scholarly journals Flagellar adhesion in chlamydomonas induces synthesis of two high molecular weight cell surface proteins

1983 ◽  
Vol 96 (3) ◽  
pp. 589-597 ◽  
Author(s):  
WJ Snell ◽  
A Clausell ◽  
WS Moore
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Adhesion Molecules ◽  
Mating Type ◽  
Surface Proteins ◽  
Cell Wall Proteins ◽  
Intact Cells ◽  
Mating Reaction ◽  
Ionic Detergent ◽  
Flagellar Proteins

Because our previous studies (Snell, W.J., and W.S. Moore, 1980, J. Cell Biol. 84:203- 210) on the mating reaction of chlamydomonas reinhardtii showed that there was an adhesion-induced turnover of proteins whose synthesis is induced during aggregation. Analysis by SDS PAGE and autoradiography showed that proteins of 220,000 M(r) and 165, 000 M(r) (designated A(1) and A(2) respectively) consistently showed a high rate of synthesis only in flagella or flagellar membrane-enriched fractions prepared from aggregating gametes. Since the two proteins were soluble in the non-ionic detergent NP-40 and were removed from intact cells by a brief pronase treatment, it is likely that A(1) and A(2) are membrane proteins expose on the cell surface. A(1) and A(2) were each synthesized by gametes of both mating types (mt(-) and mt(+)) and synthesis of these two proteins could be detected in the normal mating reaction (wild type mt(-) and mt(+)), in mixtures of mt(-) and impotent mt(+) gametes (which could aggregate but not fuse), and in mixtures of gametes of a single mating type with isolated flagella of the opposite mating type. Cells aggregating in tunicamycin, an inhibitor of protein glycosylation, lost their adhesiveness during aggregation and did not synthesize the 220,000 M(r) protein but instead produced a protein (possibly an underglycosylated form of A(1)) of slightly lower mol wt. The 220,000 and 165,000 M(R) proteins appeared to be flagellar proteins and not cell wall proteins because A(1) and A(2) did not co-migrate with previously identified cell wall proteins, and synthesis of the two proteins could not be detected in flagella-less (bald-2) mutant cells. Analysis of the adhesive activity of sucrose gradient fraction of detergent (octyl glucoside)-solubilized flagellar membranes revealed that fractions containing A(1) and A(2) did not have detectable adhesive activity. The possibility remains that A(1) and A(2) are adhesion molecules whose activity could not be measured in the assay we used. Alternatively, the 220,000 and 165,000 M(r) proteins may be inactivated adhesion molecules or else they may be flagellar surface proteins involved only indirectly in the adhesion process.

scholarly journals Clade-specific chromosomal rearrangements and loss of subtelomeric adhesins in Candida auris

Genetics ◽  
2021 ◽  
Author(s):  
José F Muñoz ◽  
Rory M Welsh ◽  
Terrance Shea ◽  
Dhwani Batra ◽  
Lalitha Gade ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Genome Instability ◽  
Chromosomal Rearrangements ◽  
Multidrug Resistant ◽  
Antifungal Drugs ◽  
Surface Proteins ◽  
Cell Wall Proteins ◽  
Candida Auris ◽  
Cell Surface Proteins

Abstract Candida auris is an emerging fungal pathogen of rising concern due to global spread, the ability to cause healthcare-associated outbreaks, and antifungal resistance. Genomic analyses revealed that early contemporaneously detected cases of C. auris were geographically stratified into four major clades. While Clades I, III, and IV are responsible for ongoing outbreaks of invasive and multidrug-resistant infections, Clade II, also termed the East Asian clade, consists primarily of cases of ear infection, is often susceptible to all antifungal drugs, and has not been associated with outbreaks. Here, we generate chromosome-level assemblies of twelve isolates representing the phylogenetic breadth of these four clades and the only isolate described to date from Clade V. This Clade V genome is highly syntenic with those of Clades I, III, and IV, although the sequence is highly divergent from the other clades. Clade II genomes appear highly rearranged, with translocations occurring near GC-poor regions, and large subtelomeric deletions in most chromosomes, resulting in a substantially different karyotype. Rearrangements and deletion lengths vary across Clade II isolates, including two from a single patient, supporting ongoing genome instability. Deleted subtelomeric regions are enriched in Hyr/Iff-like cell-surface proteins, novel candidate cell wall proteins, and an ALS-like adhesin. Cell wall proteins from these families and other drug-related genes show clade-specific signatures of selection in Clades I, III, and IV. Subtelomeric dynamics and the conservation of cell surface proteins in the clades responsible for global outbreaks causing invasive infections suggest an explanation for the different phenotypes observed between clades.

