Detergent-solubilized Cell Extracts Research Articles

Antibodies 2.0

Antibodies 2.0

Erythropoietin producing cells for potential cell therapy

Anemia is an inevitable outcome of chronic renal failure due to the kidney's decreased ability to produce erythropoietin (EPO). We examined the feasibility of isolating and expanding EPO-producing cells for cell-based therapy. Renal cells from 7- to 10-day-old mice were culture-expanded. The cells at each subculture stage were characterized for EPO expression, using immunocytochemistry, FACS, and Western Blot analysis, with EPO-specific antibodies. To assess the levels of EPO expression, cells incubated under normoxic and hypoxic conditions were analyzed. Immunocytochemical analysis of the cultured renal cells expressed EPO at each subculture stage (P1-P3). Western Blot analysis of the detergent-solubilized cell extracts detected EPO (34 kDa) protein in the kidney cells of all passages tested. These results demonstrate that EPO-producing renal cells can be grown and expanded in culture. The cells stably expressed EPO at multiple subculture stages and they are able to form tissue in vivo. This study shows that EPO-producing cells may be used as a potential treatment option for anemia caused by chronic renal failure.

Multiple interactions between pullulanase secreton components involved in stabilization and cytoplasmic membrane association of PulE.

We report attempts to analyze interactions between components of the pullulanase (Pul) secreton (type II secretion machinery) from Klebsiella oxytoca encoded by a multiple-copy-number plasmid in Escherichia coli. Three of the 15 Pul proteins (B, H, and N) were found to be dispensable for pullulanase secretion. The following evidence leads us to propose that PulE, PulL, and PulM form a subcomplex with which PulC and PulG interact. The integral cytoplasmic membrane protein PulL prevented proteolysis and/or aggregation of PulE and mediated its association with the cytoplasmic membrane. The cytoplasmic, N-terminal domain of PulL interacted directly with PulE, and both PulC and PulM were required to prevent proteolysis of PulL. PulM and PulL could be cross-linked as a heterodimer whose formation in a strain producing the secreton required PulG. However, PulL and PulM produced alone could also be cross-linked in a 52-kDa complex, indicating that the secreton exerts subtle effects on the interaction between PulE and PulL. Antibodies against PulM coimmunoprecipitated PulL, PulC, and PulE from detergent-solubilized cell extracts, confirming the existence of a complex containing these four proteins. Overproduction of PulG, which blocks secretion, drastically reduced the cellular levels of PulC, PulE, PulL, and PulM as well as PulD (secretin), which probably interacts with PulC. The Pul secreton components E, F, G, I, J, K, L, and M could all be replaced by the corresponding components of the Out secretons of Erwinia chrysanthemi and Erwinia carotovora, showing that they do not play a role in secretory protein recognition and secretion specificity.

Purification and characterization of a human bradykinin binding protein from inflammatory cells

Bradykinin (BK) is a potent mediator with a broad spectrum of pharmacological and inflammatory actions which are exerted through cell surface receptors. We report here the affinity chromatographic purification of a novel 14 kDa BK binding protein from human blood neutrophils and also peripheral blood mononuclear cells (PBMC), 80% of which are lymphocytes. Radioreceptor crosslinking experiments using bifunctional crosslinkers and radiolabelled BK identified a 14 kDa protein in these cell types both on the cell surface, in glycerol purified plasma membranes and in detergent solubilized cell extracts. Purification by BK affinity chromatography from a variety of BK responsive human cell types i.e. CCD-16Lu lung fibroblasts, HL60 promyelocytes, U937 myelomonocytes and Jurkat T lymphocytes also demonstrated a 14 kDa protein. Purified material obtained from three different BK affinity columns all demonstrated three major proteins at 190, 50 and 14 kDa when eluted with either excess BK or mild acid. Neutrophil fractions from detergent solubilized cell extracts contained an additional 150 kDa protein when eluted with mild acid. Neutrophil and PBMC crude plasma membrane BK affinity column purifications yielded only a single 14 kDa protein. Radioreceptor dot assays of the purified neutrophil eluates containing the 14 kDa protein revealed specific binding to [ 125I]-BK with a 160 fold excess signal ratio over the original membrane extract. Our data indicates that we have successfully isolated a 14 kDa novel human BK specific binding protein expressed on the surface of inflammatory cells.

