Purification and properties of a Ca2+-independent sialic acid-binding lectin from human placenta with preferential affinity to O-acetylsialic acids

A Ca2+-independent sialic acid-specific lectin from two developmental stages of human placenta was similarly purified to apparent homogeneity by DEAE-cellulose chromatography, affinity chromatography on bovine submaxillary mucin, and gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration disclosed a molecular mass of 53 kDa. The specificity of the lectin for O-acetylsialic acids was substantiated by the dependence of hemagglutination on the presence of acetylated sialic acids on the surface of mammalian erythrocytes of various sources, by hapten inhibition in hemagglutination assays with protease-treated rabbit erythrocytes and by hapten inhibition of binding of labeled N-acetylneuraminic acid-bovine serum albumin to the lectin in a solid-phase assay. Bovine and equine submaxillary mucins that contain 9(7,8)-O-acetyl and 4-O-acetylsialic acids were potent inhibitors in contrast to the non-acetylated sialic acids of ovine submaxillary mucin. Absence of inhibitory efficiency of other negatively charged substances like phosphorylated sugars, glucuronic acid, heparin, or oligodeoxynucleotides emphasized the importance of structural features instead of simple ionic interaction. In the presence of acetylation, the pattern of inhibition by gangliosides in the solid-phase assay indicated a preference to alpha-2,8- or alpha-2,6-linked sialic acids in comparison to alpha-2,3-linked moieties. Chemical modification of the lectin by group-specific reagents allowed to emphasize the role of primarily lysine residues, but also, although less pronounced, arginine, tryptophan, and carboxyl groups for ligand binding and/or maintenance of the active conformational state. Application of reagents, specific for histidine or tyrosine residues, failed to affect lectin activity.     

Purification and characterization of a lectin (plant hemagglutinin) with N blood group specificity from Vicia graminea seeds.

A lectin with N blood group specificity was isolated from Vicia graminea seeds. This lectin was purified from a crude extract by precipitation with ammonium sulfate, DEAE-cellulose chromatography and Sephadex G-150 gel filtration. Purification steps were followed by increase of specific activity. Its homogeneity was demonstrated by polyacrylamide gel electrophoresis, immunoelectrophoresis, electrofocusing and ultracentrifugation. This lectin is an acid glycoprotein with 7.3% carbohydrate, a high percentage of serine and contains no sialic acid. The native lectin has a molecular weight about 100 000 and dissociates into four subunits of 25 000 as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Preliminary hemagglutination inhibition has shown that the lectin was not inhibited by any of the monosaccharides contained in N blood group substances; however it was inhibited by the erythrocyte membrane major glycoprotein and the tryptic fragments obtained from erythrocytes.     

Specificity of the sialic acid-binding lectin from the snail Cepaea hortensis.

The specificity of the sialic acid-binding lectin from the snail Cepaea hortensis, purified by affinity chromatography on fetuin-Sepharose, was studied by hemagglutination inhibition assay applying 32 sialic acid derivatives and 14 glycoproteins. 2-alpha-Methyl-9-O-acetyl-NeuAc was the most potent inhibitor, followed closely by 2-alpha-methyl-NeuAc and 2-alpha-benzyl-NeuAc. An axially orientated carboxyl group is a prerequisite for maximal lectin-sugar binding. Neither size nor polarity of the alpha-anomeric substituent significantly influenced inhibition potency. An intact sialic acid N-acetyl group is essential for optimal lectin-sugar interaction. The trihydroxypropyl side chain also is of great importance. However, a bulky hydrophobic substituent at the side chain like a 9-O-tosyl residue did not decrease binding to the lectin. The lectin did not distinguish between NeuAc alpha 2----3Gal beta 1----4Glc and NeuAc alpha 2----6Gal beta 1----4Glc. Among other sugars tested, only N-acetylglucosamine showed inhibition, although 50-fold less. The most potent glycoprotein inhibitors were those carrying O-chains only or preferentially, as ovine submaxillary mucin, bovine submaxillary mucin, and glycophorin A. Tamm-Horsfall protein was an exception being a strong inhibitor, although carrying only N-chains. Asialoglycoproteins were inactive. Glycoproteins containing the NeuAc alpha 2----3Gal sequence inhibited the lectin as well as those with NeuAc alpha 2----6GalNAc. From the results a model of the lectin's binding site for sialic acid is suggested.     

