Differential binding of conA and WGA on the cell surface, the role of sialic acid in their expression and the increased activity of sialidase after cis-Platin treatment

It is reported that concanavalin A (conA) and wheat germ agglutinin (WGA) have a differential binding pattern on normal mouse spleen lymphocytes and the surface of Dalton's lymphoma cells. It is suggested that sialic acid on the cell surface controls the expression of lectin binding sites. Further, it has been observed that the increased release of sialic acid from cell surfaces after cis-dichlorodiammine platinum (II) (cis-Platin) treatment is due to the increased activity of sialidase.     

An agglutinin with unique specificity for N-glycolyl sialic acid residues of thyroglobulin in the hemolymph of a marine crabScylla serrata (Forskal)

A novel agglutinin with specificity for sialic acid sequence of sugars in thyroglobulin is identified in the hemolymph ofScylla serrata. The physico-chemical characteristics of its binding affinity, such as pH and temperature optima, and cationic requirements are defined. N-glycolyl neuraminic acid (NeuGc) (at 0.6 mM), in contrast to N-acetyl neuraminic acid (NeuAc) (at >5.0 mM), is the potent inhibitor of hemagglutination. Bovine and porcine thyroglobulins containing NeuGc, inhibited the agglutination. NeuGc-acid glycoprotein fraction (bovine) but not NeuAc-acid glycoprotein fraction (human) inhibited the hemagglutination. The inability of other NeuGc-glycoproteins (bovine submaxillary mucin) to inhibit the agglutination suggests that the agglutinin may also recognize glycosidic linkage associated with NeuGc. The potential of the agglutinin in identifying NeuGc containing human tumor associated antigens is discussed.     

Role of sialic acid in the maintenance of cell surface rigidity.

Removal of the cell surface sialic acid with neuraminidase brings about cell deformation in amoeba. The membranes of these deformed cells are eventually ruptured leading to the liberation of the cell mass.     

A haemagglutinin in the tissue fluid of the Pacific oyster,Crassostrea gigas,with specificity for sialic acid residues in glycoproteins

Summary An agglutinin for human red cells has a specificity for sialic acid and a high affinity for bovine salivary glycoprotein. Digestion of the glycoprotein with Pronase or neuraminidase indicated that binding of sialic acid to receptors in the agglutinin is the first tepp in the mechanism of formation of a stable complex between ligand and receptor.We wish to thank Dr. P. Walne, Conwy Laboratory, MAFF, for generous supplies of Crassostrea gigas. One of us (SWH) wishes to thank the Natural Environment Research Council for financial support.     

N-acetylmuramic acid 6-phosphate lyases (MurNAc etherases): role in cell wall metabolism, distribution, structure, and mechanism

MurNAc etherases cleave the uniqued-lactyl ether bond of the bacterial cell wall sugar N-acetylmuramic acid (MurNAc). Members of this newly discovered family of enzymes are widely distributed among bacteria and are required to utilize peptidoglycan fragments obtained either from the environment or from the endogenous cell wall (i.e., recycling). MurNAc etherases are strictly dependent on the substrate MurNAc possessing a free reducing end and a phosphoryl group at C6. They carry a single conserved sugar phosphate isomerase/sugar phosphate- binding (SIS) domain to which MurNAc 6-phosphate is bound. Two subunits form an enzymatically active homodimer that structurally resembles the isomerase module of the double-SIS domain protein GlmS, the glucosamine 6-phosphate synthase. Structural comparison provides insights into the two-step lyase-type reaction mechanism of MurNAc etherases: β-elimination of the D-lactic acid substituent proceeds through a 2,3-unsaturated sugar intermediate to which water is subsequently added. Received 31 August 2007; received after revision 12 October 2007; accepted 1 November 2007     

Sialic acid binding lectins

The literature contains several reviews on lectins in general, covering mainly those from plants and invertebrates. However, the sialic acid binding lectins have not been reviewed so far. Considering the importance of sialic acids in cell sociology, lectins which specifically recognize terminal sialic acid residues are potentially useful as analytical tools in studying the biological functions of sialoglycoconjugates. These lectins, along with monoclonal antibodies raised against sialoglycoconjugates, have been used in the detection, affinity purification, cytochemical localization and quantitation of such glycoconjugates. In this review the main emphasis has been placed on the occurrence, general purification procedures, macromolecular properties, sugar specificities and applications of these lectins.     

