DNase Activities of the Extracellular, Cell Wall-Associated, and Cytoplasmic Protein Fractions of Frankia Strain R43

DNase activities in different protein fractions of Frankia strain R43 were studied. The extracellular and the cell wall-associated fractions revealed the presence of exo- and endonucleolytic enzymes, but none was detected in the cytoplasmic fraction. The strongest DNase hydrolysis was found in the extracellular fraction, in which six DNases were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Heterogeneity of streptococcus group A cell wall protein components

Cell wall surface proteins of group A streptococcus (M 29) were isolated by mild chemical extraction with 1 M hydroxylamine pH 6.0 (37 degrees C). The proteins were purified by ammonium sulfate fractionation, gel filtration on Sephadex G-150 and ion-exchange chromatography on DEAE-Trisacryl M. Using two independent methods (disc electrophoresis in 7.5% PAAG pH 8.9 and high pressure gel filtration), it was shown that after chromatography on Sephadex G-150 the original protein fraction contains up to 8 protein components, while SDS-PAAG electrophoresis performed according to Laemmli revealed up to 25 protein components in the same fraction. During SDS-PAAG electrophoresis six protein fractions performed after ion-exchange chromatography were resolved into 40 protein components whose molecular masses vary from 13 to 80 kDa. Possible reasons for the heterogeneity of surface proteins of group A streptococcus cell wall are discussed.     

Cell surface proteins of Drosophila. II. A comparison of embryonic and ecdysone-induced proteins

The cell-surface proteins of Drosophila embryos at gastrula and myoblast fusion stages were characterized by radioiodination and two-dimensional gel electrophoresis. Over 13% of the cell surface proteins detected in gastrula embryos were not found in myoblast fusion stage embryos or in Drosophila embryonic cell line EH34A3 cells. Nearly 18% of the cell-surface proteins detected in myoblast fusion stage embryos were evident only at that stage. Embryonic cell-surface proteins were compared with cell-surface proteins from untreated EH34A3 cells and EH34A3 cells treated with 20-hydroxyecdysone, which induces cell aggregation and the expression of "new" proteins at the cell surface (D. F. Woods and C. A. Poodry, 1983, Dev. Biol. 96, 23-31). Only one of the proteins induced by ecdysone in EH34A3 cells was detected in the NP-40 soluble fraction of radioiodinated cell lysates, even after fractionation by lectin affinity chromatography and immunoprecipitation to enrich for putative ecdysone induced proteins. However, extraction of the NP-40 insoluble pellet of embryo cells revealed one additional protein that was present both in myoblast fusion stage embryos and hormone-treated culture cells. It was concluded that except for these two proteins, the cell-surface proteins induced in cultured cell lines by treatment with 20-hydroxyecdysone are not present in significant amounts in gastrula or myoblast fusion stage embryos.     

Isolation and characterization of the cell-associated region of group A streptococcal M6 protein.

DNA sequence analysis of the complete M6 protein gene revealed 19 hydrophobic amino acids at the C terminus which could act as a membrane anchor and an adjacent proline- and glycine-rich region likely to be located in the cell wall. To define this region within the cell wall and its role in attaching the molecule to the cell, we isolated the cell-associated fragment of the M protein. Assuming that the cell-associated region of the M protein would be embedded within the wall and thus protected from trypsin digestion, cells were digested with this enzyme, and the wall-associated M protein fragment was released by phage lysin digestion of the peptidoglycan. With antibody probes prepared to synthetic peptides of C-terminal sequences, a cell wall-associated M protein fragment (molecular weight, 16,000) was identified and purified. Amino acid sequence analysis placed the N terminus of the 16,000-molecular-weight fragment at residue 298 within the M sequence. Amino acid composition of this peptide was consistent with a C-terminal sequence lacking the membrane anchor. Antibody studies of nitrous acid-extracted whole bacteria suggested that, in addition to the peptidoglycan-associated region, a 65-residue helical segment of the C-terminal domain of the M protein is embedded within the carbohydrate moiety of the cell wall. Since no detectable amino sugars were associated with the wall-associated fragment, the C-terminal region of the M6 molecule is likely to be intercalated within the cross-linked peptidoglycan and not covalently linked to it. Because the C-terminal region of the M molecule is highly homologous to the C-terminal end of protein A from staphylococci and protein G from streptococci, it is likely that the mechanism of attachment of these proteins to the cell wall is conserved.     

