Difference in Topology of Normal and Tumour Cell Membranes shown by Different Surface Distributions of Ferritin-conjugated Concanavalin A

THE observations of Burger and Goldberg¹ on the differential agglutinability of malignant cells by the saccharide-binding protein, wheat germ agglutinin (WGA), have prompted other investigators to study the nature of tumour cell surfaces using similar reagents. A variety of other plant agglutinins specifically agglutinate certain oncogenic virus-transformed cells at much lower concentrations than those required to agglutinate their normal parental lines. In addition to WGA^1,2 (specific for binding N-acetyl-D-glucosamine-like residues¹), these include concanavalin A (Con A)^3,4 (specific for binding α-D-glucose or α-D-mannose-like residues⁵), soy bean agglutinin (SBA)^6,7 (specific for binding N-acetyl-D-galactosamine and D-galactose-like residues⁶) and Ricinus communis agglutinin (specific for binding D-galactose and L-arabinose-like residues) (G. L. N. and J. Blaustein, in preparation). To explain this differential agglutinability phenomenon, Burger² proposed that the agglutinin-binding sites on normal cells could have undergone three possible types of change after transformation: (a) the normal parent cell may have no agglutinin sites and after transformation a complete de novo synthesis of agglutinin sites occurs; (b) the normal parent cell may have agglutinin sites, but not enough for agglutination and transformation results in an increase in the number of agglutinin sites; or (c) the normal parent cell agglutinin sites remain constant in number after transformation; but this process results in an exposure of “cryptic” agglutinin sites and the transformed cell is rendered agglutinable. Several workers^8,10,19 postulate a fourth type of change: (d) the normal parent cell agglutinin sites remain constant in number after transformation but the topological distribution of agglutinin sites changes to a distribution more favourable for agglutination. The first and second mechanisms have proved to be unlikely as originally suggested², because agglutinin surface receptors are present on normal cells² and they are present in the same amounts on normal or transformed cells. This latter finding has been demonstrated in saturation binding experiments using purified ¹²⁵I-labelled WGA⁸, Con A^8,9 and SBA¹⁰, with results contrary to earlier reports using ⁶³Ni-labelled Con A³.     

Location of amino acid and carbohydrate transport sites in the surface membrane of normal and transformed mammalian cells

Summary Amino acid and carbohydrate transport in normal and malignant transformed hamster cells was studied after binding of the protein Concanavalin A (Con. A) to the surface membrane. Experimental conditions were used so that a similar number of Con. A molecules were bound to both types of cells. The transport of amino acids was inhibited after Con. A binding in the transformed cells but not in normal cells. This was found with the metabolizable amino acidsl-leucine,l-arginine,l-glutamic acid, andl-glutamine, and with the non-metabolizable amino acids cycloleucine and -aminoisobutyric acid. Transport ofd-glucose andd-galactose was more inhibited by Con. A in transformed than in normal cells, and in both types of cellsd-glucose was inhibited more thand-galactose. The inhibition by Con. A on transport was specific, since there was no effect on the transport ofl-fucose in either normal or transformed cells. Con. A also did not effect the entry of 3-0-methyl-d-glucose.These observations can be used to locate amino acid and carbohydrate transport sites in the surface membrane in relation to the binding sites for Con. A. The results indicate that Con. A sites are associated in normal cells with transport sites ford-glucose and to a lesser extentd-galactose, and in transformed cells with transport sites for amino acids and to a greater extent than in normal cells withd-glucose andd-galactose. Malignant transformation of normal cells therefore results in a change in the location of amino acid and carbohydrate transport sites in the surface membrane in relation to the binding sites for Con. A.     

Surface alterations in transformed epithelial and fibroblastic cells in culture: A disturbance of membrane degradation versus biosynthesis?

