Gotowa bibliografia na temat „Soybean Agglutinin (SBA)”

Colloidal gold-labeled soybean agglutinin (SBA), Helix pomatia agglutinin (HPA), Dolichos biflorus agglutinin (DBA), and Griffonia simplicifolia lectin (GS-1) were used for electron microscopic observation of blood cells. Colloidal gold-labeled SBA, HPA, and DBA showed marked deposition on eosinophil granules at all stages of maturation. Gold particles were not deposited on basophils, neutrophils, monocytes, lymphocytes, or other blood cells. Only a few colloidal gold-labeled GS-1 were deposited on eosinophil granules. Eosinophil granules are rich in N-acetyl-D-galactosamine compounds, and the colloidal gold-labeled SBA, HPA, and DBA are useful for electron microscopic detection of eosinophil granules.

We report studies demonstrating the presence on human marrow stromal cells of binding sites for the soybean lectin (SBA). Marrow cells were separated by agglutination with SBA into an agglutinated cell (SBA+) fraction containing most mature hemic cells including T lymphocytes, and an unagglutinated cell (SBA-) fraction containing the hematopoietic stem cells. The vast majority of fibroblast colony-forming units (CFU- F) (97.2% +/- 1.1%) were in the SBA+ fraction. Mixing experiments using SBA+ and SBA- cells excluded the possibility that these results were caused by an unequal distribution of accessory cells having a regulatory effect on CFU-F growth. In the heterogeneous adherent layers of long-term marrow cultures derived from SBA+ and SBA- cells, endothelial cells, and adipocytes were found almost exclusively in cultures of SBA+ marrow cells. Immunofluorescence studies with fluorescein-labeled SBA revealed the staining of cultured marrow fibroblasts. This staining was inhibited by D-galactose, which is the sugar that specifically binds to SBA. Staining of marrow-derived heterogeneous adherent layers with fluorescein-labeled SBA demonstrated that cultured endothelial cells (identified by rhodamine-labeled antibodies against factor VIII-associated protein) and early adipocytes also had surface glycoproteins binding SBA, although its density on the latter cells was much less important. Thus, our data indicate that human marrow grafts processed with soybean lectin to remove T cells are also depleted of the cellular components of the marrow stroma.

Abstract We report studies demonstrating the presence on human marrow stromal cells of binding sites for the soybean lectin (SBA). Marrow cells were separated by agglutination with SBA into an agglutinated cell (SBA+) fraction containing most mature hemic cells including T lymphocytes, and an unagglutinated cell (SBA-) fraction containing the hematopoietic stem cells. The vast majority of fibroblast colony-forming units (CFU- F) (97.2% +/- 1.1%) were in the SBA+ fraction. Mixing experiments using SBA+ and SBA- cells excluded the possibility that these results were caused by an unequal distribution of accessory cells having a regulatory effect on CFU-F growth. In the heterogeneous adherent layers of long-term marrow cultures derived from SBA+ and SBA- cells, endothelial cells, and adipocytes were found almost exclusively in cultures of SBA+ marrow cells. Immunofluorescence studies with fluorescein-labeled SBA revealed the staining of cultured marrow fibroblasts. This staining was inhibited by D-galactose, which is the sugar that specifically binds to SBA. Staining of marrow-derived heterogeneous adherent layers with fluorescein-labeled SBA demonstrated that cultured endothelial cells (identified by rhodamine-labeled antibodies against factor VIII-associated protein) and early adipocytes also had surface glycoproteins binding SBA, although its density on the latter cells was much less important. Thus, our data indicate that human marrow grafts processed with soybean lectin to remove T cells are also depleted of the cellular components of the marrow stroma.

