Determination of membrane antigens by a covalent crosslinking method with monoclonal antibodies

Monoclonal antibodies that recognize cell surface proteins may serve as very useful tools for the study of the biological functions of membrane proteins. However, solubilization of the antigens with detergents may lead to major conformational changes of the protein, making their determination with monoclonal antibodies by immune blot or ordinary immunoprecipitation methods difficult. This is especially evident when the monoclonal antibodies recognize tertiary structures of the proteins in the membrane. We have generated two monoclonal antibodies which are specific for the cell surface antigens of multidrug-resistant human cell lines. However, the antigens of both monoclonal antibodies were difficult to detect by either immune blot or ordinary immunoprecipitation methods. We used a cleavable crosslinking reagent dithiobis(succinimidyl propionate) to covalently link the monoclonal antibody with its antigenic determinant in the membrane of intact cells. By this method, we were able to detect the antigens for these two monoclonal antibodies following solubilization, immunoprecipitation, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This method should have wide applicability in determination of membrane antigens recognized by monoclonal antibodies when immune blot or ordinary immunoprecipitation methods are not successful.     

Specific monoclonal antibodies against the surface of rat islet beta cells

Type 1 diabetes arises from the autoimmune destruction of islet beta cells, with the participation of both arms of the immune system. To better characterize the beta cell membrane, we have raised monoclonal antibodies to the surface of the INS-1 insulinoma cell line. Twenty-two such antibodies were produced, 21 of the IgG class, all reactive to different cell membrane proteins from INS-1 and neonatal islet cells, yielding identical electrophoresis patterns, with molecular weights mainly between 45 and 60 kD. We have focused on three such antibodies that recognize different protein targets, and are specific for islet beta cells. The target protein of antibody AA4, also found on monkey islets, is expressed at significantly higher levels on beta cells (55.8 vs 30.6% of cells, plus 3-4 fold increase in average fluorescence intensity per cell) when neonatal rat islet cells are incubated with high (16 mM vs 3mM) glucose concentrations. Further identification of the target antigens is in progress and is expected to shed more light on the properties of beta cell membrane proteins, and their probable participation in various disease processes.     

Tube-gel digestion: a novel proteomic approach for high throughput analysis of membrane proteins

This study describes a new protein digestion protocol in which a variety of detergents can be used to solubilize membrane proteins and facilitate trypsin digestion with higher efficiency. In this protocol, proteins are dissolved in solutions containing various detergents and directly incorporated into a polyacrylamide gel matrix without electrophoresis. Detergents are subsequently eliminated from the gel matrix while proteins are still immobilized in the gel matrix. After in-gel digestion of proteins, LC-MS/MS is used to analyze the extracted peptides for protein identification. The uniqueness of the protocol is that it allows usage of a variety of detergents in the starting solution without interfering with LC-MS/MS analysis. We hereby demonstrate that different detergents, including ionic SDS, non-ionic Triton X-100 and n-octyl beta-d-glucopyranoside, and zwitterionic CHAPS, can be used to achieve maximum solubilization of membrane proteins with minimal interference with LC-MS/MS analysis. Enhanced digestions, i.e. improved number and intensity of detected peptides, are also demonstrated for digestion-resistant proteins such as myoglobin, ubiquitin, and bacteriorhodopsin. An additional advantage of the Tube-Gel digestion protocol is that, even without electrophoresis separation, it allows high throughput analysis of complex protein mixtures when coupled with LC-MS/MS. The protocol was used to analyze a complex membrane protein mixture prepared from prostate cancer cells. The protocol involves only a single digestion and 2.5 h of LC-MS/MS analysis and identified 178 membrane proteins. In comparison, the same membrane fraction was resolved by SDS-PAGE, and 20 gel slices were excised and individually digested and analyzed by LC-MS/MS. The more elaborate effort demanded more than 50 h of LC-MS/MS analysis and identified 268 proteins. The new Tube-Gel digestion protocol is an alternative method for high throughput analysis of membrane proteins.     

