Characterization of alpha-mannosidase from Erythrina indica seeds and influence of endogenous lectin on its activity

alpha-mannosidase from Erythrina indica seeds is a Zn(2+) dependent glycoprotein with 8.6% carbohydrate. The enzyme has a temperature optimum of 50 degrees C and energy of activation calculated from Arrhenius plot was found to be 23 kJ mol(-1). N-terminal sequence up to five amino acid residues was found to be DTQEN (Asp, Thr, Gln, Glu, and Asn). In chemical modification studies treatment of the enzyme with NBS led to total loss of enzyme activity and modification of a single tryptophan residue led to inactivation. Fluorescence studies over a pH range of 3-8 have shown tryptophan residue to be in highly hydrophobic environment and pH change did not bring about any appreciable change in its environment. Far-UV CD spectrum indicated predominance of alpha-helical structure in the enzyme. alpha-Mannosidase from E indica exhibits immunological identity with alpha-mannosidase from Canavalia ensiformis but not with the same enzyme from Glycine max and Cicer arietinum. Incubation of E. indica seed lectin with alpha-mannosidase resulted in 35% increase in its activity, while no such activation was observed for acid phosphatase from E. indica. Lectin induced activation of alpha-mannosidase could be completely abolished in presence of lactose, a sugar specific for lectin.     

Isolation,purification, and physicochemical characterization of a D-galactose-binding lectin from seeds of Erythrina speciosa

A lectin was isolated from the saline extract of Erythrina speciosa seeds by affinity chromatography on lactose-Sepharose. The lectin content was about 265 mg/100g dry flour. E. speciosa seed lectin (EspecL) agglutinated all human RBC types, showing no human blood group specificity; however a slight preference toward the O blood group was evident. The lectin also agglutinated rabbit, sheep, and mouse blood cells and showed no effect on horse erythrocytes. Lactose was the most potent inhibitor of EspecL hemagglutinating activity (minimal inhibitory concentration (MIC)=0.25 mM) followed by N-acetyllactosamine, MIC=0.5mM, and then p-nitrophenyl alpha-galactopyranoside, MIC=2 mM. The lectin was a glycoprotein with a neutral carbohydrate content of 5.5% and had two pI values of 5.8 and 6.1 and E(1%)(1 cm) of 14.5. The native molecular mass of the lectin detected by hydrodynamic light scattering was 58 kDa and when examined by mass spectroscopy and SDS-PAGE it was found to be composed of two identical subunits of molecular mass of 27.6 kDa. The amino acid composition of the lectin revealed that it was rich in acidic and hydroxyl amino acids, contained a lesser amount of methionine, and totally lacked cysteine. The N-terminal of the lectin shared major similarities with other reported Erythrina lectins. The lectin was a metaloprotein that needed both Ca(2+) and Mn(2+) ions for its activity. Removal of these metals by EDTA rendered the lectin inactive whereas their addition restored the activity. EspecL was acidic pH sensitive and totally lost its activity when incubated with all pH values between pH 3 and pH 6. Above pH 6 and to pH 9.6 there was no effect on the lectin activity. At 65 degrees C for more than 90 min the lectin was fairly stable; however, when heated at 70 degrees C for 10 min it lost more than 80% of its original activity and was totally inactivated at 80 degrees C for less than 10 min. Fluorescence studies of EspecL indicated that tryptophan residues were present in a highly hydrophobic environment, and binding of lactose to EspecL neither quenched tryptophan fluorescence nor altered lambda(max) position. Treating purified EspecL with NBS an affinity-modifying reagent specific for tryptophan totally inactivated the lectin with total modification of three tryptophan residues. Of these residues only the third modified residue seemed to play a crucial role in the lectin activity. Addition of lactose to the assay medium did not provide protection against NBS modification which indicated that tryptophan might not be directly involved in the binding of haptenic sugar D-galactose. Modification of tyrosine with N-acetylimidazole led to a 50% drop in EspecL activity with concomitant acetylation of six tyrosine residues. The secondary structure of EspecL as studied by circular dichroism was found to be a typical beta-pleated-sheet structure which is comparable to the CD structure of Erythrina corallodendron lectin. Binding of lactose did not alter the EspecL secondary structure as revealed by CD examination.     

