0000000000191344
AUTHOR
M V Elorza
Wall mannoproteins of the yeast and mycelial cells of Candida albicans: nature of the glycosidic bonds and polydispersity of their mannan moieties.
SUMMARY: Zymolyase released between 20 and 25% of the total protein from purified walls of yeast (Y) and mycelial (M) cells of Candida albicans. The material released contained 92% carbohydrate (86% mannose and 6% glucose) and 7:< protein. Over 85% of the carbohydrate was N-glycosidically linked to the protein and the rest (less than 15%) was linked O-glycosidically. Highly polydisperse, high molecular mass mannoproteins, resolved by electrophoresis as four defined bands in Y cells and two bands in M cells, had both types of sugar chains. A 34 kDa species found in both types of cells had a single 2.5 kDa N-glycosidically linked sugar chain and a 3 1.5 kDa protein moiety. Polydispersity in t…
Incorporation of specific wall proteins during yeast and mycelial protoplast regeneration in Candida albicans
The kinectics of incorporation of two precursor mannoproteins into the regenerating cell wall of Candida albicans protoplasts have been followed at 28°C and 37°C using two monoclonal antibodies specific for protein epitopes (MAb 1B12 and 4C12) as probes. Both molecules were secreted from the beginning of the regeneration process, and their incorporation was retarded significantly. Analysis of the secreted materials by Western immunoblotting with MAb 1B12 allowed the identification of two closely migrating bands at apparent Mr higher than 170 kDa and significant amounts of a highly polydisperse material of even greater molecular mass. Some of these mannoproteinaceous species carried both N- …
Isolation and characterization of yeast monomorphic mutants of Candida albicans.
A method was devised for the isolation of yeast monomorphic (LEV) mutants of Candida albicans. By this procedure, about 20 stable yeast-like mutants were isolated after mutagenesis with ethyl methane sulfonate. The growth rate of the mutants in different carbon sources, both fermentable and not, was indistinguishable from that of the parental strain, but they were unable to grow as mycelial forms after application of any of the common effective inducers, i.e., heat shock, pH alterations, proline addition, or use of GlcNAc as the carbon source. Studies performed with one selected strain demonstrated that it had severe alterations in the chemical composition of the cell wall, mainly in the le…
Preparation of Anti-protein and Anti-mannan Antisera against Fungal Cell Wall by Affinity Chromatography
Abstract Iranzo, M., Marcilla, A., Elorza, M. V., Mormeneo, S., and Sentandreu, R. 1994. Preparation of anti-protein and anti-mannan antisera against fungal cell wall by affinity chromatography. Experimental Mycology 18, 159-167. A novel and easy chromatographic method has been developed for the isolation of anti-protein and anti-mannan antisera from a population of polyclonal antibodies obtained against Candida albicans and Yarrowia lipolytica cell wall mannoproteins. The technique is based on the immobilization of mannan (to be used as immunoadsorbent) by Affi-Prep H z resin after the oxidation of neighboring hydroxyl groups of the polysaccharide with sodium periodate. For Y. lipolytica p…
Candida albicans mycelial wall structure: supramolecular complexes released by zymolyase, chitinase and beta-mercaptoethanol.
Different techniques released from the wall of Candida albicans mycelial cells high molecular weight mannoprotein materials with different levels of complexity. SDS solubilized among others one protein of 180 kDa which reacted with a monoclonal antibody (MAb) specific of a O-glycosylated protein secreted by regenerating mycelial protoplasts [Elorza et al. (1989) Biochem Biophys Res Commun 162:1118-1125]. Zymolyase, chitinase and beta-mercaptoethanol, released different types of high molecular highly polydisperse mannoprotein materials (greater than 180 kDa) that also reacted with the same MAb. These materials had N-glycosidically linked sugar chains, in addition to the O-glycosidically bond…
Regulation of chitin synthase activity inSaccharomyces cerevisiae: Effect of the inhibition of cell division and of synthesis of RNA and protein
The effect of pronase and trypsin on the activation or deactivation (degradation?) of chitin synthase ofSaccharomyces cerevisiae occurs faster in membranous preparations than in toluene-treated cells. When the temperature is raised, the former preparation is deactivated earlier than the latter one. The activity found in growing cells is not modified after inhibition of protein synthesis by cycloheximide or amino acid starvation or by the inhibition of RNA synthesis. It was possible to activate the chitin synthase ofS. cerevisiae cdc 25 grown at 23°C by means of pronase, whereas trypsin had no effect. After the cells were grown at 37°C, chitin synthase could not be activated either with tryp…
Cloning and characterization of CSP37, a novel gene encoding a putative membrane protein of Candida albicans.