Analysis of grape berry cell wall proteome: a comparative evaluation of extraction methods

Different methods were tested for the extraction of proteins from the cell wall-enriched fraction (CWEf) obtained from a sample formed by skin and seeds of ripe berries of Vitis vinifera L. cv. Cabernet Sauvignon. The CWEf was isolated using a disruptive approach that involves tissue homogenization and precipitation by centrifugation. To extract proteins, the CWEf was treated with CaCl(2) and LiCl in two successive steps or, alternatively, with phenol. The efficiency of the protocols was evaluated by measuring protein yield and by analyzing two-dimensional gel electrophoresis (2-DE) gels for the highest detectable spot number and the greatest spot resolution. The phenol method was also adopted for the extraction of proteins from the cytosolic fraction (CYf). The comparison of 2-DE reference maps of protein extracts from CWEf and CYf indicated the presence of both common traits and unique characteristics. To survey this aspect some spots detected in both fractions or present in only one fraction were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Of the 47 spots identified, some were found to be cell wall proteins, while others were proteins not traditionally considered as localized in the apoplastic space. The data presented here provide initial information regarding the apoplastic proteome of grape berry tissues, but also raise the issue of the technical problems that characterize the isolation of cell wall proteins from these very hardy tissues.     

Development of a sample preparation method for fungal proteomics

Since filamentous fungi including basidiomycetous fungi possess an exceptionally robust cell wall as in microorganisms, effective extraction of intracellular proteins is a key step for fungal proteomic studies. To overcome the experimental obstacle caused by cell walls, we utilized fungal protoplasts, prepared from the brown-rot basidiomycete, Tyromyces palustris. The amount and quality of proteins extracted from the protoplast cells were much higher than that from the mycelial cells. Quantitative comparisons of proteome maps prepared from mycelial and protoplast cells indicated protein spots with a wider range of molecular weights and pIs in the protoplast sample. Furthermore, no streaking or tailing was observed in the protoplasts, suggesting that effective extraction of intracellular proteins from protoplasts might help suppress degradation of proteins during this process. In addition to the efficiency of protein extraction, simple and efficient subcellular fractionation was also achieved using protoplast cells.     

Subcellular localization of the Streptococcus mutans P1 protein C terminus.

To determine the subcellular location of the Streptococcus mutans P1 protein C-terminal anchor, cell envelope fractionation experiments were conducted in combination with Western immunoblotting, using monoclonal antibody MAb 6-8C specific for an epitope that maps near the C terminus of P1 protein and also a polyclonal antibody preparation directed against the P1 C-terminal 144 amino acids (P1COOH). P1 protein was detected in cell walls but not the membrane purified from S. mutans cells by the monoclonal antibody. In contrast, P1 protein was not detected in the same cell wall preparation using the anti-P1COOH polyclonal antibody. However, proteins released from the cell walls by treatment with mutanolysin contained antigen that was recognized by the anti-P1COOH antibody, suggesting that the epitopes recognized by the antibody were masked by peptidoglycan in the cell wall preparations. When cell walls were treated with boiling trichloroacetic acid to solubilize cell-wall-associated carbohydrate, P1 antigen could not be detected in either the solubilized carbohydrate, or in the remaining peptidoglycan, regardless of whether polyclonal or monoclonal antibody was used. However, when the peptidoglycan was treated with mutanolysin, P1 antigen could be detected in the mutanolysin solubilized fraction by MAb 6-8C. Collectively, these data suggest that the C-terminal 144 amino acids of the P1 protein are embedded within the cell wall, and associated exclusively with the peptidoglycan. Furthermore, the ability of the anti-P1COOH antibody to recognize P1 antigen only after mutanolysin treatment of cell walls suggests these C-terminal 144 amino acids are tightly intercalated within the peptidoglycan strands.     