Fibroblasts which were transformed with oncogenic DNA or RNA viruses as well as by chemicals or spontaneously have, with very few exceptions, been shown to possess increased agglutinabilities with various plant agglutinins. This surface membrane alteration is now shown to hold true also for transformed epithelial cells and for tumors of epithelial origin as monitored by wheat germ agglutinin, Concanavalin A, phytohemagglutinin, Ricinus communis and Great Northern bean (GNB) agglutinin. An agglutinin from lentils did the opposite. It permitted the agglutination of normal liver cells but not that of hepatoma cells which led to the suggestion that some membrane glycoproteins or glycolipids may possibly be turned inside out while others are turned outside in (during the transformation process. Inhibition of protein synthesis in normal cells brought about the agglutinable membrane state in non-growing or resting cells but not in actively growing cells. Since this agglutinability could be inhibited by protease inhibitors and since proteolytic enzymes could bring about the agglutinable membrane state in growing normal cells, it was suggested that growing cells have as active a biosynthesis of membrane proteins as do non growing cells but that non-growing cells are actively catabolizing their membranes with proteases, much more so than non growing, stationary cells.     

Activation of chitin synthetase from Phycomyces blakesleeanus by calcium and calmodulin

Levels of basal chitin synthetase in cell-free extracts from Phycomyces blakesleeanus were reduced by breakage of cells in the presence of EDTA or EGTA. Addition of Ca²⁺ to these extracts activated chitin synthetase. Maximal activation was obtained after 2 h at a Ca²⁺ concentration of 2–5 mM. Activation by calcium was not reduced by any protease inhibitor tested but benzamidine, whereas the weak proteolytic activity of the extracts was inhibited by antipain. Larger levels of chitin synthetase activation were obtained by the simultaneous addition of calcium and calmodulin in most, but not all extracts. This further activation by calmodulin was prevented by TFP. ATP or cAMP did not stimulate activation by calcium or calcium-calmodulin.Abbreviations EGTA ethylene glycol-bis(B-aminoethylether)-N,NN-tetraacetic acid - GlcNAc N-acetyl-d-glucosamine - PMSF phenylmethylsulfonyl fluoride - SBTI soybean trypsin inhibitor - TFP trifluoperazine - TLCK N-p-tosyl-l-lysine choromethyl ketone - UDPGlcNAc uridine diphosphate N-acetyl-d-glucosamine     

Agglutinability of cattle red cells. 2. Agglutination by isologous and heterologous sera of enzyme-treated red cells

Neuraminidase treatment made cattle red cells (CRC) agglutinable by the agglutinins of the different heterologous but not of the homologous sera. These agglutinins were, however, absorbed by both the nauraminidase-treated and the intact CRC.
Proteolytic treatment made CRC agglutinable also by the normal cattle, isoimmune and autologous sera. Agglutination titres of the CRC ranged from 1: 2 to 1: 256, but the variation between CRC from members of monozygous (MZ) pairs was not greater than ± 2 agglutination score units the range of experimental error.
Treatment with trypsin made the A₁, A₂ factors more emergent on the surface of CRC for agglutination by anti-A₂, while pronase treatment had a similar effect upon agglutination of Z-positive cells by anti-Z.     

Inhibition of hamster fertilization by phytoagglutinins

Unfertilized eggs of the golden hamster were treated with phytoagglutinins and inseminated in vitro with capacitated spermatozoa. Ricinus communis agglutinin was most effective in preventing fertilization, followed by wheat germ agglutinin, Dolichos biflorus agglutinin and finally concanavalin A. The agglutinin-mediated block to fertilization is related to the saccharide-binding activities of agglutinins, because inclusion of the appropriate saccharide inhibitor counteracted the actions of the agglutinins. It is proposed that the agglutinins bind to specific oligosaccharides of the zona pellucida and induce extensive cross-linking of adjacent saccharide chains in such a way that sperm-born zona lysins can no longer depolymerize the zona material. Spermatozoa failed to bind to zona surfaces following treatment of eggs with high concentrations of wheat germ agglutinin, but not with the other agglutinins, suggesting that N-acetyl- -glucosamine-like or N-acetylneuraminic acid-like residues may be essential or sterically close to sperm attachment sites on the zona pellucida of the hamster egg.     