scholarly journals Coding Tandem Repeats Generate Diversity in Aspergillus fumigatus Genes

2007 ◽  
Vol 6 (8) ◽  
pp. 1380-1391 ◽  
Author(s):  
Emma Levdansky ◽  
Jacob Romano ◽  
Yona Shadkchan ◽  
Haim Sharon ◽  
Kevin J. Verstrepen ◽  
...  
Keyword(s):  
Cell Wall ◽  
Amino Acid ◽  
Cell Surface ◽  
Aspergillus Fumigatus ◽  
Tandem Repeats ◽  
Size Variation ◽  
Surface Proteins ◽  
Host Immune System ◽  
Cell Wall Proteins ◽  
Cell Surface Antigens

ABSTRACT Genes containing multiple coding mini- and microsatellite repeats are highly dynamic components of genomes. Frequent recombination events within these tandem repeats lead to changes in repeat numbers, which in turn alters the amino acid sequence of the corresponding protein. In bacteria and yeasts, the expansion of such coding repeats in cell wall proteins is associated with alterations in immunogenicity, adhesion, and pathogenesis. We hypothesized that identification of repeat-containing putative cell wall proteins in the human pathogen Aspergillus fumigatus may reveal novel pathogenesis-related elements. Here, we report that the genome of A. fumigatus contains as many as 292 genes with internal repeats. Fourteen of 30 selected genes showed size variation of their repeat-containing regions among 11 clinical A. fumigatus isolates. Four of these genes, Afu3g08990, Afu2g05150 (MP-2), Afu4g09600, and Afu6g14090, encode putative cell wall proteins containing a leader sequence and a glycosylphosphatidylinositol anchor motif. All four genes are expressed and produce variable-size mRNA encoding a discrete number of repeat amino acid units. Their expression was altered during development and in response to cell wall-disrupting agents. Deletion of one of these genes, Afu3g08990, resulted in a phenotype characterized by rapid conidial germination and reduced adherence to extracellular matrix suggestive of an alteration in cell wall characteristics. The Afu3g08990 protein was localized to the cell walls of dormant and germinating conidia. Our findings suggest that a subset of the A. fumigatus cell surface proteins may be hypervariable due to recombination events in their internal tandem repeats. This variation may provide the functional diversity in cell surface antigens which allows rapid adaptation to the environment and/or elusion of the host immune system.

The E-selectin-ligand ESL-1 is located in the Golgi as well as on microvilli on the cell surface

1997 ◽  
Vol 110 (6) ◽  
pp. 687-694 ◽  
Author(s):  
M. Steegmaier ◽  
E. Borges ◽  
J. Berger ◽  
H. Schwarz ◽  
D. Vestweber
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Surface Proteins ◽  
Intact Cells ◽  
Fgf Receptor ◽  
Cell Surface Biotinylation ◽  
Cell Surface Staining ◽  
Surface Staining ◽  
Selectin Ligand ◽  
Golgi Protein

Neutrophils and subsets of lymphocytes bind to E-selectin, a cytokine inducible adhesion molecule on endothelial cells. The E-selectin-ligand-1 (ESL-1) is a high affinity glycoprotein ligand which participates in the binding of mouse myeloid cells to E-selectin. The sequence of mouse ESL-1 is highly homologous to the cysteine rich FGF receptor (CFR) in chicken and the rat Golgi protein MG160. We have analysed the subcellular distribution of ESL-1 by indirect immunofluorescence, flow cytometry, various biochemical techniques and by immunogold scanning electron microscopy. We could localize ESL-1 in the Golgi as well as on the cell surface of 32Dc13 cells and neutrophils. Cell surface staining was confirmed by cell surface biotinylation and by cell surface immunoprecipitations in which antibodies only had access to surface proteins on intact cells. In addition, ESL-1(high) and ESL-1(low) expressing cells, sorted by flow cytometry, gave rise to high and low immunoprecipitation signals for ESL-1, respectively. Based on immunogold labeling of intact cells, we localized ESL-1 on microvilli of 32Dc13 cells and of the lymphoma cell line K46. Quantitative evaluation determined 80% of the total labeling for ESL-1 on microvilli of K46 cells while 69% of the labeling for the control antigen B220 was found on the planar cell surface. These data indicate that ESL-1 occurs at sites on the leukocyte cell surface which are destined for the initiation of cell contacts to the endothelium.

