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N Sharon

Publications and source records attributed to N Sharon.

At least 19 recordsLinked to original sources
PubMed1992

Immunochemical studies on the combining site of the A + N blood type specific Moluccella laevis lectin.

The specificity of the anti A+N lectin of Moluccella laevis (MLL) was examined by hemagglutination experiments with enzyme-modified human erythrocytes and by inhibition of hemagglutination. In addition, binding to various glycoproteins and inhibition by different sugars and glycoproteins were examined by enzyme immunoassay with antibodies to the lectin. Treatment of AMM erythrocytes with proteolytic enzymes increased their agglutinability by MLL 4-16-fold; similar treatment of ONN cells decreased their agglutinability 8-16-fold. This is in line with the known location and enzyme sensitivity of A and N specificity determinants. Treatment of the erythrocytes with sialidase increased their agglutinability and abolished the distinction between N and M cells. Hapten inhibition of hemagglutination of AMM and ONN erythrocytes by the lectin, and its binding to glycoproteins measured by enzyme immunoassay, confirmed the high specificity of MLL for N-acetyl-D-galactosamine (200-500 times more than for D-galactose) and suggested the presence of hydrophobic interactions around HO-2 of the D-galactose unit. The methyl alpha-glycosides of D-galactose and of N-acetyl-D-galactosamine were better inhibitors than the corresponding beta-glycosides; this preference was abolished, and sometimes reversed, when the p-nitrophenyl glycosides of the same monosaccharides were tested, stressing again the importance of hydrophobic interactions in the binding of carbohydrates to MLL. The lectin reacted well with ONN substance and with glycophorin A of the N phenotype (GPAN), but did not react with OMM substance or GPAM. The strongest inhibitor was asialo ovine submaxillary mucin, which contains many unsubstituted alpha-D-GalpNAc-(1-->3)-Ser/Thr residues; calculated per N-acetyl-D-galactosamine residue, it was 1500 stronger than free N-acetyl-D-galactosamine. In accordance with this result, it was found that the lectin strongly agglutinates Tn cells. The specificity of MLL can, thus, be defined as anti-Tn, crossreactive with blood types A and N, and with sialosyl-Tn. The N-specificity can best be explained by assuming that GPAN contains a small number of unsubstituted or partially sialylated alpha-D-GalpNAc-(1-->3)-Ser/Thr residues, which are present in smaller proportions, if at all, in GPAM.

Antibody Specificity↗
PubMed1992

Cloning, sequence analysis and expression in Escherichia coli of the cDNA encoding a precursor of peanut agglutinin.

The cDNA coding for pre-peanut agglutinin (PNA) was isolated from a bacterial expression library. It codes for a polypeptide of 273 amino acids composed of a hydrophobic signal peptide of 23 amino acids and a mature protein of 250 amino acids. The sequence of the latter is identical to that of native PNA, determined very recently by conventional methods, except that it contains 14 additional amino acids at the C-terminus. Bacterial cells harboring a plasmid with the prePNA-cDNA, produced two PNA cross-reacting proteins: one migrated on SDS-PAGE identically with the native lectin (apparent mol. wt. 31 kDa); the other, at 35 kDa, was a beta-galactosidase pre-PNA fusion protein. The former protein possessed an N-terminal sequence identical to that of the mature, native PNA, suggesting that it was processed from the 35 kDa prePNA precursor. Only the 31 kDa protein was exported into the bacterial periplasmic space, and had the ability to bind to galactose-Sepharose. The isolated processed protein had the same hemagglutinating activity as the native lectin, when assayed with sialidase-treated human erythrocytes. Like the native lectin, it did not agglutinate the untreated cells, was not inhibited by N-acetylgalactosamine, and was inhibited by Gal beta 1----3GalNAc 30-times more strongly than by galactose.

Amino Acid Sequence↗
PubMed1992

Isolation, by affinity chromatography and gel filtration in 8 M-urea, of an active subunit from the anti-(blood-group A+N)-specific lectin of Moluccella laevis.

