Tunicamycin and papulacandin B inhibit incorporation of specific mannoproteins into the wall of Candida albicans regenerating protoplasts

Regeneration of Candida albicans protoplasts began with the formation of a chitin network which was complemented after a lag of about 60 min by the deposition of beta-glucan. Proteins were incorporated early to the growing structure, beginning with the mannoproteins which are kept in place by non-covalent bonds. Incorporation of covalently linked mannoproteins took place only after deposition of glucan. The incorporation of these mannoproteins did not occur when protoplasts were incubated with papulacandin B which inhibited glucan formation, or with tunicamycin which blocked N-glycosylation of mannoproteins. In the presence of papulacandin B, large amounts of native mannoproteins accumulated in the medium. However, in the presence of tunicamycin, the large mannoprotein material found was of smaller apparent molecular weight, suggesting that it was deficient in glycosylation. Partially regenerated walls were able to incorporate 'in vitro' non-covalently bound mannoproteins, indicating that some components of very large cellular structures such as walls are capable of being articulated by a self-assembly process.     

Metabolism of Saccharomyces cerevisiae envelope mannoproteins

By pulse and chase labeling experiments, two independent mannoprotein pools have been found associated with the Saccharomyces cerevisiae envelope. One of them probably corresponds to mannoproteins localized in the periplasmic space. These molecules showed a high turnover rate at 28° C. The second pool is formed by intrinsic wall mannoproteins which are apparently stable for long periods of time, after a small initial turnover. These results suggest that at least part of the mannoproteins initially found in the periplasmic space may move into the wall.The time lag between the addition of the radioactive precursors and their incorporation in the cell envelope (20–30 min for amino acids and about 10 min for carbohydrate) indicates that protein formation and carbohydrate incorporation take place in succession. Moreover, bulk glycosylation of mannoproteins seems to occur close in time to the moment of secretion into the periplasmic space.     

Structure of the Saccharomyces cerevisiae cell wall: Mannoproteins released by zymolyase and their contribution to wall architecture

Purified zymolyase containing β-glucanase activity preferentially released a 29 kDa mannoprotein from isolated yeast cell walls and a high-molecular-mass (greater than 120 kDa) material. Endo-β-N-acetylglucosaminidase H digestion indicated that the 29 kDa mannoprotein contains a unique core coligosaccharide N-glycosidically linked to a 26 kDa peptide moiety. Cells grown in the presence of tunicamycin incorporated the nonglycosylated 26 kDa peptide into the wall, but not the large mannoprotein molecules. Treatment of isolated walls with SDS solubilized more than 30 different mannoproteins, one of tehm being the 29 kDa species, but the large-size molecules were not affected. Regenerating protoplasts incorporated into the forming walls most of the SDS-solubilizable species seen in mature cell walls, but the zymolyase-solubilizable mannoproteins were absent. Wall mannoproteins have also been compared with those of the periplasmic space, most of the species being commonly present at both compartments. Turnover of individual species has been studied by pulse and chase experiments. While mannoproteins from the walls remain stable for long periods, periplasmic molecules exhibit a rapid turnover rate.     

Dimorphism in Candida albicans: contribution of mannoproteins to the architecture of yeast and mycelial cell walls

Wall mannoproteins of the two (yeast and mycelial) cellular forms of Candida albicans were solubilized by different agents. Boiling in 2% (w/v) SDS was the best method, as more than 70% of the total mannoprotein was extracted. Over 40 different bands (from 15 to 80 kDal) were detected on SDS-polyacrylamide gel electrophoresis of this material. The residual wall mannoproteins were released after enzymic (Zymolyase and endogenous wall beta-glucanases) degradation of wall glucan, suggesting that they are covalently linked to this structural polymer. Four bands (of 160 kDal, 205 kDal and higher molecular mass) were observed in the material released from yeast walls but only the two smaller components were detected in the material obtained from mycelial walls. Moreover, the mannoproteins of high molecular mass, which are covalently linked in walls of normal cells, were not incorporated into walls of regenerating protoplasts, but non-covalently linked mannoproteins were retained from the beginning of the process.     