scholarly journals Posttranslational Modifications Required for Cell Surface Localization and Function of the Fungal Adhesin Aga1p

2003 ◽  
Vol 2 (5) ◽  
pp. 1099-1114 ◽  
Author(s):  
Guohong Huang ◽  
Mingliang Zhang ◽  
Scott E. Erdman
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Posttranslational Modifications ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
Cell Types ◽  
Fungal Species ◽  
Surface Proteins ◽  
Functional Requirements

ABSTRACT Adherence of fungal cells to host substrates and each other affects their access to nutrients, sexual conjugation, and survival in hosts. Adhesins are cell surface proteins that mediate these different cell adhesion interactions. In this study, we examine the in vivo functional requirements for specific posttranslational modifications to these proteins, including glycophosphatidylinositol (GPI) anchor addition and O-linked glycosylation. The processing of some fungal GPI anchors, creating links to cell wall β-1,6 glucans, is postulated to facilitate postsecretory traffic of proteins to cell wall domains conducive to their functions. By studying the yeast sexual adhesin subunit Aga1p, we found that deletion of its signal sequence for GPI addition eliminated its activity, while deletions of different internal domains had various effects on function. Substitution of the Aga1p GPI signal domain with those of other GPI-anchored proteins, a single transmembrane domain, or a cysteine capable of forming a disulfide all produced functional adhesins. A portion of the cellular pool of Aga1p was determined to be cell wall resident. Aga1p and the α-agglutinin Agα1p were shown to be under glycosylated in cells lacking the protein mannosyltransferase genes PMT1 and PMT2, with phenotypes manifested only in MATα cells for single mutants but in both cell types when both genes are absent. We conclude that posttranslational modifications to Aga1p are necessary for its biogenesis and activity. Our studies also suggest that in addition to GPI-glucan linkages, other cell surface anchorage mechanisms, such as transmembrane domains or disulfides, may be employed by fungal species to localize adhesins.

scholarly journals Dynamic Regulation of a GPCR-Tetraspanin-G Protein Complex on Intact Cells: Central Role of CD81 in Facilitating GPR56-Gαq/11Association

2004 ◽  
Vol 15 (5) ◽  
pp. 2375-2387 ◽  
Author(s):  
Kevin D. Little ◽  
Martin E. Hemler ◽  
Christopher S. Stipp
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Cell Activation ◽  
Surface Proteins ◽  
Membrane Microdomains ◽  
Cholesterol Depletion ◽  
Scaffolding Proteins ◽  
Intact Cells ◽  
Heterotrimeric G Protein ◽  
Vast Number

By means of a variety of intracellular scaffolding proteins, a vast number of heterotrimeric G protein–coupled receptors (GPCRs) may achieve specificity in signaling through a much smaller number of heterotrimeric G proteins. Members of the tetraspanin family organize extensive complexes of cell surface proteins and thus have the potential to act as GPCR scaffolds; however, tetraspanin-GPCR complexes had not previously been described. We now show that a GPCR, GPR56/TM7XN1, and heterotrimeric G protein subunits, Gαq, Gα11, and Gβ, associate specifically with tetraspanins and CD81, but not with other tetraspanins. CD9 Complexes of GPR56 with CD9 and CD81 remained intact when fully solubilized and were resistant to cholesterol depletion. Hence they do not depend on detergent-insoluble, raft-like membrane microdomains for stability. A central role for CD81 in promoting or stabilizing a GPR56-CD81-Gαq/11complex was revealed by CD81 immunodepletion and reexpression experiments. Finally, antibody engagement of cell surface CD81 or cell activation with phorbol ester revealed two distinct mechanisms by which GPR56-CD81-Gαq/11complexes can be dynamically regulated. These data reveal a potential role for tetraspanins CD9 and CD81 as GPCR scaffolding proteins.