Sialic acid binding lectins

Summary The literature contains several reviews on lectins in general, covering mainly those from plants and invertebrates. However, the sialic acid binding lectins have not been reviewed so far. Considering the importance of sialic acids in cell sociology, lectins which specifically recognize terminal sialic acid residues are potentially useful as analytical tools in studying the biological functions of sialoglycoconjugates. These lectins, along with monoclonal antibodies raised against sialoglycoconjugates, have been used in the detection, affinity purification, cytochemical localization and quantitation of such glycoconjugates. In this review the main emphasis has been placed on the occurrence, general purification procedures, macromolecular properties, sugar specificities and applications of these lectins.     

Diet influences the colonisation of Campylobacter jejuni and distribution of mucin carbohydrates in the chick intestinal tract

The objectives of this study were to evaluate the effect of diet on the colonisation by Campylobacter jejuni of the chick caeca, and to determine whether the viscosity of the intestinal contents and mucin carbohydrates were altered by the diet. The diets investigated were maize based, wheat-based or wheat-based supplemented with xylanase. The xylanase-supplemented diet reduced the viscosity and lowered the numbers of Camp. jejuni. Feeding the enzyme-supplemented diet increased the amount of neutral and sulphated mucins in the goblet cells of the small and large intestines and caecum. An abundance of sulphated and carboxylated mucins was seen in the surface goblet cells of the large intestine with the maize- and wheat-based diets. Both the diet supplemented with xylanase and the maize diets increased crypt-surface glycosylation of the sialic acid residues. The analysed data from the combined sites showed significant differences in the amount of neutral and acidic mucins when comparing the wheat and the wheat plus xylanase diets. However, no changes were shown in the staining intensity of sulphated mucins between the three diets. Significant differences in the glycosylation of sialic acid and in the N-acetylglucosamine residues were shown between dietary groups. These results provide evidence that the wheat diet supplemented with xylanase leads to greater changes in the mucin composition and carbohydrate expression of goblet cell glycoconjugates, which are associated with a reduction in intestinal viscosity and decreased numbers of Camp. jejuni.     

Isolated rat hepatocytes. Simultaneous study of variations in sialic acid content of glycoconjugated membranes and asialotransferrin uptake]

Hepatocytes isolated from streptozotocin treated Rats bind less asialotransferrin than hepatocytes isolated from normal rats. This decrease is parallel with a decrease in the sialic acid content. Insulin therapy restored simultaneously membrane sialic acid content and asialotransferrin binding capacity.     

The absence of gangliosides in a higher plant

Summary We report attempts to isolate and purify sialic acid-containing glycolipids (gangliosides) from etiolated hypocotyls of soybean (Glycine max) using methods developed for rat liver. The maximum amounts of ganglioside sialic acid present was found to be less than. 0.021 nmole/g fresh weight or less than 1:100,000 the amounts present in rat liver. We conclude that this tissue lacks gangliosides.     

Changes in the sialic acid content of chick thymus and bursa of Fabricius during age-involution.

The sialic acid content of both thymus and bursa of Fabricius during their growth and involution phases in chick has been reported in this study. It is observed that the sialic acid concentration is very high in 1-week-old chickens. The concentration subsequently decreases to a significant level and rises again prior to the onset of involution. In the post-involution period, a more or less minimal and constant level is maintained. The role of sialic acid in cellular activities of thymo-bursal system has been discussed.     

Changes in the sialic acid content of chick thymus and bursa of Fabricius during age-involution

Summary The sialic acid content of both thymus and bursa of Fabricius during their growth and involution phases in chick has been reported in this study. It is observed that the sialic acid concentration is very high in 1-week-old chickens. The concentration subsequently decreases to a significant level and rises again prior to the onset of involution. In the postinvolution period, a more or less minimal and constant level is maintained. The role of sialic acid in cellular activities of thymo-bursal system has been discussed.Supported by the University Grants Commission, Govt of India under the scheme Support to Research.     

Reappearance in vivo of neuraminidase-sensitive sialic acid in L 5222 rat leukemia cells

Summary Cell electrophoretic data and quantitative sialic acid determination show that, 16 to 20h after i.p. implantation of neuraminidase-treated L 5222 rat leukemia cells, the original sialic acid content at the cell periphery is reconstituted.This investigation was performed within the EORTIC Cell Surface Project Group and supported by the Swiss National Science Foundation, Grant No. 3.901.72, and by the Zürich Cancer League.     

