Purification of a plant cell wall fibronectin-like adhesion protein involved in plant response to salt stress

The structural role of extracellular-matrix (ECM) has been recognized in both plants and animals as a support and anchorage-inducing cell behavior. Unlike the animal ECM proteins, the proteins that have been identified in plant ECM have not yet been purified from whole plants and cell wall. As several immunological data indicate the presence of animal ECM-like proteins in plants cell wall, especially under salt stress or water deficit, we propose a protocol to purify a fibronectin-like protein from the cell wall of epicotyls of young germinating peas. The process consists of a combination of gelatin and heparin affinity chromatography, close to the classical one used for human blood plasma fibronectin purification. Proteins with affinity for gelatin and heparin, immunologically related to human fibronectin, are found in the cell wall of epicotyls grown under salt stress or not. Total amount of purified proteins is 3-4 times more enriched in salt stressed epicotyls. SDS-PAGE and Western blot with antibodies directed against human blood plasma fibronectin give evidence that the cell wall proteins purified by gelatin/heparin affinity chromatography are closely related to human fibronectin. The present protocol leads us to purify 17 (control) or 65 (salt stress) micrograms of protein per g of fresh starting material. Our results suggest that plant cell wall proteins can provide better anchorage of the cell to its cell-wall during salt stress or water deficit and could be considered not only as cell adhesion but also as signaling molecules.     

The augmentation of human natural killer cell activity by interferon-gamma is not associated with the induction of the interferon-alpha-inducible proteins

Treatment of partly purified large granular lymphocytes (LGL) with either IFN-alpha or IFN-gamma for 2 hr augmented their NK cell activity. This augmentation was completely inhibited by the addition of 10 micrograms/ml of cycloheximide. In contrast, when the effects of IFN-gamma on the synthesis of specific proteins in these cells was directly studied by use of two-dimensional gel electrophoresis, we found that IFN-gamma was unable to induce any of the earlier detected, IFN-alpha/IFN-beta-inducible proteins within 18 hr of incubation. No additional, IFN-gamma-induced proteins were detected in either the partly purified LGL or purified T cells. In contrast, the effects of the two factors were comparable in the glioma cell line 251 MG. This shows i) that the effects of IFN-alpha and IFN-gamma are dependent on the responder cell type, ii) that there exists at least one mechanism that can augment NK cell activity that is not dependent on the increased synthesis of the IFN-alpha-inducible proteins, and iii) that either the nine IFN-alpha-inducible proteins are not involved in any leukocyte function that is augmentable by both IFN-alpha and IFN-gamma, or that the two factors exert their actions in leukocyte through different mechanisms.     

Cell adhesion to tenascin-X mapping of cell adhesion sites and identification of integrin receptors.

Adhesive properties of tenascin-X (TN-X) were investigated using TN-X purified from bovine skin and recombinant proteins encompassing the RGD sequence located within the tenth fibronectin type-III domain, and the fibrinogen-like domain. Osteosarcoma (MG63) and bladder carcinoma cells (ECV304) cells were shown to adhere to purified TN-X, but did not spread and did not assemble actin stress fibers. Both cell types adhered to recombinant proteins harboring the contiguous fibronectin type-III domains 9 and 10 (FNX 9-10) but not to the FNX 10 domain alone. This adhesion to FNX 9-10 was shown to be mediated by alphavbeta3 integrin, was inhibited by RGD peptides and was strongly reduced in proteins mutated within the RGD site. As antibodies against alphavbeta3 integrin had no effects on cell adhesion to purified TN-X, we suggest that the RGD sequence is masked in intact TN-X. Cell attachment to the recombinant TN-X fibrinogen domain (FbgX) and to purified TN-X was greater for MG63 than for ECV304 cells. A beta1-containing integrin was shown to be involved in MG63 cell attachment to FbgX and to purified TN-X. Although the existence of other cell interaction sites is likely in this huge molecule, these similar patterns of adhesion and inhibition suggest that the fibrinogen domain might be a dominant site in the whole molecule.     

