purification and characterization of an N-acetyl-D-galactosamine-specific lectin from seeds of chickpea (Cicer arietinum L.)

A novel lectin (CAA-II) was isolated and purified from the seeds of Cicer arietinum by ammonium sulphate fractionation and affinity chromatography on an N-acetyl-D-galactosamine-linked agarose column. The lectin is composed of four identical subunits of 30 kDa and the molecular mass of the native lectin was estimated to be 120 kDa by gel filtration chromatography and confirmed by mass spectrometry. The lectin showed agglutination activity against rabbit erythrocytes (trypsin-treated and untreated) as well as against human erythrocytes. Haemagglutination inhibition assays showed that the lectin is a galactose-specific protein having a high affinity for N-acetyl-D-galactosamine. The molecular weight, haemagglutination pattern, carbohydrate specificity and N-terminal amino acid sequence indicated that the lectin is clearly distinct from the previously reported chickpea lectin CAA-I.     

Purification and characterization of a new type lactose-binding Ulex europaeus lectin by affinity chromatography

A new type lactose-binding lectin was purified from extracts of Ulex europaeus seeds by affinity chromatography on a column of galactose-Sepharose 4B, followed by gel filtration on Sephacryl S-300. This lectin, designated as Ulex europaeus lectin III (UEA-III), was found to be inhibited by lactose. The dimeric lectin is a glycoprotein with a molecular mass of 70,000 Da; it consists of two apparently identical subunits of a molecular mass of 34,000 Da. Compositional analysis showed that this lectin contains 30% carbohydrate and a large amount of aspartic acid, serine and valine, but no sulfur-containing amino acids. The N-terminal amino-acid sequences of L-fucose-binding Ulex europaeus lectin I (UEA-I) and di-N-acetylchitobiose-binding Ulex europaeus lectin II (UEA-II), both of which we have already purified and characterized, and that of UEA-III were determined and compared.     

Physicochemical properties and acute toxicity studies of a lectin from the saline extract of the fruiting bodies of the shiitake mushroom,Lentinula edodes (Berk)

A lectin (LEL) was isolated from the fresh fruiting bodies of the shiitake mushroom Lentinula edodes by a combination of gel filtration chromatography on Sephadex G-150 and affinity chromatography on an N-acetyl-Dgalactosamine-Sepharose 4B column. Its molecular mass, as determined by gel filtration, was estimated to be 71, 000 Daltons and its structure is homotetrameric with subunit molecular weight of approximately 18,000 Daltons. LEL agglutinated non-specifically red blood cells from the human ABO system as well as rabbit erythrocytes and in haemagglutination inhibition assays, exhibited sugar-binding specificity toward N-acetyl-D-galactosamine. EDTA had no inhibitory effect on its haemagglutinating activity, which was stable up to 70°C and was not affected by changes in pH. The lectin had no covalently-linked carbohydrate and amino acid composition analysis revealed that it contained 124 amino acid residues and was rich in tyrosine, proline, phenylalanine, arginine, glutamic acid and cysteine. LEL did not cause mortality neither was it observed to alter the morphology of key organs when administered intraperitoneally at concentrations up to 10,000 mg kg^-1 body weight of mice.     

Purification and characterization of a lectin from wild sunflower (Helianthus tuberosus L.) tubers

A lectin (HTTL) was isolated from Helianthus tuberosus L. (wild sunflower) tubers using ion-exchange chromatography, gel filtration, and affinity chromatography. The lectin agglutinated both untreated and trypsin-treated rabbit erythrocytes and did not agglutinate human blood cells of groups A, B, and O. The gel filtration showed the native molecular mass of 72 kDa and subunit molecular masses of 17 and 18.5 kDa on 12% SDS-PAGE. The lectin activity was inhibited by D-mannose. The tetrameric protein revealed a unique characteristic by forming a broad zone of protein in native PAGE at pH 8.3, which dissociated into seven subunits of varying e/m ratios on acid gel at pH 4.3. These seven bands revealed two polypeptide species of molecular masses 17 and 18.5 kDa on 12% SDS-PAGE, as in the case of the native protein. The result indicated that of the seven subunits, three were homotetramers of 17 kDa, one was a homotetramer of 18.5 kDa, and three were heterotetramers of 17 and 18.5 kDa. The lectin was thermostable with broad pH optima (pH 4-8) and had no requirement for divalent metal cations for its activity. The amino acid composition showed that the lectin contained higher amounts of glycine, alanine, and lysine, but no methionine. The sugar content was estimated to be 5.3% mannose equivalent. The HTTL was mitogenic to mouse spleen (total) cells at 25 microg/ml concentration. The lectin showed characteristics different from those of the earlier reported H. tuberosus tuber lectins and hence opens up a new avenue to investigate the structure-function relationship of lectin in Helianthus species.     