Role of the cell surface-exposed regions of outer membrane protein PhoE of Escherichia coli K12 in the biogenesis of the protein

To investigate the role of the cell surface-exposed regions of outer membrane protein PhoE of Escherichia coli K12 in the biogenesis of the protein, deletions were generated in two presumed cell surface-exposed regions of the protein. Intact cells expressing these mutant proteins were recognized by PhoE-specific monoclonal antibodies, which recognize conformational epitopes on the cell surface-exposed parts of the protein and/or were sensitive to a PhoE-specific phage. This shows that the polypeptides were normally incorporated into the outer membrane. When the deletions extended four amino acid residues into the seventh presumed membrane-spanning segment, the polypeptides accumulated in the periplasm. In conclusion, exposed regions of PhoE protein apparently do not play an essential role in outer membrane localization, which is consistent with the observation that these regions are hypervariable when PhoE is compared to the related proteins OmpF and OmpC. In contrast, the membrane-spanning segments are essential for the assembly process.     

Molecular organization of glutamate-sensitive chemo-stimulated nerve cell membranes. Interaction of monoclonal antibodies with glutamate-binding membrane proteins in the rat brain, human neuroblastoma and molluscan neurons

Hybrid cells obtained by fusion of myeloma PX63-Ag8-653 with immune splenocytes of BALB/c mice were found to produce monoclonal antibodies with a high degree of specificity to rat and human brain. The kinetics of specific IgG binding to purified fractions of glutamate-binding membrane proteins from rat and human brain were analyzed in Scatchard plots. The presence of a single type of binding sites with Kd = 100 nM was demonstrated. The monoclonal antibodies were shown to inhibit the specific binding of tritium-labeled L-glutamate to different brain synaptic membranes. Addition of monoclonal antibodies to the incubation medium induced a modulating effect of physiological responses to L-glutamate in Planorbarius corneus neurons. The possible use of specific antibodies to glutamate-binding proteins as immunochemical markers for the study of glutamate receptor topography on membrane surface was demonstrated with the aid of neuroblastoma cells N18 Tg2a and rat brain tissue slices. An analysis of glutamate receptor binding sites with the use of monoclonal antibodies revealed that these antibodies specifically recognize the active center in the receptor molecules which have identical antigen determinant sites in different biological systems.     

Two different cell adhesion molecules--cell-CAM 105 and a calcium-dependent protein--occur on the surface of rat hepatocytes

We have recently identified a 105000 D plasma membrane glycoprotein, denoted cell-CAM 105 (CAM, cell adhesion molecule), that is involved in intercellular adhesion of reaggregating rat hepatocytes (Ocklind &; Öbrink, J biol chem 257 (1982) 6788 [11]). In this communication we identify another cell surface protein that is also involved in hepatocyte cell-cell adhesion. This protein has an apparent molecular weight (MW) of 70000 and can be released from the surface membrane by chelation of calcium with EGTA. Results are presented indicating that it is identical with a previously discovered protein, CDP-1 (CDP, calcium-dependent protein) (Öbrink, Lindström &; Svennung, FEBS lett 70 (1976) 28 [28]). Antisera produced against either cell-CAM 105 or CDP-1 inhibit hepatocyte aggregation, but not attachment to collagen. Cell-CAM 105 and CDP-1 are present on the cell surface as separate components, as judged by the fact that both EGTA treatment and trypsin treatment of hepatocytes selectively make the cells insensitive to blocking of aggregation by antibodies against CDP-1 but not by antibodies against cell-CAM 105. However, although much less efficiently, the antibodies against CDP-1 can recognize a 105000 D protein which is also bound by the antibodies against cell-CAM 105, and under certain conditions the antibodies against cell-CAM 105 seem to recognize a 70000 D protein. CDP-1 may thus be derived from cell-CAM 105, or the two proteins might have a common precursor.     

