Encystment of zoospores of the fungus,Phytophthora cinnamomi,is induced by specific lectin and monoclonal antibody binding to the cell surface

Summary Only two of a number of macromolecules that bind to the surface of zoospores of the dieback fungus,Phytophthora cinnamomi, induce encystment when added to a suspension of actively swimming zoospores. One, the lectin Concanavalin A (ConA), binds to the entire surface of the zoospores including the surface of both flagella. Within 10 minutes more than 70% of the cells have encysted in the presence of 5 g/ml ConA. This encystment is inhibited by preincubation of the lectin with its hapten sugar, -methyl-D-mannoside. The other effective molecule, a monoclonal antibody designated Zf-1, is one of 35 that have been raised to components on the surface of zoospores and cysts ofP. cinnamomi. The antigen for Zf-1 occurs only on the surface of the two flagella. Purified Zf-1 at 15 g/ml causes encystment of 75% of the zoospores in 13minutes. To show that the induction of encystment by these two probes is not due simply to the presence of protein either in solution or bound to the zoospore a number of other proteins were tested, including other antibodies that bind to the zoospore surface. None of these other molecules caused encystment even at concentrations greater than 200 g/ml. The results are consistent with the surface components that bind ConA and Zf-1 being involved in the critical step of triggering encystment at the surface of a potential host during infection.     

Development of Synaptic Glycoproteins: Effect of Postnatal Age on the Synthesis and Concentration of Synaptic Membrane and Synaptic Junctional Fucosyl and Sialyl Glycoproteins

Abstract: Synaptic plasma membranes (SPM) and synaptic junctions (SJ) were isolated from the cortices of rats varying in age between 5 and 28 days. Gel electrophoresis of SPM and SJ indicated a marked increase in the concentration of the "PSD protein" (M. W. 52,000) with development. The biosynthesis of glycoproteins was measured following the intracranial injection of [³H]fucose or [³H] N '-acetylmannosamine. The incorporation of [³H]fucose into synaptic fractions decreased two- to threefold between 10 and 28 days whereas little change in the incorporation of [³H] N '-acetylmannosamine occurred over the same period. Gel electrophoretic analyses of labeled synaptic membranes indicated major increases in the relative incorporation of radiolabeled precursors into glycoproteins with apparent molecular weights of 74,000, 65,000, 50,000, and 40,000 with increasing age. Identification of fucosyl and sialyl glycoproteins following reaction with ¹²⁵I-fucose-binding protein or labeling of sialic acid with NaIO₄NaB[³H₄] demonstrated similar increases in the concentrations of these glycoproteins. Synaptic junctions contained three major glycoproteins with apparent molecular weights of 180,000, 130,000 and 110,000. The reaction of these glycoproteins with ¹²⁵I-fucose-binding protein increased one- to twofold between 10 and 28 days but little variation in their relative distribution or synthesis occurred over this period. The reaction of synaptic junctional glycoproteins GP 180 and GP 110 with ¹²⁵I-wheat germ ag-glutinin increased between 10 and 28 days. The results indicate that the molecular composition of the synapse continues to evolve after the initial synaptic contact has been formed.     

Developmental regulation of the carbohydrate composition of glycoproteins associated with central nervous system myelin