Immunochemical properties and intracellular localization of two molecular forms of arginine aminopeptidase in Streptococcus mitis ATCC 9811

Streptococcus mitis contains two multiple forms of arginine aminopeptidase (I and II) which differ from each other with respect to their content, immunochemical properties and cellular localization. Immunological analyses by Ouchterlony double immunodiffusion and immunoprecipitation showed an antigenic difference between each form by the use of antisera specific for each enzyme. The amounts of enzymes I and II within the cell were estimated to be 230 +/- 4.3 and 646 +/- 20 ng/mg protein (+/- S.D.), respectively, using a standard curve of purified enzyme in a single radial immunodiffusion assay. When intact cells were treated with the cell wall lytic enzyme, N-acetylmuramidase, though both enzymes were solubilized, a time lag was observed for the solubilization of enzyme II. Enzyme I was detected only in the cell wall fraction and showed no detectable associated with the membrane. Although most of the enzyme II activity was recovered in the cell wall fraction, a slight amount (7.5%) of the total activity was also found in the membrane fraction.     

PlyC, a novel bacteriophage lysin for compartment-dependent proteomics of group A streptococci

Streptococcus pyogenes (Spy) (group A streptococci) is an important and exclusively human bacterial pathogen, which uses secreted and surface-associated proteins to circumvent the innate host defense mechanisms and to adhere and internalize into host cells. Thus, investigation of the bacterial extracellular compartments, including secreted and cell wall-associated subproteomes, is crucial for understanding adherence, invasion, and internalization mechanisms as major steps of Spy pathogenesis. Here, we compared a bacteriophage encoded cell wall hydrolase, PlyC, a multimeric lysin of the C1 bacteriophage, with the established glycosidase, mutanolysin, from Streptomyces globisporus for their suitability to efficiently digest Spy cell walls and release cell wall-anchored Spy proteins for subsequent proteome research. Our results show that PlyC is superior for cell wall protein extraction compared to mutanolysin due to its higher activity and specificity as an N-acetylmuramoyl-L-alanine amidase. Furthermore, our experimental design allowed us to delineate the actual localization of the proteins despite contamination with intracellular proteins.     

An improved procedure for the isolation of plasmodesmata embedded in clean maize cell walls

A cell wall preparation of high purity was obtained using a procedure which involved repeated grindings of etiolated maize mesocotyl tissue and filtration through 200 mesh nylon cloth, followed by cell disruption via a nitrogen disruption bomb, and recovery of the cell walls via filtration. The cell wall fraction was free of particulate contaminants as determined both by phase-contrast and electron microscopy. The only membrane components found associated with the wall fraction as determined by electron microscopy were pladmodesmata embedded in the cell wall. The specific concentration of PAP26, a plasmodesmatal-associated polypeptide, was greatly increased in the cleanest cell wall fraction. A second plasmodesmatal-associated protein, PAP27, which was previously shown to be associated with the neck region of the plasmodesmata, was diminished as a result of passage through the nitrogen disruption bomb suggesting a partial fragmentation of the plasmodesmata. In addition to PAP26, the specific concentrations of at least three other cell wall-associated polypeptides with molecular weights of 80, 21 and 18 kDa, as revealed by SDS-PAGE, were also increased greatly in the cleanest cell wall fraction.     

