Intergenic and interallelic exclusion inParamecium primaurelia: Immunological comparisons between allelic and non-allelic surface antigens

InParamecium, the expression of surface antigens is regulated in such a way that only one is generally present at the cell surface under given environmental conditions. Previous analyses have indicated that the surface antigen molecules play a key role in the control of their own expression. In order to characterize the structural particularities displayed by both allelic and non-allelic surface antigen molecules, immunological; comparisons were performed in vivo and in immunodiffusion on nine G and six D allelic surface antigens inParamecium primaurelia.Our results show: (1) it is possible to distinguish two regions in the surface antigen molecule; one accessible to antibodies in vivo, carrying specific immobilization determinants, the other not accessible to antibodies in vivo, carrying common determinants shared by all the antigens of the same allelic series. Antigens coded by different loci differ in both regions. (2) The specificity of immobilization determinants is not borne by a hypothetical carbohydrate component of the molecule but by the polypeptide chain itself. (3) In heterozygotes displaying allelic exclusion the parental surface antigen phenotypically excluded in vivo at the cell surface is not present in the cytoplasm. These data permit some interpretations concerning the mechanisms of intergenic and interallelic exclusion, on the basis of the structural differences between the different surface antigens.I thank Doctors M. Weiss and L. Sperling very much for improvements to the English.     

Immunological evidence of a common structure between Paramecium surface antigens and Trypanosoma variant surface glycoproteins

The surface antigens (SAgs) of Paramecium and the variant surface antigens (VSGs) of Trypanosoma can be purified in two distinct molecular forms: a soluble form (solubilized in dilute ethanolic solution in the case of Paramecium, or in water for Trypanosoma) and a membranal form, amphiphile (solubilized in SDS). In trypanosomes, the enzymatic conversion of the membrane form into the soluble form is accompanied by the unmasking of a particular immunological determinant, called cross-reacting determinant (CRD), which is located in the COOH-terminal phospho-ethanolamine glycopeptide. We demonstrate immunological homologies between Paramecium SAgs and Trypanosoma VSGs. A determinant corresponding to the CRD of VSGs is borne by the ethanol-soluble form of the SAgs and by two cross-reacting light chains also present in ethanolic cellular extracts (together with the soluble form), and not by the membranal form of SAgs. Furthermore, we show that the membranal form of Paramecium SAgs can be converted into soluble form and that this enzymatic conversion also yields cross-reacting light chains. We also demonstrate that the membranal form is the physiological form in paramecia stably expressing a given SAg.     

Virus-Specific Antigens in Hamster Cells Transformed by Rous Sarcoma Virus

Virus-specific antigens were studied in hamster cells transformed by Rous sarcoma virus (RSV). Antigens were localized in the cytoplasm, as demonstrated by fluorescent antibody staining of fixed cells as well as by complement fixation (CF) following subcellular fractionation. Cytoplasmic extracts were analyzed by velocity and isopycnic centrifugation. CF antigens were found in a soluble form and in association with membranes and polyribosomes. Isolated plasma membranes had no CF antigen. Both soluble and particulate fractions with CF activity contained the same antigenic determinants by Ouchterlony analysis. These antigenic determinants were identical to those released by ether treatment of RSV.     

Membrane-bound and water-soluble nonclassical class I MHC antigens on rat placenta

We have used mouse monoclonal antibodies to different determinants on rat class I major histocompatibility complex (MHC) antgiens in order to identify water-soluble and membrane-bound nonclassical (i.e., non-RT1.A) class I MHC antigens on the spongiotrophoblast and labyrinthine trophoblast of rat placenta. Initial immunohistological studies with monoclonal antibodies reacting with determinant restricted to classical (RT1.A) rat class I antigens confirmed the presence of these antigens on spongiothrophoblast, but not on labyrinthine trphoblast. Staining with another monoclonal antibody, which reacts with both classical and at least some nonclassical rat class I antigens, gave strong staining of both the labyrinthine and spongiotrophoblast. To distinguish membrane-bound from water-soluble class I molecules, quantitative adsorption analyses were carried out using both placental cell membranes and ultracentrifuged aqueous extracts of placenta. The aqueous placental extract had no absorptive capacity for the RT1.A-specific antibodies, but it had very strong absorptive capacity for the more broadly reactive antibody. This strongly suggests the presence of large quantities of a soluble nonclassical class I MHC antigen in rat placenta. The placental cell membranes had four to fivefold greater absorptive capacity for the broadly reactive antibody when compared to the antibodies to classical class I antigens, a result that was consistent with the presence of membrane-bound non-classical class I antigens on rat placenta. The membrane-bound nonclassical class I antigen was purified from detergent extracts of DA rat placental membranes using monoclonal antibody affinity and lentil lectin affinity chromatography. The putative nonclassical class I antigen had a heavy chain of M _r 43 000, which is 2000 smaller than the amino acid sequence analysis demonstrated that the nonclassical placental antigen differed at three amino acid residues from the classical RT1.A class I molecule and also from the Q10-like class I molecule of the DA strain. It differed also from the pAR 1.5 cDNA sequence, the only full-length rat class I DNA sequence available so far. Address correspondence and offprint requests to: J. Fabre.     

