In silico identification of GPI-anchored proteins in Paracoccidioides

ABSTRACTGlycosylphosphatidylinositol-anchored proteins (GPI-proteins) are widely found in eukaryotic organisms. In fungi, GPI-proteins are thought to be involved in diverse cellular mechanisms such as cell wall biosynthesis and cell wall remodeling, adhesion, antigenicity, and virulence. The conserved structural domains of GPI-protein allow the utilization ofin silicoprediction approach to identify this class of proteins using a genome-wide analysis. We used different previously characterized algorithms to search for genes that encode predicted GPI-proteins in the genome ofP. brasiliensis and P. lutzii, thermal dimorphic fungi that causes paracoccidioidomycosis (PCM). By using these methods, 98 GPI-proteins were found inP. brasiliensiswith orthologs inP. lutzii. A series of 28 GPI-proteins were classified in functional categories (such as glycoside hydrolases, chitin-processing proteins, and proteins involved in the biogenesis of the cell wall). Furthermore, 70 GPI-proteins exhibited homology with hypothetical conserved proteins of unknown function. These data will be an important resource for the future analysis of GPI-proteins inParacoccidioides spp.

Cdc1 removes the ethanolamine phosphate of the first mannose of GPI anchors and thereby facilitates the integration of GPI proteins into the yeast cell wall

Temperature-sensitive cdc1tsmutants are reported to stop the cell cycle upon a shift to 30°C in early G2, that is, as small budded cells having completed DNA replication but unable to duplicate the spindle pole body. A recent report showed that PGAP5, a human homologue of CDC1, acts as a phosphodiesterase removing an ethanolamine phosphate (EtN-P) from mannose 2 of the glycosylphosphatidylinositol (GPI) anchor, thus permitting efficient endoplasmic reticulum (ER)-to-Golgi transport of GPI proteins. We find that the essential CDC1 gene can be deleted in mcd4∆ cells, which do not attach EtN-P to mannose 1 of the GPI anchor, suggesting that Cdc1 removes the EtN-P added by Mcd4. Cdc1-314tsmutants do not accumulate GPI proteins in the ER but have a partial secretion block later in the secretory pathway. Growth tests and the genetic interaction profile of cdc1-314tspinpoint a distinct cell wall defect. Osmotic support restores GPI protein secretion and actin polarization but not growth. Cell walls of cdc1-314tsmutants contain large amounts of GPI proteins that are easily released by β-glucanases and not attached to cell wall β1,6-glucans and that retain their original GPI anchor lipid. This suggests that the presumed transglycosidases Dfg5 and Dcw1 of cdc1-314tstransfer GPI proteins to cell wall β1,6-glucans inefficiently.

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

ABSTRACTAlthough 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 thecis-requirements directing localization of GPI proteins to the plasma membrane or cell wall are now understood. However, it remains to be determined howAspergillus 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 fromA. fumigatusMp1p, 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 ofAfMp1 was not required for cell wall distribution. Based on our results, thecis-requirements directing localization of GPI proteins inA. fumigatusare different from those in yeast.

Identification and Clinical Significance of Type II Granulocytes Among Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) Identified Using Multiparameter High-Sensitivity Flow Cytometry.