Efficient cell surface display of Lip2 lipase using C-domains of glycosylphosphatidylinositol-anchored cell wall proteins of Yarrowia lipolytica

2011 ◽  
Vol 91 (3) ◽  
pp. 645-654 ◽  
Author(s):  
Evgeniya Y. Yuzbasheva ◽  
Tigran V. Yuzbashev ◽  
Ivan A. Laptev ◽  
Tatiana K. Konstantinova ◽  
Sergey P. Sineoky
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Yarrowia Lipolytica ◽  
Surface Display ◽  
Cell Surface Display ◽  
Cell Wall Proteins ◽  
Lip2 Lipase

Hydrophobic cell wall protein glycosylation by the pathogenic fungus Candida albicans

1994 ◽  
Vol 40 (4) ◽  
pp. 266-272 ◽  
Author(s):  
Kevin C. Hazen ◽  
Pati M. Glee
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Cell Surface ◽  
Molecular Mass ◽  
Pathogenic Fungus ◽  
Cell Surface Hydrophobicity ◽  
Surface Hydrophobicity ◽  
Protein Glycosylation ◽  
Yeast Cells ◽  
Cell Wall Proteins

Cell surface hydrophobicity influences adhesion and virulence of the opportunistic fungal pathogen Candida albicans. Previous studies have shown that cell surface hydrophobicity is due to specific proteins that are exposed on hydrophobic cells but are masked by long fibrils on hydrophilic cells. This observation suggests that hydrophobic cell wall proteins may contain little or no mannosylation. In the present study, the glycosylation levels of three hydrophobic cell wall proteins (molecular mass range between 36 and 40 kDa) derived from yeast cells were examined. One hydrophilic protein (90 kDa) was also tested. Various endoglycosidases (endoglycosidase F – N-glycosidase F, O-glycosidase, β-mannosidase, N-glycosidase F), an exoglycosidase (α-mannosidase), and trifluoromethane sulfonic acid were used to deglycosylate the proteins. All four proteins were reactive to the lectin concanavalin A, demonstrating that they were mannoproteins. However, gel electrophoresis of the control and treated proteins revealed that mannosyl groups of hydrophobic proteins were less than 2 kDa in size, while the mannosyl group of the hydrophilic protein had a molecular mass of approximately 20 kDa. These results suggest that unlike many hydrophilic proteins, hydrophobic proteins may have low levels of glycosylation. Changes in glycosylation may determine exposure of hydrophobic protein regions at the cell surface.Key words: Candida albicans, cell wall, mannoproteins, hydrophobicity, fibrils.

scholarly journals Roles ofCandida albicansDfg5p and Dcw1p Cell Surface Proteins in Growth and Hypha Formation

2003 ◽  
Vol 2 (4) ◽  
pp. 746-755 ◽  
Author(s):  
Elisabetta Spreghini ◽  
Dana A. Davis ◽  
Ryan Subaran ◽  
Michelle Kim ◽  
Aaron P. Mitchell
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Insertional Mutagenesis ◽  
Surface Proteins ◽  
Alkaline Media ◽  
Specific Gene ◽  
External Signal ◽  
Cell Surface Proteins ◽  
Hypha Formation ◽  
Conditional Expression

TheCandida albicanscell wall participates in both growth and morphological transitions between yeast and hyphae. Our studies here focus on Dfg5p and Dcw1p, two similar proteins with features of glycosylphosphatidylinositol-linked cell surface proteins. Mutants lacking Dfg5p are defective in alkaline pH-induced hypha formation; mutants lacking Dcw1p have no detected hypha formation defect. Both homozygote-triplication tests and conditional expression strategies indicate thatdfg5anddcw1mutations are synthetically lethal. Therefore, Dfg5p and Dcw1p share a function required for growth. Epitope-tagged Dfg5p, created through an insertional mutagenesis strategy, is found in cell membrane and cell wall extract fractions, and endoglycosidase H digestion shows that Dfg5p undergoes N-linked mannosylation. Surprisingly, Dfg5p is required for expression of the hypha-specific geneHWP1in alkaline media. Because Dfg5p is a cell surface protein, it is poised to generate or transmit an external signal required for the program of hypha-specific gene expression.

Do Proline-rich Proteins Modulate a Transglutaminase Catalyzed Mechanism of Candidal Adhesion?

