Stage-specific Differences in Lectin Binding to the Surface of Anguina tritici and Meloidogyne incognita

The occurrence and distribution of several lectin binding sites on the outer surfaces of eggs, preparasitic second-stage juveniles (J2), parasitic second-stage juveniles (PJ2), females, and males of two tylenchid nematodes, Anguina tritici and Meloidogyne incognita race 3, were compared. In both species, a greater variety of lectins bound to the eggs than to other life stages; lectin binding to eggs was also more intense than it was to other life stages. Species-specific differences also occurred. More lectins bound to the amphids or amphidial secretions of M. incognita J2 than to the amphids or amphidial secretions of A. tritici J2. Lectins also bound to the amphids or amphidial secretions of adult male and female A. tritici, but binding to the cuticle occurred only at the head and tail and was not consistent in all specimens. Canavalia ensiformis and Ulex europaeus lectins bound specifically to the outer cuticle of M. incognita. Several other lectins bound nonspecifically. Oxidation of the cuticle with periodate under mild conditions, as well as pretreatment of the nematodes with lipase, markedly increased the binding of lectins to the cuticle of A. tritici J2 but not, in most cases, to M. incognita J2 or eggs of either species.     

Characterization of Carbohydrates on the Surface of Second-stage Juveniles of Meloidogyne spp.

Fluorescent conjugates of the lectins soybean agglutinin (SBA), Concanavalin A (Con A), wheat germ agglutinin (WGA), Lotus tetragonolobus agglutinin (LOT), and Limulus polyphemus agglutinin (LPA) bound primarily to amphidial openings and amphidial secretions of viable, preinfective second-stage juveniles (J2) of Meloidogyne incognita races 1 and 3 (Mil, Mi3) and M. javanica (Mj). No substantial difference in fluorescent lectin binding was observed among the populations examined. Binding of only LOT and LPA were inhibited in the presence of 0.1 M competitive sugar. Structural differences in amphidial carbohydrate complexes among populations of Mi 1, Mi3, and Mj were revealed by glycohydrolase treatment of preinfective J2 and subsequent labeling with fluorescent lectins. A quantitative microfiltration enzyme-linked lectin assay revealed previously undetected differences in lectin binding to nonglycohydrolase-treated J2. Freinfective J2 of Mj bound the greatest amount of SBA, LOT, and WGA, whereas J2 of Mil bound the most LPA.     

Scanning Electron Microscopy of Second-Stage Juvenile Cephalic Morphology in Heterodera glycines Races

Scanning electron microscopy (SEM) was used to compare juvenile cephalic morphology of the five described races of Heterodera glycines. Races 1, 2, 3, and 4 were obtained in the United States and race 5 was obtained from Japan. Differences in the gross morphology o f labial discs; ventral, dorsal and lateral lips; amphidial apertures; and fissures on the labial disc o f some specimens were observed. There was considerable interracial and intraracial variation which precluded separation o f juveniles of H. glycines races by SEM.     

Ultrastructure of the anterior sense organs of adult Gastromermis boophthorae (Nematoda: Mermithidae)

The amphids of adult Gastromermis boophthorae exhibit an organization unlike any previously reported. Each amphid consists of a distal cuticular channel which opens to the exterior, and a proximal amphidial gland. The cuticular channel is double and 15–18 cilia are held in a tight bundle by the inner channel. The amphidial gland contains an extensive reticulum, and the nerve axons which give rise to the amphidial cilia are arranged around the outside of the gland; anteriorly these axons are characteristically flattened. A granular secretion is produced by the amphidial gland and passes via the reticulum to the double cuticular channel, thus bathing the ciliary bundle. The ultrastructure of the amphids is discussed in relation to functional considerations.     

Genotypic Differentiation of Meloidogyne Populations by Detection of Restriction Fragment Length Difference in Total DNA

Detection of EcoRI restriction fragment length differences in repetitive DNA sequences permitted the rapid diagnosis, by genotype, of randomly selected populations of Meloidogyne incognita, Races 1, 2, 3, and 4; M. javanica; M. arenaria, Races 1 and 2; and M. hapla, Races A and B.     

