Cell Surface Carbohydrate Moieties Research Articles

Bovine Nebovirus Interacts with a Wide Spectrum of Histo-Blood Group Antigens.

ABSTRACTSome viruses within the Caliciviridae family initiate their replication cycle by attachment to cell surface carbohydrate moieties, histo-blood group antigens (HBGAs), and/or terminal sialic acids (SAs). Although bovine nebovirus (BNeV), one of the enteric caliciviruses, is an important causative agent of acute gastroenteritis in cattle, its attachment factors and possibly other cellular receptors remain unknown. Using a comprehensive series of protein-ligand biochemical assays, we sought to determine whether BNeV recognizes cell surface HBGAs and/or SAs as attachment factors. It was found that BNeV virus-like particles (VLPs) bound to A type/H type 2/Ley HBGAs expressed in the bovine digestive tract and are related to HBGAs expressed in humans and other host species, suggesting a wide spectrum of HBGA recognition by BNeV. BNeV VLPs also bound to a large variety of different bovine and human saliva samples of all ABH and Lewis types, supporting previously obtained results and suggesting a zoonotic potential of BNeV transmission. Removal of α1,2-linked fucose and α1,3/4-linked fucose epitopes of target HBGAs by confirmation-specific enzymes reduced the binding of BNeV VLPs to synthetic HBGAs, bovine and human saliva, cultured cell lines, and bovine small intestine mucosa, further supporting a wide HBGA binding spectrum of BNeV through recognition of α1,2-linked fucose and α1,3/4-linked fucose epitopes of targeted HBGAs. However, removal of terminal α2,3- and α2,6-linked SAs by their specific enzyme had no inhibitory effects on binding of BNeV VLPs, indicating that BNeV does not use terminal SAs as attachment factors. Further details of the binding specificity of BNeV remain to be explored.IMPORTANCE Enteric caliciviruses such as noroviruses, sapoviruses, and recoviruses are the most important etiological agents of severe acute gastroenteritis in humans and many other mammalian host species. They initiate infection by attachment to cell surface carbohydrate moieties, HBGAs, and/or terminal SAs. However, the attachment factor(s) for BNeV, a recently classified enteric calicivirus genus/type species, remains unexplored. Here, we demonstrate that BNeV VLPs have a wide spectrum of binding to synthetic HBGAs, bovine and human saliva samples, and bovine duodenal sections. We further discovered that α1,2-linked fucose and α1,3/4-linked fucose epitopes are essential for binding of BNeV VLPs. However, BNeV VLPs do not bind to terminal SAs on cell carbohydrates. Continued investigation regarding the proteinaceous receptor(s) will be necessary for better understanding of the tropism, pathogenesis, and host range of this important viral genus.

Saccharide/glycoprotein recognition inside synthetic ion channels modified with boronic acid

Here, we demonstrate a novel sensing platform for the molecular recognition of monosaccharides (glucose and fructose) and glycoprotein (ovalbumin) using synthetic ion channels fabricated in polymeric membrane. To achieve this, receptor (3-aminophenylboronic acid) is covalently attached with carboxylic groups on the surface and inner walls of the channel. The detection principle rests on the reversible binding occurring between boronic acid and diols of saccharides or glycoprotein molecules, leading to a sensible change in the ionic flux across the nanochannel. In the presence of saccharides or glycoprotein at neutral pH, saccharide–boronic acid complex changes the channel surface from neutral to negative. The binding of ovalbumin onto the channel surface also leads to the reduction of the effective channel diameter, which in turn diminishes the channel conductance. Based on the experimental results, we believe that the presented sensing platform could also be applicable in the area of biomedical sciences for the recognition of cell-surface carbohydrate moieties.