Temporal aspects of the O-glycosylation of Saccharomyces cerevisiae mannoproteins

Cleavage of the O-glycosyl bonds of Saccharomyces cerevisiae cell wall mannoproteins by β-elimination resulted in the release of about 8% of the carbohydrate in the form of mannose and other low molecular weight oligomannosaccharides (mannose to mannopentaose), leaving 92% mannose still covalently linked to the peptide, and suggesting that this alkali-resistant fraction was N-glycosidically linked. At the non-permissive temperature, S. cerevisiae sec mutants accumulated in the cytoplasm mannoproteins with different degrees of O- and N-glycosylation. The glycoproteins of mutant sec 20-1 contained 60% of the carbohydrate linked by N-bonds, the remainder being O-glycosidically linked. Strains sec 19-1 and sec 1-1 contained 80 and 87%, respectively, of the mannose as N-linked carbohydrates. In addition, when the non-permissive conditions were prolonged over 2 h, strain sec 20-1 completed the formation of the O-linked oligosaccharides, suggesting that it is the kinetics of the process that determines the final composition of the mannoproteins. Our results suggest that the glycosylation of yeast cell wall mannoproteins is probably initiated in the lumen of the endoplasmic reticulum where the O-linked oligosaccharides may be completed. Maturation of the N-linked oligosaccharides, on the other hand, probably occurs during transport of the nascent mannoproteins to the cell surface.     

Role of dimerization and modification of the CSF-1 receptor in its activation and internalization during the CSF-1 response.

We have used kinetic and cross-linking approaches to study CSF-1-induced changes in the structure and function of the CSF-1R. Addition of CSF-1 to cells stimulates or stabilizes non-covalent CSF-1R dimerization resulting in activation of the CSF-1R kinase and the tyrosine phosphorylation of the receptor and certain cytoplasmic proteins. The non-covalent dimers become covalently linked via disulfide bonds and/or are subsequently further modified. These modified forms are selectively internalized. Pre-treatment of cells with the alkylating agent, iodoacetic acid (IAA), selectively inhibits covalent dimerization, modification and internalization but enhances protein tyrosine phosphorylation. It is proposed that ligand-induced non-covalent dimerization activates the CSF-1R kinase, whereas the covalent dimerization and subsequent modification lead to kinase inactivation, phosphotyrosine dephosphorylation and internalization of the receptor--ligand complex.     

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- and O-glycosidically linked mannose residues, as deduced from their drop in apparent Mr when synthesized in the presence of tunicamycin and by their reactivity with Concanavalin A. Following secretion, the molecules reacting with MAb 1B12 were incorporated into the regenerating walls by covalent binding. Then, when the regenerating walls by covalent binding. Then, when the antigen molecules were solubilized from partially regenerated walls, their mobility differed when regeneration took place at 28°C (blastoconidia) or 37°C (mycelial cells).Abbreviations used IIF indirect immunofluorescence - MAb monoclonal antibody - PBS phosphate-buffed saline - PMSF phenyl methyl sulfonyl fluoride - SDS sodium dodecyl sulfate     

Formation of a new cell wall by protoplasts of Candida albicans: effect of papulacandin B, tunicamycin and Nikkomycin

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 relationship between the alkali-soluble and alkali-insoluble glucans existed in the wall. The results obtained with the antibiotics were confirmed and extended by cytological studies using wheat-germ agglutinin labelled with colloidal gold and concanavalin A-ferritin as specific markers of chitin and mannoproteins respectively. The results support the idea that regeneration of walls by protoplasts occurs in two steps: firstly, a chitin microfibrillar skeleton is formed, and in a later step glucan-mannoprotein complexes are added to the growing structure. The chitin skeleton probably allows the orderly spatial arrangement of the other polymers giving rise to the regenerated cell wall.     