Post-translational modifications of the cuticular proteins of Hyalophora cecropia from different anatomical regions and metamorphic stages

Post-translational modifications are a conspicuous feature of the proteins of vertebrate extracellular matrices such as cartilage. Yet this feature remains virtually unexplored with insect cuticle, a situation this paper begins to remedy. Cuticular proteins were extracted from cuticles of Hyalophora cecropia and separated on isoelectrofocusing and 2D gels. Periodic acid-Schiff reagent stained several proteins from flexible cuticles and a few proteins from rigid cuticles, indicating that some proteins were glycosylated. Elucidation of the specific nature of this glycosylation came from probing electrophoretically separated cuticular proteins blotted onto nitrocellulose with biotinylated lectins. Most major cuticular proteins did not react; minor cuticular proteins and molecules which do not stain with Coomassie blue were found to bind lectins specific for mannose and N-acetylgalactosamine. Limited binding was also detected with lectins specific for N-acetylglucosamine, galactose and fucose. No sialic acid was detected using either lectins or neuraminidase digestion. The amount of glycosylation was greatest in proteins extracted from flexible cuticles. Although several proteins stained with Alcian blue indicating presence of sulfation, ³⁵S which had been incorporated at low levels in cuticular proteins corresponded to [³⁵S]methionine. No indication of the presence of mammalian-type glycosaminoglycans in insect cuticles was obtained after treatment with chondroitinase or nitrous acid. The functional significance of the modifications detected remains unknown. No evidence for phosphorylated proteins or lipoproteins was found.     

Immunolocalization of a putative cuticular collagen protein in several developmental stages of Meloidogyne arenaria,Globodera pallida and G. rostochiensis

The monoclonal antibody IACR-CCNj.3d has previously been used to isolate a gene (gp-col-8) with strong similarity to cuticular collagen from a mixed stage Globodera pallida cDNA expression library. The antibody has also been shown to label specifically the amphidial canal of pre-parasitic second stage juveniles (J2) of several plant nematode species without any reactivity on the cuticular surface, indicating that this protein is either not present or is inaccessible on the cuticular surface. This paper investigates the cross-reactivity of Mab IACR-CCNj.3d with Meloidogyne arenaria and the localization of the putative collagen protein on the cuticular surface of parasitic stages in planta and on the cuticular surface of juveniles inside eggs. The antigen was shown to be present in all developmental stages of the two species of potato cyst nematodes and M. arenaria. The antibody bound strongly to the amphidial canal and hypodermis of pre-parasitic J2 and adult females. The antigen was present on the cuticular surface of the sausage-shaped J2 in planta and of first stage juveniles (J1) inside the eggs. The presence of collagen on the surface of the cuticle of moulting stages of plant parasitic nematodes has been observed for the first time. It is clear that this protein has a role in the construction of the cuticle of the first stage juveniles and parasitic second stage juveniles, during moulting inside the eggs and in the root tissue, respectively.     

Photochemical addition of 2-propanol and of acetone to methyl 5-deoxy-2,3-O-isopropylidene-β-d-erythro-pent-4-enofuranoside

High-capacity adsorbents for lectins, including Lotus tetragonolobusl-fucose-binding protein, were readily prepared by conjugation of monosaccharides with commercially available, epoxy-activated Sepharose. Purified, radioiodinated lectins were bound to cells of the mosquito Aedes aegyptii and of human KB tumour. Relative to human KB cells, mosquito cells bound less of lectins specific for the sugars (l-fucose and d-galactose) that are terminal residues in many mammalian glycoproteins, whereas the number of binding sites of lectins specific for core-region sugars (d-mannose and 2-acetamido-2-deoxy-d-glucose) were similar. Neuraminidase, which greatly enhanced binding of peanut agglutinin or soybean agglutinin to human KB cells, had negligible effects on binding of these lectins to mosquito cells. The comparative structures of surface oligosaccharides of mosquito and KB cells are discussed in relation to the lectin-binding studies.     

