Cloning and expression of a sialic acid-binding lectin from the snail Cepaea hortensis

Highly degenerated gene-specific oligonucleotide primers (GSPs) were constructed from the amino acid sequence of tryptic fragments produced from the purified sialic acid-specific lectin of the garden snail Cepaea hortensis. From the albumin glands, the total RNA or the mRNA was prepared. Combination of a universal primer with the GSPs delivered gene-specific fragments of about 650, 620 and 280 bp by polymerase chain reaction (PCR). These fragments were cloned into the vector pDrive (Qiagen) and sequenced. The resulting cDNA sequence consisted of 744 bp, including an open reading frame of 480 bp. The encoded protein consists of 159 amino acids, including the putative signal sequence peptide. The mature protein should comprise 141 amino acid residues with a calculated molecular mass of 15,529 Da. The expression of the recombinant lectin in Escherichia coli resulted in a soluble protein reacting specifically with rabbit antiserum raised against the native lectin.     

The sialic acid-containing lipopolysaccharides of Salmonella djakarta and Salmonella isaszeg (serogroup O: 48): Chemical characterization and reactivity with a sialic acid-binding lectin from Cepaea hortensis

Abstract Lipopolysaccharides (LPS) of Salmonella djakarta and Salmonella isaszeg , as well as of a spontaneous R-mutant of S. djakarta were investigated as to their content in neuraminic acid (Neu) and its individual linkage. The two Salmonella serovars both belong to the O:48 serogroup of Salmonella , but to two different subgroups. LPS of both S-forms contained high amounts of Neu, although in different quantities, whereas the R-form was completely devoid of it. Methylation analysis indicated that Neu is exclusively terminally linked in S. djakarta whereas both terminal and 4-linked Neu were recognized in S. isaszeg . Although terminally linked, a sialidase from Arthrobacter ureafaciens was unable to split Neu even after prolonged incubation from both S-type LPSs. When LPS was first treated by mild alkali, however, the total amount of Neu from S. djakarta LPS and about 50% from that of LPS of S. isaszeg could be removed. In contrast, alkali-treated LPS, but also the non-treated one, proved to be effective inhibitors for a sialic acid-binding lectin from Cepaea hortensis . The resistance of terminal Neu towards sialidase may be due to the presence of an O-acetyl group which would be removed during the methylation analysis but would, especially when linked to C-4, not interfere with the reactivity of the lectin.     

Temporal changes of gene frequencies in Cepaea hortensis

There have been few studies analysing long-term changes of gene frequencies in natural populations. This work is the first report of such changes in the land snail Cepaea hortensis (Mull).
Collections of C. hortensis were made at Silbury Hill, Wiltshire in 1957, 1963 and 1978. The banded phenotype significantly declined in frequency between 1963 and 1978. It is argued that the decline, which was statistically homogeneous at 15 separate sites within the area, was a consequence of natural selection. The average selection coefficient against the banded homozygote is estimated to have been greater than 1096.     

N-acetyl-D-galactosamine/N-acetyl-D-glucosamine--recognizing lectin from the snail Cepaea hortensis: purification, chemical characterization, cloning and expression in E. coli

From the albumin gland of the snail Cepaea hortensis we isolated and characterized a new N-acetyl-D-galactosamine/N-acetyl-D-glucosamine (GalNAc/GlcNAc) specific lectin (CHA-II) which was purified by a combination of affinity chromatography on GalNAc-agarose and gel filtration. The purified native lectin was found to be a multimeric protein, as revealed by SDS-PAGE and MALDI-TOF analysis. In SDS-PAGE the denatured and reduced lectin showed two bands of molecular masses with 17 and 15.5 kDa which reacted equally with anti-CHA-II rabbit antiserum. The lectin was O- and N-glycosylated with [(Gal)2-Man]2-Man-GlcNAc-GlcNAc-Asn as a probable structure for the oligosaccharide. Isoelectric focusing revealed a heterogeneous protein of at least four bands around pH 8.7. Tryptic peptides of CHA-II were N-terminally sequenced and highly degenerated gene specific oligonucleotide primers (GSPs) had been constructed. Using total RNA isolated from albumin glands, cDNAs were produced by the running race technique. Specific PCR fragments were obtained by PCR using GSPs, the universal primer and 5'- or 3'-RACE-cDNAs. The amplified fragments were cloned into the vector pDrive and were sequenced. The resulting total cDNA sequence consisted of 496 base pairs including an open reading frame of 360 base pairs which encoded a protein of 120 amino acids. The protein carried a putative signal peptide. The mature protein was predicted to comprise 99 amino acid residues with a calculated molecular weight of 11,239 Da. The PCR fragment encoding the mature protein was cloned into the vector pQE30 and expressed in E. coli. Recombinant CHA-II lectin was produced as inclusion bodies and extracted by 6 M guanidine hydrochloride. After refolding, the recombinant CHA-II agglutinated specifically human red blood cells of groups A and AB. In immunodiffusion experiments using rabbit antiserum raised against the native lectin, the protein showed a precipitation line of identity with the native lectin.     

