Purification of a PHA-like chitin-binding protein from Acacia farnesiana seeds: a time-dependent oligomerization protein.

A lectin-like protein from the seeds of Acacia farnesiana was isolated from the albumin fraction, characterized, and sequenced by tandem mass spectrometry. The albumin fraction was extracted with 0.5 M NaCl, and the lectin-like protein of A. farnesiana (AFAL) was purified by ion-exchange chromatography (Mono-Q) followed by chromatofocusing. AFAL agglutinated rabbit erythrocytes and did not agglutinate human ABO erythrocytes either native or treated with proteolytic enzymes. In sodium dodecyl sulfate gel electrophoresis under reducing and nonreducing conditions, AFAL separated into two bands with a subunit molecular mass of 35 and 50 kDa. The homogeneity of purified protein was confirmed by chromatofocusing with a pI = 4.0 +/- 0.5. Molecular exclusion chromatography confirmed time-dependent oligomerization in AFAL, in accordance with mass spectrometry analysis, which confers an alteration in AFAL affinity for chitin. The protein sequence was obtained by a liquid chromatography quadrupole time-of-flight experiment and showed that AFAL has 68% and 63% sequence similarity with lectins of Phaseolus vulgaris and Dolichos biflorus, respectively.

Lectin of Pisum arvense seeds induces in-vivo and in-vitro neutrophil migration.

PAL is a glucose/mannose-specific lectin isolated from Pisum arvense seeds. Previously, we demonstrated the capacity of other leguminous lectins to induce oedema formation and neutrophil stimulation. To investigate the potential pro-inflammatory activity of PAL, we have studied its ability to induce neutrophil migration into peritoneal cavities of rats and neutrophil chemotaxis in-vitro. The role of resident cells and sugar residues on PAL activity was analysed. PAL or saline (control) were administered intraperitoneally to rats, and total and differential leucocyte (macrophages, neutrophils and mast cells) counts were performed. The role of resident cells on the PAL effect was evaluated using three strategies: reducing the total resident cell population by lavage of rat cavities with saline; increasing macrophage population by treating animals with thioglycolate; and depleting mast cell population by subchronic treatment of rats with compound 48/80. PAL induced in-vitro and in-vivo neutrophil migration. In-vivo, PAL (50, 100, 200 and 300 microg) significantly (P < 0.05) and dose-dependently increased neutrophil migration by 600, 740, 900 and 940%, respectively, showing maximal effect 4 h after injection. PAL induced mononuclear cell migration. The neutrophil stimulatory effect of PAL was potentiated in animals treated with both thioglycolate and compound 48/ 80. The indirect lectin chemotactic effect was shown in rats injected with supernatant from cultured macrophages stimulated by PAL. In conclusion, PAL was shown to exhibit in-vivo and in-vitro proinflammatory activity. The in-vivo effect seemed to occur by a dual mechanism that was independent, but also dependent, on resident cells.

Characterization of the sugar-binding specificity of the toxic lectins isolated from Abrus pulchellus seeds.

The sugar-binding specificity of the toxic lectins from Abrus pulchellus seeds was investigated by combination of affinity chromatography of glycopeptides and oligosaccharides of well-defined structures on a lectin-Sepharose column and measurement of the kinetic interactions in real time towards immobilized glycoproteins. The lectins showed strong affinity for a series of bi- and triantennary N-acetyllactosamine type glycans. The related asialo-oligosaccharides interact more strongly with the lectins. The best recognized structures were asialo-glycopeptides from fetuin. Accordingly, the kinetic interaction with immobilized asialofetuin was by far the most pronounced. Human and bovine lactotransferrins and human serotransferrin interacted to a lesser extent. The interaction with asialofetuin was inhibited by galactose in a dose dependent manner. Lactose, N-acetyllactosamine and lacto-N-biose exhibited similar degree of inhibition while N-acetylgalactosamine was a poor inhibitor. These results suggested that the carbohydrate-binding site of the Abrus pulchellus lectins was specific for galactose and possess a remarkable affinity for the sequences lactose [beta-D-Gal-(1-->4)-D-Glc], N-acetyllactosamine [beta-D-Gal-(1-->4)-D-GlcNAc] and lacto-N-biose [beta-D-Gal-(1-->3)-D-GlcNAc].

Chemical characterization of the lectin from Amaranthus leucocarpus syn. hypocondriacus by 2-D proteome analysis.