Binding of hydrophobic ligands to plant lectins: Titration with arylaminonaphthalenesulfonates

Binding of the Hydrophobic ligands 1,8-anilinonaphthalenesulfonic acid (ANS) and 2,6-toluidinylnaphthalenesulfonic acid (TNS) to a variety of plant lectins was studied by lectin-induced alteration of the fluorescence spectra of the two ligands. With one exception, all legume lectins examined bound ANS, with affinity constants ranging from 10³ to 10⁴ M^?1. Similar ANS binding was noted for some nonlegume lectins. Titration of the five isolectins from Phaseolus vulgaris with ANS indicated positive cooperative binding of ANS to the two isolectins E₄ and E₃L₁. Titrations with TNS revealed high-affinity sites for this ligand in a number of lectins. Addition of haptenic sugars did not inhibit binding of ANS, suggesting that the hydrophobic binding sites of lectins are independent of the carbohydrate binding sites.     

Analysis of lectin-specific cell surface glycoprotein on neuroblastoma cells

The binding sites for the lectins wheat germ agglutinin, Ricinus communis agglutinin and concanavalin A on mouse neuroblastoma cell membranes were identified using SDS-gel electrophoresis in combination with fluorescent lectins. Ricinus communis agglutinin and wheat germ agglutinin were found to bind almost exclusively to a single polypeptide with an apparent molecular weight of 30 000. Concanavalin A labeled over 20 different polypeptides, most with molecular weights greater than 50 000. However, when the neuroblastoma cells were treated with concanavalin A so as to internalize all the concanavalin A binding sites visible at the level of the fluorescent microscope and the purified plasma membranes analyzed for their concanavalin A binding polypeptides, only four of the 20 glycopolypeptides were missing or significantly reduced in amount. Thus, these four high molecular weight concanavalin A-binding polypeptides appear to be the major cell surface receptors for concanavalin A. Binding studies with iodinated concanavalin A indicated that these polypeptides represented the high affinity concanavalin A binding sites $\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 2 · 10}{}^{\mathrm{?7}}\text{M}$). Low affinity concanavalin A binding sites were present on the cell surface after internalization of high affinity concanavalin A binding sites.     

Carbohydrate-mediated Recognition of Larval Mosquito Hosts by Romanomermis culicivorax

Proteases, glycosidases, and lectins were tested and the results supported a role in host recognition for glycoproteins containing β-glucose and α-mannose on the cuticular surface of host and parasite. Carbohydrates containing α-glucose, galactose, fucose, or N-acetylglucosamine residues apparently are not involved in nematode attachment. Chitin or a related N-acetylglucosamine polymer was found in R. culicivorax preparasites. Treatment of preparasites with neuraminidase, which hydrolyzes sialic acids, increased nematode attachment to Anopheles freeborni larvae.     

Separation of oyster hemocytes by density gradient centrifugation and identification of their surface receptors

Glutaraldehyde-fixed hemocytes of Crassostrea virginica were subjected to differential centrifugation on a 5, 10, 15, and 25% discontinous sucrose gradient. Five subpopulations of cells were separated by this technique. Subpopulation 1 coincides with the small granulocytes, subpopulation 2 is comprised of hyalinocytes, subpopulation 3 of medium-sized granulocytes, subpopulation 4 of large granulocytes, and subpopulation 5 of a mixture of very large granulocytes and aggregates of small cells. By using several plant and animal lectins, it was ascertained that cells of subpopulations 1, 3, and 4 were agglutinated with Con A and extracts of the albumin glands of Helix pomatia and Cepaea nemoralis while those of subpopulation 2 were agglutinated by the same three lectins as well as wheat germ agglutinin. By applying the Con A-peroxidase cytochemical technique, it was determined that approximately 20% of the granulocytes of subpopulations 1 and 3 do not possess Con A-binding sites and only 18% of the large cells comprising subpopulation 5 possess such sites. These results suggest that the subpopulations of C. virginica granulocytes distinguishable by their dimensions and densities may be further subdivided by differences in specific surface binding sites.     