Molecular cloning and characterization of a cDNA encoding a novel apoplastic protein preferentially expressed in a shikonin-producing callus strain of Lithospermum erythrorhizon

A cDNA (LEPS-2) encoding a novel cell wall protein was cloned from shikonin-producing callus tissues of Lithospermum erythrorhizon by differential display between a shikonin-producing culture strain and a non-producing strain. The LEPS-2 cDNA encoded a polypeptide of 184 amino acids. The deduced amino acid sequence exhibited no significant homology with known proteins. Expression of LEPS-2 gene as well as accumulation of LEPS-2 protein was highly correlated with shikonin production in L. erythrorhizon cells in culture. In the intact plant, expression of LEPS-2 was detected only in the roots where shikonin pigments accumulated. Cell fractionation experiments and immunocytochemical analysis showed that the protein was localized in the apoplast fraction of the cell walls. The shikonin pigments were also stored on the cell walls as oil droplets. These results indicate that expression of the LEPS-2 is closely linked with shikonin biosynthesis and the LEPS-2 protein may be involved in the intra-cell wall trapping of shikonin pigments.     

肠道病毒71型感染前后宿主细胞蛋白质组的二维液相色谱分离和比较

以肠道病毒71型及其宿主细胞为研究主体,建立了一种二维液相色谱分离和分析比较病毒感染前后细胞蛋白表达谱的方法。该方法以高效液相色谱(HPLC)为技术平台,对细胞裂解物先后进行一维色谱聚焦分离和二维反相色谱分离。利用ProteoVue软件将二维色谱数据转换成模拟胶图,再利用DeltaVue软件对感染前后的宿主蛋白表达谱进行比较和分析,找出差异蛋白。二维液相色谱分离法能够根据蛋白的等电点和疏水性建立精确的细胞蛋白表达图谱,每0．2个pH为一个收集区段,在pH8．5～3．9的范围内可见蛋白条带约1200条。该方法良好的重现性、自动化以及结果分析的简易化,使之在细胞表达谱差异显示中的应用潜力巨大,并且为研究病毒与宿主相互作用提供了新的方法和思路。     

Isolation of biochemically active chloroplasts from chlamydomonas

Chloroplasts from the cell wall mutant cw-15-2 of Chlamydomonas reinhardii were isolated by disruption of the cells in the Yeda press and fractionation through step gradients of Percoll. The resulting chloroplast fraction contained 80–85% intact chloroplasts. Electron micrographs of thin sections of the chloroplast fraction showed some cytoplasmic impurities, although almost no cytoplasmic ribosomes were detected by analysis of the ribosomal subunits.The isolated chloroplasts are active in photosynthetic O₂-evolution and CO₂-fixation, with the highest rates obtained in the presence of ATP.The chloroplast fraction also showed high rates of light-dependent in organello protein synthesis, with labelling of discrete chloroplast proteins known to be synthesized in the chloroplasts.     

Quantification of Frankia in soils using SYBR Green based qPCR

A SYBR Green based qPCR method was developed for the quantification of clusters 1 and 3 of the actinomycete Frankia in soils. Primer nifHr158 was designed to be used as reverse primer in combination with forward primer nifHf1 specifically amplifying a 191-bp fragment of the nifH gene of these Frankia. The primer combination was tested for specificity on selected pure cultures, and by comparative sequence analyses of randomly selected clones of a clone library generated with these primers from soil DNA extracts. After adjustments of DNA extraction conditions, and the determination of extraction efficiencies used for sample normalization, copy numbers of nifH genes representing Frankia of clusters 1 and 3 were quantified in different mineral soils, resulting in cell density estimates for these Frankia of up to 10(6) cells [g soil {dry weight}](-1) depending on the soil. Despite indications that the nifH gene is not a perfect target for the quantification of Frankia, the qPCR method described here provides a new tool for the quantification and thus a more complete examination of the ecology of Frankia in soils.     