Abstract: Glycoproteins from central nervous system myelin were evaluated for developmental alterations in their carbohydrate composition by autoradiographic analysis of radioiodinated lectin binding after separation by high-resolution sodium dodecyl sulfate-pore gradient slab gel electrophoresis (SDS-PGE). Sixteen lectin-binding components were assessed in highly purified myelin preparations from 15-day, 18-day, and adult rat brains, using the lectins Triticum vulgaris (wheat germ agglutinin) and Ulex europeus (gorse agglutinin I). Developmental changes in lectin binding for individual glycoproteins were evaluated semiquantitatively by comparing densitometric scans of the auto radiographs. Both increases and decreases in lectin binding for individual components were observed as a consequence of development, as well as the appearance and disappearance of lectin binding to three low-molecular-weight components. No changes in electrophoretic mobility and hence glycoprotein molecular weight were observed in any components when using these lectins. These developmental changes in lectin binding suggest that increases in glycoprotein (receptor) density occur, as well as an elaboration of oligosaccharide branching for individual glycoproteins. In addition, the appearance of a new glycoprotein in the adult myelin membrane could imply a new functional role not present in the immature membrane. These observations suggest that dynamic alterations of myelin-associated glycoproteins occur during development. Such developmental regulation of membrane glycoproteins increases the significance of their potential role in myelination and myelin maintenance.     

Lectin receptors on the peri- and early post-implantation mouse embryo

Summary Mouse embryos at the blastocyst, blastocyst outgrowth, and primitive streak (day 7.5) stages of development were analysed for expression of lectin receptors using a panel of six FITC-conjugated lectins with affinities for five distinct saccharides (BSL, ConA, DBA, LTL, UEA and WGA). Blastocyst trophoblast expressed receptors for all the lectins but later tissues of the trophectoderm lineage lost receptors for distinct but overlapping subsets of the lectin panel. The inner cell mass (ICM) of the early blastocyst lacked receptors only for UEA. Differentiation of primary endoderm was accompanied by the aquisition of UEA receptors but subsequent differentiation into visceral and parietal endoderm involved the loss of receptors for both fucose binding lectins (UEA and LTL). Embryonic ectoderm in the day 7.5 egg cylinder retained receptors only for ConA and WGA. Thus, in general, differentiation during the peri- and early post-implantation period was associated with a differential loss of lectin receptors in all cell lineages of the mouse conceptus.     

Plant made anti-HIV microbicides—A field of opportunity

HIV remains a significant global burden and without an effective vaccine, it is crucial to develop microbicides to halt the initial transmission of the virus. Several microbicides have been researched with various levels of success. Amongst these, the broadly neutralising antibodies and peptide lectins are promising in that they can immediately act on the virus and have proven efficacious in in vitro and in vivo protection studies. For the purpose of development and access by the relevant population groups, it is crucial that these microbicides be produced at low cost. For the promising protein and peptide candidate molecules, it appears that current production systems are overburdened and expensive to establish and maintain. With recent developments in vector systems for protein expression coupled with downstream protein purification technologies, plants are rapidly gaining credibility as alternative production systems. Here we evaluate the advances made in host and vector system development for plant expression as well as the progress made in expressing HIV neutralising antibodies and peptide lectins using plant-based platforms.     

Effect of a serine-to-aspartate replacement on the recognition of chitin oligosaccharides by truncated hevein. A 3D view by using NMR

The interaction of a synthetically prepared mutant peptide of hevein (a well known chitin-binding lectin) Hev32S19D with chitin oligosaccharides (and chitosan analogues) has allowed us to estimate their affinity constants and associated thermodynamic data. The mutant peptide is able to bind chitin oligomers, but with significant decreases in the association constants with chito-oligosaccharides. The determination of the three-dimensional structure of the peptide mutant, by using NMR, has permitted us to deduce that the topology of the backbone is very similar to that of the parent Hev32 peptide. The same is true regarding the orientations of the key aromatic residues Trp21, Trp23, and Tyr30. The decrease in the association constants can be attributed to the different topological orientation of key side chains and to the importance of protein-sugar intermolecular essential hydrogen bonds and CH-π stacking interactions. The analysis has permitted us to infer the free energy of binding associated with these interactions as well as to estimate the corresponding binding enthalpy.     