Podocalyxin enhances the adherence of cells to platelets

Podocalyxin (PODXL) is a mucin protein of the CD34 family expressed in kidney glomerular podocytes, vascular endothelium, progenitor bone marrow and tumor cells. It is assumed that PODXL plays an anti-adherent role in kidney podocytes. CHO cells stably expressing human PODXL (CHO-PODXL) or human tumor cells (Tera-1) inherently expressing PODXL showed increased adherence to platelets. The adherence of cells was inhibited (70%) by blockers of platelet P-selectin, prevented by the soluble ectodomain of human PODXL (PODXL-Δ) or by the arginine-glycine-aspartate (RGDS) peptide and partially impeded by inhibition of integrin αVβ3/αVβ5, suggesting a coordinated action of P-selectin and integrins. Colocalization of platelet P-selectin and PODXL expressed on CHO cells was demonstrated by confocal immunofluorescence. No adherence to platelets was observed when PODXL was expressed in glycomutant CHO cells deficient in sialic acid. Received 14 August 2007; received after revision 12 September 2007; accepted 13 September 2007     

The cytotoxicity of eosinophil cationic protein/ribonuclease 3 on eukaryotic cell lines takes place through its aggregation on the cell membrane

Human eosinophil cationic protein (ECP)/ ribonuclease 3 (RNase 3) is a protein secreted from the secondary granules of activated eosinophils. Specific properties of ECP contribute to its cytotoxic activities associated with defense mechanisms. In this work the ECP cytotoxic activity on eukaryotic cell lines is analyzed. The ECP effects begin with its binding and aggregation to the cell surface, altering the cell membrane permeability and modifying the cell ionic equilibrium. No internalization of the protein is observed. These signals induce cell-specific morphological and biochemical changes such as chromatin condensation, reversion of membrane asymmetry, reactive oxygen species production and activation of caspase-3-like activity and, eventually, cell death. However, the ribonuclease activity component of ECP is not involved in this process as no RNA degradation is observed. In summary, the cytotoxic effect of ECP is attained through a mechanism different from that of other cytotoxic RNases and may be related with the ECP accumulation associated with the inflammatory processes, in which eosinophils are present. Received 26 October 2007; accepted 23 November 2007     

Annexin V interactions with collagen

Annexin V was originally identified as a collagen-binding protein called anchorin CII and was isolated from chondrocyte membranes by affinity chromatography on native type II collagen. The binding of annexin V to native collagen type II is stable at physiological ionic strength when annexin V is reconstituted in liposomes. The binding to native collagen types II and X, and to some extent to type I as well, was confirmed using recombinant annexin V. A physiological role for annexin V interactions with extracellular collagen is consistent with the localization of annexin V on the outer cell surface of chondrocytes, microvilli of hypertrophic chondrocytes, fibroblasts and osteoblasts. A breakthrough in our understanding of the function of annexin V was made with the discovery of its calcium channel activity. At least one of several putative functions of annexin V became obvious from studies on matrix vesicles derived from calcifying cartilage. It was found that calcium uptake by matrix vesicles depend on collagen type II and type X binding to annexin V in the vesicles and was lost when collagens were digested with collagenase; calcium influx was reconstituted after adding back native collagen II or V. These findings indicate that annexin V plays a major role in matrix vesicle-initiated cartilage calcification as a collagen-regulated calcium channel.     

An agglutinin with unique specificity for N-glycolyl sialic acid residues of thyroglobulin in the hemolymph of a marine crab Scylla serrata (Forskal).

A novel agglutinin with specificity for sialic acid sequence of sugars in thyroglobulin is identified in the hemolymph of Scylla serrata. The physico-chemical characteristics of its binding affinity, such as pH and temperature optima, and cationic requirements are defined. N-glycolyl neuraminic acid (NeuGc) (at 0.6 mM), in contrast to N-acetyl neuraminic acid (NeuAc) (at greater than 5.0 mM), is the potent inhibitor of hemagglutination. Bovine and porcine thyroglobulins containing NeuGc, inhibited the agglutination. NeuGc-acid glycoprotein fraction (bovine) but not NeuAc-acid glycoprotein fraction (human) inhibited the hemagglutination. The inability of other NeuGc-glycoproteins (bovine submaxillary mucin) to inhibit the agglutination suggests that the agglutinin may also recognize glycosidic linkage associated with NeuGc. The potential of the agglutinin in identifying NeuGc containing human tumor associated antigens is discussed.     