A lectin cytochemical study of glycoconjugates in the human retina

Summary The binding to morphologically normal human retina of eleven biotin- or peroxidase-coupled lectins with different carbohydrate specificities was studied. Eight formalin-fixed and paraffin-embedded eyes were examined. Photoreceptor cells bound Lens culinaris (LCA), wheat germ (WGA), peanut (PNA) and Ricinus communis (RCAI) agglutinins, and concanavalin A (ConA). The outer segment region was labeled more strongly than the inner segment region, and PNA labeled only cones. All these lectins except PNA bound to both plexiform layers, and all but PNA and RCAI to the nuclear layers. Pretreatment with neuraminidase to remove sialic acid resulted in increased binding of RCAI and PNA, which now labeled both rods and cones, and in decreased binding of WGA. Bandeiraea simplicifolia (BSAI), Dolichos biflorus (DBA), soybean (SBA), Ulex europaeus (UEAI), and Lotus tetragonolobus (LTA) agglutinins, as well as pokeweed mitogen (PWM) reacted only with retinal vascular endothelial cells, which were also labeled with the other lectins. The results indicate that -mannose, -glucose, -galactose, N-acetyl-d-glucosamine and N-acetylneuraminic acid are present in glycoconjugates of human neuroretina.     

Lectin affinity chromatography of cell surface proteins of novikoff tumor cells

Novikoff hepatocellular carcinoma cells were radioiodinated by a cell surface-specific method using lactoperoxid ase/¹²⁵I. The iodinated proteins were solubilized in 0.5% Nonidet P-40 and subjected to affinity chromatography on Sepharose-conjugated lectins (Ricinus communis agglutinins I or II, soybean agglutinin, concanavalin A, or wheat germ agglutinin) and analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Almost all the iodinated proteins bound to one or more of the Sepharose-conjugated lectins, presumptive evidence that these peptides are glycosylated. Lectin affinity chromatography resolved defined subsets of iodinated glycoproteins and suggested that certain glycoproteins could be fractionated on the basis of heterogeneity of their heterosaccharide moieties. Incubation of the iodinated cells with neuraminidase resulted in increased binding of iodinated proteins to Sepharose-conjugated Ricinus communis agglutinins I and II and soybean agglutinin and decreased binding to Sepharose-conjugated wheat germ agglutinin. Binding of iodinated proteins to concanavalin A was unaffected by neuraminidase treatment of the cells. These studies demonstrate the utility of lectins for the multicomponent analysis of plasma membrane proteins.     

Observations on the effects of protease inhibitors on the suppression of experimental allergic encephalomyelitis

Inhibitors of proteolytic enzymes were tested for their ability to suppress the clinical signs and CNS lesions produced by injection of purified myelin in complete Freund's adjuvant into Lewis rats. Pepstatin or a series of neutral protease inhibitors including aprotinin, soybean trypsin inhibitor, leupeptin, antipain, transaminomethyl cyclohexane carboxylic acid (AMCA), -amino caproic acid (EACA) nitrophenyl guanidino benzoate (NPGB),d- andl-polylysine, or a new commercial protease inhibitor, dipropionyl Rhein (DPR) were injected daily beginning on day 7 after immunization of rats with myelin. Aprotinin and soybean trypsin inhibitor exacerbated the symptoms and lesions of experimental allergic encephalomyelitis (EAE), leupeptin and antipain had no effect, and the plasminogen activators AMCA, EACA, NPGB, as well as poly-l- and poly-d-lysine and DPR suppressed various aspects of EAE. The measurement of acid protease as a biochemical method for quantitation of the degree of cellular infiltration into the CNS is proposed, and the results with the various treatments presented. AMCA and NPGB may exert their effects at the site of entrance of the lymphoid cells into the CNS.     