Purification and carbohydrate-binding specificity of Agrocybe cylindracea lectin

A lectin was isolated from fruiting bodies of Agrocybe cylindracea by two ion-exchange chromatographies and gel filtration on Toyopearl HW55F. The lectin was homogeneous on polyacrylamide gel electrophoresis and its molecular mass was determined to be 30 000 by gel filtration, and 15 000 by sodium dodecylsulfate polyacrylamide gel electrophoresis, signifying a dimeric protein. Its carbohydrate-binding specificity was investigated both by sugar-hapten inhibition of hemagglutination and by enzyme-linked immunosorbent assay. The inhibition tests showed the affinity of the lectin to be weakly directed toward sialic acid and lactose, and the enhanced affinity toward trisaccharides containing the NeuAcα2,3Galβ-structure. Importantly, the lectin strongly interacted with glycoconjugates containing NeuAcα2,3Galβ1,3GlcNAc-/GalNAc sequences. This revised version was published online in November 2006 with corrections to the Cover Date.     

Isolation of an acidic protein from the armadillo submandibular gland

An acidic protein fraction with an apparent molecular weight of 34 000 has been isolated from the Cetavlon-treated, mucin-free supernatant of the armadillo submandibular gland 0.01 M NaCl extract. This purified material, which was obtained in a yield of 0.45%/g wet gland, contains 24 mol % acidic amino acids and 4 mol % basic amino acids. Hexosamines, sialic acid, and neutral sugars represent 7% of the dry sample weight. In polyacrylamide gel and cellulose acetate electrophoresis, a single protein band was observed. The acidic protein fraction is highly reactive with the Lowry phenol reagent, giving a protein value 83% higher than that obtained by summation of its anhydrous amino acids, and is explained by the occurrence of peptide linkages peculiar to this material. The presence of other basophilic components besides mucus glycoproteins within the salivary gland of the armadillo may have physiological significance.     

Structural parameters and natural occurrence of 2-deoxy-2,3-didehydro-N-glycoloylneuraminic acid

2-Deoxy-2,3-didehydro-N-glycoloylneuraminic acid has been found to occur in porcine, bovine and equine submandibular glands as well as in the urine of pig, horse and rat. This novel, unsaturated sialic acid was isolated by gel filtration and ion-exchange chromatography. Final purification was achieved by column chromatography or by preparative thin-layer chromatography on cellulose. The structural analysis was performed by combined capillary gas-liquid chromatography/mass spectrometry. The various data were compared with those from synthetic 2-deoxy-2,3-didehydro-N-glycoloylneuraminic acid. Besides of the unsaturated N-glycoloylated sialic acid, also the corresponding N-acetylated derivative was present in the materials analyzed. The inhibitory effect of 2-deoxy-2,3-didehydro-N-glycoloylneuraminic acid on Vibrio cholerae sialidase using N-acetylneuraminyl-(alpha 2----3)-lactose as substrate is slightly higher (50% inhibition at 10 microM) when compared with 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (50% inhibition at 15 microM).     

Sialoglycoderivatives of bovine submandibular gland identified in situ by histochemical techniques combined with lectins.

We employed sialidase procedures followed by lectin stainings combined with oxidizing and deacetylating agents to visualize the distribution and sequentiate sialoglycoconjugates in the bovine submandibular gland. In particular we evidenced in acinar and ductal cells the dishomogeneous presence of sialic acids acetylated in the polyhydroxy side chain (C7, C8, C9), whereas O-acetyl substituents at position C1 and/or C4 were not found. Sialoglycoderivatives were also differentiated by the occurrence of penultimate sugars; indeed the dimers sialic acid-(alpha 2----3,6)-beta-galactose and sialic acid-(alpha 2----6)-alpha-N-acetylgalactosamine were identified. Using such technique we supported further the possibility to develop methods for the identification of the positions of O-acetyl groups and the reconstruction of terminal disaccharides within surface and cytoplasm glycoconjugates. in situ the distribution of different O-acylated sialoglyco-derivatives in the bovine submandibular gland. To this purpose we employed oxydizing and deacetylating agents combined with sialidase digestion and lectin binding.     

Isolation and characterisation of a sialic acid-binding lectin (carcinoscorpin) from Indian horseshoe crab Carcinoscorpius rotunda cauda

A sialic acid-binding lectin, carcinoscorpin, has been purified to apparent homogeneity in 40% yield from the Indian horseshoe carb, Carcinoscorpius rotunda cauda. This glycoprotein lectin of molecular weight 420,000 was composed of two non-identical subunits of molecular weights 27,000 and 28,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. The hemagglutination activity of the lectin was susceptible to guanidine-HCl; modification of tyrosyl and tryptophanyl residues also inhibited the activity although alkylation of the -SH group, reduction of disulfide bonds or modification of amino and carboxyl groups were without any effect. The monomeric form of the lectin produced by succinylation of native protein was inactive in binding to sialoglycoconjugates.     