Two Chitotriose-Specific Lectins Show Anti-Angiogenesis,Induces Caspase-9-Mediated Apoptosis and Early Arrest of Pancreatic Tumor Cell Cycle

The antiproliferative activity of two chito- specific agglutinins purified from Benincasa hispida (BhL) and Datura innoxia (DiL9) of different plant family origin was investigated on various cancer cell lines. Both lectins showed chitotriose specificity, by inhibiting lectin hemagglutinating activity. On further studies, it was revealed that these agglutinins caused remarkable concentration-dependent antiproliferative effect on human pancreatic cancerous cells but not on the normal human umbilical vein endothelial cells even at higher doses determined using MTT assay. The GI₅₀ values were approximately 8.4 μg ml^-1 (0.247 μM) and 142 μg ml^-1(14.8 μM) for BhL and DiL9, respectively, against PANC-1 cells. The growth inhibitory effect of these lectins on pancreatic cancer cells were shown to be a consequence of lectin cell surface binding and triggering G₀/G₁ arrest, mitochondrial membrane depolarization, sustained increase of the intracellular calcium release and the apoptotic signal is amplified by activation of caspases executing cell death. Interestingly, these lectins also showed anti-angiogenic activity by disrupting the endothelial tubulogenesis. Therefore, we report for the first time two chito-specific lectins specifically binding to tumor glycans; they can be considered to be a class of molecules with antitumor activity against pancreatic cancer cells mediated through caspase dependent mitochondrial apoptotic pathway.     

Trypanosoma rhodesiense Bloodstream Trypomastigote and Culture Procyclic Cell Surface Carbohydrates1, 2, 3

Bloodstream trypomastigote and culture procyclic (insect midgut) forms of a cloned T. rhodesiense variant (WRATat 1) were tested for agglutination with the lectins concanavalin A (Con A), phytohemagglutinin P (PP), soybean agglutinin (SBA), fucose binding protein (FBP), wheat germ agglutinin (WGA), and castor bean lectin (RCA). Fluorescence-microscopic localization of lectin binding to both formalin-fixed trypomastigotes and red cells was determined with fluorescein isothiocyanate (FITC)-conjugated Con A, SBA, FBP, WGA, RCA, PNA (peanut agglutinin), DBA (Dolichos bifloris), and UEA (Ulex europaeus) lectins. Electron microscopic localization of lectin binding sites on bloodstream trypomastigotes was accomplished by the Con A-horseradish peroxidase-diaminobenzidine (HRP-DAB) technique, and by a Con A-biotin/avidin-ferritin method. Trypomastigotes, isolated by centrifugation or filtration through DEAE-cellulose or thawed after cryopreservation, were agglutinated by the lectins Con A and PP with agglutination strength scored as Con A < PP. No agglutination was observed in control preparations or with the lectins WGA, FBA or SBA. Red cells were agglutinated by all the lectins tested. Formalin-fixed bloodstream trypomastigotes bound FITC-Con A and FITC-RCA but not FITC-WGA, -SBA, -PNA, -UEA or -DBA lectins. All FITC-labeled lectins bound to red cells. Con A receptors, visualized by Con A-HRP-DAB and Con A-biotin/avidin-ferritin techniques, were distributed uniformly on T. rhodesiense bloodstream forms. No lectin receptors were visualized on control preparations. Culture procyclics lacked a cell surface coat and were agglutinated by Con A and WGA but not RCA, SBA, PP and FBP. Procyclics were not agglutinated by lectins in the presence of competing sugar at 0.25 M. The expression of lectin binding cell surface saccharides of T. rhodesiense WRATat 1 is related to the parasite stage. Sugars resembling α-D-mannose are on the surface of bloodstream trypomastigotes and culture procyclics; n-acetyl-D-galactosamine and D-galactose residues are on bloodstream forms; and n-acetyl-D-glucosamine-like sugars are on procyclic stages.     

