Molecular structure of the cell wall receptor for killer toxin KT28 in Saccharomyces cerevisiae.

The adsorption of the yeast killer toxin KT28 to susceptible cells of Saccharomyces cerevisiae was prevented by concanavalin A, which blocks the mannoprotein receptor. Certain mannoprotein mutants of S. cerevisiae that lack definite structures in the mannan of their cell walls were found to be resistant to KT28, whereas the wild-type yeast from which the mutants were derived was susceptible. Isolated mannoprotein from a resistant mutant was unable to adsorb killer toxin. By comparing the resistances of different mannoprotein mutants, information about the molecular structure of the receptor was obtained. At least two mannose residues have to be present in the side chains of the outer chain of the cell wall mannan, whereas the phosphodiester-linked mannose group is not essential for binding and the subsequent action of killer toxin KT28.     

Preliminary characterization of the material released to the culture medium byCandida albicans yeast and mycelial cells

Culture filtrate concentrates were obtained fromCandida albicans yeast and mycelial cells grown in the presence of¹⁴C-protein hydrolysate for radioactive labeling of cellular polypeptides. Both growth forms released to the medium minor but significant amounts of proteinaceous materials. The analysis of culture filtrate concentrates by means of SDS-polyacrylamide gel electrophoresis and fluorography revealed a similar and complex electrophoretic pattern, though some qualitative and quantitative differences between samples obtained from yeast and mycelial cells were observed. Materials released, mostly composed of mannoproteins as shown by their affinity towards concanavalin A, presented (i) cross-reactivity (by Western immunoblotting and ELISA) against polyclonal antisera to genuine cell wall components (among them the 58-kilodalton fibrinogen-binding mannoprotein) and (ii) high affinity for polystyrene-latex microbeads. Results presented suggest a possible common identity for the molecules shed to the medium and the cell wall protein and mannoprotein constituents.Abbreviations SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electroporesis - PAS periodic acid/Schiff method     

Saccharomyces cerevisiae structural cell wall mannoprotein

A novel mannoprotein fraction with an average molecular weight of 180 000 has been isolated from Saccharomyces cerevisiae mnn9 mutant cell wall that was solubilized by beta-glucanase digestion. The same material could be extracted from purified wall fragments with 1% sodium dodecyl sulfate. The protein component, 12% by weight, is rich in proline, whereas the carbohydrate, mainly mannose, is about evenly distributed between asparagine and hydroxyamino acids. Endoglucosaminidase H digestion of the isolated mannoprotein reduced its average molecular weight to 150 000, but the mannoprotein, while still embedded in the cell wall, was inaccessible to the enzyme. Biosynthesis and translocation of the mannoprotein were investigated by following incorporation of [3H]proline into this fraction. In the presence of tunicamycin, both mnn9 and wild-type X2180 cells made a mannoprotein fraction with an average molecular weight of 140 000, whereas in the absence of the glycosylation inhibitor, the mnn9 mutant made material with a molecular weight of 180 000 and the mannoprotein made by wild-type cells was too large to penetrate the polyacrylamide gel. Although the cell wall mannoprotein was resistant to heat and proteolytic enzymes, attempts to isolate the carbohydrate-free component failed to yield any characteristic peptide material.     

Antigenic cell wall mannoproteins in Candida albicans isolates and in other Candida species.

