Cellulose-binding proteins of Fibrobacter succinogenes and the possible role of a 180-kDa cellulose-binding glycoprotein in adhesion to cellulose

1996 ◽  
Vol 42 (5) ◽  
pp. 453-460 ◽  
Author(s):  
Jianhua Gong ◽  
Emmanuel E. Egbosimba ◽  
Cecil W. Forsberg
Keyword(s):  
Cell Surface ◽  
Binding Proteins ◽  
Cell Envelope ◽  
Immunogold Labelling ◽  
Envelope Proteins ◽  
Surface Proteins ◽  
Crystalline Cellulose ◽  
Fibrobacter Succinogenes ◽  
Endoglucanase Activity ◽  
Cellulose Binding

Fibrobacter succinogenes possesses seven cellulose-binding proteins (CBPs) of 40, 45, 50, 120, 180, 220, and 240 kDa. The 120-, 180-, 220-, and 240-kDa proteins were present in the outer membrane (OM), while the 40-, 45-, 50-, and 120-kDa proteins were either periplasmic or peripheral membrane proteins. The 120-kDa CBP, which was identified as endoglucanase 2, was a major component in both the OM and periplasm. Zymogram analysis for glucanases showed that the major membrane-associated CBPs, with the exception of endoglucanase 2, lacked endoglucanase activity. Affinity-purified antibodies against the 180-kDa CBP cross-reacted strongly with numerous cell envelope proteins of higher and lower molecular mass, including the previously characterized chloride-stimulated cellobiosidase. Treatment of the 180-kDa CBP and cell envelope proteins with periodate resulted in almost complete loss of antibody binding, suggesting that they possessed a common epitope that was carbohydrate in nature. Immunogold labelling of whole cells using antibodies against the 180-kDa CBP demonstrated that either the 180-kDa CBP or related proteins with a cross-reactive epitope were located at the cell surface. These epitopes were distributed uniformly on cells not bound to cellulose but congregated on the cell surface at sites of adhesion of cells to cellulose. Antibodies to the 180-kDa protein caused 62% inhibition of binding of F. succinogenes to crystalline cellulose, which provides evidence that either the 180-kDa CBP and (or) other related cross-reactive surface proteins have a role in adhesion to cellulose.Key words: cellulose, adhesin, adhesion, binding, Fibrobacter, succinogenes, rumen.

scholarly journals Restricted Mobility of Cell Surface Proteins in the Polar Regions of Escherichia coli

2004 ◽  
Vol 186 (9) ◽  
pp. 2594-2602 ◽  
Author(s):  
Miguel A. de Pedro ◽  
Christoph G. Grünfelder ◽  
Heinz Schwarz
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Cell Envelope ◽  
Envelope Proteins ◽  
Surface Proteins ◽  
Polar Regions ◽  
Free Medium ◽  
Cell Surface Proteins ◽  
Restricted Mobility ◽  
Murein Sacculus

ABSTRACT The polar regions of the Escherichia coli murein sacculus are metabolically inert and stable in time. Because the sacculus and the outer membrane are tightly associated, we investigated whether polar inert murein could restrict the mobility of other cell envelope elements. Cells were covalently labeled with a fluorescent reagent, chased in dye-free medium, and observed by microscopy. Fluorescent material was more efficiently retained at the cell poles than at any other location. The boundary between high and low fluorescence intensity areas was rather sharp. Labeled material consisted mostly of cell envelope proteins, among them the free and murein-bound forms of Braun's lipoprotein. Our results indicate that the mobility of at least some cell envelope proteins is restrained at regions in correspondence with underlying areas of inert murein.

scholarly journals Outer Membrane Proteins of Fibrobacter succinogenes with Potential Roles in Adhesion to Cellulose and in Cellulose Digestion

2007 ◽  
Vol 189 (19) ◽  
pp. 6806-6815 ◽  
Author(s):  
Hyun-Sik Jun ◽  
Meng Qi ◽  
Joshua Gong ◽  
Emmanuel E. Egbosimba ◽  
Cecil W. Forsberg
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Binding Proteins ◽  
Outer Membrane Proteins ◽  
Mass Spectrometry Analysis ◽  
Crystalline Cellulose ◽  
Fibrobacter Succinogenes ◽  
Essential Components ◽  
Mutant Strains ◽  
Cellulose Binding