Discovery of Human Antibodies to Cell Surface Antigens by Capture Lift Screening of Phage-Expressed Antibody Libraries

An assay for the rapid identification and cloning of antibody fragments (Fabs) reactive with cell surface antigens was established and used to identify Fabs selectively reactive with tumor cell surface antigens. The Fabs were produced by a phage expression system and screened by a modified plaque lift approach in which nitrocellulose filters were coated with an anti-immunoglobulin reagent and blocked with bovine serum albumin prior to application to the phage-infected bacterial lawn. Subsequently, capture lifts were incubated with biotinylated antigen and reactive Fabs were identified with streptavidin conjugates. This screening method, termed capture lift, results in the immobilization of greater quantities of Fab and decreases the binding of unrelated host proteins, resulting in a more sensitive plaque lift assay. The capture lift permits the simulataneous analysis of thousands of antibody clones and, more importantly, can be used with crude detergent-solubilized cell extracts, permitting the discovery of Fabs which bind integral membrane proteins present in heterogeneous mixtures of antigens. Optimal conditions were identified utilizing phage-expressed BR96 Fab and a horseradish peroxidase conjugate of Lewis Y, a soluble cross-reactive antigen. Subsequently, it was demonstrated that the assay was functional with postnuclear detergent extracts isolated from surface-biotinylated tumor cells expressing the BR96 tumor antigen. Purification of the target antigen was not required. To demonstrate the application of the capture lift assay for the discovery of Fabs reactive with novel cell surface antigens a phage-expressed human antibody library constructed from tumor-infiltrating B lymphocytes was screened. Multiple antibody clones which reacted with detergent-solubilized biotinylated surface antigens were identified. Upon further characterization a portion of these displayed selectivity for tumor cells, as demonstrated by the binding of Fab to fixed and live tumor cells but not normal fibroblasts.

Mutational analyses of the extracellular domain of the full-length lutropin/choriogonadotropin receptor suggest leucine-rich repeats 1-6 are involved in hormone binding.

Previous studies have demonstrated that the amino-terminal extracellular domain of the lutropin/choriogonadotropin receptor (LHR) is sufficient for conferring high affinity binding and binding specificity. The present study was undertaken to further delineate those regions involved in hormone binding. Since the extracellular domains of the gonadotropin receptors are defined by multiple leucine-rich repeat motifs, LHR deletion mutants were constructed in which individual or multiple leucine-rich repeats were deleted from the full-length receptor. These mutants were transiently expressed in mammalian 293 cells, assayed for protein expression by Western blotting of cell extracts, and then tested for hormone binding in both intact cells and detergent-solubilized cell extracts. Western blot analyses confirmed the expression of all LHR deletion mutant proteins in the transfected cells. Although human (h) CG binding activity was not detected for any of the mutants when intact cells were assayed, mutants in which leucine-rich repeats 9-14 were collectively deleted, or repeats 7 or 8 were individually deleted, expressed binding activity when the cells were first solubilized in detergent. Of significance, rat (r) LHR(delta LRR 9-14) exhibited high affinity hCG and hLH binding, comparable to wild type rLHR. Detergent extracts from cells expressing rLHR(delta LRR 8) and rLHR(delta LRR 7) bound hCG and hLH, albeit with reduced affinities compared with the wild type receptor. All three of these deletion mutants, rLHR(delta LRR 9-14), rLHR(delta LRR 8), and rLHR(delta LRR 7), exhibited normal binding specificity as they did not bind hFSH even at very high concentrations. Thus, deletion of these regions in the carboxyl portion of the extracellular domain of the LHR did not remove any potentially inhibitory elements that would normally prevent hFSH binding. Deletion of either the 11 amino-terminal acids before LRR 1 or LRRs 1, 2, 3, 4, 5, or 6 individually from the full-length rLHR resulted in the total absence of detectable hCG binding activity in detergent-solubilized extracts, in spite of stable protein expression. These results suggest that the amino terminus and LRRs 1-6 are absolutely essential for gonadotropin binding to the rLHR.