Immunochemical studies on the combining site of the d-galactose/N-acetyl-d-galactosamine specific lectin from Erythrina cristagalli seeds

The combining site of the Erythrina cristagalli lectin was studied by quantitative precipitin and precipitin inhibition assays. The lectin precipitated best with two fractions of a precursor human ovarian cyst blood group substance with I and i activities. A₁, A₂, B, H, Le^a, and Le^b blood group substances precipitated poorly to moderately and substances of the same blood group activity precipitated to varying extents. These differences are attributable to heterogeneity resulting from incomplete biosynthesis of carbohydrate chains. Specific precipitates with the poorly reactive blood group substances were found to be more soluble than those reacting strongly. Precipitation was minimally affected by EDTA or divalent cations. Among the monosaccharides and glycosides tested for inhibition of precipitation, p-nitrophenyl βdGal was most active and was 10 times more active than methyl βdGal, indicating involvement of hydrophobic contacts in site specificity. Methyl αdGalNAc, p-nitrophenyl αdGalNAc, methyl αdGal, N-acetyl-d-galactosamine, p-nitrophenyl αdGal, methyl βdGal, and p-nitrophenyl βdGalNAc were progressively less active than p-nitrophenyl βdGal. The best disaccharide inhibitor dGalβ1 → 4dGlcNAc was 7.5 times more potent than p-nitrophenyl βdGal. A tetraantennary and triantennary oligosaccharide containing four and three dGalβ1 → 4dGlcNAcβ1 → branches, respectively, were, because of cooperative binding effects, 1.6 and 2.5 times more active than the bi- and monoantennary oligosaccharides, respectively. dGalβ1 → 4dGlcNAcβ1 → 6dGal and dGalβ1 → 4dGlcNAcβ1 → 2dMan had the same activity, being 1.5 times more active than dGalβ1 → 4dGlcNAc, which was 2.6 and 8.5 times more active than dGalβ1 → 3dGlcNAc and dGalβ1 → 3dGlc, respectively. Substitutions by N-acetyl-d-galactos-amine or l-fucose on the d-galactose of inhibitory compounds blocked activity. These results suggest that a hydrophobic interaction with the subterminal sugar is important in the binding and that the specificity of the lectin combining site involves a terminal dGalβ1 → 4dGlcNAc and the β linkage of a third sugar.     

Crystallization of a chymotrypsin inhibitor from Erythrina caffra seeds

Crystals of a chymotrypsin inhibitor from Erythrina caffra seeds have been grown out of lithium sulfate, by the hanging drop method of vapor diffusion. The crystals belong to the rhombohedral space group R32, with a = 67.2 A and alpha = 99.4 degrees, and diffract to 3 A resolution.     

Iso-erysopinophorine,a new quaternary alkaloid from the seeds of Erythrina arborescens

The ethanolic extract of the seeds of Erythrina arborescens yielded a new quaternary alkaloid provisionally named as iso-erysopinophorine besides the other alkaloids reported previously. The new alkaloid was characterised by chemical and spectral studies.     

Crystal structure of a Kunitz-type trypsin inhibitor from Erythrina caffra seeds

The trypsin inhibitor DE-3 from Erythrina caffra (ETI) belongs to the Kunitz-type soybean trypsin inhibitor (STI) family and consists of 172 amino acid residues with two disulphide bridges. The amino acid sequence of ETI shows high homology to other trypsin inhibitors from the same family but ETI has the unique ability to bind and inhibit tissue plasminogen activator. The crystal structure of ETI has been determined using the method of isomorphous replacement and refined using a combination of simulated annealing and conventional restrained least-squares crystallographic refinement. The refined model includes 60 water molecules and 166 amino acid residues, with a root-mean-square deviation in bond lengths from ideal values of 0.016 A. The crystallographic R-factor is 20.8% for 7770 independent reflections between 10.0 and 2.5 A. The three-dimensional structure of ETI consists of 12 antiparallel beta-strands joined by long loops. Six of the strands form a short antiparallel beta-barrel that is closed at one end by a "lid" consisting of the other six strands coupled in pairs. The molecule shows approximate 3-fold symmetry about the axis of the barrel, with the repeating unit consisting of four sequential beta-strands and the connecting loops. Although there is no sequence homology, this same fold is present in the structure of interleukin-1 alpha and interleukin-1 beta. When the structure of ETI and interleukin-1 beta are superposed, the close agreement between the alpha-carbon positions for the beta-strands is striking. The scissile bond (Arg63-Ser64) is located on an external loop that protrudes from the surface of the molecule and whose architecture is not constrained by secondary structure elements, disulphide bridges or strong electrostatic interactions. The hydrogen bonds made by the side-chain amide group of Asn12 play a key role in maintaining the three-dimensional structure of the loop. This residue is in a position corresponding to that of a conserved asparagine in the Kazal inhibitor family. Although the overall structure of ETI is similar to the partial structure of STI, the scissile bond loop is displaced by about 4 A. This displacement probably arises from the fact that the structure of STI has been determined in a complex with trypsin but could possibly be a consequence of the close molecular contact between Arg63 and an adjacent molecule in the crystal lattice.     