ABSTRACT Comparative analysis of binding of intact glucose-grown Fibrobacter succinogenes strain S85 cells and adhesion-defective mutants AD1 and AD4 to crystalline and acid-swollen (amorphous) cellulose showed that strain S85 bound efficiently to both forms of cellulose while mutant Ad1 bound to acid-swollen cellulose, but not to crystalline cellulose, and mutant Ad4 did not bind to either. One- and two-dimensional electrophoresis (2-DE) of outer membrane cellulose binding proteins and of outer membranes, respectively, of strain S85 and adhesion-defective mutant strains in conjunction with mass spectrometry analysis of tryptic peptides was used to identify proteins with roles in adhesion to and digestion of cellulose. Examination of the binding to cellulose of detergent-solubilized outer membrane proteins from S85 and mutant strains revealed six proteins in S85 that bound to crystalline cellulose that were absent from the mutants and five proteins in Ad1 that bound to acid-swollen cellulose that were absent from Ad4. Twenty-five proteins from the outer membrane fraction of cellulose-grown F. succinogenes were identified by 2-DE, and 16 of these were up-regulated by growth on cellulose compared to results with growth on glucose. A protein identified as a Cl-stimulated cellobiosidase was repressed in S85 cells growing on glucose and further repressed in the mutants, while a cellulose-binding protein identified as pilin was unchanged in S85 grown on glucose but was not produced by the mutants. The candidate differential cellulose binding proteins of S85 and the mutants and the proteins induced by growth of S85 on cellulose provide the basis for dissecting essential components of the cellulase system of F. succinogenes.

scholarly journals Periplasmic Cytophaga hutchinsonii Endoglucanases Are Required for Use of Crystalline Cellulose as the Sole Source of Carbon and Energy

2016 ◽  
Vol 82 (15) ◽  
pp. 4835-4845 ◽  
Author(s):  
Yongtao Zhu ◽  
Lanlan Han ◽  
Kathleen L. Hefferon ◽  
Nicholas R. Silvaggi ◽  
David B. Wilson ◽  
...  
Keyword(s):  
Cell Surface ◽  
Outer Membrane ◽  
Soluble Sugars ◽  
Surface Proteins ◽  
Cellulolytic Enzymes ◽  
Crystalline Cellulose ◽  
Cell Fractionation ◽  
Unknown Mechanism ◽  
Content Type ◽  
Cytophaga Hutchinsonii

ABSTRACTThe soil bacteriumCytophaga hutchinsoniiactively digests crystalline cellulose by a poorly understood mechanism. Genome analyses identified nine genes predicted to encode endoglucanases with roles in this process. No predicted cellobiohydrolases, which are usually involved in the utilization of crystalline cellulose, were identified. Chromosomal deletions were performed in eight of the endoglucanase-encoding genes:cel5A,cel5B,cel5C,cel9A,cel9B,cel9C,cel9E, andcel9F. Each mutant retained the ability to digest crystalline cellulose, although the deletion ofcel9Ccaused a modest decrease in cellulose utilization. Strains with multiple deletions were constructed to identify the critical cellulases. Cells of a mutant lacking bothcel5Bandcel9Cwere completely deficient in growth on cellulose. Cell fractionation and biochemical analyses indicate that Cel5B and Cel9C are periplasmic nonprocessive endoglucanases. The requirement of periplasmic endoglucanases for cellulose utilization suggests that cellodextrins are transported across the outer membrane during this process. Bioinformatic analyses predict that Cel5A, Cel9A, Cel9B, Cel9D, and Cel9E are secreted across the outer membrane by the type IX secretion system, which has been linked to cellulose utilization. These secreted endoglucanases may perform the initial digestion within amorphous regions on the cellulose fibers, releasing oligomers that are transported into the periplasm for further digestion by Cel5B and Cel9C. The results suggest that both cell surface and periplasmic endoglucanases are required for the growth ofC. hutchinsoniion cellulose and that novel cell surface proteins may solubilize and transport cellodextrins across the outer membrane.IMPORTANCEThe bacteriumCytophaga hutchinsoniidigests crystalline cellulose by an unknown mechanism. It lacks processive cellobiohydrolases that are often involved in cellulose digestion. Critical cellulolytic enzymes were identified by genetic analyses. Intracellular (periplasmic) nonprocessive endoglucanases performed an important role in cellulose utilization. The results suggest a model involving partial digestion at the cell surface, solubilization and uptake of cellodextrins across the outer membrane by an unknown mechanism, and further digestion within the periplasm. The ability to sequester cellodextrins and digest them intracellularly may limit losses of soluble cellobiose to other organisms.C. hutchinsoniiuses an unusual approach to digest cellulose and is a potential source of novel proteins to increase the efficiency of conversion of cellulose into soluble sugars and biofuels.