Polyclonal antibodies (pAbs) and monoclonal antibodies (mAbs), raised against mannoprotein components from Candida albicans ATCC 26555 (serotype A) blastoconidia and mycelial cell walls, were used to investigate antigenic similarities among wall mannoproteins from other C. albicans serotype A and B strains, and from C. tropicalis and C. guilliermondii. Radioactively labelled walls isolated from cells grown at either 28 degrees C or 37 degrees C were digested with a beta-glucanase complex (Zymolyase 20T) to release cell-wall-bound mannoproteins. Numerous molecular species with different electrophoretic mobilities were released from the various isolates. Differences appeared to be related to both the organism and the growth temperature. Among the major protein components solubilized were mannoproteins larger than 100 kDa (high molecular mass mannoproteins), heterogeneous in size in most cases. Antigenic homology was detected among the cell wall high molecular mass mannoproteins of the two C. albicans serotype A isolates, whereas significant qualitative and quantitative differences were detected between serotype A and serotype B cell-wall-bound antigenic profiles. Moreover, C. tropicalis and C. guilliermondii wall antigenic determinants were not recognized by the preparations of pAbs and mAbs raised against C. albicans walls. A mannoprotein with a molecular mass of 33-34 kDa was present in the enzymic wall digests of all the organisms studied. When probed with pAbs raised against the protein moiety of the 33 kDa cell wall mannoprotein of Saccharomyces cerevisiae, antigenic cross-reactivity was observed in all cases except C. tropicalis. There appear to be significant antigenic differences between the mannoproteins of different isolates of C. albicans, and between those of C. albicans and other Candida species.     

Characterization with crossed immunoelectrophoresis of some antigens differentiating a virulent Candida albicans from its derived, avirulent strain

Antigenic differences between a wild-type virulent Candida albicans 4918 (wt) and its spontaneous avirulent mutant (m-10) were found with crossed immunoelectrophoresis. Yeast cell extracts as well as soluble protein and mannoprotein fractions obtained by affinity chromatography on concanavalin A (Con A) were analyzed. Sera from patients with candidiasis and antisera from rabbits infected with live wt cells and boosted with wt extracts or rabbits immunized with purified wt cell wall preparation were used as counter reactants. Qualitative differences in serum precipitins formed by patients with suspected or culture-proven candidiasis to polysaccharide antigens of wt and m-10 origin were observed. In comparison, except for a spike-formed precipitate detected only with the wt extract, the serum from infected rabbits precipitated the wt and m-10 cell wall polysaccharide antigens about equally. The same type of precipitate was also found with the Con A wt mannoprotein fractions but was again lacking with the m-10 mannoproteins. This precipitate, with extremely slow electromobility in the first dimension, may be related to some special immunodeterminant of the wt mannan molecule. No substantial differences in the precipitation patterns of the Con A wt and m-10 proteins were found when analyzed with patients' sera or rabbit anti-cell sera. However, using these protein fractions with anti-cell wall sera revealed a larger number of precipitates for the wt as opposed to the m-10 strain. The observed antigenic differences between the virulent- and the avirulent-derived strains seem to be mainly associated with cell wall determinants (components) and might be related to the greater adherence and infectivity of the wild strain.     

Leishmania braziliensis: cell surface differences in promastigotes of pathogenic and nonpathogenic strains

A comparative study of cell surface characteristics of pathogenic and nonpathogenic promastigotes of Leishmania braziliensis, NR and LBY strains, respectively, was carried out by means of concanavalin A agglutination and labeling with concanavalin A-fluorescein isothiocyanate, concanavalin A-ferritin, and cationized ferritin. Cytochemical examination showed cell surface differences in lectin receptors and negative charge moieties in the two strains of L. braziliensis. The pathogenic NR strain agglutinated with low concentrations of concanavalin A and presented abundant lectin-binding and cationized ferritin-binding surface labeling. The nonpathogenic LBY strain neither agglutinated when incubated with concanavalin A, bound lectins, or cationized ferritin at the cell surface.     

Identification of a 49-kDa hydrophobic cell wall mannoprotein present in velum yeast which may be implicated in velum formation

Analysis of velum-forming yeast cell wall components released by beta-1,3-glucanase treatment were compared with those of a non velum-forming yeast. SDS-PAGE electrophoresis and Western blotting with ConA-peroxidase staining of mannoproteins allowed us to identify a 49-kDa mannoprotein present in the cell wall of the velum-forming yeast and hardly visible in the control. The cell wall nature of this protein was confirmed by labelling with the non-permeable sulfosuccinimydiyl-6-(biotinamido)hexanoate reagent. A partial purification of this mannoprotein by anion exchange HPLC followed by surface hydrophobicity determination revealed that the fraction containing the 49-kDa mannoprotein was the most hydrophobic. Since cell surface hydrophobicity plays an important role in aggregate formation, it is likely that this mannoprotein is involved in velum formation.     