Purification and characterization of an O-acetylsialic acid-specific lectin from a marine crab Cancer antennarius

A sialic acid-binding lectin with high specificity for 9-O-acetyl- and 4-O-acetylsialic acids was purified from the hemolymph of the California coastal crab, Cancer antennarius, by affinity chromatography using bovine submaxillary mucin coupled to agarose. The binding specificity of the crab lectin distinguishes it from other known sialic acid-specific lectins from Limulus polyphemus and Limax flavus which show a broader range of specificity for sialic acids. The purified lectin is homogenous on sodium dodecyl sulfate-polyacrylamide electropherograms with a subunit molecular weight of about 36 kDa. The specificity of the lectin for O-acetylsialic acids appears to account for the fact that it agglutinates mouse, rat, rabbit, and horse erythrocytes, which contain O-acetylsialic acids on cell surface glycoconjugates, but not human monkey, sheep, goat, and chicken erythrocytes which contain only NeuAc or N-glycolylneuraminic acid (NeuGc). This conclusion was supported by the potent inhibition of hemagglutination by bovine and equine submaxillary mucins which contain 9(7,8)-O-acetyl- and 4-O-acetylsialic acids, respectively, and also by free 9-O-acetyl-N-acetylneuraminic acid (9-O-Ac-NeuAc) and 4-O-Ac-NeuAc relative to NeuAc and NeuGc. Further support for the role of O-Ac-sialic acids in hemagglutination of erythrocytes was obtained by enzymatic modification of human erythrocytes. Sialidase-treated erythrocytes were resialylated with purified sialyltransferases and various CMP-sialic acid donor substrates to contain NeuAc or NeuGc or 9-O-Ac-NeuAc in the Sia alpha 2,3Gal or Sia alpha 2,6Gal linkages. Cells resialylated to contain NeuAc or NeuGc were not agglutinated, but cells resialylated to contain 9-O-Ac-NeuAc were agglutinated with high titer, comparable to that of mice or horse erythrocytes.     

An unique specificity of a sialic acid binding lectin AchatininH, from the hemolymph of Achatina fulica snail

A sialic acid-binding lectin, AchatininH, from the hemolymph of Achatina fulica snail is found to be highly specific for 9-0-acetyl sialic acid. The binding specificity of AchatininH distinguishes it from other known sialic-acid specific lectins which usually show a broader range of specificity for sialic acid. It is even better than crab lectin which shows specificity for both 4- and 9-0-acetylated derivatives of sialic acid. This limited specificity of AchatininH appear to account for the fact that it agglutinates only rabbit, rat and guinea pig erythrocytes which contain 9-0-acetylated sialic acid but not horse (mainly contain 4-0-acetylated sialic acid), human, monkey, sheep, goat and chicken erythrocytes which contain either N-acetyl or N-glycolyl neuraminic acid but no 0-acetylated derivatives. This finding was further supported by the potent inhibition of hemagglutination by free 9-0-acetylated neuraminic acid and by several glyco shingolipids of human origin having 0-acetylated sialic acid.     

A sialic acid-binding lectin from ovine placenta: purification,specificity and interaction with actin

A sialic-acid-specific lectin from ovine placental cotyledons was purified by affinity chromatography on bovine submaxillary mucin-agarose followed by gel filtration, and it showed a molecular weight of 65 000 by sodium dodecylsulfate-polyacrylamide gel electrophoresis. This lectin has the capacity to interact with actin, since in binds to actin-F in a cosedimentation assay and it acts as a mediator in the binding of action to the affinity column. The lectin agglutinated rabbit and rat erythrocytes, but not human A, B or O erythrocytes. Haemagglutination inhibition assays of different saccharides, glycoproteins and glycolipids indicate that this lectin has affinity for sialic acid, which is enhanced by its O-acetylation. The N-terminal sequence of the protein shows 92% identity with rabbit and porcine uterine calreticulin.     

A lectin from the lichenized Basidiomycete Dictyonema glabratum

A lectin with hemagglutinating activity has been isolated from an aqueous extract of the symbiotic phenotype of Dictyonema glabratum and its cyanobacterial photobiont Scytonema sp. The purified lectin had a pI of 6.8 and its molecular mass was investigated by electrospray ionization mass spectrometry, gel filtration and SDS-PAGE, which indicated its native conformation as a dimer formed by two identical subunits of 16540 Da. The lectin is a glycoprotein with a low degree of glycosylation, containing galactose, xylose, glucose and mannose as neutral monosaccharides, in addition to glucosamine, which could indicate both N - and O -linkages. Amino acid analysis showed the predominance of nonpolar residues such as phenylalanine. Agglutination of human erythrocytes required divalent cations, which is affected by addition of EDTA. The lectin was more stable at 30 °C or less for at least 1 h and between pH 5.0 and 7.0. Among the various compounds tested for hemagglutination inhibition, N -acetylgalactosamine was the most active. The potential role of this lectin in recognition of the compatible cyanobacterial photobiont is discussed.     