Determination of the affinity constants of recombinant human galectin-1 and -3 for simple saccharides by capillary affinophoresis

The affinity constants of recombinant human galectin-1 and galectin-3 for sugars were determined by capillary affinophoresis. The monoliganded affinophore contains p-aminophenyl-beta-lactoside as an affinity ligand in the matrix of succinylglutathione and has three negative charges. An analysis of the mobility change of the lectins caused by the affinophore and its inhibition by neutral sugars allowed, for the first time, a determination of the affinity constants between the binding sites of the lectins and sugars. The relative magnitude of the affinity constants for each of the sugars in terms of dissociation constants found to be consistent with previously reported data on the concentrations of sugars that caused a 50% inhibition (I50) in the binding assay of the lectin to oligosaccharide-immobilized agarose beads but the absolute values of the dissociation constants were considerably smaller than the I50 values. Capillary affinophoresis indicated microheterogeneity of the lectin preparations and enabled the separate analysis of the affinity of each component simultaneously showing the advantage in using a separation method for analysis of bioaffinity.     

Lectin-histochemical analysis of glycans in ovine and bovine near-term placental binucleate cells

Chorionic binucleate cells (BNC) occur in several ruminants including cow, deer, goat and sheep. They migrate through the chorionic tight junction to fuse with uterine epithelial cells and discharge their granules into maternal connective tissue. We have compared the BNC of near-term, resin-embedded, ovine and bovine placentae using 15 biotinylated lectins and an avidinperoxidase revealing system. There was pronounced conservation of saccharides between the two species. Several sub-types of N-glycan were present, with highly branched structures being abundant, as shown by Galanthus nivalis, Pisum sativum and Phaseolus vulgaris (leuko) agglutinins. Among the non-reducing terminal saccharides conserved were GalNAc1,3(Fuc1,2)-Gal1,4GlcNAc1-, GalNAc1,6Gal1-, Gal1,4GlcNAc-and Gal1,3GalNAc1- shown by Dolichos biflorus, Wisteria floribunda, Erythrina cristagalli, and Maclura pomifera agglutinins, respectively. Arachis hypogaea and Glycine max agglutinins tended to bind to bovine BNC at different stages of maturity, while fucosyl residues detectable by Tetragonolobus purpureus and Ulex europaeus-1 agglutinins were not observed in either species. The only major difference related to sialyl residues, with 2,3-linked sialic acid being present in bovine (Maackia amurensis, Limax flavus) and 2,6 sialic acid being present in ovine (Sambucus nigra agglutinin) cells. This conservation of glycan may be related to glycosylation of peptide hormones in the granules, and may thus be important in the targeting of these hormones to their receptors.     

Bean lectins

Summary The relationship between the polypeptide composition and the agglutination behaviour of the lectin-containing G2/albumin protein groups has allowed the identification of the active lectin polypeptides in different cultivars of Phaseolus vulgaris (Brown et al. accompanying paper). These results were used to ascertain the particular G2/albumin group contained in the various lectin sources used previously for the purification of lectin proteins. Many studies were found to have included lectin sources which contained the same G2/albumin pattern (T_G2) and this common denominator has permitted the direct comparison of the properties reported for these purified lectins. Thus, much of the extensive literature on bean lectins is concurred.     

Immunocytochemical localisation of lectins in cells of Phaseolus vulgaris L. seeds

Antibodies against pure E₄- and L₄-lectins from the seeds of Phaseolus vulgaris L. raised in rabbits were made monospecific by immunoaffinity chromatography on E₄- or L₄-lectin Sepharose 4B columns. Localisation of lectins in bean seeds was investigated by indirect immunofluorescence and by electron microscopy on sections stained with colloidal gold particles coated with monospecific anti-E₄- and anti-L₄-IgG. In parenchyma cells from the cotyledons both E- and L-type lectins were found inside the protein bodies. Apparently the matrix of all protein bodies contained both types of lectins. On the other hand in vascular and in axis cells the two types of lectins were localised in the cytoplasm, outside the protein bodies. Thus these findings suggest different roles for the lectins: in cotyledons this may be a specific form of N storage, while in vascular and axis cells lectins may have a more direct metabolic part to play.