Saccharomyces cerevisiae mannoproteins form an external cell wall layer that determines wall porosity.

A beta-glucanase (Z-glucanase) from Zymolyase was freed from a protease (Z-protease) by affinity chromatography on alpha 2-macroglobulin-Sepharose columns and used to solubilize proteins from isolated cell walls of Saccharomyces cerevisiae. The cell wall proteins were labeled with 125I and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The bulk of the labeled material had very low mobility. Its mannoprotein nature was demonstrated by precipitation with specific antibodies and by conversion to a band with an average molecular weight of 94,000 after incubation with endo-beta-N-acetylglucosaminidase. The intact mannoproteins were hydrolyzed by Z-protease, but were resistant to the enzyme when the carbohydrate was first removed by endo-beta-N-acetylglucosaminidase. In intact cells, lysis of cell walls by Z-glucanase required a previous incubation with z-protease, which led to solubilization of most of the 125I-labeled proteins. Other proteases that did not attack the cell wall mannoproteins were unable to substitute for Z-protease. The specific effect of Z-protease is consistent with the notion that mannoproteins form a surface layer of the cell wall that penetrates the wall to some depth and shields glucans from attack by Z-glucanase. Mannoproteins, however, do not appear to cover the inner face of the cell wall, because isolated cell walls, in contrast to intact cells, were completely solubilized by Z-glucanase in the absence of protease. The function of mannoproteins in determining cell wall porosity was highlighted by the finding that horseradish peroxidase (Mr, 40,000) causes lysis of cells that had been treated with Z-protease. Depletion of mannoproteins by Z-protease also resulted in the disappearance of a darkly stained surface layer of the cell wall, as observed by electron microscopy. Other agents that facilitate cell lysis by Z-glucanase, such as 2-mercaptoethanol, digitonin, and high concentrations of salts, caused little or no solubilization of mannoprotein. We assume that they perturb and loosen the structure of the mannoprotein network, thereby increasing its porosity. The implications of our results for the construction of the yeast cell wall and the anchoring of mannoprotein to the cell are discussed.     

Fluorescence photobleaching recovery measurements of surface lateral mobilities on normal and SV40-transformed mouse fibroblasts

Lateral mobilities of fluorescent cell surface probes have been measured on normal (3T3) and transformed (SV3T3) cultured mouse fibroblasts. There is little discernible difference in the mobilities of a lipid analogue (diI), a fluorescent ganglioside derivative (GM1), and tetramethylrhodamine-labeled succinylated concanavalin A. The two cell lines showed expected differences in their abilities to grow in agar, to grow without serum, and to be agglutinated by lectins, indicating that changes of these properties in transformed cells are probably not mediated through increased overall membrane fluidity but are associated with distinct alterations in the mobilities of cell surface receptors. Both fluorescent dextran derivatives and antimouse cell surface antibodies were distinctly less mobile on SV3T3 cells, and the mobile fraction of Con A receptors was lower on SV3T3 cells.     

Agglutination of growing and differentiating cells of Dictyostelium discoideum by concanavalin A

Cells of Dictyostelium discoideum are agglutinated by by concanavalin A (Con A). Agglutination is dependent upon Con A concentration and is inhibited by preincubation with α-methyl-glucoside. Agglutination by Con A has no adverse effect on cell viability. Cells harvested from exponential growth phase are agglutinated by lower concentrations of Con A, than are cells harvested during the stationary growth phase or during differentiation. The possible significance of these findings to the process of differentiation in D. discoideum is discussed.     

Concanavalin A-induced alterations in sodium and potassium content of Ehrlich ascites tumor cells

Net fluxes of sodium and potassium were studied in Ehrlich mouse ascites tumor cells during contact with the agglutinating protein, concanavalin A. This lectin altered cation transport markedly at concentrations of 20–105 μg/ml (6–47 μg/mg cell protein). Whereas control cells extruded sodium and maintained or accumulated potassium against electrochemical gradients, in the presence of concanavalin A there was rapid net sodium entry and potassium loss. After 10–20 minutes in concanavalin A, sodium extrusion began and potassium loss diminished but these events were prevented by ouabain. The alterations in cation content induced by concanavalin A are unlikely to be the result only of agglutination since soybean agglutinin caused much smaller changes although it agglutinated the cells equally well.     

