The cell wall architecture of Candida albicans wild-type cells and cell wall-defective mutants

In Candida albicans wild-type cells, the beta1, 6-glucanase-extractable glycosylphosphatidylinositol (GPI)-dependent cell wall proteins (CWPs) account for about 88% of all covalently linked CWPs. Approximately 90% of these GPI-CWPs, including Als1p and Als3p, are attached via beta1,6-glucan to beta1,3-glucan. The remaining GPI-CWPs are linked through beta1,6-glucan to chitin. The beta1,6-glucanase-resistant protein fraction is small and consists of Pir-related CWPs, which are attached to beta1,3-glucan through an alkali-labile linkage. Immunogold labelling and Western analysis, using an antiserum directed against Saccharomyces cerevisiae Pir2p/Hsp150, point to the localization of at least two differentially expressed Pir2 homologues in the cell wall of C. albicans. In mnn9Delta and pmt1Delta mutant strains, which are defective in N- and O-glycosylation of proteins respectively, we observed enhanced chitin levels together with an increased coupling of GPI-CWPs through beta1,6-glucan to chitin. In these cells, the level of Pir-CWPs was slightly upregulated. A slightly increased incorporation of Pir proteins was also observed in a beta1, 6-glucan-deficient hemizygous kre6Delta mutant. Taken together, these observations show that C. albicans follows the same basic rules as S. cerevisiae in constructing a cell wall and indicate that a cell wall salvage mechanism is activated when Candida cells are confronted with cell wall weakening.     

Modular domain structure in the Candida glabrata adhesin Epa1p,a beta1,6 glucan-cross-linked cell wall protein

The yeast pathogen Candida glabrata adheres avidly to cultured human epithelial cells. This interaction depends on the expression of EPA1, which encodes a lectin belonging to a large family of GPI-anchored glucan-cross-linked cell wall proteins (GPI-CWPs) found in diverse fungal species. To understand the relationship between different domains of EPA1 and its function, we have mapped functional domains of Epa1p and analysed their contribution to Epa1p function. We found that the N-terminal third of the protein contains the ligand-binding domain, and that the GPI anchor is essential both for cross-linking in the cell wall and for Epa1p-mediated adherence. We also found that the C-terminal Ser/Thr-rich domain, characteristic of many GPI-CWPs, was absolutely essential for function. Although Epa1p derivatives lacking the Ser/Thr domain were expressed abundantly in the cell wall, they were localized to internal layers of the cell wall; such constructs were unable to mediate adherence. The outer layer of the yeast cell wall is known to act as a permeability barrier; we found that the C-terminal Ser/Thr-rich region was absolutely required to project the N-terminal domain of Epa1p through this permeability barrier and into the external environment. Thus, the Ser/Thr-rich domain of Epa1p and, presumably, of other related GPI-CWPs serves an essential structural role in localization of the protein at the external surface of the yeast cell where it can interact with its ligand. In conclusion, Epa1p has a modular structure, with each domain serving a distinct and essential role in the function of the adhesin.     

Novel strategy for anchorage position control of GPI-attached proteins in the yeast cell wall using different GPI-anchoring domains