scholarly journals Selective labelling of cell-surface polyamine-binding proteins on leukaemic and solid-tumour cell types using a new polyamine photoprobe

1997 ◽  
Vol 328 (3) ◽  
pp. 889-895 ◽  
Author(s):  
M. Donna FELSCHOW ◽  
Zenghui MI ◽  
J. STANEK ◽  
J. FREI ◽  
W. Carl PORTER
Keyword(s):  
Cell Surface ◽  
Tumour Cell ◽  
Solid Tumour ◽  
Mammalian Cells ◽  
Binding Proteins ◽  
Protein S ◽  
Cell Types ◽  
Biochemical Properties ◽  
Surface Proteins ◽  
Polyamine Transport

Polyamine transport is an active process which contributes to the regulation and maintenance of intracellular polyamine pools. Although the biochemical properties of polyamine transport in mammalian cells have been extensively studied, attempts to isolate and characterize the actual protein(s) have met with limited success. As one approach, photoaffinity labelling of cell surface proteins using a polyamine-conjugated photoprobe may lead to the identification of polyamine-binding proteins (pbps) associated with the transport apparatus and/or other regulatory responses. In a previous study [Felschow, MacDiarmid, Bardos, Wu, Woster and Porter (1995) J. Biol. Chem. 270, 28705-28711], we demonstrated that the photoprobes N4-ASA-spermidine and N1-ASA-norspermine [where the ASA (azidosalicylamidoethyl) group represents the photoreactive moiety] competed effectively with polyamines for transport and selectively labelled two major pbps at 118 and 50 kDa on the surface of murine and human leukaemia cells. In the present study, a new and more potent polyamine-conjugated photoprobe, N1-ASA-spermine, has been synthesized and used to develop a method based on detergent lysis for identifying putative cell-surface pbps on solid-tumour cell types. Transport kinetic assays showed that the new photoprobe competed with spermidine uptake with an apparent Ki of 1 μM, a value 20-50-fold lower than those of earlier probes. In L1210 cells, the new probe identified pbp50 and pbp118 thus reaffirming their identity as pbps. Two new bands were also detected. In A549 human lung adenocarcinoma cells, N1-ASA-spermine identified pbps at 39, 62, 73 and 130 kDa, the latter believed to be a size variant of pbp118. The presence of pbp130/118 in two very different cell types suggests the generality of the protein among mammalian cell types as well as its importance for further study. The high affinity of the photoprobe for the polyamine-transport system strongly suggests that at least some of the identified pbps may be associated with that function.

scholarly journals Collagen binding proteins derived from the embryonic fibroblast cell surface recognize arginine-glycine-aspartic acid

1989 ◽  
Vol 9 (3) ◽  
pp. 329-340 ◽  
Author(s):  
Roy C. Ogle ◽  
Charles D. Little
Keyword(s):  
Cell Surface ◽  
Aspartic Acid ◽  
Plasma Membranes ◽  
Binding Proteins ◽  
Fibroblast Cell ◽  
Surface Proteins ◽  
Collagen Molecule ◽  
Collagen Binding ◽  
Arginine Glycine Aspartic Acid ◽  
Embryonic Fibroblast

Several cell surface proteins (Mr=120,000, 90,000, 63,000 and 47,000) apparently integral to embryonic fibroblast plasma membranes were extracted with detergent and isolated by collagen affinity chromatography. Certain of these proteins (Mr=120,000, 90,000 and 47,000) were specifically eluted from collagen affinity columns by synthetic peptides containing the amino acid sequence arginyl-glycyl-aspartic acid (RGD). These data show that a number of collagen binding proteins exist on the embryonic fibroblast cell surface. Some of the proteins may be collagen receptors binding to RGD sequences in the collagen molecule while at least one of the proteins (Mr=63,000) recognizes features other than RGD.