Abstract 3015 Poster Board II-991 PNH is a hematopoietic stem cell disorder in which unregulated activation of terminal complement leads to impaired quality of life and significant ischemic morbidities with shortened lifespan. Life-threatening thromboembolism (TE) is the most feared complication of PNH, accounting for 45% of patient deaths. Thrombosis has been observed in PNH patients regardless of the level of hemolysis. Additionally, platelet activation with subsequent consumption and thrombocytopenia are observed more often in PNH patients at risk for thrombosis. Current laboratory PNH diagnostic methods rely on flow cytometry to characterize PNH clones. PNH granulocytes (Gran) are typically detected using antibodies to a variety of GPI-linked markers including CD55, CD59, CD16, CD24, and CD66b. Recently, FLAER, a fluorescent proaerolysin variant that binds directly to the GPI anchor, has been used to identify and quantify GPI-deficient WBCs at a very high level of sensitivity. Although these markers are well established to detect granulocytes with normal expression of GPI proteins (Type I cells) and complete loss of GPI proteins (Type III cells), less is known about their ability to detect granulocytes with partial loss of GPI proteins (Type II cells). The ability to detect both PNH Type II RBCs and WBCs would provide clinically important information since quantitation of only PNH RBC clones can be confounded by transfusion or hemolysis. We evaluated 2,921 consecutive patient peripheral blood samples submitted for PNH diagnostic testing with a high-sensitivity flow cytometry assay for granulocytes that includes the fluorescent proaerolysin variant (FLAER) with confirmatory lineage-specific antibodies to GPI-linked antigens to distinguish Type I, II and III Gran clones. In addition, standard CD235/CD59 analysis was performed on the RBCs and evaluation with FLAER, CD14 and lineage-specific antibodies was performed on the monocytes. 216 patient samples (7.4%) had a detectable PNH gran clone (≥ 0.01% PNH Type III granulocytes and an absolute count of at least 50 cells). Clinical information was available for 162 of these patients (Table I). Of these samples, nineteen (8.8 %) patients demonstrated a distinct Type II Gran population, ranging in size from 1.2 – 65% (median clone size = 7%). In 4/19 patients, this Type II Gran population represented >50% of the total (Type II + Type III) PNH cells. In 10/19 patients (53%), a type II monocyte population was identified. Evaluation of the granulocyte markers (Table II) showed that the type II gran population was detectable in all cases by FLAER and in decreasing percentage by CD66b (88%), CD55 (50%), CD24 (47%) and CD16 (0%). Patients with Type II Gran clones had a significantly larger median total Gran PNH clone size than those without Type II Gran clones (87% vs. 11%; p= 0.0003), as well as larger median Type II and Type III RBC clones, likely a reflection of the ability to detect type II gran PNH clones with overall larger PNH clone sizes. Patients with Type II Gran clones showed significantly lower median platelet (plt) counts (54 ×109/L) than patients without Type II Gran clones (116×109/L; p< 0.01). Patients with Type II Gran clones had similar peripheral WBC, peripheral RBC, absolute neutrophil count, and hemoglobin (Hgb) compared to patients without Type II Gran clones, suggesting that differences in platelet counts are likely not due to differences in underlying marrow blood cell production. Type II PNH cells are an important component of the PNH diagnostic evaluation and both RBC and Gran Type II clones should be enumerated. In a large population of patients tested for the presence of PNH clones using a high sensitivity flow cytometry assay, a significant proportion of patients were identified with Type II PNH Gran clones. This study identified FLAER as the best reagent to identify type II Gran PNH clones and showed CD16 was least useful. This study also identified a clinical association between the presence of significant Type II clones and thrombocytopenia, potentially indicative of terminal complement-mediated platelet consumption. These findings are consistent with an increased risk of thrombosis in patients with significant Type II PNH clones. Disclosures: No relevant conflicts of interest to declare.

Incorporation of Ceramides into Saccharomyces cerevisiae Glycosylphosphatidylinositol-Anchored Proteins Can Be Monitored In Vitro

ABSTRACT After glycosylphosphatidylinositols (GPIs) are added to GPI proteins of Saccharomyces cerevisiae, a fatty acid of the diacylglycerol moiety is exchanged for a C26:0 fatty acid through the subsequent actions of Per1 and Gup1. In most GPI anchors this modified diacylglycerol-based anchor is subsequently transformed into a ceramide-containing anchor, a reaction which requires Cwh43. Here we show that the last step of this GPI anchor lipid remodeling can be monitored in microsomes. The assay uses microsomes from cells that have been grown in the presence of myriocin, a compound that blocks the biosynthesis of dihydrosphingosine (DHS) and thus inhibits the biosynthesis of ceramide-based anchors. Such microsomes, when incubated with [3H]DHS, generate radiolabeled, ceramide-containing anchor lipids of the same structure as made by intact cells. Microsomes from cwh43Δ or mcd4Δ mutants, which are unable to make ceramide-based anchors in vivo, do not incorporate [3H]DHS into anchors in vitro. Moreover, gup1Δ microsomes incorporate [3H]DHS into the same abnormal anchor lipids as gup1Δ cells synthesize in vivo. Thus, the in vitro assay of ceramide incorporation into GPI anchors faithfully reproduces the events that occur in mutant cells. Incorporation of [3H]DHS into GPI proteins is observed with microsomes alone, but the reaction is stimulated by cytosol or bovine serum albumin, ATP plus coenzyme A (CoA), or C26:0-CoA, particularly if microsomes are depleted of acyl-CoA. Thus, [3H]DHS cannot be incorporated into proteins in the absence of acyl-CoA.