The role of microvilli in the agglutination of cells by concanavalin A

Morphological correlates of lectin agglutinability were examined in eight cell lines of varying sensitivity to agglutination by concanavalin A (ConA). The number of microvilli on the surface of cells growing in monolayers was positively correlated with agglutinability. However, when cells were brought into suspension, they all developed numerous microvilli which persisted when the cells were treated with ConA regardless of whether or not they were agglutinated by the lectin. Treatment of cells with dibutyryl cyclic AMP (db-cAMP) and theophylline caused a parallel decrease in agglutinability and numbers of microvilli in monolayer cultures, but suspended cells from control and treated cultures were identical in appearance in the absence or presence of ConA. The surface morphology of cells agglutinated by ConA was very similar to that of cells that spontaneously agglutinated in the absence of the lectin, and surface bound ConA was rapidly withdrawn from microvilli on all cell types. Neither the morphology of cells nor the surface distribution of ConA can explain observed differences in agglutinability.     

Surface Alterations of Cells Carrying RNA Tumour Virus Genetic Information

CELLS transformed by the DNA tumour viruses, polyoma virus and SV40, are agglutinated by lectins such as wheat germ agglutinin¹, concanavalin A (Con A)² and soybean agglutinin³. Agglutination in these cases presumably reflects changes in the cell surface related to the transformed properties of the cell; studies with a temperature-dependent mutant of polyoma virus has shown that cell surface changes are controlled by viral genes⁴. Here we describe experiments in which we investigated the agglutinability of cells transformed by RNA tumour viruses. One recent report had suggested that cells transformed by RNA tumour viruses were not specifically agglutinated⁵, whereas a second more recent report claimed the specific agglutination of cells transformed by RSV⁶. We find that transformed rat, mouse and cat cells that replicate the sarcoma-leukaemia virus complex of murine (MSV) and feline (FeSV) origin are strongly agglutinated by Con A, but mouse and human cells that replicate the murine and feline leukaemia virus components alone are not agglutinated. The ability to agglutinate is rapidly acquired by normal mouse cells on infection with the murine sarcoma virus at a rate that parallels virus replication. In contrast to the results obtained with cells producing virus, non-virus-producing transformed hamster and mouse cells that synthesize virus-specific RNA are either not agglutinated or are agglutinated to a lesser degree. These results suggest that the cell surface alterations responsible for agglutination are not necessarily associated with the transformed state of the cell, but rather with the possession of sarcoma virus-specific information.     

Mouse 3T3 cell filtrability correlating with concanavalin A agglutinability

In a previous study of the dextran gel sphere model system, a possible correlation between cell deformability and agglutinability by concanavalin A was indicated. Cell deformability was evaluated as filtrability, using polycarbonate membrane filtration. With 25-mm diameter filters and 5-ml cell suspensions at (0.8–16) · 10⁵ cells/ml, the filtrability at a given filter pore size was highly reproducible and was not affected by variations in cell population, viability, washings of cells retained on filter, or temperature. The filtrability of EDTA-dissociated 3T3 cells through 12-μm pore size filter was 8%, and a suspension of 10⁶ cells/ml was not agglutinated by 600 μg concanavalin A. The filtrability of trypsin-dissociated 3T3 cells was 95%, and these cells were agglutinated by 200 μg of the lectin. EDTA-dissociated SV-3T3 cells had a filtrability of 73% and were also highly agglutinable. Formalin fixation reduced the high filtrability to 6%, and also abolished the agglutinability. As a further test of the correlation, trypsin-dissociated 3T3 cells were admixed with the fixed cells. The agglutinability varied with the proportions of the two cell components, and the admixtures could be separated according to filtrability into the original components with distinctly different agglutinability. Furthermore, 25% of a random population of EDTA-dissociated SV-3T3 cells retained by the filter were found to be non-agglutinable. The separated SV-3T3 cell fractions could also form admixtures of different agglutinability. It is concluded that the agglutinability of mouse 3T3 and SV-3T3 cells by concanavalin A can be correlated with the predicted by cell filtrability.     