The presence of an endogenous lectin in early embryos ofXenopus laevis

Summary Xenopus laevis embryos were examined for the presence of endogenous carbohydrate binding proteins. Soluble extracts of cleavage, gastrula and neurula embryos are able to agglutinate trypsinized rabbit erythrocytes. Unlike other embryonic lectins this agglutination activity requires the presence of calcium ions but not of sulphydryl reducing agents. It is specifically inhibited by galactose and galactose containing derivatives. Thiodigalactoside is the most potent disaccharide inhibitor followed by lactose and melibiose respectively. Methyl -d-galactopyranoside is a more effective inhibitor than its anomer. N-acetyl-d-galactosamine, N-acetyl-d-glucosamine, methyl -d-mannopyranoside andl-fucose do not inhibit activity at concentrations at or above 25 mM. EDTA and EGTA are also strong inhibitors of this activity. -d-galactoside binding lectins present in the early chick embryo have been implicated in cell to cell and cell to substrate adhesiveness of extraembryonic chick endoderm cells. Since cells of the blastula inXenopus laevis possess surface receptors bearing terminal -d-galactoside groups it is possible that this -d-galactoside binding lectin may play a role in the control of cell surface mediated events during development.     

Visualization of chitin-wall formation in hyphal tips and anastomoses of Diplodia natalensis by fluorescein-conjugated wheat germ agglutinin and [3H] N-acetyl-D-glucosamine

Colonies of the fungus Diplodia natalensis produce ample anastomoses which are visible 0.5–1.0 mm inwards of the colony's periphery. Anastomose formation as well as other morphogenetic features, were followed by autoradiography, lectin binding and application of the chitin synthase inhibitor polyoxin D.Hyphal tips and septae were strongly labelled by short pulses of [³H] N-acetyl-D-glucosamine ([³H]GlcNAc) and were showing marked fluorescence after exposure to fluorescein isothiocyanate (FITC) conjugated wheat germ agglutinin (WGA).The dynamics of wall formation was followed by pulse and chase as well as by pulse and wash treatments in which the colony was shortly exposed to [³H]GlcNAc and then freed from the radioactive chitin precursor.Application of the chitin synthase inhibitor polyoxin D caused hyphal tip swellings as well as inflations and balloons along the hyphae at sites of initial new outgrowths and anastomoses. These structures were strongly fluorescenting after FITC-WGA application, indicating imbalance of wall formation and wall lysis.FITC-WGA binding, [³H]GlcNAc labelling and/or exposure to polyoxin D, indicated a process of anastomose formation which starts with short outgrowths of two juxtapositioned hyphae and ends with a complete bridge formation.Abbreviations FITC fluorescein isothiocyanate - WGA wheat germ agglutinin - GlcNAc N-acetyl-D-glucosamine     

Antibiotic tetaine — a selective inhibitor of chitin and mannoprotein biosynthesis in Candida albicans

The antibiotic tetaine inhibits in Candida albicans the biosynthesis of two important cell wall constituents, chitin and mannoprotein. This effect is a consequence of inactivation of the enzyme glucosamine-6-phosphate synthetase. Due to the lack of glucosamine-6-phosphate the effective secretion of mannoprotein enzymes, acid phosphatase and invertase, by Candida albicans spheroplasts is inhibited. In the presence of tetaine, probably a modified mannoprotein, lacking a branched polymannan, is synthesized. The antibiotic action decreases the viability of Candida albicans cells, especially that of mycelial forms of this fungus.Abbreviations GlcNAc N-acetyl-d-glucosamine - GlcN-6-P d-glucosamine-6-phosphate - ManNAc N-acetyl-d-mannosamine - -MM -methylmannoside - EGTA 1,2 di/2-aminoethoxy/ethane - N,N,N,N tetra-acetic acid     

Binding of concanavalin A and wheat germ agglutinin by murine peritoneal macrophages: ultrastructural and cytophotometric studies

