Molecular cloning and characterization of a mannose-binding lectin gene from Crinum asiaticum

Full-length cDNA of a mannose-binding lectin or agglutinin gene was cloned from a traditional Chinese medicinal herb Crinum asiaticum var. sinicum through RACE-PCR cloning. The full-length cDNA of C. asiaticum agglutinin (caa) was 820 bp and contained a 528 bp open reading frame encoding a lectin precursor (preproprotein) of 175 amino acid residues with a 22 aa signal peptide. The coding region of the caa gene was high in G/C content. The first 20 bp of the 5' UTR had a dC content of 50%, which was a typical feature of the leader sequence. By cutting away the signal peptide, the CAA proprotein was 15.79 kDa with a pl of 9.27 and contained 3 mannose-binding sites (QDNY). Random coil and extended strand constituted interlaced domination of the main part of the secondary structure. B-lectin conserved domain existed within N24 to G130. Predicted three-dimensional structure of CAA proprotein was very similar to that of GNA (Galanthus nivalis agglutinin). It is significant that besides certain homologies to known monocot mannose-binding lectins from Amaryllidaceae, Orchidaceae, Alliaceae and Liliaceae, caa also showed high similarity to gastrodianin type antifungal proteins. No intron was detected within the region of genomic sequence corresponding to the caa full-length cDNA. Southern blot analysis indicated that the caa gene belonged to a low-copy gene family. Northern blot analysis demonstrated that caa mRNA was constitutively expressed in all the tested tissue types including the root, bulb, leaf, rachise, flower and fruit tissues.     

Lectin histochemistry of normal human gastric mucosa

Information about the saccharides expressed in gastric mucosa is mostly limited to the glycan content of gastric mucins and there are only a few studies of the glycoprofiling of the constituent cells and their components. Knowledge of the glycan expression of normal gastric mucosa is necessary for the interpretation of the significance of changes of expression in disease. A lectin histochemical study of normal human gastric (body) mucosa was performed using 27 lectins chosen to probe for a wide range of oligosaccharide sequences within several categories of glycoprotein glycans. There were marked differences in staining reactions in the various microanatomical structures of the mucosa, particularly between pits and glands with the former more closely resembling the surface epithelium. A notable feature was the degree of difference in the staining between a substantial sub-population of cells within the neck region and the epithelium of both the pits and glands. These neck cells resembled the pit cells with some lectins, glandular cells with some others and neither with some other lectins. Overall, the differences between the pit, gland and neck epithelia were diverse and numerous, and could not be explained by altered activity of a small set of glycosyltransferases. Widespread alterations of glycans must have occurred (affecting terminal and internal parts of their structures) and the very different glycotypes of the pit, neck and gland epithelia are, therefore, suggestive of the existence of three cell lineages within normal gastric epithelium. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

Implications of oligomeric forms of POD-1 and POD-2 proteins isolated from cell walls of the biocontrol agent Pythium oligandrum in relation to their ability to induce defense reactions in tomato

The cell wall protein fraction (CWP) isolated from the biocontrol agent Pythium oligandrum induces defense reactions in tomato. CWP contains two novel elicitin-like proteins, POD-1 and POD-2, both with seven cysteines. To determine the essential structure in the defense-eliciting components of CWP, five fractions (F1, F2, F3, F4 and F5) were fractionated from CWP using cation chromatography and their components and disulfide bond compositions were analyzed. The expression levels of three defense-related genes (PR-6, LeCAS and PR-2b) were determined in tomato roots treated with each of the five fractions. Of the five fractions, F4 containing a heterohexamer of POD-1 and POD-2, and F5 containing a homohexamer of POD-1, both with disulfide bonds formed between all cysteine residues, induced the expression of three genes. F4 treatment also induced the accumulation of ethylene in tomato. The predicted three-dimensional structures of POD-1 and POD-2, and the results of SEC and MALDI-TOF MS analyses suggest that F4 consists of three POD-1 and POD-2 disulfide-bonded heterodimers that interleave into a hexameric ring through noncovalent association. These results suggest that this structure, which F5 also appears to form, is essential for stimulating defense responses in tomato.     

The titres of lectins in the haemolymph of Lacanobia oleracea and the effects of parasitism by the ectoparasitoid wasp Eulophus pennicornis

Serum from larvae of Lacanobia oleracea L. (Lepidoptera; Noctuidae) parasitized by Eulophus pennicornis (Hymenoptera; Eulophidae) and from normal non‐parasitized larvae is capable of agglutinating rabbit, sheep, calf, goat, chicken, horse and human erythrocytes, but not yeast. Studies with a range of inhibitory carbohydrates showed that serum lectins(s) had specificity for sugars containing galactose and for rhamnose, and for the glycosubstances fetuin and asialofetuin. Lectin activity is heat‐labile and is not dependent on calcium. Parasitism by E. pennicornis caused an increase in the agglutination titre of the serum from larvae of L. oleracea but not an increase in specific activity (titre per mg protein per ml). However, when venom from the venom gland of female wasps was injected into L. oleracea larvae, both the agglutinating activity and the specific activity of the larval serum increased. The possible causes of this increase are discussed. It is suggested that venom contains antigenic components which, when injected into the haemocoel of the L. oleracea larva, may be increasing lectin synthesis and/or release into the serum.     