The membrane-anchor of Paramecium temperature-specific surface antigens is a glycosylinositol phospholipid

The temperature-specific G surface antigen of Paramecium primaurelia strain 156 was biosynthetically labeled by [3H]myristic acid in its membrane-bound form, but not in its soluble form. It could be cleaved by a phosphatidylinositol-specific phospholipase C from Trypanosoma brucei or from Bacillus cereus which released its soluble form with the unmasking of a particular glycosidic immunodeterminant called the crossreacting determinant. The Paramecium enzyme, capable of converting its membrane-bound form into the soluble one, was inhibited by a sulphydril reagent in the same way as the trypanosomal lipase. From this evidence we propose that the Paramecium temperature-specific surface antigens are anchored in the plasma membrane via a glycophospholipid, and that an endogenous phospholipase C may be involved in the antigenic variation process.     

Immunochemical examination of soluble antigens in the serum of Balb/c mice infected with Rauscher leukaemia virus.

The serum of Balb/c mice infected with Rauscher leukaemia virus contained soluble antigens characterized by alpha2 and beta globulin electrophoretic mobility; their respective molecular weights, as determined with gel filtration, were 40 000 and 120 000. The antigens differed in specificity and their corresponding determinants were present on the surface of leukaemic cells. For Balb/c mice both antigens, for DBA/1 and C57B1/10Sn mice only the antigen showing alpha2 mobility was immunogenic.     

Protective and other biological properties of Bacillus anthracis soluble antigens

The soluble antigens were explored of the culture filtrate (CF) derived during static growth of B. anthracis vaccine strain 34F2 on a medium containing casein hydrolysate. Electrofocusing of CF preparations revealed that the protective activity was distributed over a wide range of pH 3-7. The most pronounced and stable protective activity was observed at pH 4.6-4.8. Following toxin factors were isolated and identified: protective antigen (87 kD), oedema factor (87 kD) and lethal factor (78-81 kD). The greatest protective activity was associated with antigens characterized by a molecular weight of 78-87 kD and toxic activity. Preparations of the oedema and lethal factors had the same protective activity as protective antigen (PA) preparations. Other CF soluble antigens protected about 30% of immunized guinea pigs. A protein was isolated with a molecular weight of 80 kD and isoelectric point at pH 5.3-5.7 which was not toxic and did not form toxic mixtures in association with other toxin factors; this protein featured a high immunogenic activity, however, it protected only 31% of immunized animals. Factors are analyzed which determine differences in the protective effects of live and chemical vaccines.     

A single gene encodes soluble and membrane-bound forms of the major histocompatibility Qa-2 antigen: anchoring of the product by a phospholipid tail

The H-2, Qa, and Tla genes of the murine major histocompatibility complex are related to each other by DNA sequence homology. The H-2 genes encode ubiquitously expressed transplantation antigens that serve as recognition structures for cytotoxic T cells. The identities of the Qa and Tla products, their sites of expression, and their functions are largely unknown. We report here that the Qa region gene Q7 encodes a membrane-bound as well as a secreted form of the serologically defined antigen Qa-2. The Q7 gene introduced into liver-derived cells is expressed as a membrane-bound and as a secreted molecule. In transfected L cells it is expressed only as a soluble protein. Biochemical analysis suggests that the Q7 product is anchored to the liver cell membranes by a phospholipid tail. This feature may be responsible for cell type-specific expression of the two forms of the Qa-2 molecules.     

The release of membrane antigens into culture by adult Schistosoma mansoni.