The relationships of the genus Cicer L. (Leguminosae): the evidence from pollen morphology

The genus Cicer has traditionally been placed in the Vicieae, although it has a number of morphological characters which suggest that it is out of place in that tribe and that its affinities lie elsewhere. A survey of pollen morphology in Cicer , the other genera of the Vicieae, the Trifolieae and the Ononideae has been carried out to help determine the relationships of Cicer. The tribe Vicieae (without Cicer) is very homogeneous and is characterized by its long, rectangular pollen grains with small, heavily thickened endoapertures. The Trifolieae is more variable in its pollen morphology but, in general, has rather similar grains to the Vicieae although the structure of the endoaperture is different. The Ononideae and Cicer have nearly spheroidal pollen with very large, unthickened endoapertures. It therefore appears that as far as pollen characters are concerned, Cicer does not belong in the Vicieae but has much more in common with the Ononideae.     

The delimitation of the tribe Vicieae (Leguminosae) and the relationships of Cicer L.

A survey of morphological, anatomical, karyological and chemical characters has been carried out, centred on the Vicieae but extending to the neighbouring tribes Trifolieae and Ononideae. The results show that Cicer , traditionally a member of the Vicieae, has more in common with genera of the Trifolieae and Ononideae than with the rest of the Vicieae. It is proposed that Cicer should be removed from the Vicieae and recognized as the monogeneric tribe Cicereae Alef. The tribe Vicieae sensu stricto, a well-defined natural group, is delimited and described. Phylogenetic relationships of the Cicereae are discussed.     

Biochemical characterization of a lectin from Delonix regia seeds

A lectin from Delonix regia (DRL) seeds was purified by gel filtration on Sephadex G-100 followed by ion-exchange chromatography on diethylaminoethyl-Sepharose and reverse-phase high-performance liquid chromatography on a C18 column. Hemagglutinating activity was monitored using rat erythrocytes. DRL showed no specificity for human erythrocytes of ABO blood groups. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a single protein in the presence of 0.1 M of dithiothreitol (DTT) and in nonreducing conditions. Native-PAGE showed that DRL is a monomer with a molecular mass of about 12 kDa, as determined by denaturing gel electrophoresis and gel filtration chromatography. An amino acid composition revealed the absence of cysteine residues, the presence of 1 mol methionine/mol protein and a high proportion of acidic amino acids and glycine. The N-terminal sequence of DRL was determined by Edman degradation, and up to 16 amino acid residues showed more than 90% homology with other lectins from the Leguminosae family. The optimal pH range for lectin activity was between pH 8.0 and 9.0, and the lectin was active up to 60°C. The lectin required Mn²⁺ for hemagglutinating activity and remained active after reduction with 0.1 M of DTT, but lost activity in the presence of 8 M of urea. Sodium metaperiodate had no effect on the activity of DRL.     

Cicerarin,a novel antifungal peptide from the green chickpea

A peptide designated cicerarin, with an N-terminal amino acid sequence VKSTGRADDDLAVKTKYLPP dissimilar from known proteins and peptides and a molecular mass of 8kDa, was isolated from seeds of the green chickpea Cicer arietinum cv green chickpea. Cicerarin was isolated with a procedure that involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, and gel filtration by fast protein liquid chromatography on Superdex 75. Cicerarin was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel in 10mM Tris-HCl buffer (pH 7.3). Cicerarin exerted antifungal activity against Botrytis cinerea, Mycosphaerella arachidicola, and Physalospora piricola. The antifungal activity was preserved after exposure to 100 degrees C for 15min.     

Seed lectin from pisum arvense: isolation, biochemical characterization and amino acid sequence

A glucose/mannose lectin was purified by affinity chromatography from Pisum arvense seeds (PAL) and the 50 kDa molecular mass in solution determined by size exclusion chromatography. SDS-PAGE and electrospray ionization mass spectrometry showed two distinct polypeptide chains: alpha (Mr. 5591 Da) and beta (19986 Da). The lectin was extensively characterized in terms of its biochemical and biological aspects. The amino acid sequence was established by Edman degradation of overlapping peptides. PAL in solution behaves as a dimer and has its monomeric structure formed by two distinct polypeptide chains named alpha (Mr. 5591 Da) and beta (19986 Da) by Electrospray ionization (ESI) mass spectrometry. PAL possesses identical amino acid sequences to that of pea seed lectin but undoubtedly does not exhibit sequence heterogeneity. It is discussed that P. arvense should be considered as a synonym of P. sativum. Furthermore, like pea lectin, PAL discriminates biantennary fucosylated glycan, determined by surface plasmon resonance.     