Studies on the Reversibility of Protein S-Thiolation in Human Endothelial Cells

Exposure of human umbilical vein endothelial cells to oxidants, such as hydrogen peroxide and diamide, has been shown to induce protein-specific S-thiolation of cellular proteins. In this study we have now identified glutathione (reduced form) as the major low molecular-weight thiol that is bound to protein substrates in human umbilical vein endothelial (HUVE) cells during oxidative stress and investigated the dose- and time-response relationship of diamide- and hydrogen peroxide-induced S-thiolation of HUVE cell protein. Intact HUVE cells are able to rapidly reduce S-thiolated proteins with almost quantitative reappearance of reduced glutathione in the cells and protection from acute, lytic cytotoxicity. Additionally, studies were performed with detergent-solubilized cell extracts to determine the nature of the reductants operating in HUVE cells to maintain protein thiol homeostasis. The results clearly show the involvement of NADH- and NADPH-dependent systems. These data suggest that the reversible S-thiolation of proteins in these human endothelial cells may represent a significant cellular antioxidant and regulatory mechanism during oxidative stress.

Human keratinocytes are a major source of cutaneous platelet-derived growth factor.

PDGF has been implicated as one of the principal mitogens involved in cutaneous wound healing. While it has been previously reported that both platelets and monocytes are a source of PDGF in human dermal wound repair, the production of PDGF by human keratinocytes has not yet been described. In this manuscript, we report the production of PDGF by cultured human keratinocytes. Both PDGF A and B chain mRNA can be detected in cultured cells. While only PDGF-AA polypeptide is found in significant levels in keratinocyte-conditioned culture media, all three PDGF isoforms (AA, AB, and BB) are present in detergent-solubilized cell extracts. No evidence of PDGF receptor expression was observed in cultured keratinocytes when analyzed for either mRNA levels or polypeptide expression, suggesting that PDGF does not play an autocrine role in keratinocyte growth. Analysis of cryosections of human cutaneous wounds by immunostaining for PDGF showed that both PDGF A and B chain is constitutively expressed in normal epidermis, as well as in newly reconstituted wound epidermis. No evidence for PDGF receptor polypeptide expression in the epidermis was detected by immunostaining of cryosections.

Platelet-derived growth factor activates membrane-associated phosphatidylinositol 3-kinase and mediates its translocation from the cytosol. Detection of enzyme activity in detergent-solubilized cell extracts

Phosphatidylinositol 3-kinase (PI 3-kinase) activity has been detected in immune complexes with active protein tyrosine kinases, and its products have been measured in intact cells in response to growth stimuli. Both methods do not directly evaluate whole cell PI 3-kinase enzymatic activity. We have developed a sensitive method to measure PI 3-kinase activity in diluted, detergent-containing whole cell extracts and used this method to determine total, soluble, and membrane-associated PI 3-kinase activity in PDGF-stimulated NIH 3T3 fibroblasts. PDGF stimulation induced a 1.4-fold increase in total Nonidet P-40-extractable PI 3-kinase activity, which occurred within 1 min and was maintained above basal levels at 10 min. At the same time, PI 3-kinase activity in the soluble fraction decreased 30-50%. However, membrane-bound PI 3-kinase activity increased 2.4-fold at 1 min and 3.1-fold at 5 min. Translocation of the p85 PI 3-kinase subunit to the membrane was maximal at 10 min. These results suggest that PDGF-mediated activation of PI 3-kinase in membrane fraction results from initial intrinsic enzymatic activation followed by translocation from the cytosol.

Dimerization of internalized epidermal growth factor receptors.