Amaranthus leucocarpus lectin (ALL) Enhances anti-CD3-Dependent Activation of Murine T Cells and Promotes Cell Survival

The Galβ1,3GalNAc-specific lectin from Amaranthus leucocarpus (ALL) shows a differential binding pattern on murine thymocytes, peripheral and activated CD4+ and CD8+ T cells. Although ALL detects activation-related changes in T cell surface carbohydrate moieties, no study has been performed to examine the effect of ALL on T cell activation. In this study, we analyzed the anti-CD3-dependent activation of murine T cells in the presence of ALL by measuring proliferation, surface activation marker expression, and IL-2 secretion using total cells from the lymph node. The results showed that ALL did not significantly induce T cell activation but did enhance anti-CD3-dependent activation of both CD4+ and CD8+ T cells. In addition, ALL protected T cells from spontaneous apoptosis and increased cell survival in serum-free culture conditions. Our findings indicate that ALL alone does not affect T cell activation, but do suggest that ALL has an anti-CD3-dependent co-stimulatory-like effect on T cell activation. Moreover, ALL promotes cell survival in regular and serum-free culture conditions. This study is the first report of a non-mitogenic T cell-binding lectin that can induce a possible costimulatory-like effect and provides a new tool for understanding how glycosylation impacts the T cell response.

Cell surface glycoconjugate abnormalities and corneal epithelial wound healing in the pax6+/- mouse model of aniridia-related keratopathy.

Congenital aniridia due to heterozygosity for Pax6 is associated with ocular surface disease, including keratopathy. This study investigated how defects in glycoconjugate component of the cell surface of Pax6+/- could cause the abnormal cellular migration phenotypes associated with the disease. Immunohistochemistry, lectin-based histochemistry, conventional staining techniques, and proteomic assays were performed on eyes and cultured corneal epithelial cells from wild-type and Pax6+/- littermates. Wild-type cells were manipulated in culture to replicate the glycoconjugate abnormalities found in Pax6 heterozygotes and determine the consequences for wound healing. Multiple glycoconjugate defects were found in Pax6-mutant cells. Lectin cytochemistry of corneal epithelial cells suggested a partial failure of glycoprotein trafficking. Blocking cell surface carbohydrate moieties in wild-type corneal cells caused wound-healing delays similar to those seen in untreated Pax6+/- cells. Alterations to the cell surface glycoconjugate signature of Pax6+/- corneal epithelia restrict the ability of cells to initiate migration in response to wounding. This underlies the observed wound-healing delay in cultured Pax6+/- epithelia.

Mode of Molecular Recognition of l-Fucose by Fucose-Binding Legume Lectins

Recognition of cell surface carbohydrate moieties by lectins plays a vital role in many a biological process. Fucosyated residues are often implicated as key recognition markers in many cellular processes. In particular, the aspects of molecular recognition of fucose by fucose-bindinglectins UEA 1 and LTA pose a special case because no crystal structure of these lectins is available. The study was conducted to elucidate the process of recognition of l-fucose by UEA1 and LTA by correlating structure-based sequence alignment and other available biochemical/biophysical data. The study points out that the mode of recognition of l-fucose is coordinated by the invariant triad of residues the asparagine 137, glycine 105, and aspartate 87. The major hydrophobic stacking residue in this case is the tyrosine 220. The study also reiterates the key role of the conserved triad of residues in the combining site which is a common feature for all legume lectins whose crystal structures are known.

Immunohistochemical and developmental studies to elucidate the mechanism of action of the snowdrop lectin on the rice brown planthopper, Nilaparvata lugens (Stal).

Rice brown planthoppers ( Nilaparvata lugens) were fed on artificial diet containing snowdrop lectin ( Galanthus nivalis agglutinin; GNA), which has been shown to be toxic towards this insect pest. In addition to decreasing survival, the lectin affected development, reducing the growth rate of nymphs by approximately 50% when present at a concentration of 5.3 μM. Immunolocalisation studies showed that lectin binding was concentrated on the luminal surface of the midgut epithelial cells within the planthopper, suggesting that GNA binds to cell surface carbohydrate moieties in the gut. Immunolabelling at a lower level was also observed in the fat bodies, the ovarioles, and throughout the haemolymph. These observations suggest that GNA is able to cross the midgut epithelial barrier, and pass into the insect's circulatory system, resulting in a systemic toxic effect. Electron microscope studies showed morphological changes in the midgut region of planthoppers fed on a toxic dose of GNA, with disruption of the microvilli brush border region. No significant proteolytic degradation of GNA was observed either in the gut or honeydew of planthoppers fed on lectin-containing diet. The presence of glycoproteins which bind GNA in the gut of the brown planthopper was confirmed using digoxigen-labeled lectins to probe blots of extracted gut polypeptides.