Relationship between plasma membrane mobility and substrate attachment in the crawling movement of spermatozoa from Caenorhabditis elegans

Caenorhabditis elegans sperm are nonflagellated cells that lack actin and myosin yet can form pseudopods to propel themselves over solid substrates. Surface-attached probes such as latex beads, lectins, and antimembrane protein monoclonal antibodies move rearward over the dorsal pseudopod surface of sessile cells. Using monoclonal antibodies against membrane proteins of C. elegans sperm to examine the role of localized membrane assembly and rearward flow in crawling movement, we determined that substrates prepared by coating glass with antimembrane protein antibodies, but not naked glass or other nonmembrane-binding proteins, promote sperm motility. Sperm locomotion is inhibited in a concentration-dependent fashion when cells are bathed with soluble antimembrane protein monoclonal antibodies but not with antimouse Ig antibodies or a monoclonal antibody against a sperm cytoplasmic protein. Our results suggest that C. elegans sperm crawl by gaining traction with substrate-attached ligands via their surface proteins and by using the motor that moves those proteins rearward on unattached cells to pull the entire cell forward. Continuous insertion of new proteins at the front of the cell and their subsequent adhesion to the substrate allows this process to continue.     

Trafficking of astrocytic vesicles in hippocampal slices

The increasingly appreciated role of astrocytes in neurophysiology dictates a thorough understanding of the mechanisms underlying the communication between astrocytes and neurons. In particular, the uptake and release of signaling substances into/from astrocytes is considered as crucial. The release of different gliotransmitters involves regulated exocytosis, consisting of the fusion between the vesicle and the plasma membranes. After fusion with the plasma membrane vesicles may be retrieved into the cytoplasm and may continue to recycle. To study the mobility implicated in the retrieval of secretory vesicles, these structures have been previously efficiently and specifically labeled in cultured astrocytes, by exposing live cells to primary and secondary antibodies. Since the vesicle labeling and the vesicle mobility properties may be an artifact of cell culture conditions, we here asked whether the retrieving exocytotic vesicles can be labeled in brain tissue slices and whether their mobility differs to that observed in cell cultures. We labeled astrocytic vesicles and recorded their mobility with two-photon microscopy in hippocampal slices from transgenic mice with fluorescently tagged astrocytes (GFP mice) and in wild-type mice with astrocytes labeled by Fluo4 fluorescence indicator. Glutamatergic vesicles and peptidergic granules were labeled by the anti-vesicular glutamate transporter 1 (vGlut1) and anti-atrial natriuretic peptide (ANP) antibodies, respectively. We report that the vesicle mobility parameters (velocity, maximal displacement and track length) recorded in astrocytes from tissue slices are similar to those reported previously in cultured astrocytes.     

Overexpressing human membrane proteins in stably transfected and clonal human embryonic kidney 293S cells

X-ray crystal structures of human membrane proteins, although potentially of extremely great impact, are highly underrepresented relative to those of prokaryotic membrane proteins. One key reason for this is that human membrane proteins can be difficult to express at a level, and at a quality, suitable for structural studies. This protocol describes the methods that we use to overexpress human membrane proteins from clonal human embryonic kidney 293 (HEK293S) cells lacking N-acetylglucosaminyltransferase I (GnTI(-)), and was recently used in our 2.1-? X-ray crystal structure determination of human RhCG. Upon identification of highly expressing cell lines, suspension cell cultures are scaled up in a facile manner either using spinner flasks or cellbag bioreactors, resulting in a final purified yield of ～0.5 mg of membrane protein per liter of medium. The protocol described here is reliable and cost effective, can be used to express proteins that would otherwise be toxic to mammalian cells and can be completed in 8-10 weeks.     

Generation and characterization of Clostridium septicum alpha toxin mutants and their use in diagnosing paroxysmal nocturnal hemoglobinuria

