The identification of the membrane proteins of human and animal viruses]

The review deals with the methods of identification of virus-specific proteins on virion and infected cell surface. The isotopic labeling of membrane proteins, their extraction by proteolytic enzymes and selective solubilization by detergents are considered. The plasmatic membrane isolation by centrifugation and by means of microcarriers is described. The methods of membrane protein localization using monoclonal antibodies and radioimmunoprecipitation are presented.     

Monoclonal antibodies that recognize lacto-N-fucopentaose III (CD15) react with the adhesion-promoting glycoprotein family (LFA-1/HMac-1/gp 150,95) and CR1 on human neutrophils

A variety of monoclonal antibodies has been used to study the roles of surface proteins in neutrophil function. Many monoclonal antibodies that bind to human neutrophils react with the oligosaccharide lacto-N-fucopentaose III. Sequential immunoprecipitation of radiolabeled proteins from extracts of neutrophils labeled at the cell surface with 125I, and partial proteolysis peptide mapping studies were used to compare the proteins recognized by several widely used monoclonal antibodies that react with human neutrophils. The monoclonal antibodies that react with lacto-N-fucopentaose III (CD15) immunoprecipitated five distinct neutrophil surface proteins. The data indicate that CD15 monoclonal antibodies react with a subset of the LFA-1/HMac-1/gp 150,95 glycoprotein family as well as with CR1 on human neutrophils. The CD15 antibodies studied differed in their avidities for these proteins. The molecules immunoprecipitated by the CD15 antibodies tested were more resistant to proteolysis than the homologous proteins immunoprecipitated by the other monoclonal antibodies studied that react directly with the alpha M (CD11) or beta (CD18) chains of the LFA-1/HMac-1/gp 150,95 glycoprotein family. Some of the differences in antibody reactivity and protease sensitivity of the membrane proteins recognized by these antibodies may be due to differences in glycosylation. The data suggest that the antibodies studied can detect differences in post-translational modification among copies of certain surface proteins.     

The isolation and purification of surface specific proteins of somatic and reproductive protoplasts of lily and rapeseed

The plasma membrane surface proteins of intact somatic (leaf) and reproductive (pollen, generative cell or sperm cell) protoplasts of lily ( Lilium longiflorum ) and rapeseed ( Brassica napus cv. Midas) were compared after probing with N-hydroxysuccinimido- (NHS) or sulfo-NHS-biotin. The plasma membranes of intact protoplasts are impermeable to these biotin probes, which bind covalently to the free amino groups of surface proteins. Enzyme-labelled streptavidin was used to detect membrane proteins after separation by SDS-PAGE and western blotting. In lily, six proteins specific to the surface membrane of leaf protoplasts were identified varying from 25–64 kDa, three proteins to pollen protoplasts in the range 35–64 kDa and two proteins to generative cell protoplasts, 63 and 67 kDa. In rapeseed leaf protoplasts, seven proteins in the range 22–69 kDa were detected, while in the sperm enriched fraction five proteins were present in the same kDa range. The proteins identified as membrane specific for generative cell protoplasts of lily have been isolated and were used as antigens for monoclonal antibody production. Preliminary results indicate the successful production of antibodies to surface antigens. These antibodies will be used to localise surface specific epitopes which are likely to be involved in cell-cell recognition at fertilization.     

A new approach to studies of cell-antibody interactions using fluorescent lipid probes

The interaction of poly- and monoclonal antibodies against the L-chain of human Ig with Burkitt lymphoma EB-3 cells was studied using a fluorescent lipid probe, anthrylvinyl-labelled sphingomyelin, incorporated into the cell plasma membrane. Binding of the antibodies to Ig receptors on the surface was shown to induce changes in the fluorescence polarization of the probe. The high sensitivity of the method allows one to detect less than 100 antibody molecules per cell. The possibility of using cells or liposomes carrying antigens and fluorescent lipids for the determination of antibodies in solution is discussed.     