1993 ◽  
Vol 4 (3) ◽  
pp. 293-299 ◽  
Author(s):  
S.D. Bradway ◽  
M.J. Levine
Keyword(s):  
Molecular Weight ◽  
Cell Wall ◽  
High Molecular Weight ◽  
Surface Proteins ◽  
Cross Linking ◽  
Cell Wall Proteins ◽  
Epithelial Surface ◽  
High Molecular Weight Complex ◽  
Sds Page ◽  
Cross Links

Previously, we reported that a membrane-bound epithelial enzyme, transglutaminase (TGase), catalyzes the covalent cross-linking of acidic proline-rich proteins (APRPs) to surface proteins of buccal epithelial cells (BECs). The purpose of this study was twofold: (1) to provide evidence that TGase stabilizes C. albicans adhesion by covalently cross-linking C. albicans and BEC surface proteins and (2) to implicate PRPs in the modulation of this adhesive mechanism. The reactivity of candidal cell wall proteins with TGase was assessed in two separate experiments. Initially, following incubation with native BECs, the cross-linking of iodinated candidal cell wall proteins into high-molecular-weight complexes, as shown by SDS-PAGE/ autoradiography, was inhibited by the TGase inhibitor iodoacetamide. Additionally, [14C]putrescine in the presence of purified TGase, but not [14C]putrescine alone, was shown by SDS-PAGE/fluorography to be cross-linked into surface proteins of both morphogenetic forms (blastospore > hyphal forms) of C. albicans. In adherence assays, a component of both blastospore and hyphal form Candida/BEC adherence was shown to be resistant to detachment by heating adherent cells in 1% SDS at 100°C. However, pretreatment of BECs with iodoacetamide decreased SDS resistant adherence of both forms of C. albicans by =75%. When incubated with [125I]APRPs and purified TGase, both morphogenetic forms of C. albicans bound dramatically more APRP than controls without TGase. [125I]APRP binding in experimental, but not control, samples was resistant to repeated extraction (48 h) with 4% SDS/10% β-mercaptoethanol at 65°C, suggesting that [125I]APRPs were cross-linked to the Candida surface. SDS-PAGE/fluorography was used to verify that APRPs, in Lyticase digests of Candida cell walls, were cross-linked into a high-molecular-weight complex. These experiments suggest that epithelial TGase may stabilize Candida adherence by cross-linking Candida and BEC surface proteins. Additionally, because TGase cross-links APRPs to candidal and epithelial surface proteins, APRPs may interfere with TGase catalyzed mechanisms of adhesion. Supported by USPHS grants DE00185, DE07585, and OSU Seed grant.

scholarly journals Screening for Glycosylphosphatidylinositol-Modified Cell Wall Proteins in Pichia pastoris and Their Recombinant Expression on the Cell Surface

2013 ◽  
Vol 79 (18) ◽  
pp. 5519-5526 ◽  
Author(s):  
Li Zhang ◽  
Shuli Liang ◽  
Xinying Zhou ◽  
Zi Jin ◽  
Fengchun Jiang ◽  
...  
Keyword(s):  
Cell Wall ◽  
Pichia Pastoris ◽  
Cell Surface ◽  
Signal Sequence ◽  
Recombinant Expression ◽  
Cell Wall Proteins ◽  
Heterologous Proteins ◽  
Content Type ◽  
Yeast Cell Surface

ABSTRACTGlycosylphosphatidylinositol (GPI)-anchored glycoproteins have various intrinsic functions in yeasts and different usesin vitro. In the present study, the genome ofPichia pastorisGS115 was screened for potential GPI-modified cell wall proteins. Fifty putative GPI-anchored proteins were selected on the basis of (i) the presence of a C-terminal GPI attachment signal sequence, (ii) the presence of an N-terminal signal sequence for secretion, and (iii) the absence of transmembrane domains in mature protein. The predicted GPI-anchored proteins were fused to an alpha-factor secretion signal as a substitute for their own N-terminal signal peptides and tagged with the chimeric reporters FLAG tag and matureCandida antarcticalipase B (CALB). The expression of fusion proteins on the cell surface ofP. pastorisGS115 was determined by whole-cell flow cytometry and immunoblotting analysis of the cell wall extracts obtained by β-1,3-glucanase digestion. CALB displayed on the cell surface ofP. pastorisGS115 with the predicted GPI-anchored proteins was examined on the basis of potential hydrolysis ofp-nitrophenyl butyrate. Finally, 13 proteins were confirmed to be GPI-modified cell wall proteins inP. pastorisGS115, which can be used to display heterologous proteins on the yeast cell surface.

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