Cloning, Expression, and Affinity Purification of RecombinantShigella flexneriInvasion Plasmid Antigens IpaB and IpaC

Shigella flexneriand related enteropathogenic bacteria are important agents of bacillary dysentery, a potentially life-threatening illness for children in underdeveloped regions of the world. Onset of shigellosis stems fromS. flexneriinvasion of colonic epithelial cells, leading to localized cell death and inflammation. Invasion plasmid antigens (Ipa) B, C, and D are three secreted proteins encoded by the large virulence plasmid ofS. flexnerithat have been implicated as essential effectors of this cell invasion process. These proteins are expressed as part of theipaoperon and are among the major targets of the host immune response to shigellosis. Biochemical characterization of the Ipa invasins has been complicated by the fact they have not been purified in the quantities needed for detailedin vitroanalysis. Here we describe the first cloning, expression, and extensive purification of IpaB and IpaC fusion proteins fromEscherichia colifor use in dissecting of the protein biochemistry ofS. flexneripathogenesis. A variety of approaches were used to prepare significant quantities of these proteins in their soluble forms, including the use of different host cell lines, modification of bacterial growth conditions, and the use of alternative plasmid expression vectors. Now that these Ipa proteins are available in a highly pure form, it will be possible to initiate studies on their important biological and immunological properties as well as their recruitment into high-molecular-weight protein complexes. Together with IpaD (purified as part of a previous study), these purified proteins will be useful for: (a) exploring properties of the host immune response toS. flexneriinvasion, (b) elucidating the specific biochemical properties that lead to pathogen internalization, (c) analyzing the importance of specific Ipa protein complexes in host cell invasion, and (d) monitoring, or perhaps even augmenting, the efficacy of live oral vaccines in human trials.     

Expression and purification of recombinant human zona pellucida proteins.

Recombinant human zona pellucida (rhZP) proteins (minus the N-terminal leader and the C-terminal transmembrane-like domain) were expressed in four different expression systems: bacteria, yeast, insect cells, and Chinese Hamster Ovary (CHO) cells. The recombinant proteins in each system were engineered with a C-terminal six histidine (His6) segment that was used to purify the proteins by metal affinity [either nickel (Ni) or cobalt (Co)] column chromatography. Each of the rhZP proteins was a candidate antigen as an immunocontraceptive vaccine. However, the rhZP proteins produced in bacteria, yeast and insect cell culture could only be purified after being solubilized by strong denaturants. After purification the final products of each of these expression systems required 6 M urea to maintain solubility. However, the rhZP proteins expressed by CHO cells were secreted into the media, and the soluble proteins could be purified to near homogeneity. In this report the expression and purification procedures used to produce and isolate these secreted proteins are described.     

Trifluoromethanesulfonic acid-based proteomic analysis of cell wall and secreted proteins of the ascomycetous fungi Neurospora crassa and Candida albicans

Cell wall proteins from purified Candida albicans and Neurospora crassa cell walls were released using trifluoromethanesulfonic acid (TFMS) which cleaves the cell wall glucan/chitin matrix and deglycosylates the proteins. The cell wall proteins were then characterized by SDS–PAGE and identified by proteomic analysis. The analyses for C. albicans identified 15 cell wall proteins and six secreted proteins. For N. crassa, the analyses identified 26 cell wall proteins and nine secreted proteins. Most of the C. albicans cell wall proteins are found in the cell walls of both yeast and hyphae cells, but some cell type-specific cell wall proteins were observed. The analyses showed that the pattern of cell wall proteins present in N. crassa vegetative hyphae and conidia (asexual spores) are quite different. Almost all of the cell wall proteins identified in N. crassa have close homologs in the sequenced fungal genomes, suggesting that these proteins have important conserved functions within the cell wall.     

Cytosolic proteins that specifically bind nuclear location signals are receptors for nuclear import.

