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N Sharon

Publications and source records attributed to N Sharon.

At least 55 records · Page 3Linked to original sources
PubMed1995

Lectins--proteins with a sweet tooth: functions in cell recognition.

Lectins, non-enzymic proteins that bind mono- and oligosaccharides reversibly and with high specificity, occur widely in nature. They come in a variety of sizes and shapes, but can be grouped in families with similar structural features. The combining sites of lectins are also diverse, although they are similar in the same family. The specificities of lectins are determined by the exact shape of the binding sites and the nature of the amino acid residues to which the carbohydrate is linked. Small changes in the structure of the sites, such as the substitution of only one or two amino acids, may result in marked changes in specificity. The carbohydrate is linked to the protein mainly through hydrogen bonds, with added contributions from van der Waals contacts and hydrophobic interactions. Coordination with metal ions may occasionally play a role too. Microbial surface lectins serve as a means of adhesion to host cells of viruses (e.g. influenza virus), bacteria (e.g. E. coli) and protozoa (e.g. amoeba): a prerequisite for the initiation of infection. Blocking the adhesion by carbohydrates that mimic those to which the lectins bind prevents infection by these organisms. The way is thus open for the development of anti-adhesive therapy against microbial diseases. Lectin-carbohydrate mediated interactions between leucocytes and endothelial cells are the first step in the recirculation of lymphocytes and in the migration of neutrophils to sites of inflammation. Such interactions may also feature highly in the formation of metastases. Studies of these processes are expected to lead to the development of carbohydrate-based anti-adhesion drugs for the treatment of inflammatory diseases as well as cancer.

Animals↗
PubMed1994

Differences in the cross-linking activities of native and recombinant Erythrina corallodendron lectin with asialofetuin. Evidence for carbohydrate-carbohydrate interactions in lectin-glycoprotein complexes.

A previous study showed that several multivalent galactose-specific lectins including the 14-kDa lectin from calf spleen and the lectins from Erythrina indica, Erythrina cristagalli, and soybean agglutinin formed specific cross-linked complexes with the glycoprotein asialofetuin (ASF) [Mandal, D. K., & Brewer, C. F. (1992) Biochemistry 31, 8465-8472]. In the present study, we have used quantitative precipitation analysis to compare the cross-linking activities of the Gal/GalNAc-specific lectin from Erythrina corallodendron (ECorL) and the recombinant protein (rECorL) which lacks the covalently linked heptasaccharide chains of the native lectin, with ASF. At low concentrations of ASF relative to the lectin, native dimeric ECorL binds to each of the three terminal Gal residues of the three N-linked triantennary chains of ASF and precipitates as a cross-linked complex at a ratio of 1:9 ASF/lectin (monomer). With increasing concentrations of ASF, the 1:9 complex changes to a 1:3 ASF/lectin complex, and at higher ASF concentrations, a 1:1 cross-linked complex forms. However, rECorL, which possesses the same specificity and binding affinity as the native lectin, forms only the 1:9 and 1:3 ASF/lectin complexes. Other Erythrina lectins examined, all of which have covalently attached carbohydrate and are structurally similar to ECorL, show the same cross-linking behavior as native ECorL. On the other hand, the dimeric 14-kDa calf spleen lectin which lacks covalently attached carbohydrate forms only 1:9 and 1:3 cross-linked complexes with ASF [Mandal, D. K., & Brewer, C. F. (1992) Biochemistry 31, 8465-8472].(ABSTRACT TRUNCATED AT 250 WORDS)

Asialoglycoproteins↗
PubMed1994

Characterization of the specificity of binding of Moluccella laevis lectin to glycosphingolipids.

The specificity of Moluccella laevis lectin was investigated by analysing its binding to glycosphingolipids separated on thin-layer chromatograms or adsorbed on microtitre wells. The binding activity of the lectin was highest for glycosphingolipids with terminal alpha-linked N-acetylgalactosamine, both in linear structures, as the Forssman glycosphingolipid, GalNAc alpha 3GalNAc beta 3Gal alpha 4Glc beta 1Cer, and in branched structures, as glycosphingolipids with the blood group A determinant, GalNAc alpha 3(Fuc alpha 2)Gal beta. In addition, the lectin bound, though considerably more weakly, to linear glycosphingolipids with terminal alpha-linked galactose. When considering the use of the M. laevis lectin for biochemical and medical purposes this cross-reactivity may be of importance.

Carbohydrate Sequence↗
PubMed1993

Protein glycosylation. Structural and functional aspects.