The lectin from Moluccella laevis seeds agglutinates specifically blood-type-A and -N erythrocytes, and both activities are inhibited by micromolar concentrations of N-acetyl-D-galactosamine. The lectin consists of three subunits: a 67 kDa heterodimer, made up of two S-S-linked polypeptides of 28 and 46 kDa, and two non-covalently linked moieties of 26 and 42 kDa, the latter migrating after reduction with an apparent molecular mass of 46 kDa. Here we demonstrate that affinity chromatography of a crude protein fraction from M. laevis seeds on immobilized D-galactose in the presence of 8 M-urea affords a fully active lectin practically devoid of the 42 kDa subunit. We also present data showing that the 26 kDa subunit is devoid of cysteine residues, that the 28 kDa subunit contains two cysteine residues engaged in S-S bonds with the 46 kDa subunit, and that the latter has, in addition, two intramolecular cystine residues. Gel filtration on Sephadex G-150 in 8 M-urea/0.2 M-D-galactose of the lectin, affinity-purified in the presence of urea, afforded a pure 26 kDa subunit which exhibited both anti-A and anti-N activity, as well as high specificity for N-acetyl-D-galactosamine. In addition to demonstrating that the lectin is unusually stable and retains its carbohydrate-binding activity in 8 M-urea, our findings also show that the activity for different blood groups resides in the same subunit.

Acetylgalactosamine↗
PubMed1992

Expression of Erythrina corallodendron lectin in Escherichia coli.

The cDNA of the Erythrina corallodendron lectin (ECorL) has been expressed in Escherichia coli. For this purpose, an NcoI site was inserted into the cDNA coding for the lectin precursor [Arango, R., Rozenblatt, S. & Sharon, N. (1990) FEBS Lett. 264, 109-112] immediately before the codon GTG (103-105) which codes for the N-terminal valine of the mature lectin. This introduced an ATG codon for a methionine preceding the valine. The mutated cDNA was ligated into pUC-8, then subcloned into the expression vector pET-3d, which carries a strong promoter derived from gene 10 of the phage T7. The recombinant plasmid was introduced into the E. coli lysogenic strain BL21(DE3). Recombinant ECorL was expressed by growing the bacteria in the presence of isopropyl beta-D-thiogalactopyranoside. Most of the recombinant lectin was found in an insoluble aggregated form as inclusion bodies and only a small part was in the culture medium in a soluble active form. Functional recombinant lectin was recovered from the inclusion bodies by solubilization with 6 M urea in cyclohexylaminopropane sulfonate pH 10.5, renaturation by 10-fold dilution in the same buffer and further adjustment of the pH to 8.0. The recombinant lectin, obtained at a yield of 4-7 mg/l culture, had, by gel filtration, a slightly lower molecular mass (56 kDa) than the native lectin, and was devoid of covalently linked carbohydrate; it was, however, essentially indistinguishable from native ECorL by other criteria, including its dimeric structure, Western blot analysis with anti-ECorL polyclonal and monoclonal antibodies, and Ouchterlony double-diffusion analysis with polyclonal antibodies, as well as hemagglutinating activity and specificity for mono- or disaccharides.

Amino Acid Sequence↗
PubMed1992

Lectinophagocytosis of type 1 fimbriated (mannose-specific) Escherichia coli in the mouse peritoneum.

Bacteria can bind specifically to phagocytic cells via lectin-carbohydrate interactions and such binding is often followed by activation and degranulation of the phagocytes, as well as uptake and killing of the bacteria, a phenomenon designated lectinophagocytosis. Although extensively studied in vitro, no direct evidence for the occurrence of lectinophagocytosis in vivo has been available. To obtain such evidence, we injected type 1 fimbriated (mannose-specific) or nonfimbriated Escherichia coli into the peritoneal cavity of mice (10(7)-10(10) bacteria/animal) in the absence or presence of sugars and quantified the phagocytic activity by assaying the release of lysosomal N-acetyl-beta-D-glucosaminidase into the peritoneal fluid, up to 45 min after injection. Following injection of the type 1 fimbriated bacteria, significant release of the enzyme was observed which was time dependent and increased with the number of bacteria injected, whereas the nonfimbriated bacteria caused only little release. Methyl alpha-D-mannoside (50 mM), but not methyl alpha-D-galactoside or sucrose, inhibited the release by 60 to 100%. No release of N-acetyl-beta-D-glucosaminidase was induced by bacteria injected into a peritoneal cavity from which the macrophages had been removed. Our findings show that lectinophagocytosis can occur in vivo and may contribute to the host's defence against type 1 fimbriated bacteria.