Antigens sharing determinants with surface membranes and soluble proteins of adult Schistosoma mansoni have been detected in culture media after incubation of radioactively labelled worms. The relative quantities of these antigens were measured with specific antisera raised in rabbits and with serum from an immune rhesus monkey. It was found that 12-16% of TCA-precipitable radioactivity in the culture medium consisted of membrane antigens and 6-8% consisted of antigens sharing determinants with proteins found in the soluble fraction of adult worms. Over half the membrane antigens were present in particulate form, while other antigens were present in solution. Surface labelling the adult worms with [125I]confirmed that some of the particles in the culture medium were derived from the surface membrane of the adult worm and electron microscope examination of such particles showed that large membrane fragments were present. These results support the hypothesis that antibodies against schistosome membrane antigens are induced by particulate membrane antigens released by the parasite.     

Subcellular distribution of calmodulin and calmodulin-binding sites in Tetrahymena pyriformis

The subcellular distribution of calmodulin and particulate calmodulin-binding activity was studied in a eukaryotic protozoan, Tetrahymena pyriformis NT-1. The particulate calmodulin-binding activity was found to be localized principally in microsomes and to some extent in cilia and surface membranes called pellicles. Nearly all (93%) of the total amount of calmodulin was recovered in two soluble compartments, the ciliary and postmicrosomal supernatant fractions.     

Antigen processing requirements for T cell activation: differential requirements for presentation of soluble conventional antigen vs cell surface MHC determinants

The present studies were undertaken to characterize the antigen-processing requirements involved in the responses to T cells to soluble antigen (antigen specific), to allogeneic cell surface MHC determinants (alloreactive), and to syngeneic MHC determinants (autoreactive). T cell clones were used that have dual cross-reactive specificities either 1) for self MHC plus soluble antigen and for allogeneic MHC products or 2) for syngeneic MHC and for allogeneic MHC, in order to permit comparison of the processing requirements for responses of the same T cell to distinct antigenic stimuli. The proliferative responses of antigen-specific, Ia-restricted T cell clones to soluble antigens were sensitive to treatment of antigen-presenting cells (APC) with 125 to 250 microM chloroquine, a lysosomotropic agent previously shown to inhibit the processing of soluble antigens. In contrast, the same T cell clones were only minimally affected in their ability to respond to similarly chloroquine-treated APC expressing allogeneic MHC products. The responses of autoreactive T cell clones to syngeneic stimulating cells and their cross-reactive responses to allogeneic cells were both resistant to chloroquine treatment of stimulating cells. The failure of chloroquine to inhibit antigen presentation to autoreactive T cell clones suggests that these clones are specific for self Ia not associated with in vitro processed foreign antigen. Thus, chloroquine sensitivity distinguishes the in vitro antigen-processing requirements for presentation of the soluble antigens tested from the requirements for presentation of syngeneic or allogeneic cell surface MHC determinants to the same T cells.     

Monoclonal antibodies for carcinoembryonic antigen (CEA) as a model system: identification of two novel CEA-related antigens in meconium and colorectal carcinoma tissue by Western blots and differential immunoaffinity chromatography

In a previous study, five monoclonal antibodies against the carcinoembryonic antigen (CEA) with different epitope specificities were delineated. One of these antibodies which exhibits a high affinity for CEA binds to different carcinoma tissues, to liver tissue, and to granulocytes. This antibody was selected for the immunoaffinity purification of CEA and related antigens from colorectal carcinoma tissue, from spleen tissues, from bile, and from meconium. After elution from the immunosorbent, the antigens were separated by SDS-PAGE, were transferred to nitrocellulose, and were incubated with the five different antibodies. Antibody T84.1 bound to the following antigens: 177 kD and 128 kD from colonic carcinoma, 81 kD from bile, 49 kD from spleen, as well as 165 kD and 100 kD from meconium. Two additional antibodies showed a similar binding pattern. The fourth antibody (CEA.11) bound to the 165 kD meconium antigen and to the two colorectal carcinoma antigens. The fifth antibody (T84.66) showed a strong reaction with the 177 kD colorectal carcinoma antigen and a faint reaction with a 183 kD antigen in meconium. As judged from m.w. and immunochemical properties, the 128 kD colorectal carcinoma antigen and the 100 kD meconium antigen are two novel CEA-related antigens. Because antibody CEA.11 did not bind to the 100 kD meconium antigen in Western blots, the 165 kD antigen could be eluted from a CEA.11 immunosorbent without contamination by the 100 kD antigen. Similarly, as predicted from the binding pattern in the Western blots, the two colorectal carcinoma antigens were separated from each other by a T84.66 immunosorbent.     