In human pancreas, soybean agglutinin (SBA) conjugated to horseradish peroxidase reacted with the acinar cells secreting blood group A and/or H antigen, but not with those secreting only B antigen. For detailed histochemical characterization of SBA staining, the effects of treatment with unlabeled lectins and of digestion of certain enzymes on SBA staining were investigated in formalin-fixed, paraffin-embedded pancreatic tissue from individuals of different blood groups. Pre-incubation of sections with unlabeled Dolichos biflorus agglutinin to block A antigen eliminated subsequent SBA staining in the cells secreting A antigen, although failing to induce any effects in those secreting H antigen. In contrast, pre-incubation with unlabeled Ulex europaeus agglutinin-I (UEA-I) to block H antigen abolished SBA staining in cells secreting H antigen but not in those secreting A antigen. Treatment with galactose oxidase yielded the same results as those with unlabeled UEA-I, i.e., SBA reactivity was significantly diminished in cells secreting H antigen but not in those secreting A antigen. Digestion with beta-galactosidase resulted in a slight decrease of SBA staining in the cells secreting H antigen. Accompanying the decrease of SBA staining, reactivity with Griffonia simplicifolia agglutinin-II (GSA-II) appeared for the first time in the enzyme-susceptible, SBA-reactive cells secreting H antigen. Pre-treatment with galactose oxidase abolished this effect of beta-galactosidase. The GSA-II reactivity disclosed by treatment with galactosidase was completely eliminated by digestion with beta-N-acetylhexosaminidase, indicating that GSA-II staining after digestion with galactosidase is due to exposed penultimate beta-N-acetyl-D-glucosamine residues. These results demonstrate that at least two substances react with SBA in acinar cells of human pancreas, one being terminal beta-N-acetyl-D-galactosamine residues of A antigen, and the other being terminal beta-D-galactose-(1----3 or 1----4)-beta-N-acetyl-D-glucosamine dimers in the precursor of blood group H antigen. Such dimers may exist in close proximity to L-fucose residues of H antigen, since unlabeled UEA-I blocked SBA staining.

To evaluate the relative mobilities of cell surface glycoconjugates during myogenesis we have studied the redistribution of fluorescein-conjugated plant lectins on L6 rat myogenic cells. Previous experiments had demonstrated that the receptors for the lectins soybean agglutinin (SBA), wheat germ agglutinin, concanavalin A and Lens culinaris agglutinin all were relatively uniformly distributed on both myoblasts and myotubes, and that SBA receptors were capable of rapid redistribution on myotubes but not myoblasts at 4 degrees C (Sawyer & Akeson, 1983). Here we show that when SBA-labelled myoblasts are incubated at 37 degrees C, or for extended times at 4 degrees C, the lectin aggregates as on myotubes. So it appears that SBA-binding components show a quantitative rather than qualitative change in their mobility during L6 differentiation. In addition, the redistribution of the three other lectins on myoblasts and myotubes was either less prominent (i.e. showing fewer apparent surface clusters) or occurred less rapidly than with SBA. None of these three lectins showed striking differences in mobility between myoblasts and myotubes. Thus, it appears that SBA binds to a subset of surface glycoconjugates that is relatively highly mobile, and that this mobility is specifically enhanced with differentiation.

Two hydrophilic, low temperature-embedding resins, Lowicryl K4M and LR White, were compared in lectin cytochemistry. Post-embedding staining of colloidal gold-labeled Griffonia symplicifolia agglutinin II (GSA-II) resulted in staining of the Golgi apparatus and mucous granules of mucous neck cells in the gastric fundic gland, pylorocytes, and Brunner's gland cells embedded in either resin, although it was much easier to make ultra-thin sections with LR White-embedded material than with the other. Post-fixation with uranyl acetate followed by LR White embedding improved general ultrastructure so that lectin binding sites were identified precisely. All examined lectins, soybean agglutinin (SBA), Maclura pomifera agglutinin (MPA), GSA-II, and Ulex europaeus agglutinin I (UEA-I), stained mucous granules and the Golgi apparatus, in which the staining pattern was characteristic of each lectin: cis cisternae were labeled with SBA and MPA, intermediate cisternae with GSA-II, and trans cisternae and mucous granules with SBA, GSA-II, UEA-I, and lightly with MPA. No labeling was observed in the rough endoplasmic reticulum with any lectin. These findings suggest that the Golgi apparatus is the site of O-linked glycosylation and can be divided into at least three distinct compartments with regard to the glycosylation.

Three lectins, wheat germ agglutinin (WGA), soybean agglutinin (SBA) and Ricinis communis agglutinin I (RCA), were used to study the basement membrane of developing chick lungs. Thinning of the basement membrane at the tips of newly formed bronchi was visualized with all three lectins, but was particularly evident using SBA. Control sections established the ability of the lectins to stain hyaluronic acid and chondroitin sulphate. Neuraminidase, bovine testes hyaluronidase and Streptomyces hyaluronidase removed some of the staining, but none were able to affect the staining of the basement membrane. Possible explanations for this are discussed in the text. Incorporation of [3H]thymidine is enhanced at the tips relative to the interbud area in stage-30 lungs, extending previous studies on stage-26 lungs. Evidence has been presented here which demonstrates that mechanisms of morphogenesis used in avian embryos are similar to those already elucidated in work on mammalian embryos.