Structural investigation of radiation-induced aggregates of ribonuclease

Following irradiation of bovine pancreatic ribonuclease in aqueous solution with 60Co gamma-rays protein aggregates are formed. The nature of the bonds linking these radiation-induced aggregates together has been investigated by chromatographic and electrophoretic methods. Thin-layer gel filtration and polyacrylamide gel electrophoresis, both in the presence of sodium dodecyl sulphate, demonstrated the existence of covalent crosslinks between the aggregates. However, non-covalent crosslinking also plays a role in the radiolysis of ribonuclease. Thin-layer gel filtration with and without 6 M urea and 2 per cent beta-mercaptoethanol added to the gel, revealed that only part of the covalent bonds between the aggregates consisted of disulphide linkages. By separation of the reduced aggregates by thin-layer gel filtration and electrophoresis, both with SDS, this finding was substantiated. Densitometric measurements indicated for example that the percentage of covalently linked dimers held together by disulphide bridges amounted to about 40-45 per cent, whereas the remaining 55-60 per cent of the dimers must be linked by other covalent bonds. The existence of covalent crosslinks other than disulphide bonds was also confirmed by isoelectric focusing. By this method definite differences were established between the proteolytic hydrolysates of the reduced aggregates and the reduced monomer of gamma-irradiated ribonuclease.     

Mutations in the thiol-disulfide oxidoreductases BdbC and BdbD can suppress cytochrome c deficiency of CcdA-defective Bacillus subtilis cells

Cytochromes of the c type in the gram-positive bacterium Bacillus subtilis are all membrane anchored, with their heme domains exposed on the outer side of the cytoplasmic membrane. They are distinguished from other cytochromes by having heme covalently attached by two thioether bonds. The cysteinyls in the heme-binding site (CXXCH) in apocytochrome c must be reduced in order for the covalent attachment of the heme to occur. It has been proposed that CcdA, a membrane protein, transfers reducing equivalents from thioredoxin in the cytoplasm to proteins on the outer side of the cytoplasmic membrane. Strains deficient in the CcdA protein are defective in cytochrome c and spore synthesis. We have discovered that mutations in the bdbC and bdbD genes can suppress the defects caused by lack of CcdA. BdbC and BdbD are thiol-disulfide oxidoreductases. Our experimental findings indicate that these B. subtilis proteins functionally correspond to the well-characterized Escherichia coli DsbB and DsbA proteins, which catalyze the formation of disulfide bonds in proteins in the periplasmic space.     

Agarose gel filtration of Triticum vulgare proteins in dissociating solvents

Certain wheat proteins (glutenins emerged from 4% agarose columns at the void volumes even in the presence of 6 M guanidine hydrochloride, 4 M urea with or without 1% sodium dodecyl sulphate, and 8 M urea. These proteins were of considerably greater molecular size than bovine thyroglobulin (sub-unit MW 335000). Urea plus sodium dodecyl sulphate was the most effective dissociating solvent. Low MW wheat flour proteins, which had been covalently labelled with a fluorescein derivative, were not incorporated through formation of new disulphide bonds into higher MW fractions during acidic extraction of flour. Limited incorporation through non-covalent association was observed. The results do not support the contention that glutenin is an artifact of extraction. It has been confirmed that all the protein of wheat flour is not extractable with water followed by 2 M urea.     

Deletion of New Covalently Linked Cell Wall Glycoproteins Alters the Electrophoretic Mobility of Phosphorylated Wall Components of Saccharomyces cerevisiae

The incorporation of radioactive orthophosphate into the cell walls of Saccharomyces cerevisiae was studied. 33P-labeled cell walls were extensively extracted with hot sodium dodecyl sulfate (SDS). Of the remaining insoluble radioactivity more than 90% could be released by laminarinase. This radioactive material stayed in the stacking gel during SDS-polyacrylamide gel electrophoresis but entered the separating gel upon treatment with N-glycosidase F, indicating that phosphate was linked directly or indirectly to N-mannosylated glycoproteins. The phosphate was bound to covalently linked cell wall proteins as mannose-6-phosphate, the same type of linkage shown previously for soluble mannoproteins (L. Ballou, L. M. Hernandez, E. Alvarado, and C. E. Ballou, Proc. Natl. Acad. Sci. USA 87:3368-3372, 1990). From the phosphate-labeled glycoprotein fraction released by laminarinase, three cell wall mannoproteins, Ccw12p, Ccw13p and Ccw14p, were isolated and identified by N-terminal sequencing. For Ccw13p (encoded by DAN1 [also called TIR3]) and Ccw12p the association with the cell wall has not been described before; Ccw14p is identical with cell wall protein Icwp (I. Moukadiri, J. Armero, A. Abad, R. Sentandreu, and J. Zueco, J. Bacteriol. 179:2154-2162, 1997). In ccw12, ccw13, or ccw14 single or double mutants neither the amount of radioactive phosphate incorporated into cell wall proteins nor its position in the stacking gel was changed. However, the triple mutant brought about a shift of the 33P-labeled glycoprotein components from the stacking gel into the separating gel. The disruption of CCW12 results in a pronounced sensitivity of the cells to calcofluor white and Congo red. In addition, the ccw12 mutant shows a decrease in mating efficiency and a defect in agglutination.     