Lectin histochemical identification of carbohydrate moieties in opossum chemosensory systems during development, with special emphasis on VVA‐identified subdivisions in the accessory olfactory bulb

Lectins, sugar-binding molecules of nonimmune origin, were used in this study to describe the development of the main olfactory and vomeronasal systems in the Brazilian gray short-tailed opossum, Monodelphis domestica. A battery of seven lectins of the N-acetylgalactosamine/galactose-binding group was used. Of the seven lectins, only two, Vicia villosa agglutinin (VVA) and Griffonia (Bandeiraea) simplicifolia lectin I-isolectin B4 (GS I-B4), were specific to the vomeronasal system. The other five lectins recognized carbohydrates in both chemosensory systems, although the binding was more intense in the accessory olfactory system. Furthermore, whereas six of the lectins stained the adult opossum accessory olfactory bulb (AOB) homogeneously, the VVA lectin distinguished two regions of the AOB. Similar to the expression of olfactory marker protein (OMP) (Shnayder et al. [1993] Neuroreport 5:193-196), the rostral half of the AOB stained much darker with VVA than the caudal half, and the onset of the restricted pattern of staining at age 45 days also coincided. We conclude that 1) GS I-B4 and VVA recognize cell surface carbohydrate moieties specific to the vomeronasal, but not to the main olfactory, system, and 2) the carbohydrate moiety that is recognized by the VVA lectin, presumably terminal N-acetyl-galactosamine, is both temporally and spatially restricted in the opossum AOB. These results are discussed in the framework of other known spatially restricted molecules of the two major nasal chemosensory systems.

Differences in cell surface carbohydrates, and in laminin and fibronectin synthesis, between adherent and non-adherent Ehrlich ascites tumor cells.

Differences in cell surface carbohydrates and in laminin and fibronectin synthesis between 2 Ehrlich ascites tumor (EAT) cell lines, the adherent and non-adherent EAT cells, have been studied. The adherent EAT (a-EAT) cells grow in monolayer in vitro in the presence of fetal bovine serum. The classical, or non-adherent EAT (na-EAT), cells grow in suspension in ascites form in the peritoneal cavity of mice, and they do not adhere when cultured in vitro. Both EAT cell lines express surface glycoproteins reactive with Maackia amurensis lectin (MAL) and Griffonia simplicifolia isolectin GS I-B4. However, only a-EAT cells react with elderberry (Sambucus nigra) bark lectin (SNA), suggesting that there are some differences in the sialylation of cell surface carbohydrate moieties between these 2 EAT cell lines. Removal of cell surface sialic acid by treating a-EAT cells with Vibrio cholerae neuraminidase did not abolish the ability of these cells to adhere to laminin- or fibronectin-coated plates, indicating that the sialic acid of the cell surface glycoproteins is not essential for their adhesion to these extracellular matrices. Therefore, the difference in sialylation of cell surface glycoproteins is not responsible for the difference in cell adhesion between these 2 lines of EAT cells. Both EAT cell lines express detectable amounts of laminin but not fibronectin on their surfaces; they both secrete fibronectin and entactin into the medium. The na-EAT cells (but not the a-EAT cells) also secrete laminin A chain into the culture medium; however, no B chain was detected in the culture medium of either cell line. The laminin isolated from the cell surface of na-EAT cells reacts with GS I and MAL lectins, but not with SNA, whereas the laminin isolated from a-EAT cells reacts with SNA, as well as GS I and MAL.

S4.11 Unsialylated PNA binding sites in human melanoma: A new marker of metastatic potential