We have purified two major polypeptides of 54 and 56 kd from bovine erythrocytes that specifically bind the nuclear location sequence (NLS) of the SV40 large T antigen. When added to a permeabilized cell system for nuclear import, the purified proteins increase by 2- to 3-fold the nuclear accumulation of a fluorescent protein containing the large T antigen NLS. The import stimulation is saturable and dependent upon the presence of cytosol. Nuclear protein accumulation in vitro is sensitive to inactivation by N-ethylmaleimide (NEM). NEM inactivation can be overcome by addition of the purified NLS-binding proteins to the import system. NEM treatment of the purified proteins abolishes their ability to stimulate import but does not affect NLS binding. Our results indicate that the NLS-binding proteins are NEM-sensitive receptors for nuclear import. At least one other NEM-sensitive cytosolic activity and an NEM-insensitive cytosolic activity are also necessary for protein import in vitro.     

Functional inactivation of the SR family of splicing factors during a vaccinia virus infection

SR proteins are essential splicing factors required for constitutive splicing and function as key regulators of alternative RNA splicing. We have shown that SR proteins purified from late adenovirus-infected cells (SR-Ad) are functionally inactivated as splicing enhancer or splicing repressor proteins by a virus-induced partial de-phosphorylation. Here, we show that SR proteins purified from late vaccinia-virus-infected cells (SR-VV) are also hypo-phosphorylated and functionally inactivated as splicing regulatory proteins. We further show that incubating SR-Ad proteins under conditions that restore the phospho-epitopes to the SR proteins results in the restoration of their activity as splicing enhancer and splicing repressor proteins. Interestingly, re-phosphorylation of SR-VV proteins only partially restored the splicing enhancer or splicing repressor phenotype to the SR proteins. Collectively, our results suggest that viral control of SR protein activity may be a common strategy used by DNA viruses to take control of the host cell RNA splicing machinery.     

Immunocytochemical evidence for a possible role of cross-linked keratinocyte envelopes in stratum corneum cohesion.

Cross-linked cornified envelopes are cell structures specifically synthesized by terminally differentiating keratinocytes. They are composed of proteins deposited at the cell periphery under the plasma membrane, and can be purified from epidermis by physicochemical extractions. The resulting keratinocyte "shells" are highly insoluble structures devoid of cytoplasmic components. The rigidity of the stratum corneum cell envelope seems to be one of the essential factors contributing to the physical resistance of this most superficial epidermal layer. We studied the purified cell envelopes from human plantar horny layer to determine their antigenic composition and protein distribution. The extraction protocol consisted of four 10-min cycles of boiling in 10 mM Tris-HCl buffer containing 2% SDS and 1% beta-mercaptoethanol. The absence of any extractable proteins persisting in the purified pellets was checked with SDS-PAGE of the sample electroeluates. Indirect immunofluorescence as well as pre- and post-embedding immunogold labeling for electron microscopy revealed the persistence of several keratinocyte antigenic determinants on the purified substrates. The antibodies directed against involucrin, keratin 10, desmoplakin I + II, desmoglein (intracellular epitope), intercellular corneodesmosome proteins, and filaggrin (a considerably weaker reactivity) labeled the cell envelopes according to the ultrastructural localization pattern characteristic for a given antigen. We conclude that the cytoskeletal and desmosomal components become "embedded" in the highly cross-linked cornified envelope structures during the process of keratinocyte terminal differentiation. This underlines the central role of cornified envelopes in the physical resistance of superficial epidermal layers and indicates a possible importance of junctional proteins in this function.     

Purification of maize pollen exines and analysis of associated proteins

Zea mays (maize) pollen exines have been purified with the use of differential centrifugation and sucrose gradients, followed by mild detergent and high salt treatment. The final exine fraction is highly purified from other organelles and subcellular structures as assayed by transmission electron microscopy. Using mature maize pollen as the starting material, 0.2 to 0.3% of the total pollen protein remained associated with the exine fraction throughout the purification. Seven abundant sodium dodecyl sulfate-extractable proteins are detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the final fraction. Amino acid analysis reveals that one of the proteins contains a substantial amount of hydroxyproline, a characteristic of some primary cell wall proteins. The amino acid composition of the 25-kD protein strongly implies that it is an arabinogalactan protein. When exines are purified from earlier pollen developmental stages, a subset of the proteins found in the mature pollen exine is seen.     

