Steven B. Oppenheimer

A glycobiology review: carbohydrates, lectins, and implications in cancer therapeutics

This review is intended for general readers who would like a basic foundation in carbohydrate structure and function, lectin biology, and the implications of glycobiology in human health and disease, particularly in cancer therapeutics. These topics are among the hundreds included in the field of glycobiology and are treated here because they form the cornerstone of glycobiology or the focus of many advances in this rapidly expanding field.

Cell surface changes occurring during sea urchin embryonic development monitored by quantitative agglutination with plant lectins

Abstract Plant lectins have been widely used to investigate the nature and functional significance of carbohydrate-containing cell surface receptor sites on normal, embryonic and tumor cells. In the present study, evidence is presented, using a quantitative electronic particle counter assay to measure agglutination, which indicated that carbohydrate-containing lectin-binding sites change during sea urchin embryonic development. Sea urchin embryo cells are 26 ± 5 % more agglutinable with ConA at early developmental stages (day 1) than at later ones with a marked decline in agglutinability between day 2 and 3. Agglutinability with Ricinus communis agglutinin (RCA) may also be greater in young embryos. Normal embryonic sea urchin cells are not agglutinated with wheat germ agglutinin (WGA) unless the cells are trypsinized. The results suggest that changes in the amount, mobility or distribution of Con A-binding (containing α- d -mannose-like or α- d -glucose-like residues) and RCA-binding (containing β- d -galactose or β- N -acetyl- d -galactosamine-like residues) receptor sites occur with differentiation in sea urchin embryos, while WGA-binding receptor sites (containing N -acetyl- d -glucosamine-like residues) are displayed in a state which precludes agglutination at all developmental stages studied. The results are discussed with respect to the cell types present during sea urchin embryo development and provide quantitative evidence for the contention that specific changes in carbohydrate-containing cell surface sites occur with differentiation and maturation.

Functional involvement of specific carbohydrate in teratoma cell adhesion factor.

Abstract Teratoma cell adhesion factor (TAF), a protein-containing molecule isolated from the fluid in which 129/J mouse ascites teratoma cells grow, may be the cell surface component which mediates cell-cell adhesion in this system. In this study, evidence suggests that TAF contains terminal d -galactosyl residues which are functionally involved in TAF cell binding activity. Purified active β-galactosidase from various sources totally destroyed TAF aggregation promoting activity. Experiments with Azocoll indicated that the enzymes were not contaminated with protease. O -Nitrophenyl-β- d -galactopyranoside, a β-galactosidase substrate, prevented the enzyme from inactivating TAF suggesting that the enzyme catalysed removal of d -galactose residues from the TAF molecule. Cells treated with the enzyme, followed by washing, aggregated fully with TAF, suggesting that the enzyme catalysed removal of d -galactosyl residues from the TAF molecule and not from the cell surface. When the teratoma cells were preincubated with a variety of monosaccharides, followed by addition of TAF, d -galactose was the most active TAF-mediated aggregation inhibitor. Sugar acids were also effective inhibitors, though the effects of these compounds may be due to non-specific charge properties. The evidence presented suggests that protein may be the structural carrier of galactose-containing carbohydrate functionally involved in TAF aggregation promoting activity.

A quantitative assay for measuring cell agglutination, agglutination of sea urchin embryo and mouse teratoma cells by concanavalin a.

Abstract Plant agglutinins are being widely used to obtain important information about cell surfaces of normal and malignant cells. In the past, agglutination has been measured using a serological estimate of cell clumping and these results suffered from a severe lack of quantification. It is important to be able to quantify the agglutination assay so that it may be used to rapidly quantify differences in cell agglutinability and correlate this with the nature and distribution of agglutinin receptor sites. In the present studies we combine the use of an electronic particle counter, a gyratory shaker with a large diameter of rotation and small volumes of cell suspensions to develop a rapid quantitative assay to measure cell agglutination with plant agglutinins. The parameters influencing agglutination are carefully studied and the results indicate that initial cell density, agglutinin concentration and temperature affect the kinetics of cell agglutination. It is suggested that some previous work be re-evaluated in light of these results. The agglutination of mouse teratoma cells and sea urchin embryo cells by concanavalin A (ConA) are also studied and carefully compared using the new assay. Teratoma cells and normal EDTA dissociated, untrypsinized, sea urchin embryo cells are agglutinated by ConA. We quantitatively show that under the same conditions equal numbers of teratoma cells and sea urchin gastrula cells agglutinate with ConA and these results are interpreted in terms of clustering of cell surface agglutinin receptor sites.

Chapter 1 Interactions of Lectins With Embryonic Cell Surfaces

Publisher Summary This chapter discusses the interactions of lectins with embryonic cell surfaces. It summarizes the information on various lectins and their sugar-binding specificity deals with the information gained through the study of the interactions of lectins with gametes. The chapter describes how lectins are being used to examine the cell surface changes that occur during development in a variety of systems. The cell surface plays an important role in morphogenesis and malignancy. Carbohydrate-containing molecules make up a part of the surface of the cells. The oligosaccharide chains of glycoproteins, glycolipids, and polysaccharides reach away from the cell surface. These sugar chains, therefore, are likely the candidates for mediating initial cell-cell surface interactions. Lectins, which are proteins or glycoproteins, usually isolated from the seeds of plants and other material bind to the cell surface sugar chains. Each lectin preferentially binds to certain terminal sugars or groups of sugars on these chains. Lectins, therefore, are useful tools for studying the surfaces of adult, embryonic, and malignant cells.