The yeast cell surface provides space to display functional proteins. Heterologous proteins can be covalently anchored to the yeast cell wall by fusing them with the anchoring domain of glycosylphosphatidylinositol (GPI)-anchored cell wall proteins (GPI-CWPs). In the yeast cell-surface display system, the anchorage position of the target protein in the cell wall is an important factor that maximizes the capabilities of engineered yeast cells because the yeast cell wall consists of a 100- to 200-nm-thick microfibrillar array of glucan chains. However, knowledge is limited regarding the anchorage position of GPI-attached proteins in the yeast cell wall. Here, we report a comparative study on the effect of GPI-anchoring domain–heterologous protein fusions on yeast cell wall localization. GPI-anchoring domains derived from well-characterized GPI-CWPs, namely Sed1p and Sag1p, were used for the cell-surface display of heterologous proteins in the yeast Saccharomyces cerevisiae. Immunoelectron-microscopic analysis of enhanced green fluorescent protein (eGFP)-displaying cells revealed that the anchorage position of the GPI-attached protein in the cell wall could be controlled by changing the fused anchoring domain. eGFP fused with the Sed1-anchoring domain predominantly localized to the external surface of the cell wall, whereas the anchorage position of eGFP fused with the Sag1-anchoring domain was mainly inside the cell wall. We also demonstrate the application of the anchorage position control technique to improve the cellulolytic ability of cellulase-displaying yeast. The ethanol titer during the simultaneous saccharification and fermentation of hydrothermally-processed rice straw was improved by 30% after repositioning the exo- and endo-cellulases using Sed1- and Sag1-anchor domains. This novel anchorage position control strategy will enable the efficient utilization of the cell wall space in various fields of yeast cell-surface display technology.     

A glucoamylase::GFP gene fusion to study protein secretion by individual hyphae of Aspergillus niger

Although Aspergillus niger is used as a host for heterologous protein production, yields are generally lower than those obtained for homologous proteins. Mechanisms of protein secretion and the secretory pathway in filamentous fungi are poorly characterised, although there is evidence to suggest that secretion occurs by a mechanism similar to that in other eukaryotes, but with proteins destined for secretion being directed to the hyphal tip. We report on a method using a glucoamylase: GFP gene fusion which allows us for the first time to monitor, in vivo, protein secretion in A. niger at the single hyphal level. A synthetic green fluorescent protein (sGFP(S65T)) was fused to truncated A. niger glucoamylase (GLA:499). Southern blot analysis of transformants confirmed that the gene fusion had successfully integrated into the A. niger genome. Confocal and fluorescence microscopy revealed that the GLA::GFP fusion protein is fluorescent in A. niger and appears to be directed to the hyphal tip. In young mycelia, hyphal cell wall fluorescence is apparent and immunogold labelling of GFP confirmed that GFP was partially localised within the hyphal cell wall. Using Western blotting, extracellular GLA::GFP was detected only in culture filtrates of young mycelia grown in a soya milk medium. The actin inhibitor latrunculin B was used to disrupt the secretion process, and its effects on the distribution of GLA::GFP were monitored.     

One Single Basic Amino Acid at the ω-1 or ω-2 Site Is a Signal That Retains Glycosylphosphatidylinositol-Anchored Protein in the Plasma Membrane of Aspergillus fumigatus

Although the plasma membrane is the terminal destination for glycosylphosphatidylinositol (GPI) proteins in higher eukaryotes, cell wall-attached GPI proteins (GPI-CWPs) are found in many fungal species. In yeast, some of the cis-requirements directing localization of GPI proteins to the plasma membrane or cell wall are now understood. However, it remains to be determined how Aspergillus fumigatus, an opportunistic fungal pathogen, signals, and sorts GPI proteins to either the plasma membrane or the cell wall. In this study, chimeric green fluorescent proteins (GFPs) were constructed as fusions with putative C-terminal GPI signal sequences from A. fumigatus Mp1p, Gel1p, and Ecm33p, as well as site-directed mutations thereof. By analyzing cellular localization of chimeric GFPs using Western blotting, electron microscopy, and fluorescence microscopy, we showed that, in contrast to yeast, a single Lys residue at the ω-1 or ω-2 site alone could retain GPI-anchored GFP in the plasma membrane. Although the signal for cell wall distribution has not been identified yet, it appeared that the threonine/serine-rich region at the C-terminal half of AfMp1 was not required for cell wall distribution. Based on our results, the cis-requirements directing localization of GPI proteins in A. fumigatus are different from those in yeast.     