Biochemical studies of the excitable membrane of paramecium tetraurelia. IX. Antibodies against ciliary membrane proteins

The excitable ciliary membrane of Paramecium regulates the direction of the ciliary beat, and thereby the swimming behavior of this organism. One approach to the problem of identifying the molecular components of the excitable membrane is to use antibodies as probes of function. We produced rabbit antisera against isolated ciliary membranes and against partially purified immobilization antigens derived from three serotypes (A, B, and H), and used these antisera as reagents to explore the role of specific membrane proteins in the immobilization reaction and in behavior. The immobilization characteristics and serotype cross- reactivities of the antisera were examined. We identified the antigens recognized by these sera using immunodiffusion and immunoprecipitation with 35S-labeled ciliary membranes. The major antigen recognized in homologous combinations of antigen-antiserum is the immobilization antigen (i-antigen), approximately 250,000 mol wt. Several secondary antigens, including a family of polypeptides of 42,000-45,000 mol wt, are common to the membranes of serotypes A, B, and H, and antibodies against these secondary antigens can apparently immobilize cells. This characterization of antiserum specificity has provided the basis for our studies on the effects of the antibodies on electrophysiological properties of cells and electron microscopic localization studies, which are reported in the accompanying paper. We have also used these antibodies to study the mechanism of cell immobilization by antibodies against the i-antigen. Monovalent fragments (Fab) against purified i- antigens bound to, but did not immobilize, living cells. Subsequent addition of goat anti-Fab antibodies caused immediate immobilization, presumably by cross-linking Fab fragments already bound to the surface. We conclude that antigen-antibody interaction per se is not sufficient for immobilization, and that antibody bivalency, which allows antigen cross-linking, is essential.     

Surface K antigen in Salmonella paratyphi B.

It was established by agar immunoelectrophoresis that Salmonella paratyphi B lysate contains a large number of soluble antigens which display a varying degree of serological specifity as well as different diffusion and electrophoretic mobility. Salmonella paratyphi B was found to possess, apart from specific O and H antigens, a surface K antigen. This is a distinct antigen having strict serological specificity. Purified K antigen displayed anodic mobility in immunoelectrophoresis. A detailed study of K antigen properties in cultures treated by different methods as well as immunochemical investigations of purified K antigen showed that the surface K antigen of S. paratyphi B differs from its O, M, Vi, H and other known antigens in terms of basic characteristics.     

Identification of mobile and fixed antigens on the plasma membrane of rat spermatozoa using monoclonal antibodies

We have used monoclonal antibodies to study the mobility and distribution of three different antigens on the cell surface of rat spermatozoa. We classified two of the antigens (designated 2B1 and 2D6) as 'mobile', since when detected by indirect immunofluorescence they were situated over the entire sperm flagellum and were susceptible to antibody-induced patching. Patching was critically dependent upon antibody concentrations and was much reduced at 4 degrees C. Patching of the 2B1 antigen was not induced by the 2B1 monoclonal antibody alone. Thus, 2B1 antibody labelled directly with fluorescein bound with a uniform distribution over the sperm flagellum, but this uniform fluorescence was made patchy on subsequent incubation in an unlabelled second antibody layer of anti-mouse IgG anti-serum. By 'Western blotting', the 2B1 antigen was found to be located to a 40 kD molecular weight polypeptide. The remaining 'fixed' antigen (designated 1B6) was not susceptible to antibody-induced patching, and was restricted to a discrete domain on the post-acrosomal region of the sperm surface. We discuss the relationship between mobility of sperm surface antigens and their segregation to discrete domains on the plasma membrane.     

Ichthyophthirius multifiliis Has Membrane-Associated Immobilization Antigens

Sera from fish that survive infections with the ciliated protozoon, Ichthyophthirius multifiliis , immobilize the parasite in vitro. In order to identify ceil surface antigens involved in the immobilization response, integral membrane proteins were extracted from tomites with Triton X-l14 and used to immunize rabbits. The rabbit antisera immobilized the parasite in vitro and antigens were localized to cell and ciliary plasma membranes by indirect immunofluorescent microscopy. The membrane protein fractions from both whole cells and tomite cilia were characterized by 1 - and 2-dimensiona! SDS-PAGE. A 43,000-dalton (D) glycoprotein with an isoelectric point of 7.0 is the predominant protein in these fractions, comprising 12% and 60% of the total protein of whole cell and ciliary membranes, respectively. Western blot analysis of ciliary proteins with immune rabbit sera indicated that the 43,000-D glycoprotein is the principal antigen.     