Formation of muscle proteins during freezing

The effect of different conditions on the formation and properties of cryogels prepared by the freezing-thawing procedure from suspensions and solutions of the carp (Cyprinus carpio) myofibrillar proteins was studied. The freezing of water solutions and suspensions of the native myofibrillar proteins resulted in the formation of the structures mainly stabilized by non-covalent bonds. When muscle proteins were denatured prior to the freezing they formed the structures stabilized by both non-covalent and covalent disulfide bonds.     

The formation of glycosidic bonds in yeast glycoproteins

Membranes of Saccharomyces cerevisiae were separated on urografin gradients. The specific activity of the light membranes (endoplasmic reticulum), the Golgi-like vesicles and the plasma membrane in transferring mannosyl residues from GDP-mannose to mannoproteins and to dolichyl monophosphate has been determined. The first mannose of the O-glycosidically linked manno-oligosaccharides is incorporated with the highest specific activity by the endoplasmic reticulum. The incorporation of the second to fourth mannosyl groups is catalysed with increasing activity also by the Golgi-like vesicles and the plasma membrane.The incorporation of mannosyl groups into weak alkali-stable positions (N-glycosidically linked chains) is carried out with almost the same specific activity by all three membrane fractions, however, dolicholdependent and-independent steps could not be distinguished as yet.The results are discussed in terms of a sequential addition of sugar residues along the route of export of the mannoproteins. The dolichol-dependent steps seem to occur on the endoplasmic reticulum and thus very carly in the event.Abbreviations GDP-mannose guanosine diphosphate mannose - Dol-P dolichyl monophosphate - Dol-P-mannose dolichyl monophosphate mannose     

Involvement of transglutaminase in the formation of covalent cross-links in the cell wall ofCandida albicans

Activity of the enzyme glutaminyl-peptide-γ-glutamylyl-transferase (EC 2.3.2.13; transglutaminase), which forms the interpeptidic cross-link N^∈-(γ-glutamic)-lysine, was demonstrated in cell-free extracts obtained from both the yeast like and mycelial forms ofCandida 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 incorporation of proteins into the cell wall. The incorporation of covalently bound high-molecular-weight proteins into the wall was sensitive to cystamine. Proteic epitopes recognized by two monoclonal antibodies, one of which is specific for the mycelial walls of the fungus, were also sensitive to cystamine. These data suggest that transglutaminase may be involved in the formation of covalent bonds between different cell wall proteins during the final assembly of the mature cell wall.     

Maturation pathway of Escherichia coli heat-stable enterotoxin I: requirement of DsbA for disulfide bond formation.

The Escherichia coli heat-stable enterotoxin STp is synthesized as a precursor consisting of pre, pro and mature regions. Mature STp is released into the culture supernatant and is composed of 18-amino-acid resides which contain three intramolecular disulfide bonds. The involvement of DsbA in the formation of the disulfide bonds of STp was examined in this study. A dsbA mutant was transformed with a plasmid harboring the STp gene, and the ST activity was significantly lower than that of the parent strain harboring the same plasmid. Furthermore, purified DsbA induced the conversion of synthetic STp peptide (inactive form) to the active form and increased the ST activity of the culture supernatant derived from the dsbA transformants. These results showed that DsbA directly catalyzes the formation of the disulfide bonds of STp. DsbA is located in periplasmic space, where STp is released as an intermediate form consisting of pro and mature regions. To examine the effect of the pro region on the action of DsbA, we replaced the cysteine residue at position 39 and tested the effect in vivo. The substitution caused a significant decrease of ST activity in the culture supernatant, the accumulation of inactive ST in periplasmic space, and an alteration in the cleavage site of the intermediate of STp. We conclude that Cys-39 is important for recognition by the processing enzymes required for the maturation of STp.     