Glycosylphosphatidylinositol (GPI) anchors various proteins to the membrane of eukaryotic cells. Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell disorder that is primarily due to the lack of GPI-anchored proteins on the surface of blood cells. To detect the GPI-deficient cells in PNH patients, we modified alpha toxin, a pore-forming toxin of the Gram-positive bacterium Clostridium septicum. We first showed that aerolysin, a homologous toxin from Aeromonas hydrophila, bound to both of Chinese hamster ovary cells deficient of N-glycan maturation as well as GPI biosynthesis at a significant level. However, alpha toxin bound to the mutant cells of N-glycosylation, but not to GPI-deficient cells. It suggested that alpha toxin could be used as a specific probe to differentiate only GPI-deficient cells. As a diagnostic probe, alpha toxin must be the least cytotoxic while maintaining its affinity for GPI. Thus, we constructed several mutants. Of these, the mutants carrying the Y155G or S189C/S238C substitutions bound to GPI as well as the wild-type toxin. These mutants also efficiently underwent proteolytic activation and aggregated into oligomers on the cell surface, which are events that precede the formation of a pore in the host cell membrane, leading to cell death. Nevertheless, these mutants almost completely failed to kill host cells. It was revealed that the substitutions affect the events that follow oligomerization. The S189C/S238C mutant toxin differentiated GPI-deficient granulocyte and PMN, but not red blood cells, of a PNH patient from GPI-positive cells at least as sensitively as the commercial monoclonal antibodies that recognize the CD59 or CD55 GPI proteins on blood cells. Thus, this modified bacterial toxin can be employed instead of costly monoclonal antibodies to diagnose PNH patients.     

Optical sensors for monitoring dynamic changes of intracellular metabolite levels in mammalian cells

Knowledge of the in vivo levels, distribution and flux of ions and metabolites is crucial to our understanding of physiology in both healthy and diseased states. The quantitative analysis of the dynamics of ions and metabolites with subcellular resolution in vivo poses a major challenge for the analysis of metabolic processes. Genetically encoded F?rster resonance energy transfer (FRET) sensors can be used for real-time in vivo detection of metabolites. FRET sensor proteins, for example, for glucose, can be targeted genetically to any cellular compartment, or even to subdomains (e.g., a membrane surface), by adding signal sequences or fusing the sensors to specific proteins. The sensors can be used for analyses in individual mammalian cells in culture, in tissue slices and in intact organisms. Applications include gene discovery, high-throughput drug screens or systematic analysis of regulatory networks affecting uptake, efflux and metabolism. Quantitative analyses obtained with the help of FRET sensors for glucose or other ions and metabolites provide valuable data for modeling of flux. Here we provide a detailed protocol for monitoring glucose levels in the cytosol of mammalian cell cultures through the use of FRET glucose sensors; moreover, the protocol can be used for other ions and metabolites and for analyses in other organisms, as has been successfully demonstrated in bacteria, yeast and even intact plants. The whole procedure typically takes ～4 d including seeding and transfection of mammalian cells; the FRET-based analysis of transfected cells takes ～5 h.     

Live-cell assay to detect antigen-specific CD4+ T-cell responses by CD154 expression

This protocol details a method to identify CD4+ T cells that respond to antigens. The method relies on detection of CD154, a costimulatory cell surface protein that is expressed by CD4+ T cells upon activation, and can be used to purify live CD4+ T cells of diverse function. To detect CD154, fluorescently labeled antibodies are cultured with cell samples, peptides (or whole antigens) and monensin during a 6- to 24-h stimulation period. (Note that the assay is not compatible with brefeldin A.) After stimulation, cells are stained with any other antibodies of interest and then are analyzed by flow cytometry or purified by cell sorting. Unlike other assays, this method allows simultaneous assessment of other cell phenotypes or functions, is compatible with downstream RNA-based assays and preserves cell viability. This protocol can be completed in 9 h.     

Neuron-specific membrane glycoproteins promoting neurite fasciculation in Aplysia californica

We have generated a library of mouse monoclonal antibodies against membrane proteins of the nervous system of the marine snail Aplysia californica. Two of these antibodies, 4E8 and 3D9, recognize a group of membrane glycoproteins with molecular masses of 100-150 kD. We have called these proteins ap100, from the molecular mass of the most abundant species. Based on Western blots, these proteins appear to be specific for the nervous system. They are enriched in the neuropil of central nervous system ganglia, and are present on the surface of neurites and growth cones of neurons in culture. They are not expressed on the surface of nonneuronal cells. Staining of living cells with fluorescently labeled mAb demonstrates that the epitope(s) are on the outside of the cell. The antibodies against the proteins defasciculate growing axons and alter the morphology of growth cones, but affect much less adhesion between neuritic shafts. In addition, the level of expression of these molecules appears to correlate with the degree of fasciculation of neurites. These observations suggest that the ap100 proteins are cell adhesion molecules that play a role in axon growth in the nervous system of Aplysia. The fact that they are enriched in the neuropil and possibly in varicosities suggest that they may also be relevant for the structure of mature synapses.     