scholarly journals Cel9D, an Atypical 1,4-β-d-Glucan Glucohydrolase from Fibrobacter succinogenes: Characteristics, Catalytic Residues, and Synergistic Interactions with Other Cellulases

2008 ◽  
Vol 190 (6) ◽  
pp. 1976-1984 ◽  
Author(s):  
Meng Qi ◽  
Hyun-Sik Jun ◽  
Cecil W. Forsberg
Keyword(s):  
Biomass Conversion ◽  
Peptide Mass Fingerprinting ◽  
Degree Of Polymerization ◽  
Exchange Chromatography ◽  
Site Directed Mutagenesis ◽  
Crystalline Cellulose ◽  
Fibrobacter Succinogenes ◽  
Endoglucanase Activity ◽  
Catalytic Residues ◽  
Peptide Mass

ABSTRACT The increasing demands of renewable energy have led to the critical emphasis on novel enzymes to enhance cellulose biodegradation for biomass conversion. To identify new cellulases in the ruminal bacterium Fibrobacter succinogenes, a cell extract of cellulose-grown cells was separated by ion-exchange chromatography and cellulases were located by zymogram analysis and identified by peptide mass fingerprinting. An atypical family 9 glycoside hydrolase (GH9), Cel9D, with less than 20% identity to typical GH9 cellulases, was identified. Purified recombinant Cel9D enhanced the production of reducing sugar from acid swollen cellulose (ASC) and Avicel by 1.5- to 4-fold when mixed separately with each of four other glucanases, although it had low activity on these substrates. Cel9D degraded ASC and cellodextrins with a degree of polymerization higher than 2 to glucose with no apparent endoglucanase activity, and its activity was restricted to β-1→4-linked glucose residues. It catalyzed the hydrolysis of cellulose by an inverting mode of reaction, releasing glucose from the nonreducing end. Unlike many GH9 cellulases, calcium ions were not required for its function. Cel9D had increased k cat /K m values for cello-oligosaccharides with higher degrees of polymerization. The k cat /K m value for cellohexaose was 2,300 times higher than that on cellobiose. This result indicates that Cel9D is a 1,4-β-d-glucan glucohydrolase (EC 3.2.1.74) in the GH9 family. Site-directed mutagenesis of Cel9D identified Asp166 and Glu612 as the candidate catalytic residues, while Ser168, which is not present in typical GH9 cellulases, has a crucial structural role. This enzyme has an important role in crystalline cellulose digestion by releasing glucose from accessible cello-oligosaccharides.

Three distinct proteases are responsible for overall cell surface proteolysis in Streptococcus thermophilus .

2021 ◽  
Author(s):  
Mylène Boulay ◽  
Coralie Metton ◽  
Christine Mézange ◽  
Lydie Oliveira Correia ◽  
Thierry Meylheuc ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Cell Surface ◽  
Lactic Acid Bacterium ◽  
Wild Type Strain ◽  
Cell Envelope ◽  
Streptococcus Thermophilus ◽  
Surface Proteins ◽  
Bacterial Vector ◽  
Cell Surface Protease ◽  
Cell Surface Proteolysis

The lactic acid bacterium Streptococcus thermophilus was believed to display only two distinct proteases at the cell surface, namely the cell-envelope protease PrtS and the house-keeping protease HtrA. Using peptidomics, we demonstrate here the existence of an additional active cell-surface protease, which shares significant homology with the SepM protease of Streptococcus mutans . Although all three proteases—PrtS, HtrA, and SepM—are involved in the turnover of surface proteins, they demonstrate distinct substrate specificities. In particular, SepM cleaves proteins involved in cell wall metabolism and cell elongation, and its inactivation has consequences for cell morphology. When all three proteases are inactivated, the residual cell-surface proteolysis of S. thermophilus is approximately 5% of that of the wild-type strain. Importance Streptococcus thermophilus is a lactic acid bacterium widely used as a starter in the dairy industry. Due to its "generally recognized as safe" status and its weak cell-surface proteolytic activity, it is also considered to be a potential bacterial vector for heterologous protein production. Our identification of a new cell surface protease made it possible to construct a mutant strain with a 95% reduction in surface proteolysis, which could be useful in numerous biotechnological applications.