Purification,some properties of a D-galactose-binding leaf lectin from Erythrina indica and further characterization of seed lectin

Lectin from a leaf of Erythrina indica was isolated by affinity chromatography on Lactamyl-Seralose 4B. Lectin gave a single band in polyacrylamide gel electrophoresis (PAGE). In SDS-gel electrophoresis under reducing and non-reducing conditions Erythrina indica leaf lectin (EiLL) split into two bands with subunit molecular weights of 30 and 33 kDa, whereas 58 kDa was obtained for the intact lectin by gel filtration on Sephadex G-100. EiLL agglutinated all human RBC types, with a slight preference for the O blood group. Lectin was found to be a glycoprotein with a neutral sugar content of 9.5%. The carbohydrate specificity of lectin was directed towards D-galactose and its derivatives with pronounced preference for lactose. EiLL had pH optima at pH 7.0; above and below this pH lectin lost sugar-binding capability rapidly. Lectin showed broad temperature optima from 25 to 50 degrees C; however, at 55 degrees C EiLL lost more than 90% of its activity and at 60 degrees C it was totally inactivated. The pI of EiLL was found to be 7.6. The amino acid analysis of EiLL indicated that the lectin was rich in acidic as well as hydrophobic amino acids and totally lacked cysteine and methionine. The N-terminal amino acids were Val-Glu-Thr-IIe-Ser-Phe-Ser-Phe-Ser-Glu-Phe-Glu-Ala-Gly-Asn-Asp-X-Leu-Thr-Gln-Glu-Gly-Ala-Ala-Leu-. Chemical modification studies of both EiLL and Erythrina indica seed lectin (EiSL) with phenylglyoxal, DEP and DTNB revealed an absence of arginine, histidine and cysteine, respectively, in or near the ligand-binding site of both lectins. Modification of tyrosine with NAI led to partial inactivation of EiLL and EiSL; however, total inactivation was observed upon NBS-modification of two tryptophan residues in EiSL. Despite the apparent importance of these tryptophan residues for lectin activity they did not seem to have a direct role in binding haptenic sugar as D-galactose did not protect lectin from inactivation by NBS.     

The reactive sites of proteinase inhibitors from Erythrina seeds

Although the Kunitz-type proteinase inhibitors from the seeds of various Erythrina species have similar molecular weights (approximately 20,000), and share many other chemical characteristics, they could nevertheless be divided into three groups on the basis of their relative abilities to inhibit chymotrypsin, trypsin and tissue plasminogen activator. Group a inhibitors were relatively specific for chymotrypsin; they were poor inhibitors of trypsin and had no apparent effect upon tissue plasminogen activator. Group b proteins inhibited trypsin strongly and chymotrypsin slightly less effectively. They had no effect upon t-PA. Group c inhibitors inhibited trypsin, chymotrypsin and t-PA. Analysis of the amino acid composition of the three groups of inhibitors revealed major differences in alanine content. Minor differences in the content of most other amino acids were also noticed. Group b and group c inhibitors had, in most cases, the same reactive sites (Arg-Ser). The sequences neighbouring the reactive sites showed a significant degree of homology. Chemical modification of arginine in proteinase inhibitors from the seeds of E. latissima and soybeans using 1-2-cyclohexanedione confirmed the presence or absence of arginine in the reactive sites.     

Preliminary crystallographic study of the Erythrina rubrinervia lectin

The galactose-specific lectin from Erythrina rubrinervia crystallizes in the hexagonal space group P6, (or P6(5)) with unit cell dimensions a = b = d = 135.1 A, c = 83.0 A. These parameters are compatible with the presence of a dimer with Mr = 60,000 in the asymmetric unit. The crystals are suitable for high-resolution X-ray analysis.     