粗提和纯化的HPV16L1重组蛋白为抗原的ELISA比较

为评价真核及原核细胞表达的HPV16L1粗提蛋白代替纯化的16L1蛋白作为ELISA检测抗原的可行性。实验分别用大肠埃希菌表达的HPV16L1纯化蛋白,表达16L1的大肠埃希菌破碎物及表达16L1的昆虫细胞破碎物作为抗原。在ELISA试验中与抗HPV16L1的单克隆抗体进行反应。结果证实3种包被抗原可以得出非常相近的OD值变化趋势。表明用表达16L1的大肠埃希菌破碎物和表达16L1的昆虫细胞破碎物可以代替纯化的HPV16L1蛋白作为抗原进行ELISA试验。     

Resistance to inhibitors of cholinesterase-8A (Ric-8A) is critical for growth factor receptor-induced actin cytoskeletal reorganization

Heterotrimeric G proteins are critical transducers of cellular signaling. In addition to their classic roles in relaying signals from G protein-coupled receptors (GPCRs), heterotrimeric G proteins also mediate physiological functions from non-GPCRs. Previously, we have shown that Gα(13), a member of the heterotrimeric G proteins, is essential for growth factor receptor-induced actin cytoskeletal reorganization such as dynamic dorsal ruffle turnover and cell migration. These Gα(13)-mediated dorsal ruffle turnover and cell migration by growth factors acting on their receptor tyrosine kinases (RTKs) are independent of GPCRs. However, the mechanism by which RTKs signal to Gα(13) is not known. Here, we show that cholinesterase-8A (Ric-8A), a nonreceptor guanine nucleotide exchange factor for some heterotrimeric G proteins, is critical for coupling RTKs to Gα(13). Down-regulation of Ric-8A protein levels in cells by RNA interference slowed down platelet-derived growth factor (PDGF)-induced dorsal ruffle turnover and inhibited PDGF-initiated cell migration. PDGF was able to increase the activity of Ric-8A in cells. Furthermore, purified Ric-8A proteins interact directly with purified Gα(13) protein in a nucleotide-dependent manner. Deficiency of Ric-8A prevented the translocation of Gα(13) to the cell cortex. Hence, Ric-8A is critical for growth factor receptor-induced actin cytoskeletal reorganization.     

Phosphorylation of Vesicular Stomatitis Virus In Vivo and In Vitro

The structural protein, NS, of purified vesicular stomatitis virus (VSV) is a phosphoprotein. In infected cells phosphorylated NS is found both free in the cytoplasm and as part of the viral ribonucleoprotein (RNP) complex containing both the 42S RNA and the structural proteins L, N, and NS, indicating that phosphorylation occurs as an early event in viral maturation. VSV contains an endogenous protein kinase activity, probably of host region, which catalyzes the in vitro phosphorylation of the viral proteins NS, M, and L, but not of N or G. The phosphorylated sites on NS appear to be different in the in vivo and in vitro reactions, and are differentially sensitive to alkaline phosphatase. After removal of the membrane components of purified VSV with a dextran-polyethylene glycol two-phase separation, the kinase activity remains tightly associated with the viral RNP. However, viral RNP isolated from infected cells shows only a small amount of kinase activity. The protein kinase enzyme appears to be a cellular contaminant of purified VSV because an activity from the uninfected cell extract can phosphorylate in vitro the dissociated viral proteins NS and M. The virion-associated activity may be derived either from the cytoplasm or the plasma membrane of the host cell since both of these cellular components contain protein kinase activity similar to that found in purified VSV.     