In the course of an analysis of the functions and assembly of the cell wall of Candida albicans, we have cloned and characterized a gene, which we designated CSP37 (cell surface protein), encoding a 37-kDa polypeptide which is a membrane-associated protein. The gene was isolated by immunological screening of a DNA library constructed from mycelial cells with a polyclonal serum raised against cell walls of this morphology. Analysis of the nucleotide sequence of a corresponding genomic DNA fragment revealed a single open reading frame which encodes a predicted protein of 321 amino acids with no significant homology to others in the databases. Disruption of the CSP37 gene by the method describ…
Possible Roles of Mannoproteins in the Construction of Candida Albicans Cell Wall
The shape of Candida albicans cells depends on their cell walls and some of their mannoproteins may act as modulators of the final molecular architecture. If that were the case, the wall mannoproteins might form part of what could be called a “morphogenetic code”.
Involvement of transglutaminase in the formation of covalent cross-links in the cell wall of Candida albicans.
Activity of the enzyme glutaminyl-peptide--glutamylyl-transferase (EC 2.3.2.13; transglutaminase), which forms the interpeptidic cross-link N epsilon-(gamma-glutamic)-lysine, was demonstrated in cell-free extracts obtained from both the yeast like and mycelial forms of Candida albicans. Higher levels of enzymatic activity were observed in the cell wall fraction, whereas the cytosol contained only trace amounts of activity. Cystamine, a highly specific inhibitor of the enzyme, was used to analyze a possible role of transglutaminase in the organization of the cell wall structure of the fungus. Cystamine delayed protoplast regeneration and inhibited the yeast-to-mycelium transition and the inc…
Relationships Between Dimorphism, Cell Wall Structure, and Surface Activities in Candida albicans
Most cells are covered with a complex network of interacting molecules that form the extracellular matrix. These molecules (proteins and polysaccharides) are secreted locally and interact among themselves to form an organized structure outside the cell plasma membrane. In unicellular eukaryotic organisms and plant cells, this structure is reinforced to withstand osmotic changes in the external environment, giving rise to the so-called cell wall.
Synthesis and Assembly of Wall Polymers on Regenerating Yeast Protoplasts
Accumulation of chitin and glucan on S. cerevisiae and C. albicans protoplasts begins shortly after resuspension in the regeneration medium, and mannoprotein molecules also appear retained by the regenerating wall after 30–60 minutes in S. cerevisiae or after a longer lag period in C. albicans. Nevertheless, a considerable fraction of the synthesized mannoproteins, which in SDS-acrylamide gels exhibit a different pattern from that of wall mannoproteins of cells, are still released to the growth medium during at least eight hours. De novo synthesis of chitin synthase, but not of glucan synthase, is observed in S. cerevisiae from about 30 minutes after initiation of the regeneration process. …
Cloning of cDNAs coding forCandida albicanscell surface proteins
Two cDNA libraries were constructed from mRNAs obtained from yeast cells and germ-tubes of Candida albicans in lambda gt11. Immunoscreening with polyclonal antibodies raised against cell wall components allowed the detection of 29 positive clones. Two of these clones were selected for their specific reactivity with antisera either from yeast (clone 11Y) or germ-tubes (clone 24M). cDNA fragments were isolated by the digestion of lambda DNA with EcoRI. Southern blot analysis with these fragments as probes demonstrated homology with C. albicans DNA, and by Northern analysis two mRNAs transcripts were detected with sizes of approximately 1·5 kb for 11Y and 1·1 kb for 24M. Both transcripts were …
Anchorage of Candida albicans Ssr1 to the cell wall, and transcript profiling of the null mutant.