Apostatic selection by song thrushes (Turdus philomelos) feeding on the snail Cepaea hortensis

There is no direct evidence that predators exert apostatic selection (the systematic overpredation of commoner forms) on live, naturally polymorphic prey. This study tested whether captive song thrushes ( Turdus philomelos ) select apostatically when presented with dimorphic populations of yellow five-banded and yellow unhanded morphs of the snail Cepaea hortensis. Four thrushes were used. Two were presented with a 9: 1 ratio of five-bandeds to unbandeds and two were presented with 1:9 ratios. Each thrush was given four trials in succession. In each trial 30 snails were presented and the trial was stopped when 15 had been eaten. There were no differences in shell size between morphs or between eaten and uneaten snails of each morph. Three thrushes selected apostatically and one thrush exerted virtually no selection. Overall, there was statistically significant apostatic selection.     

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.     

A lectin from the Chinese bird-hunting spider binds sialic acids

The affinity to sialic acid-containing oligosaccharides of the small-animal lectin SHL-I isolated from the venom of the Chinese bird-hunting spider Selenocosmia huwena is here described for the first time. By a strategic combination of NMR techniques, molecular modeling, and data mining tools it was possible to identify the crucial amino acid residues that are responsible for SHL-I’s ability to bind sialic acid residues in a specific way. Furthermore, we are able to discuss the role of the functional groups of sialic acid when bound to SHL-I. Also the impact of Pro31 in its cis- or trans-form on SHL-I’s ligand affinity is of special interest, since it answers the question if Trp32 is a crucial amino acid for stabilizing complexes between SHL-I and sialic acid. SHL-I can be considered as a proper model system that provides further insights into the binding mechanisms of small-animal lectins to sialic acid on a sub-molecular level.     

Food overlap in co-existing populations of the land snails Cepaea nemoralis (L.) and Cepaeahortensis (Müll)

Faecal analysis of adult Cepaea nemoralis and Cepaea hortensis from a mixed population on chalk grassland shows that the two snail species select the same plant material as food. Herbs are selected in preference to grasses and Urtica dioica is particularly favoured. C. hortensis has the more pronounced preference for senescent material. These results are discussed in relation to competition between the two species.     

Wild birds prefer the familiar morph when feeding on pastry-filled shells of the landsnail Cepaea hortensis (Müll.)

Colour polymorphisms in prey could be maintained if predators concentrate on common morphs and confer a selective advantage on rare morphs. We describe experiments to test whether wild birds feed on pastry-stuffed shells of Cepaea hortensis in a manner that might lead to such apostatic selection. The birds were first given a 'pre-training' choice test of a shell population with equal numbers of yellow unbandeds and yellow five-bandeds; they were then trained on one morph alone, given a second choice test, trained on the other morph and, finally, given a third choice test. The birds preferred five-bandeds in five of the six pre-training tests. In all six experiments the first training session increased the birds' preferences for the morph that was familiar. The results were less clear-cut when selection during pre-training was compared with selection after the second training session. However, a comparison between selection after each of the two training sessions showed that in all six experiments the results were in the direction predicted from the hypothesis that familiar morphs are preferred. This set of experiments is one of the few in which behaviour which could lead to apostatic selection has been tested with morphs that differ in pattern. The findings support the idea that polymorphism in Cepaea could be maintained by apostatic selection.     

Purification and physicochemical characterization of a cotyledonary lectin from Luetzelburgia auriculata