Amaranthus caudatus lectin with polyproline II fold: conformational and functional transitions and molecular dynamics

Polyproline II (PPII) fold, a peculiar structural element was detected in the Amaranthus caudatus seed lectin (ACL) based on far UV circular dichroism spectrum, conformational transitions of the lectin, and a distinct isodichroic point in thermal denaturation. It was confirmed using PolyprOnline database to estimate the percentage of amino acids contributing to PPII fold and showed the values as 13.5 and 13.9% for PROSS and XTLSSTR, respectively. Investigations of the functional and conformational transitions of ACL during thermal-, pH-, and guanidine hydrochloride (GdnHCl)-induced denaturation were carried out using biochemical and biophysical techniques and molecular dynamics (MD) simulations approach. The lectin got aggregated at 60°C with instantaneous structural alterations. The aggregation-prone regions in ACL were predicted using online servers viz. AGGRESCAN, AmylPred, FoldAmyloid, and Waltz that were represented by Visual Molecular Dynamics tools. Nine conserved regions were identified by these softwares as being ‘hot-spots’ for aggregation. MD simulation studies of the lectin at 60°C revealed increase in radius of gyration. The loss of PPII fold in 2.0 M GdnHCl was reversible. The partially unfolded intermediate of ACL with diminished PPII fold formed at pH 1.0 was stable up to 90°C. The polyproline II fold has been rarely detected in lectins, ACL being the second after the potato lectin.     

Fractionation and characterization of boar seminal plasma spermadhesin PSP-II glycoforms reveal the presence of uncommon N-acetylgalactosamine-containing N-linked oligosaccharides

Lectin mapping, carbohydrate analysis and electrospray mass spectrometry of boar seminal plasma PSP-II glycoforms show that its single N-glycosylation site displays a repertoire of carbohydrate structures consisting of the basic pentasaccharide core Manα 1–6[Manα 1–3]Manβ1-4GlcNAcβ1-4GlcNAc with a fucosyl residue α1-6-linked to the innermost N-acetylglucosamine residue. Other glycoforms display fucosylated hybrid-type or monoantennary complex-type chains, some of which contain α2-6-linked sialic acid. N-acetylgalactosamine, possibly in Galβ1-3GalNAc sequence, is present in most of the PSP-II glycoforms. Abbreviations: PSP-I and PSP-II, porcine seminal plasma proteins I and II; PNGaseF, peptide-N4-(N-acetyl-β-D-glucosaminyl) asparagine amidase (EC 3.5.1.52) from Flavobacterium meningosepticum; ConA, Cannavalia ensiformis (jack bean) agglutinin; GNA, Galanthus nivalis (snowdrop) agglutin; SNA, Sambucus nigra (elderberry) agglutinin; MAA, Maackia amurensis (maakia) agglutinin; PNA, Arachis hypogaea (peanut) agglutinin; DSA, Datura stramonium (jimson weed) agglutinin; AAA, Aleuria aurantia agglutinin This revised version was published online in November 2006 with corrections to the Cover Date.     

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.     

Optimizing lectin-carbohydrate interactions: improved binding of divalent α-mannosylated ligands towards Concanavalin A

The synthesis and binding properties to Jack bean phytohaemagglutinin in (Concanavalin A, Con A) of a new family of divalent α-D-mannopyranoside ligands are described. The synthesis of these ligands is based on the coupling of commercially available diamines to p-isothiocyanatophenyl 2,3,4,6 tetra-O-acetyl-α-D-mannopyranoside (4). The resulting dimers 6, 15 to 22 and 30 were tested for their relative inhibitory potency by solid-phase enzyme-linked lectin assays (ELLA) using methyl α-D-mannopyranoside as standard. Divalent mannosylated ligand 35 bearing a non-aromatic aglycon was also tested for comparison purposes. Concentrations necessary for 50% inhibition (IC50s) of binding of yeast mannan to Jack bean phytohaemagglutinin (Con A) were determined. The inhibitions showed dimers to be approximately 10- to 90-fold more potent than methyl α-D-mannopyranoside. Variations in the intra-mannosyl distance proved to be an important factor for optimum binding. This revised version was published online in November 2006 with corrections to the Cover Date.     

CBP70, a glycosylated nuclear lectin

Some years ago, a lectin designated CBP70 that recognized glucose (Glc) but had a stronger affinity for N-acetylglucosamine (GlcNAc), was first isolated from HL60 cell nuclei. Recently, a cytoplasmic form of this lectin was described, and one 82 kDa nuclear ligand was characterized for the nuclear CBP70. In the present study, the use of Pronase digestion and the trifluoromethanesulphonic acid (TFMS) procedure strongly suggest that the nuclear and the cytoplasmic CBP70 have a same 23 kDa polypeptide backbone and, consequently, could be the same protein. In order to know the protein better and to obtain the best recombinant possible in the future, the post-translational modification of the nuclear and cytoplasmic CBP70 was analyzed in terms of glycosylation. Severals lines of evidence indicate that both forms of CBP70 are N- and O-glycosylated. Surprisingly, this glycosylation pattern differs between the two forms, as revealed by β-elimination, hydrazinolysis, peptide-N-glycosydase F (PNGase F), and TFMS reactions. The two preparations were analyzed by affinity chromatography on immobilized lectins [Ricinus communis-I agglutinin (RCA-I), Arachis hypogaea agglutinin (PNA), Galanthus nivalis agglutinin (GNA), and wheat germ agglutinin (WGA)] and by lectin-blotting analysis [Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), Lotus tetragonolobus (Lotus), succinylated-WGA, and Psathyrella velutina agglutinin (PVA)]. Both forms of CBP70 have the following sugar moities: terminal βGal residues, Galβ1–3 GalNAc, Man α1–3 Man, sialic acid α2–6 linked to Gal or GalNAc; and sialic acid α2–3 linked to Gal. However, only nuclear CBP70 have terminal GlcNAc and α-L-fucose residues. All these data are consistent with the fact that different glycosylation pattern found for each form of CBP70 might act as a complementary signal for cellular targeting. J. Cell. Biochem. 66:370–385, 1997. © 1997 Wiley-Liss, Inc.