Cross-linking of insulin receptors to MHC antigens in human B lymphocytes: evidence for selective molecular interactions

Molecular interactions between insulin receptors and MHC antigens were investigated in human B cells. Two B lymphoblastoid cell lines, IM-9 and 526, chosen for their high insulin binding capacity, were found to express 15,000 and 25,000 insulin receptors per cell, respectively. Insulin receptors were labeled with a 125I-photoreactive insulin analogue, and all other surface proteins by lactoperoxidase-catalyzed radioiodination. Neighbor proteins were cross-linked with a cleavable homobifunctional reagent dithio-bis-(succinimidyl propionate) (DSP) and solubilized before immunoprecipitation by anti-HLA monoclonal antibodies. Gel analysis of the precipitated proteins showed that 90% of insulin receptors precipitable by anti-insulin receptor antibodies were precipitated by anti-class I antibodies (anti-heavy chain and anti-beta 2-microglobulin) after cross-linking with 2 mM DSP. In neither IM-9- nor 526 cells could HLA antigens be precipitated by anti-insulin receptor antibodies, suggesting that the concentration of class I antigens largely exceeds the concentration of insulin receptors at the cell surface. In 526 lymphocytes, class I MHC antigens were also found to adjoin class II antigens, since both molecules could be coprecipitated with anti-HLA A, B, C and with anti-HLA-DR antibodies after chemical cross-linking. Down-regulation of insulin receptors by chronic exposure of IM-9 cells to insulin did not affect the amount of MHC molecules present on the cell surface, and conversely, class I MHC molecules were internalized in 526 cells irrespective of the presence of insulin. These results thus show that insulin receptors and MHC antigens form multimolecular complexes in the plasma membrane of cultured human B cells. These interactions, which do not appear to influence the regulation of these proteins on the cell surface, may be involved in the mechanism of hormone signaling.     

Surface and internal antigens of rat spermatozoa distinguished using monoclonal antibodies

Fluorescent antibody labeling techniques are frequently used to investigate the topography of antigens on spermatozoa. It is generally assumed that these procedures detect molecules only on the sperm surface but we now show that this assumption is not always valid. Using monoclonal antibodies that recognize either surface or internal antigens we demonstrate how spurious conclusions can be made, and we suggest simple procedures for assigning the position of an antigen to the cell surface or to an intracellular organelle. Antibodies against plasma membrane antigens should stain 100% of normal intact spermatozoa, but this proportion should be greatly reduced if the spermatozoa have previously been demembranated. If ? 100% of spermatozoa are stained but the proportion increases following permeabilization, then the possibility should be considered that the antigens are intracellular. We conclude that assignment of an antigen to a regional domain on the sperm surface using fluorescent antibody techniques should be validated by a demonstration that the antigen is actually located on the cell surface.     

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.     

Generation of monoclonal antibodies against chromosomal antigens that have a high sequence similarity between human and mouse

We raised monoclonal antibodies by immunizing mice with total chromosome proteins extracted from isolated human metaphase chromosomes. The indirect immunofluorescence screening of hybridoma cell lines provided 15 monoclonal antibodies against the chromosomal antigens. The antigen proteins of the mAbs were identified by immunoblotting as core histones or by immunoprecipitation followed by a peptide mass fingerprinting method as nuclear mitotic apparatus protein, heterogeneous nuclear ribonucleoprotein A2/B1, ribosomal protein S4, linker histone and beta-actin. During mitosis, localizations of these proteins on chromosomes were clearly observed using the obtained antibodies. These results indicate that the current strategy is effective for obtaining monoclonal antibodies useful for immunoblotting and/or immunofluorescent staining of human proteins, using the antigens with high homology to mouse proteins.     

Biochemical studies of the human thymocyte cell-surface antigens T6, T9 and T10

Three human thymic cell-surface antigens T6, T9 and T10, previously defined by monoclonal antibodies, were analyzed using immunoprecipitation techniques. The antigen T6 was found to be a 49,000 dalton glycoprotein, which is associated with β2-microglobulin, the small subunit (12,000 daltons) of the HLA-A, -B, and -C antigens. The target antigen for the monoclonal reagent anti-T9 was found to be a glycoprotein of 94,000 daltons, which appears as a disulfide-linked dimer of 190,000 daltons on the cell surface. The antigen precipitated by the anti-T10 antibody is a 45,000 dalton glycoprotein. We present preliminary evidence that all three cell-surface proteins may be integral membrane proteins. These findings, in addition to the distribution patterns, suggest that the T6 antigen is the human homolog of the murine thymus leukemia (TL) antigen.     