A lectin was purified from the cotyledons of Luetzelburgia auriculata (Fr. All) Ducke by affinity chromatography on agarose-N-acetyl-D-galactosamine. The lectin is a potent agglutinin for rabbit erythrocytes, reacts with human red cells, but is inactive against cow, sheep, and goat erythrocytes. Hemagglutination of rabbit erythrocytes was inhibited by either 0.39 mM N-acetyl-neuraminic acid or N-acetyl-D-galactosamin, 12.5 mM D-lactose or D-melibiose, 50 mM D-galactose or raffinose. Its hemagglutinating activity was lost at 80 degrees C, 5 min, and the activation energy required for denaturation was 104.75 kJ mol(-1). Chromatography on Sephadex G-100, at pH 7.6, showed that at this hydrogenic ionic concentration the native lectin was a homotetramer (123.5 kDa). By denaturing SDS-PAGE, LAA seemed to be composed of a mixture of 29 and 15 kDa polypeptide subunits. At acidic and basic pHs it assumed different conformations, as demonstrated by exclusion chromatography on Superdex 200 HR 10/30. The N-terminal sequence of the 29 kDa band was SEVVSFSFTKFNPNQKDII and the 15 kDa band contained a mixture of SEVVSFSFTKFNPNQKDII and KFNQIVAVEEDTDXESQPQ sequences, indicating that these bands may represent full-length and its endogenous fragments, respectively. The lectin is a glycoprotein having 3.2% neutral carbohydrate, with a pI of 5.8, containing high levels of Asp+Asn and Glu+Gln and hydroxy amino acids, and low amount or absence of sulfur amino acids. Its absorption spectrum showed a maximum at 280 nm and a epsilon (1%) x (1cm) of 5.2. Its CD spectrum was characterized by minima near 228 nm, maxima near 196 nm and a negative to positive crossover at 210 nm. The secondary structure content was 6% alpha-helix, 8% parallel beta-sheet, 38% antiparallel beta-sheet, 17% beta-turn, 31% unordered and others contribution, and 1% RMS (root mean square). In the fluorescence spectroscopy, excitation of the lectin solution at 280 nm gave an emission spectrum in the 285-445 nm range. The wavelength maximum emission was in 334.5 nm, typical for tryptophan residues buried inside the protein.     

A sialic acid-specific lectin from the slug Limax flavus

The slug, Limax flavus, contains a lectin that appears to be highly specific for sialic acid residues of glycoproteins. The carbohydrates which inhibited the hemagglutinating activity of the slug lectin and the concentration of the carbohydrate which gave a 50% inhibition are as follows: N-acetylneuraminic acid, 0.13 mm; N-glycolylneuraminic acid, 0.90 mm; d-glucosamine, 4.9 mm; d-galactosamine, 7.6 mm; N-acetyl-d-glucosamine, 23 mm; and N-acetyl-d-galactosamine, 24 mm. d-Galactose, d-glucose, d-mannose, α-methyl-d-glucoside, α-methyl-d-mannoside, l-arabinose, d-xylose, l-fucose, d-glucuronic acid, lactose, and sucrose were found to be ineffective as inhibitors of the hemagglutinating activity of the slug lectin. Hemagglutination by slug lectin was strongly inhibited by bovine submaxillary mucin and fetuin but not by sialic acid-free bovine submaxillary mucin or fetuin.     

The glycosylation status of murin postnatal thymus: a study by histochemistry and lectin blotting

During the intrathymic development, the fate of the thymocytes depends largely on variable expression of CD4/CD8 markers and T cell receptor protein expressions. In addition, changes of cell surface glycosylation status also affect the thymocyte maturation. In this study the glycosylation alterations in thymic tissues from 1, 9, 13 and 16 days old mice were evaluated by histochemical and lectin blotting techniques. With alcian blue (AB) at pH 5.7/periodic acid-Schiff (PAS) stainings, it was shown that thymic microenvironments contained carboxlylated and sulfated glycosaminoglycans (GAGs). Strong positivity to AB at pH 2.5, which specific for sialomucins, was seen in some medullary thymocytes. Similarly, it was shown that with Maackia amurensis agglutinin (MAL) medullary thymocytes, but not cortical ones, contained alpha(2 --> 3) linked sialic acid structures. On the other hand, while reaction with peanut agglutinin (PNA), which specific for core disaccharide galactose beta(1 --> 3) N-acetylgalactosamine, was only seen in cortical thymocytes, reaction with Galanthus nivalis agglutinin (GNA), which specific for terminal mannose residues, was seen in both cortex and medulla. However, Datura stramonium agglutinin (DSA), which recognizes galactose beta(1 --> 4) N-acetylglucosamine, was not only cell-specific, but it was bound some thymic vessels. With lectin blotting studies, five glycoprotein bands of molecular weights ~39, ~54, 100, ~110 and ~212 were found which reacted with MAL, PNA and DSA as well as GNA. These results suggest that glycosylation patterns of cell surface glycoconjugates are modified during thymocyte selection processes of postnatal days.     

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.     

The karyotype of Cepaea sylvatica (Pulmonata: Helicidae) and its relationship to those of C. hortensis and C. nemoralis

The karyotypes of Cepaea nemoralis (L.) and C. hortensis (Müller), with 2n=44 and a conspicuously large pair of chromosomes, are described and compared with that of C. sylvatica (Draparnaud) which has 2n=50. The karyotype of C. sylvatica also has a conspicuously large pair of chromosomes but the comparison suggests that these have an independent origin from those in the 2n = 44 species. There is no evidence that the large chromosomes in C. nemoralis and C. hortensis have originated from simple fusion of chromosomes from a 2n=50 karyotype with chromosomes all sub-equal such as is reported for C. vindobonensis. It may be that such a karyotype with little size differentiation amongst the chromosomes is not a primitive feature in the Helicinae. The relationship of shell colour and banding polymorphism to the chromosome architecture is discussed.     