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.     

Crystallization and preliminary X-ray diffraction studies of the lectin from Erythrina corallodendron

The lectin from Erythrina corallodendron, specific for N-acetyllactosamine, crystallizes in the hexagonal space group P6(1) (P6(5)) with unit cell dimensions a = b = 136.3 A, c = 83.2 A and one dimer of Mr 60,000 in the asymmetric unit. The crystals are suitable for high-resolution work.     

Properties of a lectin purified from the seeds of Cicer arietinum

A lectin was isolated from seed extracts of Cicer arietinum by (NH4)2SO4 precipitation and subsequent ion exchange chromatography and gel filtration. Affinity chromatography on desialylated human IgM coupled to AH-Sepharose was also performed, but the amount bound was very low. The lectin has a molecular mass of about 44000 Da, as determined by ultracentrifugation and gel filtration. Dodecyl sulphate polyacrylamide-gel electrophoresis showed one band corresponding to a molecular mass of 26000 Da. N-Terminal amino acid sequence analyses indicate only one type of chain, suggesting that the lectin is probably dimeric. The amino acid composition is given. Papainized human erythrocytes of the different ABO groups were agglutinated equally well by the Cicer lectin, whereas untreated cells reacted weakly and only in the presence of bovine serum albumin. Simple sugars did not inhibit the agglutination, but some glycoproteins did inhibit. The lectin is probably nonmitogenic against human lymphocytes. Antigenic analyses in an enzyme-linked immunosorbent assay (ELISA) showed only a weak cross-reaction between Cicer and the lectins in the Vicieae tribe. Thus, our physicochemical and antigenic studies of the Cicer lectin support the botanical reasons recently given for removing the genus Cicer from the Vicieae tribe.     

Precipitation of the D-galactose specific lectin from Erythrina indica by a triantennary complex type oligosaccharide

We have previously demonstrated that a high mannose type glycopeptide is bivalent for binding Concanavalin A (Con A) and can precipitate the lectin (Bhattacharyya L. and Brewer, C.F. (1986) Biochem. Biophys. Res. Commun. 137, 670-674). The present results show that a triantennary complex type oligosaccharide containing nonreducing terminal galactose residues can precipitate the D-galactose/N-acetyl-D-galactosamine specific lectin from Erythrina indica (EIL). The interactions of the oligosaccharide with EIL was investigated by quantitative precipitin analysis. The equivalence point of the precipitin curve indicated that the glycopeptide is trivalent for EIL binding. These results indicate that each arm of the oligosaccharide can independently bind separate lectin molecules leading to precipitation of the complex. These findings are discussed in terms of the possible biological structure-function properties of complex type oligosaccharides.     

Purification and characterization of a lectin from Annona coriacea seeds

A novel lectin, denominated ACLEC, was isolated from Annona coriacea seeds, belonging to the Annonaceae family. The lectin presented one protein band in SDS-PAGE of 14 kDa. Of the sugars tested, Dglucose and D-mannose were the best inhibitors. A search sequence database showed that ACLEC had homology with other plant lectins, belonging to leguminous lectin family.     

Isolation and characterization of a lectin from Annona muricata seeds

A lectin with a high affinity for glucose/mannose was isolated from Annona muricata seeds (Annonaceae) by gel filtration chromatography on Sephacryl S-200, ion exchange chromatography on a DEAE SP-5 PW column, and molecular exclusion on a Protein Pak Glass 300 SW column. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE) yielded two protein bands of approximately 14 kDa and 22 kDa. However, only one band was seen in native PAGE. The Mr of the lectin estimated by fast-performance liquid chromatography-gel filtration on Superdex 75 was 22 kDa. The lectin was a glycoprotein with 8% carbohydrate (neutral sugar) and required divalent metal cations (Ca2+, Mg2+, and Mn2+) for full activity. Amino acid analysis revealed a large content of Glx, Gly, Phe, and Lys. The lectin agglutinated dog, chicken, horse, goose, and human erythrocytes and inhibited the growth of the fungi Fusarium oxysporum, Fusarium solani, and Colletotrichum musae.     