Binding of epidermal growth factor (EGF) to cell surface EGF receptors initiates the formation of the receptor homodimers that can be detected by covalent cross-linking in intact cells or in detergent-solubilized cell extracts. Low pH dissociation of EGF from surface receptors results in immediate monomerization of receptor dimers. Using chemical cross-linking during mild permeabilization or cell solubilization, we have detected dimers of internalized EGF receptors in human carcinoma A-431 cells and transfected NIH 3T3 cells that express human EGF receptors. The percentage of internalized cross-linked receptor dimers was similar to that observed for surface EGF receptors. Furthermore, at the time of maximal accumulation of EGF-receptor complexes within the endosomal compartment (10-15 min of incubation at 37 degrees C), both the dimeric and monomeric forms of the EGF receptor are tyrosine-phosphorylated to the same extent as surface dimer and monomer species. In transfected NIH 3T3 cells, the level of dimerized and internalized kinase-negative EGF receptors was not different from that observed for wild-type receptors. These data suggest that for some time after internalization EGF does not dissociate from its receptor and indicate that a receptor conformation is preserved intracellularly that allows maintenance of receptor-receptor interactions and tyrosine kinase activity.

The IL-2 receptor beta-chain (p70). Ligand binding ability of the cDNA-encoding membrane and secreted forms.

The high affinity IL-2R is composed of at least two distinct subunits; alpha (p55 or Tac)- and beta (p70)-chains. Recent cDNA expression studies with lymphoid cells unequivocally demonstrated that the IL-2R beta is an indispensable component for the ligand internalization and the signal transduction via the high affinity IL-2R. Furthermore, these studies confirmed that the IL-2R beta singly expressed on T lymphoid cells represents the intermediate affinity IL-2R. In the present study, however, we show 1) the IL-2R beta expressed on a mouse fibroblast line, NIH-3T3, did not bind IL-2 under the intermediate affinity conditions, 2) nevertheless, the IL-2R beta coexpressed with the IL-2R alpha not only formed the high affinity receptor but also internalized IL-2. By the use of competitive sandwich ELISA to study the IL-2 binding ability of the IL-2R beta molecule itself, we also show that the IL-2R beta present in the detergent-solubilized cell extracts and the secreted IL-2R beta (p37) produced by the truncated cDNA are actually capable of binding IL-2, but with a much reduced affinity compared with the intact IL-2R beta expressed on the lymphoid cells. These findings lead us to postulate a more complex feature of the IL-2R than ever speculated; the IL-2R beta molecule having on its own minimal IL-2 binding ability requires a putative gamma-chain specifically present on lymphoid cells to generate the functional intermediate affinity IL-2R. The gamma-chain, however, seems not to be required as far as the formation of the high affinity IL-2R by the alpha- and beta-chains and the ligand internalization through it are concerned.

Export of Escherichia coli Alkaline Phosphatase Attached to an Integral Membrane Protein, SecY

The SecY protein is a membrane-bound factor required for bacterial protein export and embedded in the cytoplasmic membrane by its 10 transmembrane segments. We previously proposed a topology model for this protein by adapting the Manoil-Beckwith TnphoA approach, a genetic method to assign local disposition of a membrane protein from the enzymatic activity of the alkaline phosphatase (PhoA) mature sequence attached to the various regions. SecY-PhoA hybrid proteins with the PhoA domain exported to the periplasmic side of the membrane have been obtained at the five putative periplasmic domains of the SecY sequence. We now extended this method to apply it to follow export of the newly synthesized PhoA domain. Trypsin treatment of detergent-solubilized cell extracts digested the internalized (unfolded) PhoA domain but not those exported and correctly folded. One of the hybrid proteins was cleaved in vivo after export to the periplasm, providing a convenient indication for the export. Results of these analyses indicate that export of the PhoA domain attached to different periplasmic regions of SecY occurs rapidly and requires the normal functioning of the secY gene supplied in trans. Thus, this membrane protein with multiple transmembrane segments contains multiple export signals which can promote rapid and secY-dependent export of the PhoA mature sequence attached to the carboxyl-terminal sides.