Isolation and characterization of receptor sialoglycoprotein for hemagglutinating virus of Japan (Sendai virus) from bovine erythrocyte membrane

Sialoglycoprotein which exhibits inhibitory activity for hemagglutination by Hemagglutinating Virus of Japan (HVJ, Sendai virus) was isolated from the membrane of bovine erythrocytes. Purification steps for this sialoglycoprotein included extraction with lithium diiodosalicylate, phenol partition, precipitation with ethanol, and chromatography on a phosphocellulose column and an SDS-Sepharose CL-4B column. Purified sialoglycoprotein (GP-2) has high specific activity for inhibiting the hemagglutination with HVJ, and a lesser activity for that with Newcastle disease virus, but it does not inhibit the hemagglutination by influenza A virus. Inhibitory activity of GP-2 on hemagglutination by HVJ is 2,500-fold higher than that of fetuin. Liposomes containing a 10,000-fold larger amount of ganglioside mixture of bovine erythrocytes and those containing a 5,000-fold larger amount of each ganglioside of bovine erythrocytes, N-glycolylneuraminosyl-lactosyl ceramide, sialosyllacto-N-neotetraosyl- and sialosyl-lacto-N-norhexaosyl ceramide, had no inhibitory activity toward hemagglutination with HVJ. GP-2 (mol. wt. 250 K daltons) behaved homogeneously in SDS-polyacrylamide gel electrophoresis. It contained 70% carbohydrate and 30% protein, by weight. N-Acetylgalactosamine, N-acetylglucosamine, galactose, sialic acid (N-glycolylneuraminic acid, 96%; N-acetylneuraminic acid, 4%) were identified as carbohydrate components, in molar ratios of 1.0:4.0:5.2:2.9. All the oligosaccharides of GP-2 appeared to be linked to polypeptide chains by alkali-labile O-glycosidic linkages. Sialidase treatment of GP-2 and conversion of sialic acid residue of the glycoprotein to C8 and C7 analogues resulted in the loss of the inhibitory activity on hemagglutination by HVJ. Oligosaccharides isolated by gel filtration after treatment of GP-2 with alkaline borohydride had also lost the ability to inhibit the hemagglutination by HVJ. The above results indicate that isolated sialoglycoprotein is the endogenous receptor in bovine erythrocyte membrane specific to HVJ, and the hydroxy group linked to the 9-carbon atom of sialic acid and probably also the hydrophobic protein moiety are important for the recognition of HVJ attachment.     

Analysis of the carbohydrate binding specificity of the mushroom Pleurotus ostreatus lectin by surface plasmon resonance

The sugar binding specificity of the mushroom Pleurotus ostreatus lectin (POL) was analyzed by surface plasmon resonance. The lectin was immobilized to a sensor chip, and asialo-bovine submaxillary mucin (asialo-BSM), one of the most potent inhibitors in the hemagglutination inhibition assay, tightly bound to the lectin. The binding specificity of various mono- or oligosaccharides to the lectin was evaluated by the coinjection method. The dissociation of asialo-BSM was promoted by injection of some haptenic saccharides. For the most part, the order of acceleration ability of the sugars to the dissociation in the coinjection experiment agreed with that of the inhibitory potency of each sugar evaluated by the hemagglutination inhibition assay. In conclusion, POL recognized a galactosyl residue, and the specificity was increased by substitution at the C-2 position of the galactosyl residue with a fucosyl or acetylamino group. This method using the coinjection method proved useful in analysis of carbohydrate-lectin binding specificity.     