In this work, we characterized chemically the N-acetyl-D-galactosamine specific lectin from Amaranthus leucocarpus syn hypocondriacus lectin (ALL). It is a dimeric glycoprotein composed by three isoforms with pl at 4.8, 4.9, and 5.2. Circular dichroism analysis indicated that the secondary structure of ALL contains 45% of \bibeta-sheet and 5% of \bialpha-helix. Amino acid sequence of the purified lectin and its isoforms was determined from peptides obtained after trypsin digestion by MALDI-TOF (Matrix assisted laser desorption ionization-time of flight). The tryptic peptides prepared from the purified lectin and the three isoforms showed different degrees (80 to 83%) of identity with the amino acid sequence belonging to a previously described high nutritional value protein from A. hypocondriacus not shown at the time to be a lectin. Furthermore, analysis of tryptic peptides obtained from ALL previously treated with peptide N-glycosidase, revealed a 93% identity with the aforementioned protein. Presence of N-glycosidically linked glycans of the oligomannosidic type and, in minor proportion, of the N-acetyllactosaminic type glycans was determined by affinity chromatography on immobilized Con A.

Isolation of the receptor for Amaranthus leucocarpus lectin from murine naive thymocytes.

From murine medullary thymocytes we purified the receptor for the Amaranthus leucocarpus lectin (ALL) using a complex with the biotin-labeled lectin and avidin-agarose as the affinity matrix. Most ALL(+)thymocytes (83%) are naive cells with the CD4(+)CD8(-)CD45RB(+)phenotype. The receptor for this lectin is a 70 kDa glycoprotein that contains 20% of sugar by mass. It is constituted mainly by aspartic and glutamic acids, serine, proline, and glycine; its glycosidic portion contains mainly O-glycosidically linked glycans with Gal, GalNAc and NeuAc residues as well as one N-glycosidically linked glycan per molecule. Ionic strength chromatography revealed that the ALL-thymocyte receptor (ALLTr) is made up by three isoforms, which possess similar amino acid composition but show slight differences in their sugar composition. The N-terminal amino acid residues are blocked both in the receptor and its purified isoforms. Analyses of the receptor's peptides, obtained by trypsin digestion with MALDI-TOF (matrix assisted laser desorption ionization-time of flight), were compared with the relative values obtained from the NCBInr (Swiss-Prot 10/01/99) database. Our results indicate that the peptides of ALLTr show low homology (<17%) with the human KIIA protein, the Fas-associated death domain protein, and the transforming growth factor-beta type II receptor. Our results suggest that the ALL thymocyte receptor could be considered a novel phenotypic marker specific for naive T cells.

Purification and characterization of an adhesin from Pasteurella haemolytica.

We purified an adhesin from Pasteurella. haemolytica by affinity chromatography using glutaraldehyde treated rabbit erythrocytes stroma. The adhesin is a protein of 68 kDa, as determined by SDS-PAGE, and the most abundant amino acids constituting this protein were Gly, Ser, Glx, and Ala, and low concentrations of Cys, Met, and Tyr residues were also found. The N-terminal sequence of the adhesin is ANEVNVYIYKQPYLI. No carbohydrate residues were detected. The adhesin agglutinated rabbit erythrocytes but when the latter were desialylated or pronase treated the agglutinating activity was abolished. The agglutinating activity of the adhesin was inhibited with N-acetyl-D-glucosamine (GlcNAc), and in a lesser degree with N-acetyl-neuraminic acid (NeuAc). GalNAc, N-glycolyl-neuraminic acid, N-deacetylated GlcNAc, or neutral sugars do not modify the activity of the adhesin. The equatorial -OH on C4 and the NH-acetylated group on C2 from GlcNAc, as well as the 4-OH and NH-acetylated group on C5 from NeuAc seem to be responsible for the interaction with the adhesin. The protein is divalent cation-dependent and thermolabile. As for the agglutinating activity, the adhesion of P.haemolytica to tracheal cell-cultures was inhibited by GlcNAc, NeuAc or the purified adhesin, strongly suggesting that the P.haemolytica adhesin plays an important role in infection.

A comparative study on the purification of the Amaranthus leucocarpus syn. hypocondriacus lectin.