A novel homodimeric lectin from Astragalus mongholicus with antifungal activity

A novel lectin (AMML) was isolated from a Chinese herb, i.e., the roots of Astragalus mongholicus, using a combination of ammonium sulfate fraction and ion exchange chromatographies. The molecular mass of intact AMML was determined to be 66,396 Da by MALDI-TOF mass spectrometry and 61.8 kDa by gel filtration, respectively. AMML was a dimeric protein composed of two identical subunits each with a molecular mass of 29.6 kDa. The lectin was a glycoprotein with a neutral carbohydrate content of 19.6%. The purified lectin hemagglutinated both rabbit and human erythrocytes, and showed preference for blood types O (native) and AB (trypsin-treated). Among various carbohydrates tested, the lectin was best inhibited by D-galactose and its derivatives with pronounced preference for lactose (3.13 mM). N-terminal amino acid sequence of AMML was determined as ESGINLQGDATLANN. The optimal pH range for lectin activity was between pH 4.5 and 7.5, and the lectin was active up to 65 degrees C. It also exerted antifungal activity against Botrytis cincerea, Fusarium oxysporum, Colletorichum sp., and Drechslera turcia but not against Rhizoctonia solani and Mycosphaerella arachidicola.     

Purification and some characteristics of a beta-galactoside binding soluble lectin from amphibian ovary

Soluble extracts of Bufo ovaries agglutinate sialidase-treated rabbit erythrocytes. Unlike other amphibian lectins this agglutination activity does not require the presence of calcium ions. It is specifically inhibited by D-galactose and its derivatives. Thiodi-D-galactoside is the most potent saccharide inhibitor followed by lactose and methyl-beta-D-galactoside, respectively. D-Fucose, D-glucose and D-mannose do not inhibit the activity at concentrations at or above 100 mM. The lectin has been purified 500-fold to apparent homogeneity from the ovaries by salt extraction and affinity chromatography on lactose-aminophenyl-agarose, with a yield of about 0.2%. The molecular mass determined by gel filtration under native conditions was 30 kDa; polyacrylamide gel electrophoresis in SDS gave a molecular mass of 15 kDa, suggesting that the lectin is a dimer. The lectin has an isoelectric point of 40 and contains a high proportion of acidic amino acids.     

Purification of a sialic acid-specific lectin from the Indian scorpion Heterometrus granulomanus

A sialic acid-specific lectin, scorpin, has been purified to apparent homogeneity from the Indian scorpion Heterometrus granulomanus by affinity chromatography on equine submandibular gland glycopeptides linked to Sepharose and gel filtration on Sephadex G-200. The lectin has a molecular mass of 500 000 Da and was dissociated into single polypeptide chains of 15 000 Da, as determined by SDS gel electrophoresis in the presence of 2-mercaptoethanol. Scorpin is a glycoprotein containing 2.8% sugars. Its specificity was investigated by the inhibition of hemagglutination with various derivatives of sialic acid and other sugars. N-Acetylneuraminic acid gave better inhibition than N-glycoloylneuraminic acid but showed less inhibitory effect than sialyl-alpha(2----3)-lactose and disialyllactose. Among the sialoglycoconjugates tested, equine submandibular gland glycopeptide was found to be the most potent inhibitor. Scorpin showed a strong tendency to bind to carboxyl groups, since reduction of the carboxyl group of N-acetylneuraminic acid destroyed the inhibitory potency of this sugar. Furthermore, D-glucuronic acid inhibited hemagglutination whereas N-acetylglucosamine or N-acetylgalactosamine were not inhibitors.     