Purification and molecular characterization of a sialic acid specific lectin from the phytopathogenic fungus Macrophomina phaseolina

A lectin was isolated and purified from the culture filtrate of the plant pathogenic fungus Macrophomina phaseolina by a combination of ammonium sulfate precipitation, affinity chromatography on fetuin-Sepharose 4B and ion-exchange chromatography on DEAE-A 50. The lectin designated MPL was homogeneous by PAGE and HPLC and a monomeric protein with a molecular weight of approximately 34 kDa as demonstrated by SDS-PAGE. It is a glycoprotein and agglutinated human erythrocytes regardless of the human blood type. Neuraminidase treatment of erythrocytes reduced the agglutination activity of the lectin. It is thermally stable and exhibits maximum activity between pH 6 and 7.2. Its carbohydrate binding specificity was investigated both by hapten inhibition of hemagglutination and by enzyme-conjugated lectin inhibition assay. Although, M. phaseolina lectin bound sialic acid, it exhibited binding affinity towards neuraminyl oligosaccharides of N-linked glycoproteins, alpha-Neu5Ac-(2-->3)-beta-Gal-(1-->4)-GlcNAc being maximum.     

Isolation and characterization of a novel lectin from the mushroom Armillaria luteo-virens

From the dried fruiting bodies of the mushroom Armillaria luteo-virens, a dimeric lectin with a molecular mass of 29.4 kDa has been isolated. The purification procedure involved (NH(4))(2)SO(4) precipitation, ion exchange chromatography on DEAE-cellulose, CM-cellulose, and Q-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The hemagglutinating activity of the lectin could not be inhibited by simple sugars but was inhibited by the polysaccharide inulin. The activity was stable up to 70 degrees C but was acid- and alkali-labile. Salts including FeCl(3), AlCl(3), and ZnCl(2) inhibited the activity whereas MgCl(2), MnCl(2), and CaCl(2) did not. The lectin stimulated mitogenic response of mouse splenocytes with the maximal response achieved by 1microM lectin. Proliferation of tumor cells including MBL2 cells, HeLa cells, and L1210 cells was inhibited by the lectin with an IC(50) of 2.5, 5, and 10 microM, respectively. However, proliferation of HepG2 cells was not affected. The novel aspects of the isolated lectin include a novel N-terminal sequence, fair thermostability, acid stability, and alkali stability, together with potent mitogenic activity toward spleen cells and antiproliferative activity toward tumor cells.     

老鸦烟筒花的化学成分及药理作用研究进展

通过查阅近年来国内外的文献,对紫葳科植物老鸦烟筒花(Millingtonia hortensis L.)的化学成分及药理活性研究现状进行了整理和总结。老鸦烟筒花的主要化学成分和有效成分均为黄酮类化合物,另外还包括生物碱类、环己乙醇类、苯丙素苷类和三萜类等多种类型的化合物,药理活性显著,具有止咳、抗菌、消炎、抗癌、抗突变、驱虫、治疗糖尿病等多种作用。     

Neutral N-glycan patterns of the gastropods Limax maximus, Cepaea hortensis, Planorbarius corneus, Arianta arbustorum and Achatina fulica

The N-glycosylation potentials of Limax maximus, Cepaea hortensis, Planorbarius corneus, Arianta arbustorum and Achatina fulica were analysed by investigation of the N-glycan structures of the skin and viscera glycoproteins by a combination of HPLC and mass-spectrometry methods. It is one of the first steps to enlarge the knowledge on the glycosylation abilities of gastropods, which may help to establish new cell culture systems, to uncover new means for pest control for some species, and to identify carbohydrate-epitopes which may be relevant for immune response. All snails analysed contained mainly oligomannosidic and small paucimannosidic structures, often terminated with 3-O-methylated mannoses. The truncated structures carried modifications by β1-2-linked xylose to the β-mannose residue, and/or an α-fucosylation, mainly α1,6-linked to the innermost N-acetylglucosaminyl residue of the core. Many of these structures were missing the terminal N-acetylglucosamine, which has been shown to be a prerequisite for processing to complex N-glycans in the Golgi. In some species (Planorbarius corneus and Achatina fulica) traces of large structures, terminated by 3-O-methylated galactoses and carrying xylose and/or fucose residues, were also detected. In Planorbarius viscera low amounts of terminal α1-2-fucosylation were determined. Combining these results, gastropods seem to be capable to produce all kinds of structures ranging from those typical in mammals through to structures similar to those found in plants, insects or nematodes. The detailed knowledge of this very complex glycosylation system of the gastropods will be a valuable tool to understand the principle rules of glycosylation in all organisms.