Induction of apoptosis and CD10/neutral endopeptidase expression by jaspamide in HL-60 line cells

Jaspamide (jasplakinolide) is a natural peptide isolated from marine sponges of Jaspis species and has fungicidal and growth-inhibiting activities. We characterized the jasplakinolide-induced loss of viability by programmed cell death in the HL-60 human promyelocytic leukemia cell line and found that this process was accompanied by neutral endopeptidase (NEP)/CD10 expression on the surface of the apoptotic cells. HL-60 cells do not normally express detectable amounts of NEP/CD10 on their surface or intracytoplasmically, but upon jaspamide treatment, CD10 was synthesized de novo, its expression being inhibited by cycloheximide pretreatment. Once synthesized, NEP/CD10 interfered with the jasplakinolide signal delivered to HL-60 cells. Inhibition of NEP/CD10 by the NEP inhibitor phosphoramidon or by an anti-CD10 monoclonal antibody significantly increased apoptosis induction. The appearance of CD10 on the cell surface was blocked by preincubation of the cells with the monocytic/macrophage-differentiating agents vitamin D3 and phorbol 12-myristate 13-acetate, but not by the granulocytic differentiating agents retinoic acid or dimethyl sulfoxide. Moreover, in the promonocytic U937 and mature monocytic THP-1 cell lines, jaspamide induced apoptosis but not CD10 expression. In HL-60 cells, CD10 expression was partially but not totally blocked by the broad-spectrum caspase inhibitor benzyloxacarbonyl-Val-Ala-Asp-fluoromethylketone, indicating a connection between apoptosis induction and CD10 synthesis. Our findings suggest that the CD10 expression is related to the programmed cell death induction by jaspamide, and also with the process of granulocytic differentiation in HL-60 cells. Received 22 April 2002; received after revision 8 June 2002; accepted 10 June 2002     

Orotic acid prevents changes in cardiac sarcolemmal glycoproteins and contractility associated with muscular dystrophy in hamsters

Summary Orotic acid included in the diet of cardiomyopathic hamsters during the myolytic phase of the disease (30–60 days of age) prevented the reduction in cardiac sarcolemmal sialic acid, calcium binding, sialyltransferase activity and contractile activity associated with the cardiomyopathy.This work was supported by research grants from the MRC of Canada, the Muscular Dystrophy Association of Canada and the Nova Scotia Heart Foundation. I thank Dr Peter E. Dresel for his encouragement and Ms Jane Benjamin and Mr Steven Couban for their technical assistance.     

Loss of Siglec-14 reduces the risk of chronic obstructive pulmonary disease exacerbation

Chronic obstructive pulmonary disease (COPD) is a leading cause of mortality worldwide. COPD exacerbation, or episodic worsening of symptoms, often results in hospitalization and increased mortality rates. Airway infections by new bacterial strains, such as nontypeable Haemophilus influenzae (NTHi), are a major cause of COPD exacerbation. NTHi express lipooligosaccharides that contain sialic acids, and may interact with Siglec-14, a sialic acid recognition protein on myeloid cells that serves as an activating signal transduction receptor. A null allele polymorphism in SIGLEC14 may attenuate the inflammatory responses to NTHi by eliminating Siglec-14 expression. We asked if the loss of Siglec-14 attenuates the inflammatory response by myeloid cells against NTHi, and if the SIGLEC14-null polymorphism has any effect on COPD exacerbation. We found that NTHi interacts with Siglec-14 to enhance proinflammatory cytokine production in a tissue culture model. Inhibitors of the Syk tyrosine kinase suppress this response. Loss of Siglec-14, due to SIGLEC14-null allele homozygosity, is associated with a reduced risk of COPD exacerbation in a Japanese patient population. Taken together, Siglec-14 and its downstream signaling pathway facilitate the “infection–inflammation–exacerbation” axis of COPD disease progression, and may represent promising targets for therapeutic intervention.