Purification and primary structure of a mannose/glucose‐binding lectin from Parkia biglobosa Jacq. seeds with antinociceptive and anti‐inflammatory properties

Parkia biglobosa (subfamily Mimosoideae), a typical tree from African savannas, possess a seed lectin that was purified by combination of ammonium sulfate precipitation and affinity chromatography on a Sephadex G‐100 column. The P. biglobosa lectin (PBL) strongly agglutinated rabbit erythrocytes, an effect that was inhibited by d ‐mannose and d ‐glucose‐derived sugars, especially α‐methyl‐d ‐mannopyranoside and N‐acetyl‐d ‐glucosamine. The hemagglutinating activity of PBL was maintained after incubation at a wide range of temperature and pH and also was independent of divalent cations. By sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis, PBL exhibited an electrophoretic profile consisting of a single band with apparent molecular mass of 45 kDa. An analysis using electrospray ionization–mass spectrometry indicated that purified lectin possesses a molecular average mass of 47 562 ± 4 Da, and the analysis by gel filtration showed that PBL is a dimer in solution. The complete amino acid sequence of PBL, as determined using tandem mass spectrometry, consists of 443 amino acid residues. PBL is composed of a single non‐glycosylated polypeptide chain of three tandemly arranged jacalin‐related domains. Sequence heterogeneity was found in six positions, indicating that the PBL preparations contain highly homologous isolectins. PBL showed important antinociceptive activity associated to the inhibition of inflammatory process. Copyright © 2013 John Wiley & Sons, Ltd.     

Purification and partial characterization of a tripeptidase from Pediococcus pentosaceus K9.2.

A tripeptidase was purified from the cytoplasm of Pediococcus pentosaceus K9.2 by anion-exchange chromatography, gel filtration chromatography, and high-performance liquid chromatography. The molecular mass of the enzyme was estimated by gel filtration at 100,000 Da. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified peptidase showed one protein band of 45,000 Da. Optimal enzyme activity was obtained at pH 7.0 and at 50 degrees C. The peptidase hydrolyzed all tripeptides tested. Cleavage was not observed with dipeptides, oligopeptides, or amino acid-p-nitroanilide derivatives. Strong inhibition of activity was caused by EDTA, 1,10-phenanthroline, dithiothreitol, and beta-mercaptoethanol, whereas phenylmethylsulfonyl fluoride and sulfur-reactive reagents had no effect on peptidase activity. Mg2+, Mn2+, and Ca2+ stimulated the hydrolyzing activity of the enzyme. The 20 N-terminal amino acids of the tripeptidase from P. pentosaceus had 84% identity with those from the corresponding N-terminal region of the tripeptidase from Lactococcus lactis subsp. cremoris Wg2.     

Chemical and physicochemical characterization of the sialic acid-specific lectin from Cepaea hortensis

A sialic acid-specific lectin was isolated from the albumin glands of the garden snail Cepaea hortensis by affinity chromatography on fetuin-Sepharose following gel filtration on Superdex 200. The purified native lectin showed a molecular mass of about 95 kDa by gel filtration and 100 kDa by SDS electrophoresis. It was cleaved by boiling in buffer containing SDS in three serological identical bands corresponding to molecular masses of about 24, 20 and 16 kDa, respectively. From these three fragments, only the 24- and the 20-kDa bands were found to be glycosylated. Only the three sugars mannose, galactose and N-acetylglucosamine could be detected in a molar ratio of 3:8.6:2. The oligosaccharide moieties seem to be N- and partially O-glycosidic bound. Isoelectric focusing (IEF) of the purified lectin revealed a heterogeneous pattern with bands in the pH range of 4.3-5.0. Isolated bands of different isoelectric points showed in SDS electrophoresis the same three fragments with molecular masses of 24, 20 or 16 kDa. The heterogeneity of the lectin was revealed either by IEF or amino acid sequencing of internal tryptic peptides.     

Isolation of a novel legumin-like lectin with potent hemagglutinating activity from seeds of the Chinese chestnut Castanea mollisima

A novel mannose- and glucose-specific lectin with high hemagglutinating activity was isolated from seeds of the Chinese chestnut Castanea mollisima. The lectin possessed a molecular mass of 140 kDa and was made up of two subunits, one with a molecular mass of 31 kDa and another with a molecular mass of 32 kDa. They exhibited substantial homology in N-terminal sequence to the storage protein legumin. The lectin was unstable in the presence of acid and alkali and at temperatures above 50 degrees C, but it was unaffected by various salts. The lectin was purified with a procedure involving ion exchange chromatography on CM-Sepharose, Q-Sepharose and Resource Q and gel filtration on Superose 12.     