Overproduction of bacterial protein disulfide isomerase (DsbC) and its modulator (DsbD) markedly enhances periplasmic production of human nerve growth factor in Escherichia coli

Production of eukaryotic proteins with multiple disulfide bonds in the Escherichia coli periplasm often encounters difficulty in obtaining soluble products with native structure. Human nerve growth factor beta (NGF) contains three disulfide bonds between nonconsecutive cysteine residues and forms insoluble aggregates when expressed in E. coli. We now report that overexpression of Dsb proteins known to catalyze formation and isomerization of disulfide bonds can substantially enhance periplasmic production of NGF. A set of pACYC184-based plasmids that permit dsb expression under the araB promoter were introduced into cells carrying a compatible plasmid that expresses NGF. The efficiency of periplasmic production of NGF fused to the OmpT signal peptide was strikingly improved by coexpression of DsbCD or DsbABCD proteins (up to 80% of total NGF produced). Coexpression of DsbAB was hardly effective, whereas that of DsbAC increased the total yield but not the periplasmic expression. These results suggest synergistic roles of DsbC and DsbD in disulfide isomerization that appear to become limiting upon NGF production. Furthermore, recombinant NGF produced with excess DsbCD (or DsbABCD) was biologically active judged by the neurite outgrowth assay using rat PC12 cells.     

Preparation and structure of the charge-transfer intermediate of the transmembrane redox catalyst DsbB

Disulfide bond formation is a critical step in the folding of many secretory proteins. In bacteria, disulfide bonds are introduced by the periplasmic dithiol oxidase DsbA, which transfers its catalytic disulfide bond to folding polypeptides. Reduced DsbA is reoxidized by ubiquinone Q8, catalyzed by inner membrane quinone reductase DsbB. Here, we report the preparation of a kinetically stable ternary complex between wild-type DsbB, containing all essential cysteines, Q8 and DsbA covalently bound to DsbB. The crystal structure of this trapped DsbB reaction intermediate exhibits a charge-transfer interaction between Q8 and the Cys44 in the DsbB reaction center providing experimental evidence for the mechanism of de novo disulfide bond generation in DsbB.     

A disulfide relay system in the intermembrane space of mitochondria that mediates protein import

We describe here a pathway for the import of proteins into the intermembrane space (IMS) of mitochondria. Substrates of this pathway are proteins with conserved cysteine motifs, which are critical for import. After passage through the TOM channel, these proteins are covalently trapped by Mia40 via disulfide bridges. Mia40 contains cysteine residues, which are oxidized by the sulfhydryl oxidase Erv1. Depletion of Erv1 or conditions reducing Mia40 prevent protein import. We propose that Erv1 and Mia40 function as a disulfide relay system that catalyzes the import of proteins into the IMS by an oxidative folding mechanism. The existence of a disulfide exchange system in the IMS is unexpected in view of the free exchange of metabolites between IMS and cytosol via porin channels. We suggest that this process reflects the evolutionary origin of the IMS from the periplasmic space of the prokaryotic ancestors of mitochondria.     

Purification of neuronal inclusions of patients with Huntington's disease reveals a broad range of N-terminal fragments of expanded huntingtin and insoluble polymers

Huntington's disease resulting from huntingtin containing an expanded polyglutamine is associated with aggregates largely confined to neuronal inclusions, and with neuronal death. Inclusions are thought to originate from discrete N-terminal fragments of expanded huntingtin produced by specific endopeptidases. We have now purified the neuronal inclusions of Huntington's disease brain. When incubated in concentrated formic acid, purified inclusions release a polymer, an oligomer and a broad range of N-terminal fragments of expanded huntingtin. The fragments and the polymeric forms are linked to each other by non-covalent bonds as they are both released by formic acid, whereas the polymeric forms themselves are presumably stabilized by covalent bonds, as they are resistant to formic acid. We also demonstrate the presence in affected areas of the brain but not in unaffected areas of a broad range of soluble N-terminal fragments of expanded huntingtin not yet associated with the inclusions and which are likely to be the precursors of the inclusions. Fragmentation of expanded huntingtin in Huntington's disease must result from the operation of multiple proteolytic activities with little specificity and not from that of a specific endopeptidase; subsequent aggregation of the fragments by covalent and non-covalent bonds leads to the formation of the inclusions.