Two novel, putatively cell wall-associated and glycosylphosphatidylinositol-anchored alpha-glucanotransferase enzymes of Aspergillus niger

In the genome sequence of Aspergillus niger CBS 513.88, three genes were identified with high similarity to fungal alpha-amylases. The protein sequences derived from these genes were different in two ways from all described fungal alpha-amylases: they were predicted to be glycosylphosphatidylinositol anchored, and some highly conserved amino acids of enzymes in the alpha-amylase family were absent. We expressed two of these enzymes in a suitable A. niger strain and characterized the purified proteins. Both enzymes showed transglycosylation activity on donor substrates with alpha-(1,4)-glycosidic bonds and at least five anhydroglucose units. The enzymes, designated AgtA and AgtB, produced new alpha-(1,4)-glycosidic bonds and therefore belong to the group of the 4-alpha-glucanotransferases (EC 2.4.1.25). Their reaction products reached a degree of polymerization of at least 30. Maltose and larger maltooligosaccharides were the most efficient acceptor substrates, although AgtA also used small nigerooligosaccharides containing alpha-(1,3)-glycosidic bonds as acceptor substrate. An agtA knockout of A. niger showed an increased susceptibility towards the cell wall-disrupting compound calcofluor white, indicating a cell wall integrity defect in this strain. Homologues of AgtA and AgtB are present in other fungal species with alpha-glucans in their cell walls, but not in yeast species lacking cell wall alpha-glucan. Possible roles for these enzymes in the synthesis and/or maintenance of the fungal cell wall are discussed.     

Fungal α-arabinofuranosidases of glycosyl hydrolase families 51 and 54 show a dual arabinofuranosyl- and galactofuranosyl-hydrolyzing activity

Abstract Aspergillus niger possesses a galactofuranosidase activity, however, the corresponding enzyme or gene encoding this enzyme has never been identified. As evidence is mounting that enzymes exist with affinity for both arabinofuranose and galactofuranose, we investigated the possibility that α-l-arabinofuranosidases, encoded by the abfA and abfB genes, are responsible for the galactofuranosidase activity of A. niger. Characterization of the recombinant AbfA and AbfB proteins revealed that both enzymes do not only hydrolyze p-nitrophenyl-α-l-arabinofuranoside (pNp-α-Araf) but are also capable of hydrolyzing p-nitrophenyl-β-d-galactofuranoside (pNp-β-Galf). Molecular modeling of the AbfB protein with pNp-β-Galf confirmed the possibility for AbfB to interact with this substrate, similarly as with pNp-α-Araf. We also show that galactomannan, a cell wall compound of A. niger, containing β-linked terminal and internal galactofuranosyl moieties, can be degraded by an enzyme activity that is present in the supernatant of inulin-grown A. niger. Interestingly, purified AbfA and AbfB did not show this hydrolyzing activity toward A. nigergalactomannan. In summary, our studies demonstrate that AbfA and AbfB, α-l-arabinofuranosidases from different families, both contain a galactofuranose (Galf)-hydrolyzing activity. In addition, our data support the presence of a Galf-hydrolase activity expressed by A. niger that is capable of degrading fungal galactomannan.     

Expression of transglutaminase substrate activity on Candida albicans germ tubes through a coiled, disulfide-bonded N-terminal domain of Hwp1 requires C-terminal glycosylphosphatidylinositol modification

By serving as a microbial substrate for epithelial cell transglutaminase, Hwp1 (Hyphal wall protein 1) of Candida albicans participates in cross-links with proteins on the mammalian mucosa. Biophysical properties of the transglutaminase substrate domain were explored using a recombinant protein representative of the N-terminal domain of Hwp1 and were similar to other transglutaminase substrates, the small proline-rich proteins of cornified envelopes found in stratified squamous epithelia. Recombinant Hwp1 lacks alpha and beta structures by circular dichroism and likely exists as a disulfide-cross-linked coiled-coil. The transglutaminase substrate property prompted a unique approach for investigating the features of surface Hwp1 on germ tubes. A lysine analog, 5-(biotinamido)pentylamine, was cross-linked to germ tubes catalyzed by transglutaminase 2 prior to cell fractionation, immunoprecipitation, and detection with streptavidin conjugates. The majority of the transglutaminase-modifiable Hwp1 was covalently attached to the beta-glucan of hyphae by the C terminus of Hwp1 via a glycosylphosphatidylinositol remnant anchor. A putative precursor of cell wall forms of Hwp1 was identified in the cell extract and in the culture medium. Hwp1 was modified by relatively short N-linked glycans, and the molecular size of the protein was reduced by hypomannosylation when expressed in O-glycosylation mutant strains. Hwp1 combines features of mammalian transglutaminase substrate proteins with characteristics of fungal cell wall proteins to form an unconventional adhesin at the hyphal wall of C. albicans.     