Summary Concanavalin A and wheat germ agglutinin were employed in conjunction with the horseradish peroxidase-diaminobenzidine method for the detection of sugar residues on the surface coat of exudate and resident murine peritoneal macrophages. Electron microscopical and cytophotometric techniques were used for the visualization and quantification of the final reaction product on the surface of cells. After incubation with concanavalin A and wheat germ agglutinin, both exudate and resident macrophages showed readily detectable final reaction product indicating the presence of numerous, easily accessible, -methyl-d-mannosyl andN-acetyl-d-glucosaminyl residues on their surface. The binding of concanavalin A was higher with resident than with exudate macrophages. With wheat germ agglutinin, a different pattern of lectin binding was observed: more electron-dense product was deposited on exudate than on resident macrophage surfaces. The binding of concanavalin A and wheat germ agglutinin to macrophages was inhibited by the competing sugars -methyl-d-mannoside andN-acetyl-d-glucosamine, respectively.     

Nature of the receptor sites for galactosyl-specific lectins on human lymphocytes

The nature of the receptors for four lectins specific for -galactosyl residues was examined in human lymphocytes. The cells were fixed with formaldehyde to avoid subsequent cell lysis, treated with pronase, sialidase and organic solvents, and the binding of the lectins to the treated cells measured. The results show that the bulk of the receptors for peanut agglutinin (PNA) and ricin (RCA 60) are glycoproteins, whereas those for Ricinus communis agglutinin (RCA 120) and soybean agglutinin (SBA) are distributed nearly equally between membrane glycoproteins and glycolipids.     

Lectin binding by microvillous membranes and coated-pit regions of human syncytial trophoblast

Summary The distribution of saccharides on the microvillous membrane of the human syncytial trophoblast was investigated using ferritin conjugates of four lectins: concanavalin A (specific for -d-manno- and -d-glucopyranosyl residues), wheatgerm agglutinin (specific forN-acetylglucosamine),Limulus polyphemus lectin (specific forN-acetylneuraminic acid), andLotus tetragonolobus lectin (specific for -l-fucose). Concanavalin A and wheatgerm agglutinin (WGA) reacted strongly with the surface membrane and ferritin deposits were also observed in coated pit regions of the membrane. Lectins fromL. polyphemus andL. tetragonolobus, however, reacted only weakly with the microvillous border and neither reacted with coated pits.Enhanced agglutinability of trophoblast cells in comparison with other foetal cells from the same conceptus was seen with WGA. This agglutination was inhibited by addition of acetylglucosamine or by a solubilized membrane fraction which was bound by a column of WGA-Sepharose. The membrane fraction which did not bind to the column did not inhibit agglutination. Electrophoresis of the WGA-bound membrane proteins revealed six subunits, the major band having an apparent mol. wt. of 55 000. A protein of this mol. wt was also seen in coated vesicles isolated from equivalent human placentae.     

On the importance of the binding of lectins to cell surface receptors at low lectin concentrations

The binding of soybean agglutinin to human and rabbit erythrocytes, before and after treatment with trypsin, was reinvestigated with special emphasis on measurements at very low lectin concentrations. This communication presents two features of the binding that are observed only at the low concentrations used. (1) The trypsinized erythrocytes bind more lectin molecules than untreated cells at low concentrations (0.1–1.0 μg/ml), even though the total number of binding sites appears to be the same for both treated and untreated cells. It is suggested that this difference could explain, at least in part, the much higher susceptibility of the trypsin-treated cells to agglutination by soybean agglutinin. (2) At low site occupancy the binding of soybean agglutinin exhibits positive cooperativity, indicating a conformational change in the membrane. Trypsin-treated cells exhibit this effect at much lower lectin concentrations than untreated cells.     