Temporal changes in lectin binding of peri-implantation mouse blastocysts

Mouse blastocysts were exposed to a series of ferritin-conjugated lectins during Day 5 (preadhesive) and Day 6 (adhesive; collected Day 5, 24 hr in vitro) of embryogenesis to determine whether there were any changes in lectin binding characteristics that coincided with the acquisition of adhesiveness. After exposure to lectin, the blastocysts were processed for electron microscopy and lectin binding sites were determined by visualization of ferritin particles with the electron microscope. No binding sites were observed for either Dolichos biflorus agglutinin or soybean agglutinin on blastocysts from either stage examined. Binding sites for Ulex europaeus agglutinin, Con A, and wheat germ agglutinin were seen on blastocysts from both stages without apparent increase or reduction in binding sites from either stage. Ricinus communis agglutinin-I (RCA-I) bound heavily to the surface of Day 5 blastocysts and did not bind at all to $\text{3}\text{12}$ Day 6 blastocysts and did bind, though with apparent diminution, to $\text{9}\text{12}$ Day 6 blastocysts, as compared with the binding observed on Day 5 blastocysts. Peanut agglutinin (PNA) did not bind at all to Day 5 blastocysts but did bind heavily to the surface of Day 6 blastocysts. Both RCA-I and PNA bound to the surface of embryos during Day 5 of delayed implantation, thus indicating that neither the appearance of PNA binding sites on Day 6 blastocysts nor the apparent reduction of RCA-I binding sites on Day 6 blastocysts could be solely implicated in the acquisition of adhesiveness. PNA binding sites were abolished from the surface of Day 6 blastocysts by treatment with Pronase, indicating that the PNA binding molecule was associated with a glycoprotein rather than a glycolipid.     

Guanine nucleotides modulate cell surface cAMP-binding sites in membranes from Dictyostelium discoideum

D. discoideum contains kinetically distinguishable cell surface cAMP binding sites. One class, S, is slowly dissociating and has high affinity for cAMP (K_d = 15 nM, $\text{t}\text{1}\text{2}\text{= 15 s}$). A second class is fast dissociating ($\text{t}\text{1}\text{2}$ about 1 s) and is composed of high affinity binding sites H (K_d ≈ 60 nM), and low affinity binding sites L (K_d = ≈ 450 nM) which interconvert during the binding reaction. Guanine nucleotides affect these three binding types in membranes prepared by shearing D.discoideum cells through Nucleopore filters. The affinity of S for cAMP is reduced by guanine nucleotides from 13 nM to 25 nM, and the number of S-sites is reduced about 50%. The number of fast dissociating sites is not altered by guanine nucleotides, but these sites are mainly in the low affinity state. Half-maximal effects are obtained at about 1 μM GTP, 2 μM GDP and 10 μM Gpp(NH)p(guanyl-5′-yl-imidodiphosphate); ATP and ADP are without effect up to 1 mM. These results indicate that D.discoideum cells have a functionally active guanine nucleotide binding protein involved in the transduction of extracellular cAMP signals via cell surface cAMP receptors.     

Multiple interactions between human vitronectin and Staphylococcus aureus

Multiple interactions between human vitronectin and Staphylococcus aureus strain V8 were observed. An upward-curved Scatchard plot indicated both high-affinity binding (K_d1 = 7.4 · 10^?10 M) with 260 binding sites per bacterial cell and moderate-affinity binding (K_d2 = 7.4 · 10^?8 M) with 5240 copies per cell. Negative cooperativity of this binding was characterized by its Hill coeffiocient of less than unity (0.70 ± 0.08). Up to 60% of the vitronectin-bacteria interaction was unaffected by high ionic strength (i.e., 2.4 M NaCl), and was not inhibited by highly-charged heparin oligosaccharides. Various oligosaccharides (4–20 monosaccharide units) generated by partial deaminative cleavage of heparin were found to affect vitronectin binding to S. aureus. Short-chain-length oligosaccharides increase and long oligosaccharides inhibit vitronectin binding, in accordance with direct association of these saccharides with multimeric vitroectin. A protein having a molecular mass of 60 kDa was identified as a putative high-affinity staphylococcal vitronectic-binding protein. These results indicate that interaction of multimeric vitronectin, mostly present at extracellular matrix sites with multiple recognition sites on the S. aureus surface, may contribute to bacterial colonisation.     