Icthyophthirius multifiliis has membrane-associated immobilization antigens

Sera from fish that survive infections with the ciliated protozoon, Ichthyophthirius multifiliis, immobilize the parasite in vitro. In order to identify cell surface antigens involved in the immobilization response, integral membrane proteins were extracted from tomites with Triton X-114 and used to immunize rabbits. The rabbit antisera immobilized the parasite in vitro and antigens were localized to cell and ciliary plasma membranes by indirect immunofluorescent microscopy. The membrane protein fractions from both whole cells and tomite cilia were characterized by 1- and 2-dimensional SDS-PAGE. A 43,000-dalton (D) glycoprotein with an isoelectric point of 7.0 is the predominant protein in these fractions, comprising 12% and 60% of the total protein of whole cell and ciliary membranes, respectively. Western blot analysis of ciliary proteins with immune rabbit sera indicated that the 43,000-D glycoprotein is the principal antigen.     

Expression of differentiation and age-related antigens on chicken erythroleukemia cells transformed by avian erythroblastosis virus (AEV)

Immature circulating chicken red cells express on their surface two antigenic molecules referred to as Im 48 kD and Im 140 kD antigens. The Im 140 kD antigen is not present beyond the erythroblast stage while the expression of Im 48 kD antigenic molecule remains detectable on circulating erythrocytes of embryos and young chickens, but not on erythrocytes of adult animals. In addition to Im 48 kD and Im 140 kD antigens, the avian erythroblastosis virus (AEV)-transformed erythroid cells express two novel high molecular weight (MW) immature antigens referred to as Im 150 kD and Im 160 kD. Since the transformed erythroid cells are apparently blocked at a stage close to the colony-forming units erythrocytic (CFU-E), these molecules might be expressed on these progenitor cells. The age-related antigenic molecules referred to as E1 48 kD and A 40 kD/A 85 kD antigens are detected on erythrocytes of embryos (and young chickens) and adult animals respectively. The E1 48 kD antigen as well as an antigen related to the A 40 kD were also detected on AEV-transformed erythroid cells deriving from both young chicken bone marrow and yolk sac. The presence of an adult antigen on the embryonic cells might well be related to the transformation by AEV, since the yolk sac CFU-E progenitor cells do not bear the adult antigenicity.     

Analysis of the Soluble and Membrane-bound Immobilization Antigens of Ichthyophthirius multifiliis

ABSTRACT. The immobilization antigens (i-antigens) are a class of highly abundant surface membrane proteins found on a number of holotrich ciliates. In Ichthyophthirius multifiliis (an obligate parasite of fish) these antigens appear to be targets of the host immune response. While the i-antigens of Ichthyophthirius are predominantly membrane-associated proteins, we now find that they are released into the water surrounding the parasite in a highly enriched form. The membrane-associated and water soluble proteins appear indistinguishable by antigenic means, as well as by several biochemical criteria including peptide mapping, mobility in reducing and non-reducing SDS-polyacrylamide gels, and relative glycosylation. Antibodies raised against the membrane-associated antigens react with the water soluble proteins on Western blots. Not surprisingly, immunocytochemical localization studies show binding of these antibodies to surface membranes of the cell. In addition, however, antibody binding is also detectible on the membranes of a secretory organelle (that is, mucocysts) present in the cortical cytoplasm. The significance of these findings with regard to the potential role of the i-antigens in infection and immunity is discussed.     

Isolation and Immunological Characterization of a Glycoprotein from Adrenal Chromaffin Granules

A glycoprotein (s-GP III) was isolated from the soluble lysate of chromaffin granules by chromatography with immunoaffinity and lectin columns. An identical protein (m-GP III) was shown to be present in the granule membranes. The apparent molecular weight of these glycoproteins as determined by the electrophoresis system of Laemmli (1970) was 43,000 under reducing conditions. In the absence of mercaptoethanol they aggregated to dimers. Antisera were raised against both the soluble and the membrane-bound forms of this glycoprotein. With these antisera GP III was further characterized: Immunoreplicas were obtained after two-dimensional electrophoresis of soluble and membrane-bound proteins of chromaffin granules. GP III was identified as a protein with a rather broad pI (4.6-5.3), indicating microheterogeneity. As shown by subcellular fractionation, m-GP III is specifically confined to chromaffin granules. GP III can therefore be used as a marker for the membranes of these organelles. The soluble form is secreted from adrenal medulla during stimulation with carbamylcholine chloride. An immunologically identical antigen was detected in adeno- and neurohypophysis. The physiological function of GP III is still unknown. It does not demonstrate any of the enzymatic activities so far known to occur in chromaffin granules.