Characterization of T cell antigens associated with the cell wall protein-peptidoglycan complex of Mycobacterium tuberculosis

Mycobacterium tuberculosis cell walls are likely to contain critical T cell Ag capable of inducing protective immunity against the development of tuberculosis in animal models. Therefore, we characterized cell wall-associated Ag that stimulate T lymphocytes in tuberculosis patients and clinically well tuberculin-positive individuals. A protein-peptidoglycan complex isolated from the M. tuberculosis cell wall had potent immunologic activity, evoking PBMC proliferative responses similar to those induced by sonicated whole M. tuberculosis. In order to characterize the immunoreactive protein determinants associated with the protein-peptidoglycan complex, T cell lines were established to cell wall Ag and used to probe M. tuberculosis proteins separated by SDS-PAGE. These T cell lines proliferated primarily to protein Ag of 10, 19, 23, 28, 30, 40 to 50, and 65 kDa. Cell wall-reactive T cell clones that recognized the 10-, 23-, 28-, and 30-kDa proteins as single bands on SDS-PAGE did so under reducing and nonreducing conditions, suggesting that these are not proteolytic fragments or subunits of larger protein aggregates. We propose that these protein monomers, when post-translationally complexed with peptidoglycan, are the key ingredients of the immunogenic protein-peptidoglycan complex. In order to assess the relationship of the cell wall-associated Ag to those secreted proteins from "early culture filtrates" of actively growing M. tuberculosis recently implicated in eliciting protective immunity, cell wall-reactive T cell clones were tested for their ability to recognize early culture filtrates. Results revealed that at least three proteins shared with the cell wall complex are contained within early culture filtrates. Our data indicate that antigenic determinants associated with the protein-peptidoglycan complex of the M. tuberculosis cell wall may be involved in protective immunity and hence are potential candidates for inclusion in an effective antituberculosis vaccine.     

Proteins from nuclear fractions with different contents of active genes

The protein content in four nuclear fractions was compared. The nuclear fraction of rat liver deficient in active genes was characterized by a very low content of non-histone proteins whose mobility is less than that of histone H1.. The predominant protein of this fraction is an acid-soluble protein (Mr = 41 +/- 1 kD) designated as 41K. This protein was detected in acid nuclear extracts of rat lungs, kidney and spleen but was absent (or practically absent) in four murine and rat hepatomas under study. The decreased content of protein 41K was correlated with the diminution of the content of histone H1(0) fraction. It was shown that proteins HMG 14 and 17 are readily washed off during fractionation of nuclei and they bind to DNA fragments passing into solution irrespective of whether they contain active or inactive genes. The nuclear matrix fraction rich in active genes was heterogeneous according to its protein composition. Differences in the intensity of staining and in electrophoretic mobility of some polypeptides of this nuclear fraction in normal and hepatoma cells were revealed.     

A periplasm in Bacillus subtilis.

The possibility of there being a periplasm in Bacillus subtilis, in the distinct cell compartment bounded by the cytoplasmic membrane and the thick cell wall, has been investigated quantitatively and qualitatively. Cytoplasmic, membrane, and protoplast supernatant fractions were obtained from protoplasts which were prepared isotonically from cells grown under phosphate limitation. The contents of the protoplast supernatant fraction represent an operational definition of the periplasm. In addition, this cell fraction includes cell wall-bound proteins, exoproteins in transit, and contaminating cytoplasmic proteins arising through leakage from, or lysis of a fraction of, protoplasts. The latter, measured by assay of enzyme markers and by radiolabeled RNA and protein, was found to represent 7.6% of total cell protein, yielding a mean of 9.8% +/- 4.8% for B. subtilis 168 protein considered periplasmic. Qualitatively, after subjection of all cell fractions to polyacrylamide gel electrophoresis, RNase and DNase, zymographs revealed that (i) each cell fraction had a unique profile of nucleases and (ii) multiple species and a major fraction of both nucleases were concentrated in the periplasm. We conclude that the operationally defined periplasmic fraction corresponds closely, both quantitatively and qualitatively, to the contents of the periplasm of Escherichia coli. We discuss evidence that the maintenance of the components of this surface compartment in B. subtilis is compatible with the thick negatively charged cell wall acting as an external permeability barrier.     