Effect of phorbol esters on down-regulation and redistribution of cell surface receptors for tumor necrosis factor-alpha.

The effects of various phorbol esters on the interaction of human cells with recombinant human tumor necrosis factor-alpha (rTNF-alpha) was investigated. Preexposure of several different types of cells with only biologically active tumor promoter, i.e. 4 beta-phorbol 12-myristate 13-acetate (PMA), inhibited the specific binding of rTNF-alpha to its receptor. The reduction in specific binding of TNF-alpha was observed only by PMA but not with several other phorbol esters tested. 1-oleoyl-2-acetylglycerol, which is an analogue of the natural protein kinase C activator, diacylglycerol, was active in down-regulating TNF-alpha receptors but only at 1000 times concentration than PMA. Scatchard analysis of the binding data on U-937 cells revealed that PMA caused a decrease in high affinity cell surface receptor number (approximately 8300 versus approximately 2500 binding sites/cell) without any significant change in the dissociation constant (0.38 nM versus 0.32 nM). This decrease in receptor number is dependent on temperature, the time of exposure, and dose of PMA. Greater than 95% of the specific binding of 125I-TNF-alpha could be abolished within 10 min by preexposure of cells to 10 nM PMA at 37 degrees C. The down-regulation of receptors by PMA occurred only at 37 degrees C but not at 4 degrees C, suggesting a probable internalization of the receptors. The specific binding of TNF-alpha to detergent-solubilized cell extracts remained unchanged after exposure of cells to PMA. The rates of dissociation of TNF-alpha from the cell surface and the rate of internalization was not significantly affected by PMA, but the rate of disappearance from cell interior and its appearance into the medium was slightly enhanced by PMA. PMA did not alter the rate of degradation of the TNF-alpha nor cause the shedding of receptors into the medium. Approximately 70% of TNF-alpha cell surface receptors could be regenerated within 16 h after PMA removal. These results suggest the involvement of PMA-activated protein kinase C in down-regulation and redistribution of TNF-alpha receptors.

Monoclonal Antibodies to a Human Islet Cell Surface Glycoprotein: 4F2 and LC7-2*

Monoclonal antibodies 4F2 and LC7-2 react with a cell surface differentiation antigen expressed by the endocrine cells of the human pancreatic islet, but not by the acinar pancreatic, ductular, vascular, or stromal connective tissue cells. Western immunoblotting procedures demonstrate the reactivity of the monoclonal antibody 4F2 with a 120 kilodalton islet cell protein in detergent-solubilized cell extracts. These two monoclonal antibodies have potential for application in many aspects of islet cell research and diabetes in general.

Soluble interleukin 2 receptors are released from activated human lymphoid cells in vitro.

With the use of an enzyme-linked immunoabsorbent assay to measure soluble human interleukin 2 receptors (IL 2R), certain human T cell leukemia virus I (HTLV I)-positive T cell lines were found to spontaneously release large quantities of IL 2R into culture supernatants. This was not found with HTLV I-negative and IL 2 independent T cell lines, and only one of seven B cell-derived lines examined produced small amounts of IL 2R. In addition to this constitutive production of soluble IL 2R by certain cell lines, normal human peripheral blood mononuclear cells (PBMC) could be induced to release soluble IL 2R by plant lectins, the murine monoclonal antibody OKT3, tetanus toxoid, and allogeneic cells. Such activated cells also expressed cellular IL 2R measurable in detergent solubilized cell extracts. The generation of cellular and supernatant IL 2R was: dependent on cellular activation, rapid, radioresistant (3000 rad), and inhibited by cycloheximide treatment. NaDodSO4-polyacrylamide gel electrophoresis analysis of soluble IL 2R released from either the HTLV I-positive T cell line HUT 102B2 or normal phytohemagglutinin-activated PBMC demonstrated molecules of apparent Mr = 35,000 to 40,000, and 45,000 to 50,000, respectively, somewhat smaller than the mature surface receptor on these cells. The release of soluble IL 2R appears to be a characteristic marker of T lymphocyte activation and might serve an immunoregulatory function during both normal and abnormal cell growth and differentiation.