Isolation, characterization, and localization of antigen gamma, a serine proteinase of the "kallikrein-family" in the rat submandibular gland

A trypsin-like serine proteinase, antigen gamma, immunologically partially identical to glandular kallikrein when run against anti-rat glandular kallikrein antiserum in immunoelectrophoresis, was purified from the rat submandibular gland. The enzyme was purified by a two-step chromatography procedure, ionexchange chromatography followed by gel filtration. The criteria for purity were one band in SDS-polyacrylamide gel electrophoresis and in immunoelectrophoresis, respectively. Antigen gamma had a molecular mass of 25,000 Da and consisted of two polypeptide chains with molecular masses of 14,000 and 11,000 Da. The preparation contained several isoenzymes with pI ranging from 4.1 to 4.5. The enzyme showed high specific enzyme activity against the substrate D-valyl-L-leucyl-L-arginine-4-nitroanilide (S-2266), some trypsin-like and kininogenase activity, but no angiotensin converting enzyme, kininase, or tonin activity. Amidolytic activity was increased and stabilized by the presence of detergent in the assay buffer. The pH-optimum of antigen gamma amidolytic activity was about 10. Antigen gamma was inhibited by SBTI and PMSF, whereas aprotinin had to be added in a more than 100 times higher concentration than for glandular kallikrein. The binding pattern of antigen gamma to plasma proteins was different from that of tonin and glandular kallikrein. Antiserum against antigen gamma was raised in rabbits and characterized against rat submandibular gland homogenate. Immunohistochemistry showed antigen gamma in the secretory granules of the submandibular gland granular tubular cells but only adhering to the luminal cell wall in the striated and main excretory ducts. Antigen gamma was not detected in the sublingual or parotid gland or in the kidney. Antigen gamma was demonstrated by immunoelectrophoresis in rat submandibular gland saliva. The concentration was higher in sympathetically than in parasympathetically induced secretion.     

Purification of a Sperm Lectin Extracted from Spermatozoa of the Sea Urchin Hemicentrotus pulcherrimus

Hemagglutinating activity for human type A erythrocytes was detected in a sperm extract obtained by treatment with Triton X-100 of spermatozoa from the sea urchin Hemicentrotus pulcherrimus. Among tested sugars only N-acetyl-D-galactosamine had any inhibitory effect on the hemagglutinating activity of the sperm extract. The lectin was purified by a combination of affinity chromatography and ion-exchange chromatography. A single band was obtained after SDS-polyacrylamide gel electrophoresis of the purified lectin, corresponding to an apparent molecular weight of 15,000 daltons. Trypsin-generated fragments of the surface of eggs significantly inhibited hemagglutination of erythrocytes by the purified lectin. The biological role of the sperm lectin is discussed.     

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.     

Isolation and Characterization of a Sialic Acid-specific Lectin from Hemolymph of the Southeast Asian Horseshoe Crab Tachypleus gigas

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

Purification and characterization of a β-galactoside-binding soluble lectin from rat and bovine brain

A β-galactoside-binding activity has been detected in mammalian brain extracts using a hemagglutination test and a nerve cell aggragation assay. Inhibition studies suggested the involvement of lectin-carbohydrate interactions in these processes. In an attempt to explore further the biological role of brain lectins, the β-galactoside-binding activity has been purified to apparent homogeneity from bovine and rat brain by salt extraction of the brain tissue and affinity chromatography on asialofetuin-agarose. The molecular weights determined by gel filtration, under native conditions on Ultrogel AcA-34, were 30 000 for the bovine brain lectin and 32 000 for the rat brain lectin; polyacrylamide gel electrophoresis in SDS gave molecular weights of 15 000 and 16 000, respectively, suggesting that the two brain lectins are dimers. Both lectins have an isoelectric point of 3.9. Amino acid composition data indicate that both lectins contain high proportions of glycine and acidic amino acids. The lectins are specific for β-D-galactosides and related sugars and the configuration of carbon atoms 1, 2 and 4 seems of primary importance. Moreover, the nerve cell aggregation-promoting activity of the purified lectin is 300-fold that of the crude extracts.     

N-glycolylneuraminic acid specific lectin from Pila globosa snail

A N-glycolylneuraminic acid-specific lectin (PAL) has been purified from an albumin gland extract of the apple snail, Pila globosa. Purification is conducted on a bovine submaxillary mucin-Sepharose 4B affinity matrix followed by gel filtration on a Sepharose 6B column. The lectin agglutinates rabbit erythrocytes. The hemagglutination activity is dependent on Ca2+ concentration in a significant manner but with a remarkable behaviour. The lectin is a trimeric glycoprotein of native Mr 440 kDa with 25% carbohydrate and is composed of three nonidentical subunits of molecular weights 190, 145, and 105 kDa. It has a pI of 7.0. The lectin exhibits a unique and strict specificity toward N-glycolylneuraminic acid and this phenomenon discriminates it from other known sialic acid binding lectins. The uniqueness indicates the absolute need for a glycolyl substitution on the amino residue and of a glyceryl side chain on the exocyclic part and an axial -COOH group in neuraminic acid. The presence of an acetyl substitution on the exocyclic part impedes lectin-ligand interaction.