Isolation, purification and characterization of a lectin from the seed of Erythrina costaricensis (Leguminosae)]

A lectin was isolated from the crude extract prepared from the seeds of E. costaricensis. It was purified by gel filtration on Sephadex G-100 followed by DEAE-Sephadex A-50 chromatography. PAGE revealed only one protein band, while analytical isoelectric focusing revealed four bands. The protein is a dimer with M(r) 58kda not united by disulfide bridges. It is a glycoprotein with 6.5% of neutral sugars, stable at 70 degrees C and at a pH range between 2 to 10. The protein exhibited a non specific agglutination of human erythrocytes, nevertheless it differentiated between erythrocytes of animal origin, agglutinating those of rabbit and chicken and not those from horse, goat, sheep or rat. Galactose, N-acetyl-D-galactosamine, lactose and EDTA are inhibitors while Ca++ and Mn++ acted as activators of the agglutination. No change in the blood pressure was observed when the lectin was intravenously injected in rats.     

Effect of glycosylation on the structure of Erythrina corallodendron lectin

The three-dimensional structure of the recombinant form of Erythrina corallodendron lectin, complexed with lactose, has been elucidated by X-ray crystallography at 2.55 A resolution. Comparison of this non-glycosylated structure with that of the native glycosylated lectin reveals that the tertiary and quaternary structures are identical in the two forms, with local changes observed at one of the glycosylation sites (Asn17). These changes take place in such a way that hydrogen bonds with the neighboring protein molecules in rECorL compensate those made by the glycan with the protein in ECorL. Contrary to an earlier report, this study demonstrates that the glycan attached to the lectin does not influence the oligomeric state of the lectin. Identical interactions between the lectin and the non-covalently bound lactose in the two forms indicate, in line with earlier reports, that glycosylation does not affect the carbohydrate specificity of the lectin. The present study, the first of its kind involving a glycosylated protein with a well-defined glycan and the corresponding deglycosylated form, provides insights into the structural aspects of protein glycosylation.     

Purification and some properties of a lectin from the seeds ofTrichosanthes anguina

A galactose-binding lectin was isolated in electrophoretically pure form from the seeds of the snake gourd,Trichosanthes anguina, by affinity chromatography on an immobilised lactose column, as well as on a cross-linkedGuar Gum column. The lectin agglutinates native erythrocytes of human A, B and 0 phenotypes and of rabbit, rat and mouse. The molecular mass of the lectin, as estimated bySephadex G-200 gel chromatography, is 49 kDa. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis, after reduction with β-mercaptoethanol, revealed two polypeptide chains linked by disulphide bonds in the lectin molecule. It contains no covalently linked sugars. Amino acid analysis of the lectin revealed a high content of acidic amino acids, relatively lower proportion of basic amino acids and traces of cysteine and methionine. The lectin has good thermal stability, and is inactivated when oxidised by metaperiodate.     

A mannose-specific lectin from Vicia villosa seeds

A lectin specific to mannose has been purified from Vicia villosa seed by (NH₄)₂SO₄ fractionation, GalNAc-Sepharose and Man-Sepharose affinity chromatography. It was defined as VVL_M, which showed a single band on an acidic-PAGE stained with Coosmassie brilliant blue. The molecular weight of VVL_M was 50 kDa as determined by gel filtration on Biogel P-100 column. The VVL_M molecule consists of 2 distinct subunits with apparent molecular weight of 30 kDa and 22kDa determined by SDS-PAGE. VVL_M has at least four isolectins with similar haemagglutinating activity. Its extinction coefficient is calculated as A_1cm¹ = 16.4 at 280 nm. Sugars could not be detected phenol-sulfuric acid method. The circular dichroism analysis at far UV indicated that VVL_M was a β-sheet-rich protein, and gave no α-helix, 69% β-sheet, 14% β-turn by Provencher and Glockner method. The lectin was inhibited by α-methyl-d-mannose at 12.5 mM and glucose or GlcNAc at 50 mM. The carbohydrate binding specificity of VVL_M was investigated by using affinity chromatography on a VVL_M-Sepharose column. Among various Asn-linked oligosaccharides, core structure Manα1→3(Manα1→6)Manβ1→4GlcNAcβ1→4GlcNAc_OT were found to have high affinity for VVL_M-Sepharose. The antisera of VVL_M did not produce precipitin line with VVL_G in agar double diffusion plate indicating so serological relationship between VVL_M and VVL_G. However VVL_M showed similar immunodeterminants of some other lectins of mannose specificity such as Con A, PSL, LCA and VFL.