Green fluorescent protein expression in the symbiotic basidiomycete fungus Hebeloma cylindrosporum

The symbiotic basidiomycete Hebeloma cylindrosporum is a model fungal species used to study ectomycorrhizal symbiosis at the molecular level. In order to have a vital marker, we developed a green fluorescent protein (GFP) reporter system efficiently expressed in H. cylindrosporum using the sgfp coding region bordered by two introns fused to the saprophytic basidiomycete Coprinopsis cinerea cgl1 promoter. Expression of this reporter system was tested under different environmental conditions in two transformants, and glucose was shown to repress gfp expression. Such a reporter system will be used in plant-fungus interaction to evaluate sugar supply by the plant to the compatible mycorrhizal symbiont and to compare the expression of various genes of interest in the free-living mycelia, in the symbiotic (mycorrhizas) and the reproductive (fruit bodies) structures formed by H. cylindrosporu

Molecular characterization of caveolin-induced membrane curvature

The generation of caveolae involves insertion of the cholesterol-binding integral membrane protein caveolin-1 (Cav1) into the membrane, however, the precise molecular mechanisms are as yet unknown. We have speculated that insertion of the caveolin scaffolding domain (CSD), a conserved amphipathic region implicated in interactions with signaling proteins, is crucial for caveola formation. We now define the core membrane-juxtaposed region of Cav1 and show that the oligomerization domain and CSD are protected by tight association with the membrane in both mature mammalian caveolae and a model prokaryotic system for caveola biogenesis. Cryoelectron tomography reveals the core membrane-juxtaposed domain to be sufficient to maintain oligomerization as defined by polyhedral distortion of the caveolar membrane. Through mutagenesis we demonstrate the importance of the membrane association of the oligomerization domain/CSD for defined caveola biogenesis and furthermore, highlight the functional significance of the intramembrane domain and the CSD for defined caveolin-induced membrane deformation. Finally, we define the core structural domain of Cav1, constituting only 66 amino acids and of great potential to nanoengineering applications, which is required for caveolin-induced vesicle formation in a bacterial system. These results have significant implications for understanding the role of Cav1 in caveola formation and in regulating cellular signaling events

MURC/Cavin-4 and cavin family members form tissue-specific caveolar complexes

Polymerase I and transcript release factor (PTRF)/Cavin is a cytoplasmic protein whose expression is obligatory for caveola formation. Using biochemistry and fluorescence resonance energy transfer–based approaches, we now show that a family of related proteins, PTRF/Cavin-1, serum deprivation response (SDR)/Cavin-2, SDR-related gene product that binds to C kinase (SRBC)/Cavin-3, and muscle-restricted coiled-coil protein (MURC)/Cavin-4, forms a multiprotein complex that associates with caveolae. This complex can constitutively assemble in the cytosol and associate with caveolin at plasma membrane caveolae. Cavin-1, but not other cavins, can induce caveola formation in a heterologous system and is required for the recruitment of the cavin complex to caveolae. The tissue-restricted expression of cavins suggests that caveolae may perform tissue-specific functions regulated by the composition of the cavin complex. Cavin-4 is expressed predominantly in muscle, and its distribution is perturbed in human muscle disease associated with Caveolin-3 dysfunction, identifying Cavin-4 as a novel muscle disease candidate caveolar protein

Missense mutations in PIEZO1, which encodes the Piezo1 mechanosensor protein, define Er red blood cell antigens

Despite the identification of the high-incidence red cell antigen Era nearly 40 years ago, the molecular background of this antigen, together with the other 2 members of the Er blood group collection, has yet to be elucidated. Whole exome and Sanger sequencing of individuals with serologically defined Er alloantibodies identified several missense mutations within the PIEZO1 gene, encoding amino acid substitutions within the extracellular domain of the Piezo1 mechanosensor ion channel. Confirmation of Piezo1 as the carrier molecule for the Er blood group antigens was demonstrated using immunoprecipitation, CRISPR/Cas9-mediated gene knockout, and expression studies in an erythroblast cell line. We report the molecular bases of 5 Er blood group antigens: the recognized Era, Erb, and Er3 antigens and 2 novel high-incidence Er antigens, described here as Er4 and Er5, establishing a new blood group system. Anti-Er4 and anti-Er5 are implicated in severe hemolytic disease of the fetus and newborn. Demonstration of Piezo1, present at just a few hundred copies on the surface of the red blood cell, as the site of a new blood group system highlights the potential antigenicity of even low-abundance membrane proteins and contributes to our understanding of the in vivo characteristics of this important and widely studied protein in transfusion biology and beyond

Novel method for the measurement of erythrocyte acetylcholinesterase activity in the Yt blood group system using amperometry in conjunction with screen-printed carbon electrodes