T-cell surface antigens defined by monoclonal antibodies, involved in the induction of human interferon-gamma and interleukin 2

Monoclonal antibodies specific for human T-cell-differentiation antigens were used to investigate the mechanism of induction of interferon-gamma (IFN-gamma) and interleukin 2 (IL-2). High levels of IFN-gamma, accompanied by IL-2 production, were detected in the lymphocyte cultures stimulated by pan T monoclonal antibodies that were mitogenic. These antibodies recognize an antigen complex Tp 19-29 (a complex of T-cell proteins of 19-29 kDa). However, it was possible to induce IL-2 without concomitant production of IFN-gamma using some antibodies specific for other T-cell surface antigens, e.g., Tp 32-45, Tp 41, and Tp 100-120. These antibodies were not mitogenic. The production of lymphokines, therefore, appears to be regulated at the cell surface by receptors or interaction molecules involved in cell triggering. Binding of antibodies to T3 receptor was obligatory for both IFN-gamma induction and mitogenesis but was not required in the induction of IL-2 activity.     

Monoclonal antibodies to cell surface antigens involved in sex pheromone induced mating in Streptococcus faecalis

Hybridoma cell lines producing monoclonal antibodies to Streptococcus faecalis cell surface antigens were constructed. Some of the antibodies isolated were directed against surface components involved in pheromone-induced mating. This paper describes the use of the monoclonal antibodies to identify antigenically related surface components detected by immunoprecipitation and Western blotting, their use in pheromone response assays, and their use as functional inhibitors in mating experiments.     

Structural characterization of developmentally expressed antigenic markers on chicken erythrocytes using monoclonal antibodies

Monoclonal antibodies selected for embryonic and adult erythrocyte specificity have been used to characterize developmentally expressed markers on the surfaces of mature circulating erythrocytes of young and adult chickens. The data presented demonstrate that the antigenic changes which occur on the avian erythrocyte membrane with organismic maturation can be accounted for, at least in part, by changes in the expression of structurally different, but possibly related polypeptides. Monoclonal antibodies selected for specific reactivity with the erythrocytes of newly hatched chicks recognize a glycoprotein of 48,000 daltons apparent molecular weight. On two-dimensional isoelectric focusing gels, this antigen, which appears identical in all strains studied, displays microheterogeneity; consisting of eight to nine closely spaced spots with an isoelectric midpoint of approximately 5.5. This antigen is not expressed on the circulating erythrocytes of mature birds; however, an antigen with similar, but perhaps not completely identical structure, can be detected within the adult bone marrow. The monoclonal antibodies which show preferential binding to the circulating erythrocytes of adult birds also immune precipitate an antigen of 48,000 daltons apparent molecular weight, but this antigen has a more basic isoelectric point. The adult antigen is polymorphic. Slightly different patterns were obtained on two-dimensional gels with erythrocytes from inbred birds having different major histocompatibility genotypes. It has a major component near pH 7.0 and additional focusing spots usually occurring at a slightly lower molecular weight near pH 6.8 or 6.6 depending upon strain. Competitive radiobinding assays with B-system-specific alloantisers suggest that these antigens may in fact be antigens of the polymorphic BG locus of the chicken major histocompatibility complex. One-dimensional peptide mapping of the immune precipitated embryonic and adult erythrocyte polypeptides demonstrate that the antigens are borne on distinct but possibly related polypeptides. Both common and unique peptide fragments are found in the digestion products. Selective solubilization of the chicken erythrocyte membrane suggests that the antigens are integral membrane proteins extractable with nonionic detergent but not with reagents which remove peripheral proteins.     