1012 SOLUBLE INTERLEUKIN-2 RECEPTORS (IL-2R) ARE PRODUCED BY ACTIVATED CORD BLOOD CELLS IN VITRO

The interaction the lymphokine IL-2 with its cellular receptor (IL-2R) plays a central role in the maturation and immunoregulation of the immune response. Using monoclonal antibodies to the human IL-2R, we have developed a quantitative assay to measure soluble IL-2R. To study the maturation of IL-2R expression, cord blood mononuclear cells (CBMC) were cultured in vitro under various conditions and the IL-2R in cell-free culture supernatants and in detergent solubilized cell extracts were measured. Cultures were stimulated with PHA or the monoclonal antibody OKT3 reacting with the T-cell antigen-receptor complex. Soluble IL-2R were first detected.in culture supernatants 48 hours following activation with either stimulus and the amount of receptor had increased roughly 10-fold by day 7 of culture. Supernatants of cells stimulated with PHA consistently contained 2-5 times more soluble IL-2R by day 7 than those stimulated with OKT3. The culture supernatants and solublized cell extracts of activated CBMC contained amounts of IL-2R comparable to similarly activated adult MNC. When stimulated with pokeweed mitogen, adult MNC secreted both IL-2R and IgM while CBMC secreted only IL-2R suggesting differential maturation and/or immunoregulation of the two responses. However cord blood cells are immunocompetent for IL-2R production in vitro. Soluble IL-2R may have an immunoregulatory role and abnormal levels of soluble IL-2R may also accompany cellular activation in vivo.

Repeated isolation of unique Qa2+ Ia+ clonally derived cell lines from Qa2- mice.

Qa2+ tumor cell lines were previously isolated from individual BALB/cBy (Qa2-) splenic lymphomas induced by murine sarcoma virus-murine leukemia virus-Moloney (MSV-MuLV-M). Two clonally derived cell lines, ORA I-a and Thorbly, and one noncloned cell line, BOMS, expressed Qa2, but neither Ly-1 nor Ly-2 were detected. In order to determine whether eight cloned and two noncloned tumor cell lines all represented a unique population of transformed cells, the presence of a series of surface differentiation antigens as studied. In addition to Qa2, each cell line examined expressed IAd, IEd, H-2Kd, H-2Dd, and receptors for C3b and the Fc portion of immunoglobulin (Ig). Neither Thy-1.2 nor Ig were detected on the cell surfaces, and cytoplasmic Ig was not precipitated from metabolically radiolabeled and detergent-solubilized cell extracts. However, monocyte specific alpha-napththyl acetate esterase-containing granules were present in all cell lines examined. Therefore, a unique Qa2+ monocytic cell is repeatedly isolated after chronic MSV-MuLV-M infection. Further analysis of these cells may provide insight into both the regulation of Qa2 expression and the interactions between monocytes and lymphocytes during leukemogenesis.

Serological and biochemical characterization of rat (RT1) class II molecules with restricted mouse anti-Ia sera.

Murine anti-Ia sera were tested for their reactivity with rat peripheral lymphocytes. Extensive cross-reactions were observed in a complement-dependent cytotoxicity assay. In the majority of strain combinations, these reactions were specific for B cells. We demonstrated by absorption analyses that (1) the reaction was specific, (2) B cells from all rat strains reacted with anti-I-Ek serum, and (3) the anti-I-A sera defined five new specificities. These antisera can also immunoprecipitate class II (Ia-like) molecules from radiolabeled, detergent-solubilized cell extracts. The rat equivalents of molecules encoded in the I-A and I-E subregions in mice can be isolated from all rat strains studied. The alpha and beta subunits of the rat class II molecules were polymorphic when analyzed by sodium dodecyl sulfate (SDS) polyacrylamide electrophoresis. This observation allows the characterization of rat alloantisera or monoclonals of unknown specificity by comparing them to the defined reactivity of the mouse anti-Ia sera.