Activation of Endothelial Cells by β2-Glycoprotein I (β2GPI) Antibodies Is Mediated by Annexin II Cross Linking and May Involve TLR4.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 278-278 ◽  
Author(s):  
Jianwei Zhang ◽  
Kristen A. Lieske ◽  
Brett A. McCrae ◽  
Keith R. McCrae
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Surface ◽  
Cell Activation ◽  
Binding Proteins ◽  
Surface Proteins ◽  
Annexin Ii ◽  
Cross Linking ◽  
Endothelial Cell Activation ◽  
Glycoprotein I

Abstract The antiphospholipid antibody syndrome (APS) is characterized by the presence of circulating antiphospholipid antibodies (APLA) in association with thrombosis and/or recurrent fetal loss. Although initially thought to directly recognize anionic phospholipids, most APLA actually recognize phospholipid binding proteins, most commonly β2-glycoprotein I (ß2GPI). β2GPI binds with high affinity to annexin II on the surface of endothelial cells (Ma et al, JBC, 2000), and β2GPI-dependent “APLA” activate endothelial cells in a β2GPI-dependent manner (Simantov et al., JCI, 1995). Moreover, a preliminary report from our laboratory has demonstrated that cross-linking of annexin II bound β2GPI by APLA/anti-β2GPI antibodies leads to endothelial cell activation through a pathway involving NF-κB (Zhang et. al, Blood 2003). However, the mechanism by which annexin II cross-linking might induce signaling responses is uncertain, as annexin II is not a transmembrane protein. We thus have investigated the hypothesis that activation of endothelial cell signaling pathways by annexin II cross-linking might require a transmembrane “adaptor” protein that spans the plasma membrane, yet associates with cell surface annexin II. First, human umbilical vein endothelial cell (HUVEC) surface proteins were biotin labeled using the membrane impermeable biotinylation reagent NHS-LC-biotin, and labeled annexin II binding proteins were affinity purified on immobilized annexin II. Purified annexin II binding proteins were detected following 10% SDS-PAGE, transfer to PVDF and development of the membranes using streptavidinperoxidase and chemiluminescence. These studies revealed bands of ~83, ~79, ~62 and ~34 kD. None of these bands were affinity-purified on immobilized bovine serum albumin, suggesting that specific annexin II binding proteins were present on the surface of endothelial cells. To identify these proteins, affinity-purification of annexin II binding proteins from ~40 x 106 HUVEC was undertaken using Affi-Gel HZ to which 10 mg of recombinant annexin II was coupled. Elution of bound proteins from this column followed by SDS-PAGE and staining with Coomassie brilliant blue revealed proteins of similar Mr as those identified using cell-surface labeled HUVEC. These proteins were excised from the gel, and analyzed by LC-MS following in gel tryptic digestion. Results of these studies revealed that the ~83 kD band was the Toll-like receptor 4 (TLR-4), the ~79 kD band was nucleolin, the ~62 kD band was calreticulin, and the ~34 kD band was annexin II. While we have not yet analyzed the role of each of these proteins in APLA/anti-β2GPI antibody-mediated endothelial cell activation, we have performed preliminary studies to address the potential involvement of TLR4, as a recent report demonstrated potential involvement of MyD88, a downstream mediator of TLR4-dependent signaling, in the activation of endothelial cell lines by APLA/anti-β2GPI antibodies (Raschi et al., Blood 2003). Preliminary studies suggest that TLR4 co-immunoprecipitates with annexin II, and that APLA/anti-β2GPI antibody induced endothelial cell activation, but not that caused by TNF-α, was partially blocked following transfection of endothelial cells with TLR4 siRNA. In conclusion, these studies confirm that cross-linking of endothelial annexin II initiates APLA/anti-β2GPI antibody-induced endothelial cell activation, and suggests the involvement of TLR4, and perhaps other endothelial cell surface proteins.

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