Cell surface glycoconjugates influence the metastatic behaviour of tumor cells. Using a recently developed model of human tumor spontaneous metastasis (1), we examined changes in cell surface carbohydrate moieties in melanoma cells associated with metastasis. Lectin binding patterns of seven human melanoma clones and variants, selected from the same parental cell line (2), were compared by flow cytometry and Scatchard analysis. Human melanoma clones/variants with high (HM) and low metastatic (LM) potential could be distinguished by their PNA binding, but not by their WGA, UEA I and SBA binding. LM clones/variants proved to be made up from a single cell population, poorly labeled by PNA (2 .2 10 6 sites/cell). In contrast, HM clones/variants were constituted by two cellular subpopulations exhibiting a moderate (2 .6 -3 .7 10 6 sites/cell) and a high PNA staining (17,7 18.8 10 6 sites/cell). Using an EPICS V cytometer, these two subpopulations were sorted and tested for their metastatic abilities. Cells with high PNA binding generate a higher frequency of metastases than cells with moderate PNA binding. Following treatment with Vibrio Cholerae neuraminidase, all cells from all variants and clones were brightly labeled by PNA, collecting in a single peak with similar fluorescence intensity. We have identified 2 major PNA reactive glycoproteins of 110 and 140 kDa, expressed only in HM cells, which became strongly labeled in the LM cells only after neuraminidase treatment. These results suggest that the expression of terminal unsialylated Gal/31-3GalNAc structure, specifically positioned on two glycoproteins 140 kDa and 110 kDa, play a key role in the metastatic potential of human melanoma ceils. These carbohydrate alterations detected in ceils in vitro are also expressed by tumor ceils in vivo. In 37 specimens tested, PNA appears to react selectively with invasive tumors, since none of the four early primary melanomas (Clark I-II) reacted while 10/16 late primary melanomas (Clark Il l-V) and 3/5 melanoma metastases were reactive. Only 1/12 benign nevi bound PNA. Our data gave evidence that the non sialylation of terminal Gal/31-3GalNAc carbohydrate may be markers of invasive melanoma, applicable on an individual patient basis. (1) Bailly M., Bertrand S., Dor6, J. F., Int J Cancer 49:457466, 1991. (2) Bailly M., Dor6 J. F., Int J Cancer 49:750-757, 1991. $4.12 Polylactosaminoglycans of Lysosomal Membrane Glycoproteins Lamp-1 and Lamp-2 in Human Milk

Role of asparagine-linked carbohydrates in pulmonary metastasis of B16-F10 murine melanoma cells: implication through glycosylation inhibition by nojirimycin.

N-linked oligosaccharide moieties of cell surface glycoconjugates may be involved in the metastatic potential of tumour cells. Many studies have examined the anti-metastatic properties of inhibitors of carbohydrate synthesis or processing in vitro. However, there has so far been little evidence of such inhibitory factors on metastasis in vivo because of the general high toxicity of the inhibitors. In this study, we found nojirimycin (NM) to be a substantially non-toxic carbohydrate synthesis inhibitor that inhibited the experimental metastasis of B16-F10 melanoma cells not only in vitro but also in vivo. When NM was administered intraperitoneally to syngeneic C57BL/6 mice, the inhibition was dose-dependent, with 40-75% suppression of pulmonary colonization observed after 5 days exposure to NM (at 0.4 or 4 mg/day). An in vitro lectin sensitivity assay showed that NM-treated B16-F10 cells were less susceptible to the lectin concanavalin-A, suggesting alteration or decrease of high mannose-type carbohydrate moieties on the cell surface. Further in vitro analysis of adhesiveness between B16-F10 cells and endothelial cells demonstrated that NM treatment causes reduced binding of B16-F10 cells to endothelial cells. We have also studied the effect of NM on natural killer (NK) cells in mice. NM treatment elicited no substantial increase in the cytotoxic activity of splenic NK cells against YAC-1 target cells. These results indicate that NM acts on the process of adhesion and that specific structures of the cell surface carbohydrate moieties may be involved in the colonization phase of metastasis in vivo.

Crystallization of the B chain of Shiga-like toxin I from Escherichia coli

Shiga-like toxin I (SLT-I) is produced by several pathogenic strains of Escherichia coli associated with diarrheal disease. The toxin consists of an A chain, which attacks eukaryotic ribosomes, inhibiting protein synthesis, and multiple copies of a 69 ammo acid B chain. The B subunit mediates cell binding and uptake through its interactions with cell surface carbohydrate moieties. Here we report that the B chain has been crystallized in a form suitable for high-resolution X-ray analysis. The space group is P2 12 12 1, with a = 56.2 A ̊ , b = 59.9 A ̊ and c = 102.5 A ̊ . A rotation function using three-dimensional diffraction data suggests that the asymmetric unit is a tetramer.

Role of cell surface carbohydrate moieties in monocytic cell adhesion to endothelium in vitro.