Incorporation into the wall of Candida albicans Ssr1, a GPI-dependent protein, was investigated by construction of different truncated genes for which the three potential omega sites (S199, S215 and G216) and the corresponding omega+1 and omega+2 were eliminated or modified. Cells of the C. albicans ssr1Delta mutant were transformed with pADH-pl harboring the truncated versions of CaSSR1, pADH-DeltaCaSSR1t(217-234) (lacking a C-terminal hydrophobic stretch of 18 aa including the putative omega+2 and omega+1, omega+2 of S215 and G216) or pADH-DeltaCaSSR1t(199-201) (lacking three serine residues), and their walls were analyzed for the protein. Results suggested that the three serine residues …
Yeast Cell Wall Glycoproteins
In higher cells, glycoproteins play a variety of functions as surface receptors, cell-cell mediators, carriers of enzyme activities, components of the extracellular matrix, etc. In most glycoproteins, the protein moiety will be the functional part whereas the carbohydrate moiety would contribute to the attainment of an adequate tertiary structure, modify the glycoprotein molecule making it more resistant to degradation, and facilitate its secretion.
Role of Pir1 in the construction of the Candida albicans cell wall
Searches in a Candida albicans database (http://genolist.pasteur.fr/CandidaDB/) identified two Individual Protein Files (IPF 15363 and 19968) whose deduced amino acid sequences showed 42 % and 45 % homology with Saccharomyces cerevisiae Pir4. The two DNA sequences are alleles of the same gene (CaPIR1) but IPF 19968 has a deletion of 117 bases. IPF 19968 encodes a putative polypeptide of 364 aa, which is highly O-glycosylated and has an N-mannosylated chain, four cysteine residues and seven repeats. Both alleles are expressed under different growth conditions and during wall construction by regenerating protoplasts. The heterozygous mutant cells are elongated, form clumps of several cells an…
Formation of a new cell wall by protoplasts of Candida albicans: effect of papulacandin B, tunicamycin and Nikkomycin.
SUMMARY: Incorporation of polysaccharides into the walls of regenerating protoplasts of Candida albicans was followed in the presence of papulacandin B, tunicamycin and nikkomycin. With the first drug, chitin was incorporated normally whereas incorporation of glucans and mannoproteins was significantly decreased. Tunicamycin decreased incorporation of all wall polymers when added at the beginning of the regeneration process but blocked only mannan and alkali-insoluble glucan incorporation when added after 5 h. Nikkomycin inhibited chitin synthesis, and the walls formed by the protoplasts were enriched in alkali-soluble glucan. Pulse-chase experiments suggested that a precursor-product relat…
Cell wall composition and protoplast regeneration in Candida albicans
The transition of blastospores to the mycelial phase in Candida albicans was induced after the blastospores were kept at 4°C for several hours and then transferred to a fresh medium prewarmed at 37°C. Glucan was the most abundant polymer in the wall in the two morphogenetic forms but the amount of chitin was higher in the mycelial form than in blastospores. Efficient protoplasting required reducing agents and proteases together with β-glucanases (zymolyase). Protein synthesis in regenerating protoplasts was initiated after about 30 min. Chitin synthetase, initially very low, was incorporated in important amounts into cell membranes mainly in a zymogenic state. After a few hours chitin was t…
Wall formation by Candida albicans yeast cells: synthesis, secretion and incorporation of two types of mannoproteins.
SUMMARY: The mannoprotein components solubilized from the walls of Candida albicans blastoconidia following degradation of the glucan network with β-glucanase (Zymolyase) have higher molecular masses than their probable precursors present in the supernatant of regenerating protoplasts. It therefore appears that the mannoproteins are released from the walls as part of supramolecular complexes. Immunological analysis using both polyclonal and monoclonal antibodies has demonstrated the probable relationship between molecules found in a mixed membrane preparation, those secreted by regenerating protoplasts, and those present in yeast cell walls. Some mannoproteins secreted by protoplasts incuba…