Utilization of l-glutamine in intercellular adhesion: Ascites tumor and embryonic cells

Abstract l -Glutamine is required for the synthesis of complex carbohydrates required for the intercellular adhesion of mouse teratoma cells. It remained to be seen if these pathways were of general importance in the adhesion of other cell types. In this study, using an electronic particle counter assay to measure cell adhesion, Ehrlich ascites, Sarcoma 180 and Taper liver ascites tumor cells require exogenous l -glutamine to aggregate. This effect is concentration dependent and the amino sugar, d -glucosamine, replaces the glutamine requirement. Structural analogs of the active compounds are substantially less effective and metabolic inhibitors block the activity of the effective compounds. Two specific glutamine antagonists, DON (6-diazo-5-oxo- l -norleucine) and azaserine (O-diazoacetyl-serine) decrease the action of l -glutamine but not of d -glucosamine. Trypsin dissociated six day old chick embryo neural retina cells do not require l -glutamine to reaggregate, though the rate of aggregation is enhanced after preincubation with glutamine. Dissociation of small clumps of neural retina and inhibition of reaggregation of these cells are facilitated by preincubation with azaserine for 3–5 h. l -Glutamine reduces the effect of azaserine on retina cells. These results are consistent with known metabolic pathways and suggest that l -glutamine is involved in the synthesis of complex carbohydrates necessary for adhesion in a variety of cell types. The defective adhesion of the tumor cells examined may result from inability to produce glutamine synthetase, or effectively store cr transport l -glutamine.

On cell membrane lipid fluidity and plant lectin agglutinability. A spin label study of mouse ascites tumor cells

The fluidity of the plasma membrane of Sarcoma 180 mouse ascites tumor cells has been studied in viable cells using fatty acid spin labels. The order parameter was found to vary from 0.61, approximately four carbon bond lengths removed from the membrane surface, to 0.47 approximately eleven bond lengths removed at 22 degrees C and from 0.55 to 0.33 at 37 degrees C. Thus these cells show similar membrane fluidity to that found in other mammalian cells with the exception of human erythrocytes which are less fluid. The concanavalin A mediated agglutinability of Sarcoma 180 cells was altered by the addition of cytochalasin B and the fluidity was found to be the same as in unaltered cells.

Quantitative agglutination of specific populations of sea urchin embryo cells with concanavalin A

Abstract It has been demonstrated that specific changes in carbohydrate-containing cell surface lectin receptor sites occur with differentiation and maturation of sea urchin embryo cells. In this study, evidence is presented, using a quantitative electronic particle counter assay to measure agglutination, which indicates that concanavalin A (Con A) mediated agglutination of dissociated 32 64 cell sea urchin embryos differs dramatically with respect to specific cell populations. The migratory cell type, the micromere, is significantly more agglutinable with Con A than the other cell types and colchicine treatment markedly increases sea urchin embryo cell agglutinability. The results indicate that like many malignant cells which display extensive migratory behavior, specific migratory populations of embryonic cells are agglutinable with Con A. The results are discussed with respect to the possible nature of lectin receptor sites on specific populations of embryonic cells and the possible role of colchicine-sensitive structures in controlling the display patterns of these sites.

Quantitative evaluation of concanavalin A receptor site distributions on the surfaces of specific populations of embryonic cells

MORPHOGENESIS and malignant invasion seem to be dependent on cellular migration and changes in cellular adhesiveness1–3. Plant lectins such as concanavalin A (con A) have been extensively used to study the nature and distribution of lectin receptor sites on the surfaces of normal, embryonic and tumour cells. These sites may be involved in controlling cellular migration and adhesiveness2,4,5.

A rapid lectin receptor binding assay: comparative evaluation of sea urchin embryo cell surface lectin receptors

Lectin receptor binding assays, such as those that utilize fluorescence, radioactivity or electron microscopy are not designed for rapidly screening hundreds of cell types for the presence or absence of specific lectin receptors. An assay is described here that is designated for this purpose. It utilizes lectins derivatized to agarose beads and can be used to screen many cell types in min. This assay was used to examine lectin receptors on the surfaces of 1-8 cell stage Strongylocentrotus purpuratus sea urchin embryos. The same cells were also assayed using standard fluorescence and agglutinability procedures to ascertain the type of information obtained by the new assay and how it correlates with results from the standard methods. The bead results correlated well with results using fluorescent lectin. Only wheat germ agglutinin bound very strongly in both bead and fluorescence assays, while concanavalin A, Dolichos biflorus, Lens culinaris and Tetragonolobus purpureas did not bind or bound weakly using both methods. Results using a third method, lectin mediated cell agglutination, did not correlate with the bead or fluorescence assays. Lectin receptors were also examined on embryos prepared by two different methods of preventing formation of fertilization membranes, so that coat-free cell surfaces could be studied, the standard dithiothreitol method and a new method using alpha-amylase. Lectin receptors on the cell surfaces of embryos prepared by both methods were nearly identical. The possible functions of WGA receptors, the most prevalent lectin receptors of those studied, that were uniformly present throughout early development of this sea urchin species, are considered.(ABSTRACT TRUNCATED AT 250 WORDS)