Differential binding of lectins to haemocytes of the mussel Mytilus edulis

Summary Pre- and post-embedding techniques were used to investigate the ultrastructural binding of a range of lectins to the haemocytes of the mussel Mytilus edulis. Direct and indirect labelling procedures were employed using colloidal gold and ferritin-labelled lectins, or biotinylated lectins followed by gold-labelled streptavidin. Cell surface receptors were present for lectins from Helix pomatia (HPA), Helix aspersa (HAA), Triticum vulgaris (WGA) and Tetragonolobus purpureas (TPA). Double labelling of haemocytes with HPA and WGA demonstrated binding sites for both lectins on the plasma membrane of the majority of haemocytes. Endocytosis of colloidal gold-labelled HPA was observed for unfixed haemocytes. Three classes of haemocyte were identified by use of morphological criteria: hyalinocytes; granulocytes containing small granules; and granulocytes containing large granules. Lectin binding showed the small granules of the granulocytes to be HPA-positive and the large granules of the granulocytes to be WGA-positive. The WGA-positive granules demonstrated a differential pattern of binding according to granule size. Binding sites for the lectin from Arachis hypogaea (PNA) were not demonstrated on the cell surface, but did show an affinity for the heterochromatin region of the nucleus in post-embedding protocols.     

Wheat Germ Agglutinin Bound to the Outer Cuticle of the Seed Gall Nematodes Anguina agrostis and A. tritici

The presence of wheat germ agglutinin (WGA) on the cuticular surface of the seed gall nematodes Anguina agrostis and Anguina tritici was demonstrated, and the nature of its binding was examined. Crude extracts from the cuticles of A. tritici agglutinated human red blood cells, and only N-acetylglucosamine (GlucNAc) inhibited the agglutination. Distribution of the lectin was visualized by treating live infective juveniles (J2) with rabbit anti-WGA antibody and staining with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG. The lectin bound to the outer cuticular surface of the whole body wall. Pretreatment with GlucNAc oligomers did not reduce the fluorescence created by the anti-WGA-WGA binding, indicating at least a partial nonspeciflc adhesion of the WGA to the nematode surface. Proteolytic enzyme pretreatments diminished the fluorescence, whereas lipase and periodate pretreatments increased the fluorescence. Adult females and males were labeled only on the head and tail, whereas eggs were not labeled at all. It was concluded that the WGA on the J2 cuticle originates from the host.     

Multiplicity of carbohydrate-binding sites in <Emphasis Type="Italic">β</Emphasis>-prism fold lectins: occurrence and possible evolutionary implications

The β-prism II fold lectins of known structure, all from monocots, invariably have three carbohydrate-binding sites in each subunit/domain. Until recently, β-prism I fold lectins of known structure were all from dicots and they exhibited one carbohydrate-binding site per subunit/domain. However, the recently determined structure of the β-prism fold I lectin from banana, a monocot, has two very similar carbohydrate-binding sites. This prompted a detailed analysis of all the sequences appropriate for two-lectin folds and which carry one or more relevant carbohydrate-binding motifs. The very recent observation of a β-prism I fold lectin, griffithsin, with three binding sites in each domain further confirmed the need for such an analysis. The analysis demonstrates substantial diversity in the number of binding sites unrelated to the taxonomical position of the plant source. However, the number of binding sites and the symmetry within the sequence exhibit reasonable correlation. The distribution of the two families of β-prism fold lectins among plants and the number of binding sites in them, appear to suggest that both of them arose through successive gene duplication, fusion and divergent evolution of the same primitive carbohydrate-binding motif involving a Greek key. Analysis with sequences in individual Greek keys as independent units lends further support to this conclusion. It would seem that the preponderance of three carbohydrate-binding sites per domain in monocot lectins, particularly those with the β-prism II fold, is related to the role of plant lectins in defence.