Mechanisms for quality control of misfolded transmembrane proteins

To prevent the accumulation of misfolded and aggregated proteins, the cell has developed a complex network of cellular quality control (QC) systems to recognize misfolded proteins and facilitate their refolding or degradation. The cell faces numerous obstacles when performing quality control on transmembrane proteins. Transmembrane proteins have domains on both sides of a membrane and QC systems in distinct compartments must coordinate to monitor the folding status of the protein. Additionally, transmembrane domains can have very complex organization and QC systems must be able to monitor the assembly of transmembrane domains in the membrane. In this review, we will discuss the QC systems involved in repair and degradation of misfolded transmembrane proteins. Also, we will elaborate on the factors that recognize folding defects of transmembrane domains and what happens when misfolded transmembrane proteins escape QC and aggregate. This article is part of a Special Issue entitled: Protein Folding in Membranes.     

Cytokines for the induction of antitumor effectors: The paradigm of Cytokine-Induced Killer (CIK) cells

Cytokine-Induced killer (CIK) cells are raising growing interest in cellular antitumor therapy, as they can be easily expanded with a straightforward and inexpensive protocol, and are safe requiring only GMP-grade cytokines to obtain very high amounts of cytotoxic cells. CIK cells do not need antigen-specific stimuli to be activated and proliferate, as they recognize and destroy tumor cells in an HLA-independent fashion through the engagement of NKG2D. In several preclinical studies and clinical trials, CIK cells showed a reduced alloreactivity compared to conventional T cells, even when challenged across HLA-barriers; only in a few patients, a mild GVHD occurred after treatment with allogeneic CIK cells. Additionally, their antitumor activity can be redirected and further improved with chimeric antigen receptors, clinical-grade monoclonal antibodies or immune checkpoint inhibitors. The evidence obtained from a growing body of literature support CIK cells as a very promising cell population for adoptive immunotherapy. In this review, all these aspects will be addressed with a particular emphasis on the role of the cytokines involved in CIK cell generation, expansion and functionalization.     

Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using affinity chromatography. While affinity chromatography using porous protein A agarose beads packed in columns is arguably the most common method of laboratory scale isolation of antibodies and recombinant proteins expressing Fc fragments of IgG, it can be a time consuming and expensive process. Time and financial constraints are especially daunting in small basic science labs that must recover hundreds of micrograms to milligram quantities of protein from dilute solutions, yet lack access to high pressure liquid delivery systems and/or personnel with expertise in bioseparations. Moreover, product quantification and characterization may also excessively lengthen processing time over several workdays and inflate expenses (consumables, wages, etc.). Therefore, a fast, inexpensive, yet effective protocol is needed for laboratory scale isolation and characterization of antibodies and other proteins possessing an Fc fragment. To this end, we have devised a protocol that can be completed by limited-experience technical staff in less than 9 hr (roughly one workday) and as quickly as 4 hr, as opposed to traditional methods that demand 20+ work hours. Most required equipment is readily available in standard biomedical science, biochemistry, and (bio)chemical engineering labs, and all reagents are commercially available. To demonstrate this protocol, representative results are presented in which chimeric murine galectin-1 fused to human Fc (Gal-1hFc) from cell culture supernatant was isolated using a protein A membrane adsorber. Purified Gal-1hFc was quantified using an expedited Western blotting analysis procedure and characterized using flow cytometry. The streamlined workflow can be modified for other Fc-expressing proteins, such as antibodies, and/or altered to incorporate alternative quantification and characterization methods.     

Cell receptors for baboon endogenous virus recognized by monoclonal antibodies.