Amaranthus leucocarpus lectin is a homodimeric glycoprotein of 35 kDa per sub-unit, which interacts specifically with N-acetyl-galactosamine. In this work, we compared different glycoproteins that contain Galbeta1-3 GalNAcalpha1-3 Ser/Thr or GalNAcalpha1-3 Ser/Thr in their structure as ligands to purify the A. leucocarpus lectin. From the glycoproteins tested, fetuin was the most potent inhibitor of the hemagglutinating activity and the better ligand for lectin purification; however, the use of desialylated stroma from erythrocytes represented the cheapest method to purify this lectin. O-linked glycans released from the glycoproteins used as affinity matrix and those from different erythrocytes were less inhibitory than parental glycoproteins. The NH2-terminal of the lectin is blocked; moreover, this is the only example of a lectin isolated from this genus to be a glycoprotein. Analysis of the glycoprotein sequences with inhibitory activity for the lectin, showed a different pattern in the O-glycosylation, which confirms that A. leucocarpus lectin recognizes conformation and, probably, distances among O-linked glycans moieties.

Characterization and molecular cloning of the lectin from Helianthus tuberosus.

A lectin called Helianthus tuberosus agglutinin or Heltuba has been isolated from tubers of the Jerusalem artichoke, a typical representative of the Asteraceae family. Heltuba is a tetrameric protein composed of four identical subunits of 15.5 kDa and exhibits a preferential specificity towards oligomannosides. Cloning of the corresponding cDNAs revealed that the mature lectin polypeptide comprises the entire open reading frame of the cDNA suggesting that the primary translation product is not processed and that the lectin is a cytosolic protein. Searches in the databases revealed sequence similarity with lectins from the taxonomically unrelated Convolvulaceae and Moraceae species. Therefore, the discovery of Heltuba is of great importance in view of the occurrence and molecular evolution of the jacalin-related lectins.

Isolation of isolectins from Vitis vinifera L. Cv. Chardonnay grape berries.

A lectin fraction from Chardonnay grape juice has been isolated by affinity chromatography on a column of p-aminophenyl beta-D-glucoside-derivatized agarose. The lectin fractions agglutinate rabbit and human erythrocytes without serological specificity. None of the usual monosaccharides, glycosides, or glycoproteins inhibit the hemagglutinating activity. Erythroagglutination is only inhibited by nitrophenyl glycosides, p-nitrophenyl beta-D-glucoside being the strongest inhibitor. In SDS-PAGE in the presence of 2-mercaptoethanol and gel filtration HPLC, the lectin fraction gave a single band or peak corresponding to M(r) 13.2-11.9 kDa, thus indicating it to be a monomer. Three bands were observed by isoelectric focusing with pI values of 4.1, 4. 4, and 4.9. The isolectins seem to be glycoproteins since they are bound on a concanavalin A-Sepharose column.

Specificity of the isolectins from the plant cactus Machaerocereus eruca for oligosaccharides from porcine stomach mucin.

Sugar specificity of the Machaerocereus eruca isolectins, MeAI and MeAII, has been determined by comparing the capacity of glycans with well defined structures to inhibit their haemagglutinating activity. Both are galactose-specific isolectins with high affinity for O-glycans. However, the two M. eruca isolectins recognize different oligosaccharidic sequences belonging to O-glycosidically linked glycans from porcine stomach mucin. The minimal structure recognized by MeAI on the porcine mucin glycans is the O-glycan core Gal beta 1,3GalNAc-ol, whereas MeAII has a more extended site and interacts with a biantennary O-glycan possessing the terminal trisaccharide Fuc alpha 1,2 (GalNAc alpha 1,3) Gal beta 1,4.

Flow cytofluorimetric analysis of young and senescent human erythrocytes probed with lectins. Evidence that sialic acids control their life span.

Comparing the properties of 'young' and senescent ('aged') O+ erythrocytes isolated by applying ultracentrifugation in a self-forming Percoll gradient, we demonstrate that the sialic acids of membrane glycoconjugates control the life span of erythrocytes and that the desialylation of glycans is responsible for the clearance of the aged erythrocytes. This capture is mediated by a beta-galactolectin present in the membrane of macrophages. The evidence supporting these conclusions is as follows: (1) Analysis by flow cytofluorimetry of the binding of fluorescein isothiocyanate labelled lectins specific for sialic acids shows that the aged erythrocytes bind less WGA, LPA, SNA and MAA than young erythrocytes. The binding of DSA and LCA is not modified. On the contrary, the number of binding sites of UEA-I specific for O antigen and of AAA decreases significantly. PNA and GNA do not bind to erythrocytes. (2) RCA120 as well as Erythrina cristagalli and Erythrina corallodendron lectins specific for terminal beta-galactose residues lead to unexpected and unexplained results with a decrease in the number of lectin binding sites associated with increasing desialylation. (3) The glycoconjugates from the old erythrocytes incorporate more sialic acid than the young cells. This observation results from the determination of the rate of transfer by alpha-2,6-sialyltransferase of fluorescent or radioactive N-acetylneuraminic acid, using as donors CMP-9-fluoresceinyl-NeuAc and CMP-[14C]-NeuAc, respectively. (4) Microscopy shows that the old erythrocytes are captured preferentially by the macrophages relative to the young ones. Fixation of erythrocytes by the macrophage membrane is inhibited by lactose, thus demonstrating the involvement of a terminal beta-galactose specific macrophage lectin. (5) Comparative study of the binding of WGA, LPA, SNA and MAA to the aged erythrocytes and to the in vitro enzymatically desialylated erythrocytes shows that the desialylation rate of aged cells is low but sufficient to lead to their capture by the macrophages.