Modification of tryptophan side chains of soybean agglutinin (SBA) with N-bromosuccinimide results in a loss of the hemagglutinating and carbohydrate binding activities of the protein. One residue/subunit is probably essential for the binding activity. Modification leads to a large decrease in the fluorescene of the protein accompained by a blue shift. Iodide ion quenching of the protein fluorescence shows that saccharide binding results in a decreased accessibility of some of the tryptophan side chains. These results strongly point towards the involvement of tryptophan residues in the active site of SBA.

The lepidopteran-specific P1 delta-endotoxin of Bacillus thuringiensis var. kurstaki HD-1 was activated in vitro using insect gut proteases and found to be highly specific for the lepidopteran cell line Choristoneura fumiferana CF1 among a wide range of lepidopteran and dipteran cell lines tested. The toxicity of P1 against CF1 cells is inhibited by N-acetylgalactosamine (GalNAc), and the lectins soybean agglutinin (SBA) and wheat-germ agglutinin. Protein blotting was used to identify a glycoprotein of 146 X 10(3) Mr in the plasma membrane of CF1 cells, capable of binding both the toxin and SBA, which is specific for GalNAc. This glycoprotein was labelled using galactose oxidase and sodium boro-[3H]hydride and solubilized in Triton X-100 before partial purification by affinity chromatography on SBA-agarose. We propose that this glycoprotein is a good candidate for the cellular receptor of the lepidopteran-specific P1 delta-endotoxin of B. thuringiensis var. kurstaki HD-1.