Production and purification of novel secreted human proteins

Our objective during the last year was to produce and purify 50–80 novel, secreted human proteins identified via high throughput cDNA sequencing and computer analysis. We chose the baculovirus expression vector system in order to obtain secreted, correctly folded, bioactive proteins. Recombinant (re-)baculoviruses (BV) were plaque purified, and pulse-labeling was used to verify the synthesis and secretion of the re-proteins. N-terminal microsequencing was performed to simultaneously confirm the identity of the protein(s) as well as the signal peptide (SP) cleavage site(s). Following sequence confirmation, the proteins were purified to homogeneity and functional assays carried out to determine potential therapeutic applications. We identified proteins with antiviral activity, several novel growth factors, proteins influencing the differentiation of specific cell types, novel proteases and protease inhibitors among others. Certain proteins were expressed both in insect cells and in CHO stable cell lines. In the cases analyzed, we found that the same SP cleavage site was utilized in the two expression systems. Significant differences were observed in the carbohydrate moieties attached to the proteins, though no effects on the biological activity due to these differences have been demonstrated. The BV system has served as a viable alternative for the high throughput, high fidelity expression of many novel secreted human genes. To date, more than 75 new genes have been expressed, and the re-proteins purified. This expression system combines many favorable traits including relative speed, moderate cost but perhaps most importantly, the production of biologically active proteins.     

Calcium signaling in dendritic cells by human or mycobacterial Hsp70 is caused by contamination and is not required for Hsp70-mediated enhancement of cross-presentation

Extracellular heat shock proteins (HSPs) can stimulate antigen-specific immune responses. Using recombinant human (rhu)Hsp70, we previously demonstrated that through complex formation with exogenous antigenic peptides, rhuHsp70 can enhance cross-presentation by antigen-presenting cells (APCs) resulting in stronger T cell stimulation. T cell stimulatory activity has also been described for mycobacterial (myc)Hsp70. MycHsp70-assisted T cell activation has been reported to act through the binding of mycHsp70 to chemokine receptor 5 (CCR5), calcium signaling, phenotypic maturation, and cytokine secretion by dendritic cells (DCs). We report that highly purified rhuHsp70 and mycHsp70 proteins both strongly enhance cross-presentation of exogenous antigens. Augmentation of cross-presentation was seen for different APCs, irrespective of CCR5 expression. Moreover, neither of the purified Hsp70 proteins induced calcium signals in APCs. Instead, calcium signaling activity was found to be caused by contaminating nucleotides present in Hsp70 protein preparations. These results refute the hypothesis that mycHsp70 proteins require CCR5 expression and calcium signaling by APCs for enhanced antigen cross-presentation for T cell stimulation.     

Distinct restriction of complement- and cell-mediated lysis

Complement- and cell-mediated killing utilize related effector proteins (C8/C9 and perforin, respectively), suggesting that proteins which protect cells against complement- and cell-mediated attack may also be similar. In homologous complement-mediated killing two protective proteins, which are anchored to the cell membrane by phosphatidylinositol glycan (PIG) tails, are known. To study whether similar PIG-tailed proteins protect against lymphocyte-mediated killing, nucleated cell lines with a mutation in the biosynthesis of the PIG anchor were used. It was found that PIG-tailed membrane proteins restrict homologous complement-mediated lysis but not three different types of cell-mediated killing or lysis by purified perforin. Furthermore, E from patients with an acquired defect in PIG tail biosynthesis did not differ from normal E in sensitivity to antibody-dependent cell-mediated cytotoxicity, in spite of their increased sensitivity to human C8 and C9.     

Comparative toxicity of Bacillus thuringiensis var. israelensis crystal proteins in vivo and in vitro

Bacillus thuringiensis var. israelensis crystal proteins were purified by FPLC on a Mono Q column to yield 130, 65, 28, 53, 30-35 and 25 kDa proteins. All the purified proteins killed Aedes aegypti larvae after citrate precipitation, but the 65 kDa protein was the most toxic. A precipitated mixture of 27 and 130 kDa proteins was almost as toxic as solubilized crystals. In assays against a range of insect cell lines, the activated form (25 kDa) of the 27 kDa protein was generally cytotoxic with the lowest LC50 values in vitro. By contrast, the activated forms of the 130 kDa and 65 kDa protoxins (53 kDa and 30-35 kDa proteins, respectively) were much more specific than the 25 kDa protein in their action on dipteran cells, and each showed a unique toxicity profile which, in the case of the 130 kDa preparation, was restricted to Anopheles and Culex cell lines.     