GPI-anchor synthesis is indispensable for the germline development of the nematode Caenorhabditis elegans

Glycosylphosphatidylinositol (GPI)-anchor attachment is one of the most common posttranslational protein modifications. Using the nematode Caenorhabditis elegans, we determined that GPI-anchored proteins are present in germline cells and distal tip cells, which are essential for the maintenance of the germline stem cell niche. We identified 24 C. elegans genes involved in GPI-anchor synthesis. Inhibition of various steps of GPI-anchor synthesis by RNA interference or gene knockout resulted in abnormal development of oocytes and early embryos, and both lethal and sterile phenotypes were observed. The piga-1 gene (orthologue of human PIGA) codes for the catalytic subunit of the phosphatidylinositol N-acetylglucosaminyltransferase complex, which catalyzes the first step of GPI-anchor synthesis. We isolated piga-1-knockout worms and found that GPI-anchor synthesis is indispensable for the maintenance of mitotic germline cell number. The knockout worms displayed 100% lethality, with decreased mitotic germline cells and abnormal eggshell formation. Using cell-specific rescue of the null allele, we showed that expression of piga-1 in somatic gonads and/or in germline is sufficient for normal embryonic development and the maintenance of the germline mitotic cells. These results clearly demonstrate that GPI-anchor synthesis is indispensable for germline formation and for normal development of oocytes and eggs.     

Spatially resolving the secretome within the mycelium of the cell factory Aspergillus niger

Aspergillus niger is an important cell factory for the industrial production of enzymes. These enzymes are released into the culture medium, from which they can be easily isolated. Here, we determined with stable isotope dimethyl labeling the secretome of five concentric zones of 7-day-old xylose-grown colonies of A. niger that had either or not been treated with cycloheximide. As expected, cycloheximide blocked secretion of proteins at the periphery of the colony. Unexpectedly, protein release was increased by cycloheximide in the intermediate and central zones of the mycelium when compared to nontreated colonies. Electron microscopy indicated that this is due to partial degradation of the cell wall. In total, 124 proteins were identified in cycloheximide-treated colonies, of which 19 secreted proteins had not been identified before. Within the pool of 124 proteins, 53 secreted proteins were absent in nontreated colonies, and additionally, 35 proteins were released ≥4-fold in the central and subperipheral zones of cycloheximide-treated colonies when compared to nontreated colonies. The composition of the secretome in each of the five concentric zones differed. This study thus describes spatial release of proteins in A. niger, which is instrumental in understanding how fungi degrade complex substrates in nature.     

Functional analysis of Candida albicans GPI-anchored proteins: roles in cell wall integrity and caspofungin sensitivity

The outer layer of the Candida albicans cell wall is enriched in highly glycosylated proteins. The major class, the GlycosylPhosphatidylInositol (GPI)-anchored proteins are tethered to the wall by GPI-anchor remnants and include adhesins, glycosyltransferases, yapsins and superoxide dismutases. In silico analysis suggested that C. albicans possesses 115 putative GPI anchored proteins (GpiPs), almost twice the number reported for Saccharomyces cerevisiae. A global approach to characterise in silico predicted GpiPs has been initiated by generating a library of 45 mutants. This library was subjected to a screen for cell wall modifications by testing the cell wall integrity (SDS and Calcofluor White sensitivity) and response to caspofungin. We showed that, when caspofungin sensitivity was modified, in more than half of the cases the susceptibility can be correlated to the level of chitin and cell wall thickness: sensitive strains have low level of chitin and a thin cell wall. We also identified, for the first time, genes that when deleted lead to decreased caspofungin sensitivity: DFG5, PHR1, PGA4 and PGA62. The role of two unknown GpiPs, Pga31 and Pga62 in the cell wall structure and composition was clearly demonstrated during this study.     