Monocyte adhesion to endothelium represents the first step in the emigration of this leukocyte from blood to tissue during such pathologic and physiologic processes as atherosclerotic plaque development, wound healing, and inflammation. We have examined the role of carbohydrate moieties in the binding of mononuclear cells to endothelium in vitro. Wheat germ agglutinin (WGA) completely inhibited binding of the human monocytic cell line U937 to pig or human endothelial cells (EC). The inhibition was abolished by the presence of N-acetyl glucosamine, a preferred ligand for WGA. This sugar itself, however, had no effect on monocytic cell binding to EC, suggesting that WGA is inhibiting the cell-cell interaction by binding to a distinct sugar moiety. We tested a series of simple and phosphorylated sugars for the ability to inhibit U937 cell binding to EC. Two phosphorylated disaccharides, lactose-1-phosphate and maltose-1-phosphate, but not 14 other sugars, caused complete suppression of monocyte adhesion to EC. Among the inactive sugars were mannose-6-phosphate and fructose-1-phosphate, which have been shown by others to markedly suppress lymphocyte adhesion to EC. A nonionic detergent, n-octyl-beta-D-glucopyranoside (octyl glucoside), which contains a sugar group as a hydrophilic moiety, also inhibited U937 cell or human monocyte binding to human or porcine EC. The inhibition was observed at a nontoxic concentration of octyl glucoside and appeared to be due to an effect on the monocytic cell rather than the EC. When suboptimal doses of WGA and octyl glucoside were added in combination to the U937 cell-EC adhesion assay, the level of inhibition was greatly reduced when compared with either of the inhibitors alone, suggesting an interaction between these two blocking agents. Lactose-1-phosphate, but not octyl glucoside or WGA, blocked neutrophil adhesion to EC. In summary, our results indicate that specific cell surface carbohydrate groups are required for the adhesion of monocytes to the endothelium.

Age- and region-dependent concanavalin A reactivity of chick wing-bud mesoderm cells.

MORPHOGENETIC potential of embryonic chick limb mesoderm cells declines with time beginning at approximately stage 24 of development1. This decline occurs in a proximo-distal direction2. Accompanying the loss of morphogenetic potential there is an age-dependent decline in the ability of limb and axial mesoderm to sort from one another in a randomised mixture of cells1,3,4. Due to the age-dependent decline in both morphogenetic potential and the sorting ability of the limb mesoderm cells, it has been suggested that the decline in morphogenetic potential may be related to changes occurring at the cell surface5. Cell surface carbohydrate moieties have been postulated to play an integral part in cellular interactions6. Because of their high degree of carbohydrate-binding specificity7, plant lectins, such as concanavalin A (con A), Ricinus communis agglutinin (RCA), and wheat germ agglutinin (WGA), have been used to monitor cell surface changes which occur during development7–13. These changes manifest themselves as differences in lectin-induced agglutinability of cells, and changes in number and distribution of lectin binding sites on the cell surface. This study was designed to determine if there were cell surface changes accompanying the decline in morphogenetic potential of the limb mesoderm cells, which could be detected by the use of plant lectin probes. We report here that there is a slight increase in RCA-induced agglutination of chick wing-bud mesoderm cells with increasing developmental age. There is also a significant age-dependent, proximo-distal decline in the susceptibility of chick wing-bud mesoderm cells to con A-induced agglutination between stages 19 and 26 of development. As such, there is a spatial as well as a temporal correlation between the decline of con A agglutinability and the decline of the morphogenetic potential of the chick limb-bud mesoderm cells.

LIGAND-INDUCED MOVEMENT OF LYMPHOCYTE MEMBRANE MACROMOLECULES

The fate of different complexes on the membrane of thymocytes and spleen lymphocytes was studied with the use of both immunofluorescence and ultrastructural radioautography. The complexes of anti-immunoglobulin (Ig) with the surface Ig of B lymphocytes were present all around the membrane at 4 degrees C; an increase in temperature produced a rapid aggregation of the complex into a cap which was readily interiorized in vesicles. Ultrastructural details of this process were given. The movement of the complexes depended upon the amount of anti-Ig and the temperature. The complexes of anti-lymphocyte antibody with surface antigen(s) did not result in formation of a single large aggregate (or cap) unless an anti-antibody was brought into the reaction. The caps formed by this trilayered complex were not interiorized. Concanavalin A (Con A) bound to cell surface carbohydrate moieties and the complexes of Con A readily formed a cap and were interiorized. Finally, antibodies to H-2 determinants did not form in most instances a single cap aggregate even when anti-antibodies were used. With time the H-2 complexes tended to form several large aggregates with some endocytosis.