Isolation and characterization of Sertoli cell plasma membranes and associated plasminogen activator activity

Plasma membranes were isolated from the cultured Sertoli cells of 20-day-old rat testes by differential centrifugation and sucrose density fractionation. The distribution and purity of subcellular components was determined by marker enzyme analysis of gradient fractions. The plasma membrane fraction showed an enrichment in two plasma membrane marker enzymes, 5'-nucleotidase and ouabain-sensitive Na+/K+-ATPase-specific activities, of 9- and 23-fold, respectively. Forty-two percent and 52% of the total cellular 5'-nucleotidase and ouabain-sensitive Na+/K+-ATPase activities, respectively, were found in the membrane fraction. The protein yield of plasma membrane was approximately 6% of the total cellular protein. Two-dimensional polyacrylamide gel electrophoresis was used to compare [35S] methionine- and [3H] glucosamine-labeled membrane proteins. The incorporation of [35S] methionine and [3H] glucosamine was increased in several proteins when the cultured Sertoli cells were treated with follicle-stimulating hormone, insulin, retinol, and testosterone. Isolated Sertoli cell membranes contained a membrane-associated form of plasminogen activator. Analysis of this plasminogen activator demonstrated that the membrane-associated enzyme existed primarily as a single 38,000-40,000-Mr form.     

Cell surface expression of bacterial esterase A by Saccharomyces cerevisiae and its enhancement by constitutive activation of the cellular unfolded protein response

Yeast cell surface display is a powerful tool for expression and immobilization of biocatalytically active proteins on a unicellular eukaryote. Here bacterial carboxylesterase EstA from Burkholderia gladioli was covalently anchored into the cell wall of Saccharomyces cerevisiae by in-frame fusion to the endogenous yeast proteins Kre1p, Cwp2p, and Flo1p. When p-nitrophenyl acetate was used as a substrate, the esterase specific activities of yeast expressing the protein fusions were 103 mU mg(-1) protein for Kre1/EstA/Cwp2p and 72 mU mg(-1) protein for Kre1/EstA/Flo1p. In vivo cell wall targeting was confirmed by esterase solubilization after laminarinase treatment and immunofluorescence microscopy. EstA expression resulted in cell wall-associated esterase activities of 2.72 U mg(-1) protein for Kre1/EstA/Cwp2p and 1.27 U mg(-1) protein for Kre1/EstA/Flo1p. Furthermore, esterase display on the yeast cell surface enabled the cells to effectively grow on the esterase-dependent carbon source glycerol triacetate (Triacetin). In the case of Kre1/EstA/Flo1p, in vivo maturation within the yeast secretory pathway and final incorporation into the wall were further enhanced when there was constitutive activation of the unfolded protein response pathway. Our results demonstrate that esterase cell surface display in yeast, which, as shown here, is remarkably more effective than EstA surface display in Escherichia coli, can be further optimized by activating the protein folding machinery in the eukaryotic secretion pathway.     

A comparison of proteins from the developing xylem of compression and non-compression wood of branches of sitka spruce (Picea sitchensis) reveals a differentially expressed laccase

Soluble and cell wall-associated proteins were extracted from the developing xylem of the compression and non-compression sides of branches of Sitka spruce (Picea sitchensis (Bong) Carr.) by an identical procedure. Equal amounts of proteins were separated by SDS-PAGE, and polypeptides were identified that were more abundant in soluble and cell wall-associated extracts from the developing xylem of either compression or non-compression wood. Two polypeptides (at apparent M(r)s of 48 kDa and 120 kDa) that were more abundant in cell wall-associated extracts of the developing xylem of the compression tissues were selected for amino-terminal protein sequencing. The 48 kDa polypeptide yielded an amino-terminal sequence that had no homology with known protein, gene or EST database sequences. The amino-terminal sequence of the 120 kDa polypeptide was homologous to a number of laccase-type polyphenol oxidases (EC 1.10.3.2) thought to be involved in lignin biosynthesis in trees. Using non-denaturing SDS-PAGE, the 120 kDa laccase was confirmed as a major oxidase activity in extracts of lignifying compression xylem but it was barely detectable in the non-compression extracts where an 85 kDa oxidase was the predominant activity. The differential expression of oxidases in compression and non-compression xylem is discussed.