Acetylglucosaminidase↗
PubMed1992

Synthesis of methyl alpha- and beta-N-dansyl-D-galactosaminides, probes for the combining sites of N-acetyl-D-galactosamine-specific lectins.

The synthesis of the methyl alpha- and beta-N-dansyl-D-galactosaminides is described using methyl alpha,beta-2-azido-2-deoxy-D-galactopyranoside as starting material. This was reduced to the corresponding methyl alpha,beta-2-amino-2-deoxy-D-galactopyranoside and then treated with dansyl chloride to yield a mixture of methyl alpha,beta-N-dansyl-D-galactosaminides which was separated into individual anomeric forms by flash chromatography on silica gel. Methyl alpha-N-dansyl-D-galactosaminide was used as a fluorescent indicator ligand in continuous substitution titrations to determine the association constants of nonchromophoric carbohydrates with the N-acetyl-D-galactosamine specific lectin from Erythrina corallodendron.

Acetylgalactosamine↗
PubMed1991

Structure of a legume lectin with an ordered N-linked carbohydrate in complex with lactose.

The three-dimensional structure of the lactose complex of the Erythrina corallodendron lectin (EcorL), a dimer of N-glycosylated subunits, was determined crystallographically and refined at 2.0 angstrom resolution to an R value of 0.19. The tertiary structure of the subunit is similar to that of other legume lectins, but interference by the bulky N-linked heptasaccharide, which is exceptionally well ordered in the crystal, forces the EcorL dimer into a drastically different quaternary structure. Only the galactose moiety of the lactose ligand resides within the combining site. The galactose moiety is oriented differently from ligands in the mannose-glucose specific legume lectins and is held by hydrophobic interactions with Ala88, Tyr106, Phe131, and Ala218 and by seven hydrogen bonds, four of which are to the conserved Asp89, Asn133, and NH of Gly107. The specificity of legume lectins toward the different C-4 epimers appears to be associated with extensive variations in the outline of the variable parts of the binding sites.

Amino Acid Sequence↗
PubMed1991

The glycosylation of glycoprotein lectins. Intra- and inter-genus variation in N-linked oligosaccharide expression.

Glycosylated lectins represent a series of glycoproteins with related activities and, in the case of the Leguminosae, related amino acid sequences. Therefore, they offer a model system in which to study the diversity of N-linked oligosaccharide structures of plant glycoproteins. The influence of the polypeptide on the type of oligosaccharide substitution and the problem of inter- and intra-genus variation in glycosylation can also be addressed. Analysis of the glycosylation of 18 lectins has shown that they can be classified into four qualitatively similar groups on the basis of the Bio-Gel P-4 elution profiles of the oligosaccharides released by hydrazinolysis: (a) The Erythrina cristagalli profile, with a major component at 8.8 glucose units (gu) and minor components at 8.0, 7.2, and 5.8 gu. The major component is the heptasaccharide, alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-[beta-D-Xyl p-(1----2)]- beta-D-Manp-beta-D-GlcpNAc-(1----4)-[alpha-L-Fucp-(1----3)]- D-GlcNAc. (b) The Phaseolus vulgaris profile, which was characterized by peaks at 12.5, 11.7, 10.8, and 9.9 gu, in addition to the peaks at 8.8, 8.0, 7.2, and 5.8 gu mentioned above. These higher-mol.-wt. components were oligo-D-mannose oligosaccharides containing 9, 8, 7, and 6 D-mannose residues, respectively. (c) The Lonchocarpas capassa profile, which had a major peak at approximately 8 gu. (d) The soybean agglutinin profile, which has a single peak at 12.5 gu. This peak consisted solely of an oligomannose undecasaccharide containing 9 D-mannose residues. This lectin is unique in that it shows no microheterogeneity.

Carbohydrate Conformation↗
PubMed1991

Identification of the leukocyte adhesion molecules CD11 and CD18 as receptors for type 1-fimbriated (mannose-specific) Escherichia coli.