Background: The five antigens of the Yt blood group system are located on the membrane GPI‐linked erythrocyte acetylcholinesterase (AChE‐E), which is implicated in a range of clinical issues, including sleep apnoea and neurotoxin poisoning. Conformational changes associated with different Yt antigens, as well as the storage of red cells, may affect AChE‐E activity. Direct analysis of AChE‐E presents unique challenges that may be overcome by electrochemical measurement using low‐cost electron‐mediated screen‐printed carbon electrodes (SPCEs) under suitable amperometric conditions.Aims: We present the electrochemical analysis of AChE‐E activity of five different Yt phenotypes, the effect on AChE‐E of red cell refrigeration over a four‐week timeframe and storage by freezing. The efficacy of electron‐mediated SPCEs is examined in relation to the potential interferences and electrode‐fouling.Methods: SPCEs containing the electron mediator (cobalt phthalocyanine) were manufactured on a plastic substrate, each in association with a screen‐printed Ag/AgCl counter/reference electrode in a two‐electrode format. Whole blood samples were initially washed to remove butyrylcholinesterase and resuspended in PBS. AChE‐E activity was assessed at 37⁰C using chronoamperometry at + 0 mV following the addition of acetylthiocholine chloride to the cell suspension. Ten Yt(a+b‐) and four Yt(a‐b+) donor samples were tested after collection and at two and four weeks storage at 4⁰C. Five Yt(a+b‐) and twenty Yt(a‐b+) were tested following freezing in liquid nitrogen. Samples with confirmed rare Yt phenotypes were evaluated to determine if the phenotype effected AChE‐E activity. The assay longevity and electrode viability were examined using eight of the samples representing the different Yt phenotypes over an extended duration of measurement.Results: SPCEs gave a consistent response in all samples tested without evidence of interference or fouling by the adherence of the cells to the electrode surface. The sensors were shown to operate continuously for a minimum of 60 minutes until the substrate became limiting. The reproducibility of the sensors (six replicates each of five samples) was 14% (RSD), which is typical for SPCEs in biological samples. An optimal assay duration of 120 seconds was selected.Compared to fresh samples, storage by freezing resulted in a loss of approximately 40% AChE‐E activity in Yt(a+b‐), whereas Yt(a‐b+) samples showed a 40% increase in enzyme activity. These differences occurred despite the almost identical conformation of the Yta and Ytb AChE‐E structure as studied by molecular dynamic calculations. Samples stored at 4⁰C showed a significant deterioration in AChE‐E activity within the first 14 days; a reduction of 35% for Yt(a+b‐) and 28% for Yt(a‐b+) (P < 0.01), followed by no significant change between 14 and 28 days of storage.The effect of rare Yt phenotypes was examined using stored frozen samples. AChE‐E activity differed markedly when compared to Yt(a+b‐); 82% higher in Yt(a‐b+) samples, 39% in YTLI– and 210% in YTOT–. An exception was YTEG–, which remained comparable to Yt(a+b‐).Summary/Conclusions: Mediated SPCEs were used effectively in the electrochemical measurement of AChE‐E activity, showing their potential for rapid low‐cost analysis of red cells. We showed that the activity of the enzyme was reduced with refrigeration, although the consequences of freezing were more varied in terms of Yt phenotype

Green fluorescent protein expression in the symbiotic basidiomycete fungus Hebeloma cylindrosporum

ISSN:0378-1097ISSN:1574-6968ISSN:0168-6496ISSN:0920-8534ISSN:0168-644

Structural basis for chitotetraose coordination by CGL3, a novel galectin-related protein from Coprinopsis cinerea

Recent advances in genome sequencing efforts have revealed an abundance of novel putative lectins. Among these, many galectin-related proteins, characterized by many conserved residues but intriguingly lacking critical amino acids, have been found in all corners of the eukaryotic superkingdom. Here we present a structural and biochemical analysis of one representative, the galectin-related lectin CGL3 found in the inky cap mushroom Coprinopsis cinerea. This protein contains all but one conserved residues known to be involved in β-galactoside binding in galectins. A Trp residue strictly conserved among galectins is changed to an Arg in CGL3 (R81). Accordingly, the galectin-related protein is not able to bind lactose. Screening of a glycan array revealed that CGL3 displays preference for oligomers of β1-4-linked N-acetyl-glucosamines (chitooligosaccharides) and GalNAcβ1-4GlcNAc (LacdiNAc). Carbohydrate-binding affinity of this novel lectin was quantified using isothermal titration calorimetry, and its mode of chitooligosaccharide coordination not involving any aromatic amino acid residues was studied by X-ray crystallography. Structural information was used to alter the carbohydrate-binding specificity and substrate affinity of CGL3. The importance of residue R81 in determining the carbohydrate-binding specificity was demonstrated by replacing this Arg with a Trp residue (R81W). This single-amino-acid change led to a lectin that failed to bind chitooligosaccharides but gained lactose binding. Our results demonstrate that, similar to the legume lectin fold, the galectin fold represents a conserved structural framework upon which dramatically altered specificities can be grafted by few alterations in the binding site and that, in consequence, many metazoan galectin-related proteins may represent lectins with novel carbohydrate-binding specificities