During the last decade, there have been enormous advances in our knowledge of glycoproteins and the stage has been set for the biotechnological production of many of them for therapeutic use. These advances are reviewed, with special emphasis on the structure and function of the glycoproteins (excluding the proteoglycans). Current methods for structural analysis of glycoproteins are surveyed, as are novel carbohydrate-peptide linking groups, and mono- and oligo-saccharide constituents found in these macromolecules. The possible roles of the carbohydrate units in modulating the physicochemical and biological properties of the parent proteins are discussed, and evidence is presented on their roles as recognition determinants between molecules and cells, or cell and cells. Finally, examples are given of changes that occur in the carbohydrates of soluble and cell-surface glycoproteins during differentiation, growth and malignancy, which further highlight the important role of these substances in health and disease.

Amino Acid Sequence↗
PubMed1993

Modification by site-directed mutagenesis of the specificity of Erythrina corallodendron lectin for galactose derivatives with bulky substituents at C-2.

Examination of the three-dimensional structure of Erythrina corallodendron lectin (ECorL) in complex with a ligand (lactose), the first of its kind for a Gal/GalNAc-specific lectin [(1991) Science 254, 862-866], revealed the presence of a hydrophobic cavity, surrounded by Tyr108 and Pro134-Trp135, which can accommodate bulky substituents such as acetamido or dansylamido (NDns) at C-2 of the lectin-bound galactose. Comparison of the primary sequence of ECorL with that of soybean agglutinin, specific for galactose and its C-2 substituted derivatives, and of peanut agglutinin, specific for galactose only, showed that in soybean agglutinin, Tyr108 is retained, and Pro134-Trp135 is replaced by Ser-Trp, whereas in peanut agglutinin, the former residue is replaced by Thr and the dipeptide by Ser-Glu- Tyr-Asn. Three mutants of ECorL were therefore constructed: L2, in which Pro134-Trp135 was replaced by Ser-Glu-Tyr-Asn; Y108T, in which Tyr108 was replaced by Thr and the double mutant L2; Y108T. They were expressed in Escherichia coli, as done for recombinant ECorL [(1992) Eur. J. Biochem. 205, 575-581]. The mutants had the same hemagglutinating activity as native or rECorL. Their specificity for galactose, GalNAc and Me beta GalNDns was examined by inhibition of hemagglutination and of the binding of the lectin to immobilized asialofetuin; in addition, their association constants with Me alpha GalNDns and Me beta GalNDns were measured by spectrofluorimetric titration. The results showed that Y108T had essentially similar specificity as the native and recombinant lectins. The affinity of L2 and L2;Y108T for galactose was also the same as ECorL, but they had a lower affinity for GalNAc and markedly diminished affinity for the dansyl sugars (up to 43 times, or 2 kcal, less). This appears to be largely due to steric hindrance by the two additional amino acids present in the cavity region in these mutants. Our findings also provide an explanation for the inability of PNA to accommodate C-2-substituted galactose derivatives at its primary subsite.

Amino Acid Sequence↗
PubMed1993

The leukocyte surface antigens CD11b and CD18 mediate the oxidative burst activation of human peritoneal macrophages induced by type 1 fimbriated Escherichia coli.

Analysis by immunofluorescence-activated cell sorting of human peritoneal macrophages from patients undergoing intermittent peritoneal dialysis revealed that they express the CD11/CD18 surface antigens, with CD11b and CD18 as the predominant ones. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates obtained from lysates of 125I-labeled macrophages with rabbit polyclonal antibodies against the CD11a-c/CD18 complex or against CD18, revealed four radioactive bands corresponding to CD11a, CD11b, CD11c, and CD18. Monoclonal antibodies against CD11b and CD18 inhibited by 80 and 90%, respectively, the oxidative burst activation of the macrophages by type 1 fimbriated Escherichia coli, whereas monoclonal antibodies against CD11a, CD11c, and CD43 were without effect. Our results suggest that CD11b and CD18 (receptors for C3bi) serve also as receptors for mannose-specific E. coli on human peritoneal macrophages and may be involved in the lectinophagocytosis of the bacteria by these cells.

Antibodies, Monoclonal↗
PubMed1993

Developmental changes in neutral glycosphingolipids of mouse placenta.