Analysis of cell wall material from plant tissues: Extraction and purification

The alcohol-insoluble residue (AIR) of immature and mature runner beans contains co-precipitated cytoplasmic proteins, nucleic acids, starch and polyphenols, which contaminate the isolated polysaccharide fractions and their binding is sufficiently tenacious to resist complete extraction with the usual protein solubilizing reagents. Therefore, a method was developed for preparation of “cell wall material” from plant tissues in which the contamination with cytoplasmic constituents was minimal. Alternative solvents for cell disruption and protein extraction have been compared. The method depended for its success on the selective removal of the contaminants from fresh ball-milled tissue by sequential treatments with 1% aq. Na deoxycholate, PhOH-HOAc-H₂O followed by α-amylase digestion. Ball-milling the tissue ensured almost complete rupture of the cells and organelles and allowed the solvents to penetrate the sample fully and dissolve the cytoplasmic constituents. The purified “cell wall material” has protein contents varying from 2.5 to 5.5% depending on the type and maturity of the tissue. The residual proteins are resistant to pronase, rich in hydroxyproline and have the amino acid composition of purified cell wall proteins, showing that the wall preparations are relatively pure.     

Proteins Specified by Herpes Simplex Virus VI. Viral Proteins in the Plasma Membrane

Previous papers in the series have shown that the surface membranes of herpesvirus-infected cells acquire new immunological specificities and that purified infected cell membrane preparations, characterized by their physical properties rather than topology in the cell, contain new glycoproteins genetically determined by the virus. In this study, we prepared purified plasma membrane identified by its 5' nucleotidase, fucose, and reduced nicotinamide adenine dinucleotide-diaphorase content. Analysis of the membrane proteins and glycoproteins by electrophoresis in acrylamide gels indicated the following. (i) Purified plasma membranes from infected cells contained two sets of proteins, i.e., host proteins were present both before and after infection and viral proteins were present only after infection. (ii) After infection, no appreciable selective or nonselective loss of host proteins from membranes was demonstrable. However, no new host proteins were made. (iii) Electropherograms of plasma membrane proteins from infected cells indicated the presence of at least 12 virus-specific proteins ranging in molecular weight from 25 x 10(3) to 126 x 10(3) daltons. Of these, at least nine were glycosylated. Proteins and glycoproteins with similar electrophoretic mobilities but in somewhat different ratios were also present in preparations of highly purified virions.     

Quantification by affinity perfusion chromatography of phosphorylated BRCAl and BRCA2 proteins from tumor cells after lycopene treatment

A new procedure for the quantification of phosphorylated BRCA1 (P-BRCA1) and BRCA2 (P-BRCA2) proteins in breast cell lines after different treatments was carried out. Cells were cultivated with [35S]-methionine and extracts subjected to three perfusion chromatographies. First heparin affinity chromatography purified cellular DNA-binding proteins. Subsequent specific immunoprecipitation of BRCA1 and BRCA2 proteins was performed with antibodies raised against BRCA1 or BRCA2. The immune complexes were isolated by protein A affinity chromatography. Phosphorylated BRCA1 or BRCA2 proteins were then purified with a Poros 20 AL column where anti-phosphothreonine and anti-phosphoserine antibodies were previously bound. The percentage of phosphorylated BRCA1 or BRCA2 proteins was calculated as follows: 100 x dpm of P-BRCA1 or P-BRCA2 eluted from the POROS 20AL column/total dpm eluted from POROS 20AL column. Treatment with 10 microM lycopene increased P-BRCA1 and P-BRCA2 in the breast tumor cell line MCF7 but not in MDA-MB-231 or MCF-10a, breast tumor or fibrocystic cell lines, respectively.