Distribution of concanavalin A binding sites on the surface of dissociated rat submandibular gland acinar cells.

The submandibular glands of 4-week-old rats were dissociated by a procedure involving digestions with collagenase and hyaluronidase, chelation of divalent cations and mechanical force. A suspension of single cells was obtained in low yield by centrifugation in a Ficoll-containing medium. Immediately after dissociation and after a culture period of 16-18 hr the dissociated cells were tested for agglutinability by concanavalin A (Con A). Using ferritin (tfer)-conjugated Con A the lectin binding by the isolated acinar cells was also studied. The dissociated cells were agglutinated by low concentrations of Con A and bound Fer-Con A molecules on their entire surface without any indication of polarization of the cell membrane. There was a considerable cell to cell variation in the amount of Fer-Con A binding which was, in general, sparse and patchy. The contact surfaces between agglutinated cells revealed a dense binding of Fer-Con A molecules irrespective of the types of cells participating in the agglutination reaction. Cells cultured for 16-18 hr were no longer agglutinated by Con A. As compared to the freshly dissociated cells the cultured acinar cells revealed a more uniform and denser binding of Fer-Con A molecules. Furthermore, there were more lectin molecules bound to the cell surface corresponding to the basal part of the cell, where the nucleus and most of the rough surface endoplasmic reticulum were located, than to the apical cell surface. It is suggested that the higher density of lectin-binding sites on the cell surface in the vicinity of the cisternae of the rough endoplasmic reticulum indicates insertion sites of newly synthesized membrane glycoproteins.     

Characterization of a CV-1 cell cycle. III. Biophysical parameters.

Membrane preparations from three independently selected concanavalin A-resistant cell lines incorporated si$\text{g}$nificantly less GDP-[¹⁴C]mannose into lipid, oligosaccharide-lipid and protein fractions than preparations obtained from parental wild populations. The results from experiments with membranes from a revertant concanavalin A-resistant line more closely resembled the wild-type populations. The amount of mannose label incorporated into glycoprotein in the variant cells was higher than expected if it is assumed that the pathway GDP-mannose → mannolipid → oligosaccharide-lipid → mannoprotein is functioning in these cells. Evidence is presented to suggest that conversion of mannose label to fucose occurs in wild-type and variant cell lines and that this pathway may be of greater importance in the variant cells; this result could explain at least in part, the higher than expected levels of ¹⁴C-label in glycoprotein in the variant cell lines. The changes in the glycosyl transferase activities in these lectin-resistant cell lines are probably involved in determining the concanavalin A-resistant property and the accompanying complex phenotype exhibited by these variant cell lines.     

C. albicans increases cell wall mannoprotein, but not mannan, in response to blood, serum and cultivation at physiological temperature

The cell wall of Candida albicans is central to the yeasts ability to withstand osmotic challenge, to adhere to host cells, to interact with the innate immune system and ultimately to the virulence of the organism. Little is known about the effect of culture conditions on the cell wall structure and composition of C. albicans. We examined the effect of different media and culture temperatures on the molecular weight (Mw), polymer distribution and composition of cell wall mannan and mannoprotein complex. Strain SC5314 was inoculated from frozen stock onto yeast peptone dextrose (YPD), blood or 5% serum agar media at 30 or 37°C prior to mannan/mannoprotein extraction. Cultivation of the yeast in blood or serum at physiologic temperature resulted in an additive effect on Mw, however, cultivation media had the greatest impact on Mw. Mannan from a yeast grown on blood or serum at 30°C showed a 38.9 and 28.6% increase in Mw, when compared with mannan from YPD-grown yeast at 30°C. Mannan from the yeast pregrown on blood or serum at 37°C showed increased Mw (8.8 and 26.3%) when compared with YPD mannan at 37°C. The changes in Mw over the entire polymer distribution were due to an increase in the amount of mannoprotein (23.8-100%) and a decrease in cell wall mannan (5.7-17.3%). We conclude that C. albicans alters the composition of its cell wall, and thus its phenotype, in response to cultivation in blood, serum and/or physiologic temperature by increasing the amount of the mannoprotein and decreasing the amount of the mannan in the cell wall.     