Attachment of bacteria to phagocytic cells may be mediated by lectin-carbohydrate interactions, resulting in lectinophagocytosis. The best-studied system is the interaction of type 1-fimbriated (mannose-specific) Escherichia coli with human phagocytic cells. Here we demonstrate that the leukocyte integrins CD11 and CD18 (CD11/CD18) constitute the major receptors for type 1-fimbriated E. coli. Bacteria were bound in a dose-dependent and saturable manner to CD11/CD18, which was immobilized to microwells, whereas nonfimbriated E. coli cells failed to bind. The binding was efficiently inhibited (82 to 85%) by methyl-alpha-mannoside but not by galactose, and it was reduced by treatment of the immobilized CD11/CD18 with sodium metaperiodate, endoglycosidase H, or a mixture of endoglycosidase F and N-glycosidase. The fimbriated bacteria also bound to CD11a,b,c and CD18 separated by polyacrylamide gel electrophoresis with sodium dodecyl sulfate and blotted onto nitrocellulose paper. This binding was inhibited specifically by methyl-alpha-mannoside and was significantly diminished by treatment of the blots with sodium metaperiodate. Only minimal binding to the blotted CD11/CD18 that had been deglycosylated enzymatically prior to electrophoresis was observed. On blots of granulocyte lysates, specific binding to two glycoproteins (Mrs, 90,000 to 100,000 and 165,000) with mobilities similar to that of CD11/CD18 was observed. Monoclonal antibodies to CD11a, CD11b, or CD18 inhibited the binding of the bacteria to intact human granulocytes by 55 to 80%, whereas antibodies against other leukocyte surface antigens were not inhibitory. We conclude that type 1-fimbriated E. coli binds to human granulocytes via the oligomannose and hybrid N-linked units of CD11/CD18. Since CD11b/CD18 and CD11c/CD18 are known to serve as receptors for complement fragment iC3b, this study provides a link between opsonophagocytosis and lectinophagocytosis.

Antibodies, Monoclonal↗
PubMed1991

Lectinophagocytosis of encapsulated Klebsiella pneumoniae mediated by surface lectins of guinea pig alveolar macrophages and human monocyte-derived macrophages.

Macrophages express a mannose/N-acetylglucosamine-specific lectin which serves as a receptor for nonopsonic phagocytosis of mannose-coated particles. We have examined the binding to guinea pig alveolar macrophages in a serum-free medium of 16 Klebsiella pneumoniae serotypes and of the capsular polysaccharides isolated from 7 of these serotypes. Only five polysaccharides containing the repeating sequence Man alpha 2/3Man or L-Rha alpha 2/3-L-Rha bound to the macrophages. Of the 11 bacterial serotypes expressing such disaccharides in their capsular polysaccharides, 7 bound efficiently, 2 bound poorly, and 2 did not bind at all. No binding occurred with five serotypes lacking these disaccharides. Binding of the bacteria was inhibited by homologous and heterologous capsular polysaccharides that contain the disaccharide sequences, by mannan, and by (Man)25BSA (where BSA is bovine serum albumin). Man alpha 2/3Man-containing oligosaccharides were potent inhibitors compared with monosaccharides. Binding was dependent on Ca2+, modulated by cultivating the macrophages on mannan-coated surfaces, and increased in human monocyte-derived macrophages compared with monocytes. The bulk of the bacteria bound to the macrophages was internalized and killed. The data taken together suggest that Klebsiella pneumoniae cells undergo lectinophagocytosis mediated by capsular disaccharides recognized by the mannose/N-acetylglucosamine-specific lectin of macrophages. This may enhance clearance of the organisms from the serum-poor environment of the lung.

Animals↗
PubMed1990

Cloning and sequence analysis of the Erythrina corallodendron lectin cDNA.

Examination of the hemagglutinating activity of extracts from seeds of Erythrina corallodendron at various maturation stages revealed that the level of lectin increases markedly past mid-maturation. Seeds at this stage of maturation served as a source of mRNA for the construction of an expression cDNA library in the vector lambda Zap, which generates fusion proteins with an N-terminal portion of beta-galactosidase. The library was screened with rabbit polyclonal anti-ECorL antiserum. Four immunopositive clones were isolated. Western blot analysis of cell extracts from one of the clones (pIEcL-B) showed a 36 kDa protein that reacted with the antiserum, as well as with a mouse monoclonal antibody raised against the lectin. DNA sequence analysis by the chain termination method revealed that clone pIEcl-C has an insert of 1017 bp with the entire coding sequence of ECorL, beginning with an initiation codon ATG at position 26 and ending with stop codon TAA at position 868. This fragment encodes a polypeptide of 281 amino acids consisting of a signal leader sequence of 25 amino acids and a mature protein of 256 amino acids. The deduced amino acid sequence from this fragment is identical to the sequence of the first 244 amino acids of ECorL, as determined at the protein level, except at 7 positions.