Shared carbohydrate epitopes on distinct surface and secreted antigens of the parasitic nematode Toxocara canis

The nematode parasite Toxocara canis is found in all dog populations and poses a poorly defined health hazard to humans. We have studied excretory-secretory antigen (ES) and surface antigens of the infective larval stage which is tissue-invasive in mammalian hosts. Antigens were probed with a panel of eight monoclonal antibodies raised in mice to whole ES. Six of eight antibodies reacted with periodate-sensitive carbohydrate epitopes on ES molecules, and the remaining two (Tcn-3 and Tcn-6) recognized either peptide or periodate-resistant sugar determinants. By immunoprecipitation and immunoblotting, the anti-carbohydrate monoclonals each reacted with several distinct ES molecules, known from previously published work to possess contrasting biochemical properties. Tcn-3 and -6 were directed predominantly against 32,000 and 120,000 m.w. molecules, respectively. Iodinated surface antigens of similar m.w. were precipitated by each antibody after detergent solubilization, but only two clones (Tcn-2 and -8) were able to bind exposed sites on the epicuticle of intact Toxocara larvae. Significantly, these antibodies do not bind to newly hatched larvae, and their target antigens are poorly expressed until the second day of in vitro cultivation. The specificities of the monoclonals were further studied by cold antibody inhibition of radiolabeled monoclonal binding, and by a matrix of two-site binding assays. These data show that Tcn-2, -4, -5, and -8 recognize a related group of repetitive carbohydrate epitopes, whereas Tcn-1, -6, and -7 bind discrete determinants on the same molecules. These studies are being continued to define further the structure of antigenic Toxocara carbohydrates and to compare the diagnostic utility of carbohydrate and peptide antigens.     

Adaptive surface antigen variation in Mycoplasma bovis to the host immune response

Abstract The variability of predominant Mycoplasma bovis surface antigens in the presence of specific immune pressure was analyzed in an in vitro assay to determine if M. bovis could escape immune destruction. We have shown that serum antibodies from immunized or experimentally infected calves and monoclonal antibodies which specifically react with previously characterized or as yet undefined major M. bovis membrane surface proteins cause repression of expression or shortening of the target protein, or induce switching to expression of an antigenically distinct variant protein. We have further demonstrated that removal of the inducing antibody results in reversion to the original phenotype. These results suggest that the level of expression and the length of M. bovis surface antigens in the host is modulated by cognate antibodies. According to the surface antigenic variation systems, random selection of preexisting variants resistant to antibody-mediated inhibition or direct regulation of gene expression may be means by which this organism evades host immune defences.     

Antigenic variability of the outer membrane antigens of Legionella pneumophila serogroups 1 to 8

The outer membrane proteins of Legionella pneumophila serogroups 1 to 8 were prepared from broken cells by selective solubilization using sodium lauryl sarcosinate. The isolated proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose sheets. Rabbit antisera against each of the eight serogroups of L. pneumophila were obtained by immunizing each animal with live bacteria. The transferred proteins were revealed using these antisera and peroxidase-labeled swine anti-rabbit immunoglobulins. Antigenic determinants common to all eight serogroups were found in at least three outer membrane antigens (19, 29, and 45 kilodaltons (kDa)). However, cross-absorption experiments revealed that these three antigens were immunologically related, but not identical among serogroups. The antigenic relationships observed with two of these three antigens correlated well with cross-reactions observed in immunofluorescence. When a monoclonal antibody directed against L. pneumophila serogroup 1 lipopolysaccharide was used to reveal a blot of serogroup 1 outer membrane antigens, the 29- and 45-kDa bands appeared. This demonstrates a strong association between lipopolysaccharide and outer membrane proteins.     

Molecular properties of ECMA 2 and ECMA 3 antigens defined by monoclonal antibodies against embryonal carcinoma cells

ECMA 2 and ECMA 3 antigens defined by two monoclonal antibodies are preferentially expressed in early embryonic cells of the mouse. The antigens were isolated from F9 embryonal carcinoma cells by detergent solubilization followed by indirect immunoprecipitation. Both antigens were glycoproteins, which, upon extensive pronase digestion, released the high-molecular-weight glycan (embryoglycan). The immunoprecipitation reactions were inhibited by the glycan, indicating that the two antigens were carried by it. Furthermore, binding of anti-ECMA 2 antibody to the glycan was directly demonstrated by a modified Farr's assay. The antigenic determinant of ECMA 2 antigen was found to involve an alpha-galactosyl residue, since alpha-galactosidase from coffee bean, but not other glycosidases, abolished the antigenic activity. Serological experiments indicated that ECMA 2 antigen is different from other alpha-galactosyl antigens, namely blood group B and P1 antigens and an antigen defined by antibodies in the sera of patients with ovarian germ cell tumors.     