Fine sugar specificity of the mistletoe (Viscum album) lectin I.

The behaviour of N-acetyllactosamine-type oligosaccharides and glycopeptides on a column of mistletoe lectin I (MLI) immobilized on Sepharose 4B was examined. The immobilized lectin does not show any affinity for asialo-N-glycosylpeptides and related oligosaccharides, which possess one to four unmasked N-acetyllactosamine sequences. However, substitution of at least one of the N-acetyllactosamine sequences by sialic acid residues, either at O-3 or O-6 of galactose, slightly enhances the affinity of the lectin. Such sialylated N-glycosylpeptides or oligosaccharides are eluted from the lectin column by the starting buffer as retarded fractions. Surprisingly, the affinity of the immobilized MLI is higher for P1 antigen-containing glycopeptide isolated from turtle-dove ovomucoid and for glycopeptides from bovine thyroglobulin containing terminal non-reducing Gal alpha 1-3Gal sequences. These structures are strongly bound on the lectin column and their elution is obtained with 0.15 M galactose in the starting buffer.

Fast purification of Phaseolus vulgaris isolectins.

Phytohemagglutinin from red kidney bean has been purified by affinity chromatography on a human alpha 1-acid glycoprotein Sepharose 4B column. Further purification of the hemagglutinin's five isolectins was achieved on a Mono S column with an 86% protein recovery. Each sequentially eluted isolectin from the ion exchange column displayed either hemagglutinating or mitogenic activity. The main activity of each fraction was the result of the combination of varying proportions of the L and E subunits.

Structural analysis of the carbohydrate chains isolated from mistletoe (Viscum album) lectin I.

Two glycopeptide fractions prepared from mistletoe (Viscum album) lectin I by Pronase digestion were fractioned by affinity chromatography on a concanavalin A-Sepharose column. With 400-MHz 1H NMR spectroscopy, in conjunction with sugar analysis, the following oligosaccharide structures could be determined: two oligomannose-type glycans in the ratio 4:1, one containing six mannose and the other containing five mannose units, both with two 2-acetamido-2-deoxyglucose units. In addition, a mannotriosyl-->N,N'-diacetylchitobiose glycan containing a xylosyl group and an alpha-fucosyl group (1-->3)-linked to the 2-acetamido-2-deoxyglycosyl-1 residue, a common core element of many plant glycoproteins, was also observed.

Specificity of Amaranthus leucocarpus lectin.

We have demonstrated that Amaranthus leucocarpus lectin hemagglutinating activity was powerfully inhibited by the T-antigen, containing Gal(beta 1-3)GalNAc(alpha 1-3)Ser/Thr, and the Tn-antigen, which contains GalNAc(alpha 1-3)Ser/Thr. This suggests that the acetamido group at C-2 and the axial -OH at C-4 of the N-acetyl-D-galactopyranosylamine ring are important for lectin binding. The hemagglutination assays also established that desialylated and Pronase-treated human type O erythrocytes with an M phenotype were better recognized than erythrocytes from all other blood groups. The recognition was dependent on pH and ionic strength.

Fine sugar specificity of the Butea frondosa seed lectin.

Various monosaccharides and oligosaccharides were used to define the specificity of the Butea frondosa lectin using the hapten inhibition technique of human erythrocyte agglutination. Although B. frondosa lectin exhibited higher affinity for N-acetylgalactosamine, lactose and N-acetyllactosamine appeared to be relatively good inhibitors of haemagglutination. The behaviour of N-acetyllactosamine-type oligosaccharides and glycopeptides on a column of B. frondosa lectin immobilized on Sepharose 4B showed that the sugar-binding specificity of the lectin is directed towards unmasked N-acetyllactosamine sequences. Substitution of these N-acetyllactosamine sequences by sialic acid residues completely abolished the affinity of the lectin for the saccharides. The presence of one or several alpha Fuc(1-3)GlcNAc groups completely inhibited the interaction between the glycopeptides and the lectin. Substitution of the core beta-mannose residue by an additional bisecting beta(1-4)GlcNAc residue decreases the affinity of the lectin for these structures as compared with the unsubstituted ones.