Hybridomas from mice immunized with baboon endogenous virus (BaEV) from A204(M7) cells produced several antiviral monoclonal antibodies and, in addition, antibodies D-12 and E-4, which appeared to be virus specific because they reacted with BaEV but not with Mason-Pfizer virus or RD-114 virus. However, they also bound to human virus-free cells, and they did not recognize BaEV from bat or canine host cells. Cell membrane targets for these antibodies comigrated with an 18,000-dalton protein, which may contain specific determinants of BaEV receptors since antibody masking of these cell sites prevented BaEV but not Mason-Pfizer virus or RD-114 virus adsorption. However, RD-114 virus interfered with BaEV adsorption. Thus, the two viral receptors must be adjacent, but the antibody D-12 and E-4 targets are not within the active site of RD-114 virus receptor. Conversely, cell coating with BaEV from bat or canine hosts inhibited antibody D-12 binding. Noncultivated human lymphocytes and cells from fetal organs bound much less antibody D-12 than did cells from established cell lines, with a correlation between amounts of antibody D-12 acceptor sites and BaEV receptors. Thus, in vivo, BaEV infection of human cells may be inefficient. In vitro, antibody D-12 treatment of chronically infected A204(M7) cells caused intracellular accumulation of viral proteins and decreased virus release, with no such effect on RD-114 virus-producing cells. Canine cells bound antibody D-12 only if coated with BaEV from A204(M7) cells, indicating that the human determinant coadsorbed with the virions to animal cells. Possibly, determinants of cell receptors participate in BaEV maturation and become associated with the virions.     

A method for imaging single molecules at the plasma membrane of live cells within tissue slices

Recent advances in light microscopy allow individual biological macromolecules to be visualized in the plasma membrane and cytosol of live cells with nanometer precision and ∼10-ms time resolution. This allows new discoveries to be made because the location and kinetics of molecular interactions can be directly observed in situ without the inherent averaging of bulk measurements. To date, the majority of single-molecule imaging studies have been performed in either unicellular organisms or cultured, and often chemically fixed, mammalian cell lines. However, primary cell cultures and cell lines derived from multi-cellular organisms might exhibit different properties from cells in their native tissue environment, in particular regarding the structure and organization of the plasma membrane. Here, we describe a simple approach to image, localize, and track single fluorescently tagged membrane proteins in freshly prepared live tissue slices and demonstrate how this method can give information about the movement and localization of a G protein–coupled receptor in cardiac tissue slices. In principle, this experimental approach can be used to image the dynamics of single molecules at the plasma membrane of many different soft tissue samples and may be combined with other experimental techniques.     

Immunochemical characterization of the outer membrane complex of Serratia marcescens and identification of the antigens accessible to antibodies on the cell surface

Serratia marcescens New CDC O14:H12 contains major outer membrane proteins of 43.5 kDal, 42 kDal (the porins) and 38 kDal (the OmpA protein) which can be separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Immunoblotting of whole cell or outer membrane preparations using antiserum raised against the whole cells revealed similar complex patterns of antigens. The OmpA protein was the major immunogen, although six other outer membrane proteins were also detected; the porins reacted only weakly with antibodies in this system. Immunoabsorption of antisera with whole cells showed that only the O antigenic chains of lipopolysaccharide and the H (flagella) antigens were accessible to antibody on the cell surface. Failure to detect the OmpA protein and other envelope antigens in this way suggests that their antigenic sites are not able to react with antibodies and are possibly masked by the O antigen.     

Ex vivo time-lapse confocal imaging of the mouse embryo aorta

Time-lapse confocal microscopy of mouse embryo slices was developed to access and image the living aorta. In this paper, we explain how to label all hematopoietic and endothelial cells inside the intact mouse aorta with fluorescent directly labeled antibodies. Then we describe the technique to cut nonfixed labeled embryos into thick slices that are further imaged by time-lapse confocal imaging. This approach allows direct observation of the dynamic cell behavior in the living aorta, which was previously inaccessible because of its location deep inside the opaque mouse embryo. In particular, this approach is sensitive enough to allow the experimenter to witness the transition from endothelial cells into hematopoietic stem/progenitor cells in the aorta, the first site of hematopoietic stem cell generation during development. The protocol can be applied to observe other embryonic sites throughout mouse development. A complete experiment requires ～2 d of practical work.