Characterization of antigens recognized by new monoclonal antibodies raised against culture filtrate proteins of Mycobacterium bovis bacillus Calmette-Guérin

Effective protection against Mycobacterium tuberculosis may be achieved in experimental animals by immunization with proteins secreted by tuberculous bacilli in the extracellular milieu during growth. In this study, monoclonal antibodies were raised against Mycobacterium bovis bacillus Calmette-Guérin (BCG) culture filtrate proteins or live BCG, in an attempt to identify novel mycobacterial secretion antigens: the localization of the antigens recognized by the monoclonal antibodies within the mycobacterial cell was studied and interspecies reactivity was also investigated. The monoclonal antibodies obtained recognized proteins of molecular mass ranging from 5 to 82 kDa, with a prevailing frequency in the 30 kDa region. Three of the monoclonal antibodies recognized proteins present only in culture filtrates, one reacted with a cytoplasmic antigen, while the remaining antibodies recognized components which were mainly associated with the cell wall and the cytoplasmic membrane. The chemical nature and possible identity of the antigens was checked. Three monoclonal antibodies are likely to react with novel mycobacterial antigens of 5, 42 and 82 kDa, respectively.     

A duplex approach for immunochemical staining and typing of proteins in Western blots

The qualitative and semiquantitative Western blotting technique enables the detection of separate proteins and the determination of subtypes and fragments by specific immunological reactions. Protein typing on immunoblots is restricted to antibody-specific determination, with the result of a specific banding pattern. For protein characterization, several antibodies that recognize different epitopes within the protein sequence are used. However, repeated or parallel gel runs are needed. Here we describe a sequential determination of prion proteins in healthy and pathological states that both consist of di-, mono-, and nonglycosylated isoforms using a single blot with two antibodies from two species that recognize one antigen with two epitopes. The band signals are visualized by using different chemiluminescent substrate reactions. This application can be used in the fields of diagnostics and public health to detect full-length and fragmented proteins and can also be used for characterization of overlaying proteins.     

Generation and characterisation of rabbit monoclonal antibodies against the native cell surface antigens of embryonic stem cells

Embryonic stem (ES) cells are potent resources for cell therapy, and monoclonal antibodies (mAbs) against native cell surface markers of ES cells could be useful tools for therapeutic applications. Here, we report the development of a feasible approach, which could be used in mass production, for experimentally producing rabbit mAbs against native cell surface antigens on the cell surface. Two of the 14mAbs, which were selected at random, could be bound to the cell surface antigens of mES cells. The immunocytochemistry (ICC) and Western blot results showed that mAb 39 recognises conformational epitopes. The target antigen of mAb 39 was then successfully purified using an improved immunoprecipitation approach in which mAb was bounded to intact mES cells before the cells were lysed. The LC-LTQ mass spectrum analysis showed that the target antigen of mAb 39 was Glut3. This result was further confirmed by Western blot using commercially available antibodies against Glut3. Further experiments showed that mAb 39 exhibited an antiproliferative effect on mES cells. We also found that Glut3 was differentially expressed among the mES cell population as detected by flow cytometry.     

H-2 histocompatibility antigens of subcellular membranes of mouse liver

Purified fractions of plasma membrane, Golgi apparatus, rough endoplasmic reticulum vesicles, nuclear envelope, and mitochondria were isolated from mouse liver and the distribution of H-2 histocompatibility antigens determined by indirect radioimmunoassay before and after membrane disruptive treatments. Fractions enriched in plasma membrane (surface membrane) revealed H-2 antigens in highest concentration; disruptive treatments were not necessary to reveal H-2 antigens with surface membranes. In contrast, internal membranes did not possess H-2 antigens which were accessible to antibody. Golgi apparatus fractions or some component of these fractions (e.g. secretory vesicles) possessed the antigens but in a latent form where accessibility was provided by simple rupture of the membrane vesicles. With endoplasmic reticulum, detergent solubilization of the membranes was required before H-2 antigen could be detected. Nuclear envelope preparations contained little or no demonstrable H-2 activity. These results were confirmed by several techniques including immunoprecipitation of labelled solubilized membrane components with anti-H-2 serum and subsequent analysis in SDS-PAGE.