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N Sharon

Publications and source records attributed to N Sharon.

At least 73 records · Page 4Linked to original sources
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

Involvement of protein kinase C in activation of human granulocytes and peritoneal macrophages by type 1 fimbriated (mannose specific) Escherichia coli.

Specific binding of bacteria to phagocytic cells mediated by antibody and complement (opsonins) or by lectin-carbohydrate interactions is required for their efficient uptake and killing by opsonophagocytosis or lectinophagocytosis, respectively (Ofek and Sharon, Infect. Immun. 56, 539, 1988). An early step in these processes is activation of the phagocytes by the bound bacteria, as evidenced by appearance of an oxidative burst. Previous work has shown that protein kinase C (PKC) is involved in activation of human granulocytes by opsonized Escherichia coli. In the present study, we used three inhibitors of PKC to examine the possible involvement of the enzyme in activation of human granulocytes and peritoneal macrophages by type 1 fimbriated (mannose-specific) Escherichia coli in the absence of opsonins. Activation, as measured by chemiluminescence, was completely inhibited by sphingosine (50 microM) and only partially (50%) by 100 microM H-7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine]; in both cases it was fully reversible. The third inhibitor, K252a, also inhibited almost completely the activation at 1 microM. The inhibitors acted similarly on activation of the phagocytic cells by opsonized bacteria or by phorbol-12-myristate-13-acetate (0.1 microM). Down regulation of the kinase, by pretreatment of the human granulocytes or macrophages with a high concentration (1.6 microM) of phorbol myristate acetate, abolished their ability to respond to stimulation by the bacteria. Our findings provide evidence for the involvement of PKC in the activation of phagocytic cells by type 1 fimbriated bacteria.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

The amino acid sequence of Erythrina corallodendron lectin and its homology with other legume lectins.

The primary sequence of Erythrina corallodendron lectin was deduced from analysis of the peptides derived from the lectin by digestion with trypsin, chymotrypsin, Staphylococcus aureus V8 protease, elastase and lysylendopeptidase-C, and of fragments generated by cleavage of the lectin with dilute formic acid in 6 M guanidine hydrochloride. Purification of the individual peptides was achieved by gel filtration, followed by reverse phase HPLC. The glycosylation site (Asn17-Leu18-Thr19) was deduced from analysis of the glycopeptide isolated from a pronase digest of the lectin before and after deglycosylation of the glycopeptide with endoglycosidase F. Comparison of the sequence of 244 residues thus obtained with those of 9 other legume lectins revealed extensive homologies, including 39 invariant positions and 60 partial identities. These data provide further evidence for the conservation of the lectin gene in leguminous plants.

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

Lectin-mediated, nonopsonic phagocytosis of type 1 Escherichia coli by human peritoneal macrophages of uremic patients treated by peritoneal dialysis.

Human peritoneal macrophages isolated from uremic patients undergoing peritoneal dialysis bind type 1 fimbriated Escherichia coli in the absence of opsonins. The number of bacteria bound per macrophage was 6.9, as determined by microscopic examination. Methyl alpha-mannoside (0.1 mM) and p-nitrophenyl alpha-mannoside (0.01 mM) inhibited this binding by about 66%. The ability of peritoneal macrophages to bind E. coli in a mannose-specific manner was confirmed in further experiments using an enzyme-linked immunosorbent assay (ELISA) with an antibacterial antibody, radiolabelled E. coli, and counts of colony-forming units (CFU). The number of bacteria bound per macrophage was 7 to 12 in the ELISA and 5.5-8.5 in the CFU assay. Methyl alpha-mannoside caused 70% inhibition of binding in the ELISA and 84% in the CFU assay, whereas p-nitrophenyl alpha-mannoside showed inhibition of 79% and 90%, respectively. Most bound bacteria (76-80%) were subsequently killed. Nonfimbriated E. coli 827 bound poorly to the macrophages (approximately 22%) as compared to that of the fimbriated bacteria. Although this binding was not inhibited by methyl alpha-D-mannoside or p-nitrophenyl alpha-mannoside, the percentage of bacteria killed was similar to that of the fimbriated phenotype. The peritoneal macrophage is thus able to phagocytose E. coli in the absence of opsonins. This may explain the relative rarity of E. coli as an etiologic agent of peritoneal infections in the dialysed patient.

Adult↗
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↗
PubMed1989

Enhancement of human monocyte cytotoxicity by both interferon-gamma and -beta and comparison to other stimuli.

Recombinant interferon preparations caused a dose-dependent increase of human monocyte cytotoxicity to the K562 and Daudi cell lines. Both rIFN-gamma and rIFN-beta enhanced this function to a similar extent, while rIFN-alpha c had less effect when compared on the basis of their anti-viral effects. Endotoxin and concanavalin A increased basal monocyte cytotoxicity while phagocytosis of latex particles had no effect. The increased monocyte cytotoxic effect of rIFN-beta was completely abrogated by monoclonal antibody to IFN-beta, while monoclonal antibody to IFN-gamma had no effect. However, monoclonal antibody to IFN-gamma only reduced the increased cytotoxic effect caused by rIFN-gamma by 25%. Catalase inhibited both basal monocyte cytotoxicity and the increase in cytotoxicity following addition of rIFN-gamma only slightly, suggesting that mechanisms other than the oxidative burst were active and could be induced by rIFN-gamma.

Antibodies, Monoclonal↗
PubMed1989

Inhibitory activity of cranberry juice on adherence of type 1 and type P fimbriated Escherichia coli to eucaryotic cells.

Inhibition of bacterial adherence to bladder cells has been assumed to account for the beneficial action ascribed to cranberry juice and cranberry juice cocktail in the prevention of urinary tract infections (A. E. Sobota, J. Urol. 131:1013-1016, 1984). We have examined the effect of the cocktail and juice on the adherence of Escherichia coli expressing surface lectins of defined sugar specificity to yeasts, tissue culture cells, erythrocytes, and mouse peritoneal macrophages. Cranberry juice cocktail inhibited the adherence of urinary isolates expressing type 1 fimbriae (mannose specific) and P fimbriae [specific for alpha-D-Gal(1----4)-beta-D-Gal] but had no effect on a diarrheal isolate expressing a CFA/I adhesin. The cocktail also inhibited yeast agglutination by purified type 1 fimbriae. The inhibitory activity for type 1 fimbriated E. coli was dialyzable and could be ascribed to the fructose present in the cocktail; this sugar was about 1/10 as active as methyl alpha-D-mannoside in inhibiting the adherence of type 1 fimbriated bacteria. The inhibitory activity for the P fimbriated bacteria was nondialyzable and was detected only after preincubation of the bacteria with the cocktail. Cranberry juice, orange juice, and pineapple juice also inhibited adherence of type 1 fimbriated E. coli, most likely because of their fructose content. However, the two latter juices did not inhibit the P fimbriated bacteria. We conclude that cranberry juice contains at least two inhibitors of lectin-mediated adherence of uropathogens to eucaryotic cells. Further studies are required to establish whether these inhibitors play a role in vivo.

Agglutination↗