Contact Inhibition of Transformed Cells Incompletely Restored by Dibutyryl Cyclic AMP

INCREASED levels of cyclic AMP have been found in normal cells as compared with malignant cells^1,2. Several types of malignant cells become morphologically similar to untransformed cells when incubated in media containing cyclic AMP or its derivative dibutyryl adenosine 3′:5′-cyclic monophosphate (dibutyryl cyclic AMP)^3,4. Sheppard reported that 3T3 mouse fibroblasts, transformed by polyoma virus, grew to low saturation density and became less agglutinable with wheat germ agglutinin if theophylline and dibutyryl cyclic AMP were added to the medium⁵.     

Isolation of a crustaceann-acetyl-d-glucosamine-1-phosphate transferase and its activation by phospholipids

Summary Then-acetyl-d-glucosamine-1-phosphate: dolichol phosphate transferase fromArtemia has been partially purified and characterized. The enzyme is solubilized from crude microsomes using Triton X-100, and after detergent removal appears to be associated with phospholipids. Using dolichol phosphate and UDP-n-acetyl-d-glucosamine as substrates, the enzyme catalyzes the formation of dolichol-pyrophosphate-n-acetyl-d-glucosamine. the product identity has been verified by TLC and paper chromatography following mild acid hydrolysis. Under the incubation conditions used only one product is made, i.e., Dol-p-p-GlcNAc. The formation of product is linear with increasing amounts of added protein and with time of incubation. The enzyme requires magnesium ions for activity. Activity of the enzyme is stimulated 6-fold by exogenous dolichol phosphate and is also stimulated by added phospholipids, with optimal activity being obtained in the presence of mixtures of phosphatidylcholine and phosphatidylglycerol. Enzymatic activity is not increased upon addition of GDP-mannose or dolichol phosphate mannose. The enzyme is rapidly inactivated by exposure to several detergents, including Triton X-100 and deoxycholate. The activity is inhibited by tunicamycin and by the purified B₂ homologue of this antibiotic. Other antibiotic inhibitors such as diumycin and polyoxin D have little effect on the enzyme. Both the microsomal and solubilized enzyme preparations are inactivated by 70% upon treatment with phospholipase A₂; activity may be restored by addition of phospholipids. Following hydrophobic interaction chromatography on Phenyl Sepharose, gel filtration chromatography on Sepharose CL-4B indicated that the enzyme, purified 81-fold, contained phophatidylcholine and phosphatidyl-ethanolamine.Abbreviations SDS sodium dodecyl sulfate - PMSF phenyl methanesulfonylfluoride - HEPES 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid - GlcNAc N-acetyl-d-glucosamine - Dol-PP-GlcNAc dolichol pyrophosphate N-acetyl-d-glucosamine - Dol-P-man dolichol-phosphate-mannose - Dol-PP- (GlcNAc)₂ dolichol-pyrophosphate-di-N- acetylchitobiose - DMSO dimethylsulfoxide - C:M (2:1) chloroform:methanol (2:1) - C:M:W (10:10:3) chloroform:methanol:water (10:10:3) - GlcNAc-1-P N-acetyl-d-glucosamine-1-phosphate - Dol-P dolichol phosphate - EGTA ethylene glycol bis (b-aminoethyl ether)-NNNN tetraacetic acid     

Chitinolytic properties of Streptomyces lividans

Streptomyces lividans TK24, an established host for genetic and molecular studies in actinomycetes, is able to use chitin as sole carbon and nitrogen source. Extracellular chitinase and N-acetyl--d-glucosamidinase (chitobiase) activities were detected in liquid cultures. Chitinase production was inducible by chitin and its low molecular weight derivatives. Low levels of chitinase were also produced in the absence of chitin. Production of extracellular N-acetylglucosaminidase was correlated with the beginning of the stationary phase of growth and was independent of the presence of chitin. Beside highly N-acetylated chitin, supernatants of chitin-induced cultures were able to hydrolyse chitosans with a wide range of degrees of N-acetylation.Abbreviations MS minimal salts - GlcNAc N-acetyl--d-glucosamine - pNP-GlcNAc p-nitrophenyl-2-acetamido-2-deoxy--d-glucopyranoside - d.a. degree of N-acetylation - TLC thin-layer chromatography