The 65 kDa mannoprotein gene of Candida albicans encodes a putative beta-glucanase adhesin required for hyphal morphogenesis and experimental pathogenicity

Mannoproteins are fungal cell wall components which play a main role in host-parasite relationship. Camp65p is a putative beta-glucanase mannoprotein of 65 kDa which has been characterized as a main target of human immune response against Candida albicans. However, nothing is known about its specific contribution to the biology and virulence of this fungus. We constructed CAMP65 knock-out mutants including null camp65/camp65 and CAMP65/camp65 heterozygous strains. The null strains had the same growth rate and morphology under yeast form as the wild-type strain but they were severely affected in hyphal morphogenesis both in vitro and in vivo. Hyphae formation was restored in revertant strains. The null mutants lost adherence to the plastic, and this was in keeping with the strong inhibition of fungal cell adherence to plastic exerted by anti-Camp65p antibodies. The null mutants were also significantly less virulent than the parental strains, and this loss of virulence was observed both in systemic and in mucosal C. albicans infection models. Nonetheless, the virulence in both infectious models was regained by the CAMP65 revertants. Thus, CAMP65 of C. albicans encodes a putative beta-glucanase, mannoprotein adhesin, which has a dual role (hyphal cell wall construction and virulence), accounting for the particular relevance of host immune response against this mannoprotein.     

Comparative lectin-binding and agglutination properties of the strain-specific,TA3-St,and the non-strain-specific,TA3-Ha,murine mammary carcinoma ascites sublines. Further studies of receptors in epiglycanin

The complex carbohydrates at the cell surfaces of two TA3, murine mammary carcinoma ascites sublines (the strain-specific, TA3-St subline and the nonstrain-specific, TA3-Ha line) were compared by binding studies with ¹²⁵I-labelled concanavalin A (con A), Ricinis communis agglutinin (RCA), and eel-serum agglutinin (ESA). The TA3-Ha cell bound equal amounts of con A, 1.5-fold more RCA, and 4-fold more ESA than the TA3-St cell. Binding-inhibition studies by these lectins and two others [wheat-germ agglutinin (WGA) and potato lectin (STA)] suggest complementary binding-sites between con A and both RCA and ESA. Quantitative agglutination studies with the five lectins, and inhibition determinations by both neuraminidase-treated and untreated epiglycanin revealed that TA3-St, but not TA3-Ha, cells were agglutinated by con A, and that epiglycanin inhibited this agglutination, as well as the agglutination of rabbit erythrocytes by con A. The presence of a con A receptor on epiglycanin was also suggested by the binding of epiglycanin to con A-Sepharose, and its specific elution with methyl α-d-manno-pyranoside. TA3-St cells were agglutinated at a 10-15-fold lower concentration of either STA or RCA than TA3-Ha cells, but both cells were agglutinated by the same concentration of WGA and ESA. Inhibition by epiglycanin of agglutination of TA3-St cells by either STA or ESA occurred at a concentration lower than that of TA3-Ha cells, but epiglycanin inhibited RCA agglutination of TA3-Ha cells at a concentration     

The effect of temperature on concanavalin A-mediated agglutination of cells with rigid receptors.

The agglutination of a yeast, Candida albicans, by concanavalin A has been described. The agglutination was cell-number dependent. Prolonged incubation (60 min) was needed to reach maximum agglutination at 37 degrees C. The rate but not the extent of agglutination was temperature dependent. The dimeric forms of concanavalin A, obtained either at low pH or after succinylation, agglutinated the yeast cells as well as the tetramer. Temperature changes affected the agglutination of yeast cells by dimers and by tetramers to the same extent.