Amino Acid Sequence↗
PubMed1990

Improved detection of metastases to lymph nodes and estrogen receptor determination.

We present a new method for detection of micrometastases to axillary lymph nodes and estrogen receptor determination. Cellular suspensions from primary infiltrating ductal breast carcinoma or level I axillary lymph nodes of patients who underwent mastectomies were obtained, by loosely grinding fresh tumors or lymph nodes through a grid and then transferring the matrix to a slide using cytocentrifugation. Tumor samples were analyzed for estrogen receptor status using an immunocytochemical kit and compared with the dextran-coated charcoal method. Thirty-eight of 48 correlated (20 were estrogen positive, and 18 were estrogen negative). Seven of 46 were estrogen positive while results from the dextran-coated charcoal method were estrogen negative. One of 46 was estrogen negative, while the results from the dextran-coated charcoal method were estrogen positive. Lymph node slide preparations were stained to detect tumor cells using antikeratin monoclonal antibodies. Three of 8 node-negative patients were found to have micrometastases. Four of 15 node-positive patients had additional nodes with tumor. Our method combines the advantages of serial sectioning and immunohistochemical staining.

Antibodies, Monoclonal↗
PubMed1990

Legume lectins--a large family of homologous proteins.

More than 70 lectins from leguminous plants belonging to different suborders and tribes have been isolated, mostly from seeds, and characterized to varying degrees. Although they differ in their carbohydrate specificities, they resemble each other in their physicochemical properties. They usually consist of two or four subunits (25-30 kDa), each with one carbohydrate binding site. Interaction with carbohydrates requires tightly bound Ca2+ and Mn2+ (or another transition metal). The primary sequences of more than 15 legume lectins have been established by chemical or molecular genetic techniques. They exhibit remarkable homologies, with a significant number of invariant amino acid residues, among them most of those involved in metal binding. The 3-dimensional structures of the legume lectins are similar, too, and are characterized by a high content of beta-sheets and a lack of alpha-helix. The location of the metal and carbohydrate binding sites, established unequivocally in concanavalin A by high resolution X-ray crystallography, appears to be the same in the other legume lectins. Several of the lectin genes have been cloned and expressed in heterologous systems. This opens the way for the application of molecular genetics to the investigation of the atomic structure of the carbohydrate binding sites of the lectins, and of the relationship between their structure and biological activity. The new approaches may also provide information on the mechanisms that control gene expression in plants and on the role of lectins in nature.

Amino Acid Sequence↗
PubMed1989

Lectins as cell recognition molecules.

Lectins on cell surfaces mediate cell-cell interactions by combining with complementary carbohydrates on apposing cells. They play a key role in the control of various normal and pathological processes in living organisms.

Animals↗
PubMed1989

Binding and precipitating activities of Erythrina lectins with complex type carbohydrates and synthetic cluster glycosides. A comparative study of the lectins from E. corallodendron, E. cristagalli, E. flabelliformis, and E. indica.

Erythrina lectins possess similar structural and carbohydrate binding properties. Recently, tri- and tetra-antennary complex type carbohydrates with non-reducing terminal galactose residues have been shown to be precipitated as tri- and tetravalent ligands, respectively, with certain Erythrina lectins [Bhattacharyya L, Haraldsson M, Brewer CF (1988) Biochemistry 27:1034-41]. The present work describes a comparative study of the binding and precipitating activities of four Erythrina lectins, viz., E. corallodendron, E. cristagalli, E. flabelliformis, and E. indica, with multi-antennary complex type carbohydrates and synthetic cluster glycosides. The results show that though their binding affinities are very similar, the Erythrina lectins show large differences in their precipitating activities with the carbohydrates. The results also indicate significant dependence of the precipitating activities of the lectins on the core structure of the carbohydrates. These findings provide a new dimension to the structure-activity relationship of the lectins and their interactions with asparagine-linked carbohydrates.

Carbohydrate Conformation↗