Biochemical properties of the major proteins from Rhodnius prolixus eggshell

Two proteins from the eggshell of Rhodnius prolixus were isolated, characterized and named Rp30 and Rp45 according to their molecular masses. Purified proteins were used to obtain specific antiserum which was later used for immunolocalization. The antiserum against Rp30 and Rp45 detected their presence inside the follicle cells, their secretion and their association with oocyte microvilli. Both proteins are expressed during the final stage of vitellogenesis, preserved during embryogenesis and discarded together with the eggshell. The amino terminals were sequenced and both proteins were further cloned using degenerated primers. The amino acid sequences appear to have a tripartite arrangement with a highly conserved central domain which presents a repetitive motif of valine-proline-valine (VPV) at intervals of 15 amino acid residues. Their amino acid sequence showed no similarity to any known eggshell protein. The expression of these proteins was also investigated; the results demonstrated that this occurred strictly in choriogenic follicles. Antifungal activity against Aspergillus niger was found to be associated with Rp45 but not with Rp30. A. niger exposed to Rp45 protein induced growth inhibition and several morphological changes such as large vacuoles, swollen mitochondria, multi-lamellar structures and a disorganized cell wall as demonstrated by electron microscopy analysis.     

Prion rods contain an inert polysaccharide scaffold

A polysaccharide consisting of mainly 1,4-linked glucose units was found associated with prion rods, which are composed mainly of insoluble aggregates of the N-terminally truncated prion protein (PrP 27-30) exhibiting the ultrastructural and tinctorial properties of amyloid. The polysaccharide differs in composition from the Asn-linked oligosaccharides and the GPI-anchor of the prion protein. Prion rods were prepared from scrapie-infected hamster brains using two different purification protocols. Prolonged digestion of rods with proteinase K reduced PrP by a factor of at least 500, leaving about 10% (w/w) of the sample as an insoluble remnant. Only glucose was obtained by acid hydrolysis of the remnant and methylation analysis showed 80% 1,4-, 15% 1,6- and 5% 1,4,6-linked glucose units. The physical and chemical properties as well as the absence of terminal glucose units indicate a very high molecular mass of the polysaccharide. No evidence was found for covalent bonds between PrP and the polysaccharide. The polysaccharide certainly contributes to the unusual chemical and physical stability of prion rods, acting like a scaffold. A potential structural and/or functional relevance of the polysaccharide scaffold is discussed.     

Mapping N-linked glycosylation sites in the secretome and whole cells of Aspergillus niger using hydrazide chemistry and mass spectrometry

Protein glycosylation (e.g., N-linked glycosylation) is known to play an essential role in both cellular functions and secretory pathways; however, our knowledge of in vivo N-glycosylated sites is very limited for the majority of fungal organisms including Aspergillus niger. Herein, we present the first extensive mapping of N-glycosylated sites in A. niger by applying an optimized solid phase glycopeptide enrichment protocol using hydrazide-modified magnetic beads. The enrichment protocol was initially optimized using both mouse blood plasma and A. niger secretome samples, and it was demonstrated that the protein-level enrichment protocol offered superior performance over the peptide-level protocol. The optimized protocol was then applied to profile N-glycosylated sites from both the secretome and whole cell lysates of A. niger. A total of 847 N-glycosylated sites from 330 N-glycoproteins (156 proteins from the secretome and 279 proteins from whole cells) were confidently identified by LC-MS/MS. The identified N-glycoproteins in the whole cell lysate were primarily localized in the plasma membrane, endoplasmic reticulum, Golgi apparatus, lysosome, and storage vacuoles, supporting the important role of N-glycosylation in the secretory pathways. In addition, these glycoproteins are involved in many biological processes including gene regulation, signal transduction, protein folding and assembly, protein modification, and carbohydrate metabolism. The extensive coverage of N-glycosylated sites and the observation of partial glycan occupancy on specific sites in a number of enzymes provide important initial information for functional studies of N-linked glycosylation and their biotechnological applications in A. niger.     