Purification and characterization of acid trehalase from the yeast suc2 mutant

Acid trehalase was purified from the yeast suc2 deletion mutant. After hydrophobic interaction chromatography, the enzyme could be purified to a single band or peak by a further step of either polyacrylamide gel electrophoresis, gel filtration, or isoelectric focusing. An apparent molecular mass of 218,000 Da was calculated from gel filtration. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate suggested a molecular mass of 216,000 Da. Endoglycosidase H digestion of the purified enzyme resulted after sodium dodecyl sulfate gel electrophoresis in one distinct band at 41,000 Da, representing the mannose-free protein moiety of acid trehalase. The carbohydrate content of the enzyme was 86%. Amino acid analysis indicated 354 residues/molecule of enzyme including 9 cysteine moieties and only 1 methionine. The isoelectric point of the enzyme was estimated by gel electrofocusing to be approximately 4.7. The catalytic activity showed a maximum at pH 4.5. The activity of the enzyme was not inhibited by 10 mM each of HgCl2, EDTA, iodoacetic acid, phenanthrolinium chloride or phenylmethylsulfonyl fluoride. There was no activation by divalent metal ions. The acid trehalase exhibited an apparent Km for trehalose of 4.7 +/- 0.1 mM and a Vmax of 99 mumol of trehalose min-1 X mg-1 at 37 degrees C and pH 4.5. The acid trehalase is located in the vacuoles. The rabbit antiserum raised against acid trehalase exhibited strong cross-reaction with purified invertase. These cross-reactions were removed by affinity chromatography using invertase coupled to CNBr-activated Sepharose 4B. Precipitation of acid trehalase activity was observed with the purified antiserum.     

Purification and characterization of a new antiviral protein from the leaves of Pandanus amaryllifolius (Pandanaceae)

A lectin, designated Pandanin, was isolated from the saline extract of the leaves of Pandanus amaryllifolius, using ammonium sulfate precipitation, affinity chromatography on mannose-agarose and molecular size exclusion by gel filtration. Pandanin is an unglycosylated protein with a molecular mass of 8.0 kDa both after gel filtration and on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that it is a single polypeptide chain. The first 10 residues of the N-terminal amino acid sequence are DNILFSDSTL. An analysis of the sequence of first 30 amino acids at the N-terminal region shows that Pandanin has about 50-60% homology to those of mannose-specific lectins reported from monocot plants. Pandanin exhibits hemagglutinating activity toward rabbit erythrocytes, and its activity could be reversed exclusively by mannose and mannan. Pandanin also possesses antiviral activities against human viruses, herpes simplex virus type-1 (HSV-1) and influenza virus (H1N1) with 3 day's EC50 of 2.94 and 15.63 microM, respectively.     

Primary structure and characteristics of a lectin from skin mucus of the Japanese eel Anguilla japonica

Two types of lactose-binding lectins, AJL-1 and AJL-2, were purified from the skin mucus extract of the Japanese eel Anguilla japonica by lactose affinity chromatography and subsequent gel filtration. The molecular masses of AJL-1 and AJL-2 were 16,091 and 31,743 Da, respectively. Intact AJL-1 was comprised of two identical 16-kDa subunits having blocked N termini and no disulfide bonds. AJL-2 was a homodimer with disulfide bonds. Based on the N-terminal amino acid sequence of the AJL-2 monomer, the nucleotide sequence of cDNA encoding this lectin was determined by 3'- and 5'-rapid amplification of cDNA ends. The deduced amino acid sequence showed approximately 30% homology with C-type lectins, which bind to carbohydrates in a Ca(2+)-dependent manner. In addition, AJL-2 exhibited highly conserved consensus amino acid residues of the C-type carbohydrate recognition domain, although this lectin showed Ca(2+)-independent activity. Gene expression of AJL-2 was detected only in the skin by Northern blot analysis, and this lectin localization was demonstrated in the club cells by immunohistochemistry. These results indicate that AJL-2 is secreted on the body surface and function as a component of skin mucus. AJL-2 agglutinated Escherichia coli and suppressed its growth, suggesting that this lectin is involved in host defense.     

Mannose-binding lectin from<Emphasis Type="Italic">Curcuma zedoaria</Emphasis> Rosc

A mannose-binding lectin was isolated from rhizomes of the medicinal plantCurcuma zedoaria. We used extraction with 20 mM phosphate buffer, ammonium sulfate precipitation, ion exchange chromatography on Q-Sepharose, gel filtration chromatography on Superdex 75, and reverse-phase HPLC. The purified lectin yielded a single band on SDS-PAGE that corresponded to a molecular mass of 13 kDa. This lectin exhibited hemagglutinating activity toward rabbit erythrocytes, which could be inhibited by mannose only. The lectin was digested with trypsin and its digests were analyzed using MALDI-TOF/TOF. Partial amino acid sequences were obtained from tandem mass spectra via automatedde novo sequencing, and were then identified by MS-BLAST homology searches to enable recognition of related proteins in other species. Inferred peptide sequences exhibited similarity to a mannose-binding lectin fromEpipactis helleborine, a member of the Orchidaceae.