Structures of the alpha(1-3)-galactose-containing asparagine-linked glycans of a Lewis lung carcinoma cell subline resistant to Aleuria aurantia agglutinin: elucidation by 1H-NMR spectroscopy.

Four bi-antennary glycan fractions of the N-acetyllactosamine-type, derived from a Lewis lung carcinoma (LL2) cell subline resistant to the Aleuria aurantia agglutinin were studied by 400 MHz 1H-NMR spectroscopy. By this method, their antennae were found to be terminated either by alpha(2-3 or 6)-linked N-acetylneuraminic acid or alpha(1-3)-linked galactose residues. The primary structure of glycans of these four glycopeptide or derived oligosaccharide-alditols has been determined in full detail.

[Treatment of vomiting in infants and children induced by acute infectious pathology. A comparative study of alizapride versus metopimazine]. / Traitement des vomissements du nourrisson et de l'enfant liés à une pathologie infectieuse aiguë. Etude comparative alizapride versus métopimazine.

The effectiveness and tolerance of alizapride and metopimazine used to treat vomiting induced by acute infectious diseases were evaluated in 47 infants and children seen in five hospitals. Patients were randomized to alizapride (n = 23) or metopimazine (n = 24). Medications were given orally (drops) for 3 to 5 days. All the patients were monitored until the end of the study period. Effectiveness was excellent or good in both groups with no statistically significant difference. Clinical tolerance was outstanding in both groups; one patient in the alizapride group exhibited transient, mild drowsiness after the doses. This study confirms the good risk/benefit ratio of alizapride in the treatment of emesis in infants and children.

Lectin-resistant variants of mouse Lewis lung carcinoma cells. I. Selection and in vivo properties.

The availability of lectin-resistant cell lines with altered carbohydrate moieties in cell surface glycoproteins and glycolipids has greatly facilitated study of the involvement of cellular glycoconjugates in tumor growth and metastasis. We present here a new animal model for metastasis study based on mouse Lewis lung carcinoma LL2 in vitro cell line. From this line, five lectin-resistant variant sublines were selected with the following lectins: wheat germ agglutinin (WGAR), Ricinus communis agglutinin II (RCA IIR) and Aleuria aurantia agglutinin (AAAR). The correlation of the lectin resistance with their in vitro and in vivo growth properties, and especially lung colonizing ability, were investigated. Three WGAR variants with well-preserved tumorigenicity revealed reduced metastatic ability, both spontaneous, after subcutaneous (s.c.) administration and experimental, after intravenous (i.v.) administration. The RCA IIR variant also possessed reduced spontaneous and experimental metastatic ability, but exhibited higher growth rate of local s.c. tumors. The AAAR variant possessed reduced spontaneous metastatic ability but its ability to colonize the lungs after i.v. administration was five-fold higher than that of the parent LL2 line, whereas its tumorigenicity remained unchanged. The relative differences among WGAR variants and parent LL2 line, concerning their experimental metastatic ability, remained similar in cyclophosphamide-modified mice to those in normal recipients.

Lectin-resistant variants of mouse Lewis lung carcinoma cells. II. Altered glycosylation of membrane glycoproteins.

Lectin-resistant variants of mouse Lewis lung carcinoma LL2 cell line, selected with wheat germ agglutinin (WGAR), Ricinus communis agglutinin II (RCA IIR) and Aleuria aurantia agglutinin (AAAR) were studied. Total cellular glycopeptides of the parent LL2 line and of the five lectin-resistant variants were analyzed by gel filtration and affinity chromatography on immobilized concanavalin A and Lens culinaris agglutinin. The results revealed that low-metastatic WGAR and RCA IIR variants possessed less highly branched tri- and tetra-antennary N-acetyllactosaminic type glycans with a simultaneous increase in biantennary N-acetyllactosaminic type, oligomannosidic type or hybrid type glycans, as compared to the parent metastasizing LL2 cell line. These findings imply that cell surface carbohydrate changes may possibly be relevant for metastasis. However, the AAAR variant, which possessed reduced spontaneous metastatic ability after s.c. administration, but increased experimental metastatic ability after i.v. inoculation, exhibited apparently the same glycan pattern than the parent LL2 line. This particular variant is under investigation in order to find specific modification(s) of glycan(s) which could play a specific role in the metastatic process.