The glycosyl phosphatidylinositol anchor is critical for Ly-6A/E-mediated T cell activation.

Ly-6E, a glycosyl phosphatidylinositol (GPI)-anchored murine alloantigen that can activate T cells upon antibody cross-linking, has been converted into an integral membrane protein by gene fusion. This fusion product, designated Ly-6EDb, was characterized in transiently transfected COS cells and demonstrated to be an integral cell surface membrane protein. Furthermore, the fusion antigen can be expressed on the surface of the BW5147 class "E" mutant cell line, which only expresses integral membrane proteins but not GPI-anchored proteins. The capability of this fusion antigen to activate T cells was examined by gene transfer studies in D10G4.1, a type 2 T cell helper clones. When transfected into D10 cells, the GPI-anchored Ly-6E antigen, as well as the endogenous GPI-anchored Ly-6A antigen, can initiate T cell activation upon antibody cross-linking. In contrast, the transmembrane anchored Ly-6EDb antigen was unable to mediate T cell activation. Our results demonstrate that the GPI-anchor is critical to Ly-6A/E-mediated T cell activation.     

Processing and localisation of a GPI-anchored Plasmodium falciparum surface protein expressed by the baculovirus system

We describe the expression, in insect cells using the baculovirus system, of two protein fragments derived from the C-terminus of merozoite surface protein 1(MSP-1) of the human malaria parasite Plasmodium falciparum, and their glycosylation and intracellular location. The transport and intracellular localisation of the intact C-terminal MSP-1 fragment, modified by addition of a signal sequence for secretion, was compared with that of a similar control protein in which translation of the GPI-cleavage/attachment site was abolished by insertion of a stop codon into the DNA sequence. Both proteins could only be detected intracellularly, most likely in the endoplasmic reticulum. This lack of transport to the cell surface or beyond, was confirmed for both proteins by immunofluorescence with a specific antibody and characterisation of their N-glycans. The N-glycans had not been processed by enzymes localised in post-endoplasmic reticulum compartments. In contrast to MSP-1, the surface antigen SAG-1 of Toxoplasma gondii was efficiently transported out of the endoplasmic reticulum of insect cells and was located, at least in part, on the cell surface. No GPI-anchor could be detected for either of the MSP-1 constructs or SAG-1, showing that the difference in transport is a property of the individual proteins and cannot be attributed to the lack of a GPI-anchor. The different intracellular location and post-translational modification of recombinant proteins expressed in insect cells, as compared to the native proteins expressed in parasites, and the possible implications for vaccine development are discussed.     

Identification of three novel mutations in the PIG-A gene in paroxysmal nocturnal haemoglobinuria (PNH) patients

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired haemolytic disorder caused by the absence of glycosyl phosphatidylinositol (GPI)-anchored surface proteins resulting from a defect in one step of GPI-anchor biosynthesis. Recent analysis has shown that mutations at the PIG-A (phosphatidylinositoglycan-class A) gene are responsible for GPI-anchor deficiency in all PNH patients. In the current study, we describe three new mutations of the PIG-A gene in Italian patients with PNH. The analysis has been performed by RNA/single-strand conformation polymorphism using genomic DNA purified from nucleated peripheral blood cells. An abnormal pattern of migration of polymerase chain reaction amplified fragments containing exons 2 and 5 was observed. Sequencing analysis led to the identification of three mutations: a transversion C-to-A creating a stop codon (Y98X), an A insertion at position 460 (460insA), and a C deletion (1114delC). All the mutations cause a premature termination of the translation of the PIG-A protein.