The mammalian placenta is a unique organ for the study of developmental changes. Placentas of laboratory animals such as the mouse allow for the determination of the exact stage of pregnancy, which cannot be achieved with human placenta. In this study, neutral glycosphingolipids were isolated from mouse (inbred strain C57BL/6) placentas, from day 10 to day 18 of gestation, and were separated by high performance thin layer chromatography. Densitometric measurements after orcinol staining showed, at day 10 of gestation, the presence of mono-, tetra-, tri- and dihexosylceramide in decreasing quantities, as well as four unidentified spots. On day 12, the glycosphingolipid composition changed with the disappearance of the unidentified spots and the appearance of an orcinol positive migrating similarly to the Forssman antigen; no further changes occurred between days 12 and 18 of gestation. The identity of the Forssman-like glycosphingolipid with the Forssman antigen was established by binding of 125I labelled Helix pomatia agglutinin (alpha-GalNAc specific) to glycosphingolipids separated on high performance thin layer chromatography plates, and by the reaction of the isolated glycosphingolipid with a monoclonal anti-Forssman antibody. The appearance of the Forssman antigen at day 12 of gestation coincided with the day of final maturation of the mouse placenta and subsequent cessation of growth, suggesting a possible role of the glycosphingolipid during embryonic development.

Animals↗
PubMed1993

Use of O-glycosylation-defective human lymphoid cell lines and flow cytometry to delineate the specificity of Moluccella laevis lectin and monoclonal antibody 5F4 for the Tn antigen (GalNAc alpha 1-O-Ser/Thr).

The Tn antigen (GalNAc alpha 1-O-Ser/Thr) is a disease-related O-linked (mucin-type) carbohydrate neoantigen which is expressed in idiopathic Tn syndrome, AIDS, T-cell lymphoma and in many carcinomas. In the present study, we took advantage of a Tn antigen expressing T-lymphocyte clone derived from a patient with the idiopathic form of the Tn syndrome and the Tn+ Jurkat cell line to characterize new reagents that should identify Tn antigens (monoclonal antibody 5F4 and a lectin newly isolated from Moluccella laevis seeds). Flow cytometry revealed that both reagents strongly bound to Tn antigen expressing T lymphocytes but not to normal donor T cells, which are Tn negative. In contrast to mAb 5F4, Moluccella laevis lectin weakly bound to normal donor cells after sialidase pretreatment, indicating its broader specificity. N-Acetyl-D-galactosamine at a concentration of 100 mM significantly reduced antibody binding and abolished lectin binding, completely demonstrating the sugar specificity of both reagents. These reagents should be useful tools in glycobiology and for clinical purposes.

Antibodies, Monoclonal↗
PubMed1993

Lectin-carbohydrate complexes of plants and animals: an atomic view.

Lectins are a structurally diverse class of proteins, their only common features being the ability to bind carbohydrates specifically and reversibly, and to agglutinate cells. Some, however, can be grouped together into distinct families, such as those of the legumes or the cereals that are structurally similar, or the C-type (Ca(2+)-dependent) animal lectins that contain homologous carbohydrate recognition domains. Recent high-resolution X-ray crystallographic studies have revealed the structures of the sugar complexes of over half a dozen lectins. These studies demonstrate that the combining sites of lectins are also structurally diverse, although they may be similar in the same family.

Animals↗
PubMed1993

Carbohydrates in cell recognition.

Telltale surface sugars enable cells to identify and interact with one another. New drugs aimed at those carbohydrates could stop infection and inflammation.

Animals↗
PubMed1993

Shigella flexneri transformants expressing type 1 (mannose-specific) fimbriae bind to, activate, and are killed by phagocytic cells.

Shigella flexneri M90T (invasive) and BS176 (noninvasive) are typical nonfimbriated organisms that do not bind to or activate phagocytic cells. We demonstrate that S. flexneri M90Tp and BS176p, obtained by transformation of the strains named above with the cluster of genes encoding type 1 (mannose-specific) fimbriae of Escherichia coli, express the functional fimbriae, as shown by electron microscopy, by binding of antifimbria antibodies and by yeast cell aggregation. The transformants, but not the parental strains, bound to human granulocytes and mouse peritoneal macrophages. This binding was inhibited by methyl alpha-D-mannoside but not by methyl alpha-D-galactoside. The bound bacteria induced oxidative burst activation and degranulation of the granulocytes in vitro. With mouse peritoneal macrophages, the binding of the fimbriated bacteria induced degranulation in vitro. Injection of the bacteria into mouse peritoneum also induced degranulation of the macrophages in vivo; no such effect was observed with the nonfimbriated strains. The bound fimbriated transformants were effectively killed by the human granulocytes in vitro in the absence of opsonins or after opsonization with human anti-S. flexneri antiserum. The nonfimbriated strains were killed only after opsonization. These results provide further evidence for the role of type 1 fimbriae in lectin-mediated nonopsonic phagocytosis.

Animals↗
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↗