Carbohydrates are vital to life. In their naive form, they serve as a primary energy source for supporting life. However, in most cases carbohydrates do not exist as simple sugars in nature. Instead they occur as more complex molecular conjugates known as the glycans and Glycobiology is the study of the roles of these glycans in various biological events. The study of sugars is gaining importance in all strata of today’s scientific world, be it cell biology, immunology or even neurobiology. These glycans do not exist at the cell surface or in the extracellular matrix as free-standing polymers. Rather, they are organized onto specific proteins to form protein-glycan conjugates. One such specific class of proteins is the lectins. Lectins are ubiquitously distributed in nature and are recognized universally by their property of carbohydrate recognition. Much work has been done in the field of lectins to delineate the roles of different lectins in their recognition events and proper propagation of the protein folding machinery. However, these studies mainly concentrate on the animal lectins. Nonetheless, a good deal of structural work has been done on plant lectins and these plant lectins serve as excellent paradigms to work on the ever-exciting problem of protein folding. This thesis primarily concentrates on a glycoprotein plant lectin, Soybean agglutinin. I have mainly addressed the issues of glycosylation and oligomerization on the stability of the protein. Chapter 1 is a prelude to glycosylation in proteins and their functions in vivo. Glycosylation, particularly the N-linked kind, profoundly affects protein folding, oligomerization, and stability. The increased efficiency of folding of glycosylated proteins could be due to the chaperone like activity of glycans, which is observed even when the glycan is not attached to the protein. Covalently linked glycans could also facilitate oligomerization by mediating inter-subunit interactions in the protein or stabilizing the oligomer in other ways. Glycosylation is also seen to affect the rate of fibril formation in prion proteins, with N-glycans reducing the rate of fibril formation, and O-glycans affecting the rate either way based on factors such as position and orientation. While it is still not apparent if there is a common theme in the significance of glycosylation sites in multiply glycosylated proteins or in the conservation of glycans in a related family of glycoproteins, it is evident that glycosylation is a multifaceted post translational modification that incredibly affects proteins. This is followed by a brief description on lectins, their structure, and functions with the emphasis on the various kinds of quaternary associations that they are involved in. Further I have also included a very concise discussion on the major forces involved in protein folding and stability. Chapter 2 deals with the stability studies of two structurally similar legume lectins, Soybean agglutinin and Concanavalin A. The unfolding pathway of these two legume lectins are determined using GdnCl induced denaturation. Both displayed a reversible two-state unfolding mechanism. The analysis of isothermal denaturation data provided values for conformational stability of the two proteins. It was found that the DG of unfolding of SBA was much higher than Con A at all the temperatures at which the experiments were done. Con A had a Tg 18°C lower than SBA. The higher conformational stability of SBA in comparison to Con A is largely due to substantial differences in their degrees of subunit interactions. Ionic interactions at the interface of the two proteins especially at the non-canonical interface seem to play a significant role in the observed stability differences between these two proteins. Further, SBA is a glycoprotein with a GlcNac2Man9 chain attached to Asn-75 of each subunit. The sugar chain in SBA spans the entire non-canonical interface of the protein. However, the entire glycan chain in SBA is not seen in its crystal structure. Only the two proximal GlcNac residues are visible in the crystal structure of the protein. I probed the interactions of these two sugar residues with the amino acid residues of the protein and determined that there were a number of inter-subunit interactions. These interactions further stabilize SBA with respect to Con A, which is not glycosylated. In Chapter 3 I have presented my investigations on the role of glycosylation in the stability of the glycoprotein SBA. I have used the non-glycosylated recombinant form of the protein rSBA expressed in E. coli cells for the stability studies and compared it with the native glycosylated form. The non-glycosylated form features a lower stability when compared to the glycosylated form. Further the unfolding pathways in the two are widely different. While the glycosylated form undergoes a simple two state unfolding, the non-glycosylated species unfolds via a compact monomeric intermediate which is not a molten globule. Representative isothermal and thermal denaturation profiles show that glycosylation accounts for a stabilization of nearly 9 kcal/mol of the tetramer, while the difference in Tm in the two forms is 26oC. To my knowledge this is the first report which shows that glycosylation imparts huge conformational stability to the macromolecule to which it is attached. As already mentioned, the density for the entire oligomannosidic chain is not observed in the crystal structure of the protein. Hence, I attempted to obtain a three-dimensional structure of the glycoprotein by combining the structure of the protein (from the protein data bank) and the glycan (from the www.glycosciences.de database). The glycoprotein thus obtained portrayed substantial inter-subunit glycan protein interactions (hydrogen bonding and hydrophobic) which is perhaps the major cause for the stability of the glycoprotein tetramer. In Chapter 4 I present the thermodynamic analysis on the monomer of SBA and estimate the stability of SBA monomer. Almost all legume lectins show oligomerization-deoligomerization with change in pH. However, pH dependent association/dissociation was not reported for SBA. As evident from size exclusion chromatographic and dynamic light scattering studies, I show that the monomeric form of the protein is existent at pH 1.9. The analyses of CD and fluorescence spectroscopy suggest that the monomer is well folded, and it has a very different characteristic feature when compared to the tetramer. The conformational stabilities of the tetramer and the monomer at the temperature of their maximum stabilities were widely different indicating that oligomerization contributes huge stability to the native molecule. Also, the Tg difference in the two forms of the protein is~40K, while the difference in DCp is only 1.6kcal/mol/K. This suggests that the major hydrophobic core is present in the monomer and oligomerization involves mainly ionic interactions. The thesis ends with a summary of all the findings in Chapter 5. I have tried to review the vital factors that contribute to the stability and quaternary integrity of the protein. Brief discussions involving the roles of oligomerization and glycosylation have been discussed. Further I have also tried to focus on the various unfolding thermodynamic parameters obtained by solution denaturation studies of glycosylated SBA, non-glycosylated SBA and the monomer of SBA and give a comparative account of all with the possible rationale for the variations. Appendix A deals with one of the much-discussed issues of the recent times: the use of quantum dots in biology as imaging and targeting agents. The quantum dots surpass the already accessible existing organic fluorophores by virtue of fluorescence, resistance to photo bleaching and optical properties that facilitate the simultaneous imaging of multiple fluorophores. One-pot synthesized neoglycoconjugates with reactive thiol group are introduced here for functionalization with carbohydrates for solubilization and stabilization of CdSe-ZnS quantum dots (QDs) in aqueous solution. Three different sizes of QDs with lactose, melibiose and maltotriose on them surface have been utilized, for the first time, for lectin detection through agglutination assay. Agglutination of Sugar-QDs (S-QDs) by three different lectins occurred through specific carbohydrate-lectin interactions and was studied extensively by monitoring the scattered light at 600 nm. This assay is very selective, which has been demonstrated by a more selective binding of Soybean agglutinin (SBA) with melibiose-QD as compared to lactose- QD, and specific de-agglutination caused by a-D-galactose while a-Dmannose did not show any effect. The detection sensitivity of the maltotriose-QD was tested with Concanavalin A (ConA), and as low as 100 nM of the lectin was detected using light scattering. Furthermore, the visual detection sensitivity of S-QDs has been enhanced by the emission properties of semiconductor QDs.