An antigenic analysis for membranes of Mycoplasma hominis by cross-absorption

Antigenic components at the outer surface membranes of seven serotypes of Mycoplasma hominis were analysed by the mycoplasmacidal reaction and the agglutination during growth reaction. Antibody absorbing capacities of the mycoplasma cells were compared with absorbing capacities of membranes. It was shown that serologically active membrane antigens were mainly heat-labile proteins. No major antigens common to all seven serotypes were detected and each strain had its own specific antigens at the cell surface. Results of analysis indicate that there is a complex antigenic structure exposed in M. hominis and that 7 to 14 cross-reacting antigens may be present at the outer surface in the different serotypes examined. Additional cross-reacting antigens, presumably inner membrane in origin and not exposed at the cell surface, were also demonstrable.     

Comparison of surface proteomes of enterotoxigenic (ETEC) and commensal Escherichia coli strains

Pathogenesis of enterotoxigenic Escherichia coli (ETEC) infections involves colonization of the small intestine mediated by cell-surface fimbriae (CS) or colonization fimbriae antigens (CFA). However, protection against reinfection of ETEC is also conferred by somatic antigens rather than by virulence factors. To discover ETEC specific somatic antigens, the surface proteome of the ETEC H10406 strain was compared with that of non-pathogenic E. coli K12 strains. In this study, we were using stable isotope labelling with amino acids in cell culture (SILAC) technology for the labelling and relative quantification of surface proteins in order to identify polypeptides that are specifically present on ETEC strains. Outer membrane proteins were isolated, separated by gel electrophoresis, and identified by mass spectrometry. Twenty-three differentially expressed cell-surface polypeptides of ETEC were identified and evaluated by bioinformatics for protein vaccine candidates. The combination of being surface-exposed and present differentially makes these polypeptides highly suitable as targets for antibodies and thus for use in passive or active immunisation/vaccination.     

Multiple forms of polyoma virus tumor antigens from infected and transformed cells.

At least three distinct forms of polyoma virus tumor antigens were isolated from productively infected and transformed hamster cells by immunoprecipitation with anti-T serum. These proteins had approximate molecular weights of 105,000 (large T antigen), 63,000 (middle T antigen), and 20,000 (small T antigen) as estimated by acrylamide gel electrophoresis. An examination of the appearance of these antigens in polyoma-infected mouse cells showed that all three polypeptides were synthesized maximally at approximately the same time after infection. Analysis of the methionine-containing tryptic peptides of these proteins indicated that the large, middle, and small forms of polyoma T antigens contained five similar or identical peptides. In addition, the 63,000- and 20,000-dalton antigens contained two other methionine peptides absent from the large T-antigen species. Other methionine peptides were found only in the large or middle T-antigen forms. These results and results obtained previously suggested that the three T-antigen species have the same NH2-terminal end regions but different COOH termini. A model is presented describing the synthesis of these polypeptides from different regions of the polyoma virus genome.     

Cell surface proteins of encapsulated Streptococcus cremoris: identification and immunochemical characterization

Cell surface proteins of two slime-forming, encapsulated Streptococcus cremoris strains, MLS96 and T₅ from the fermented milk product viili, were extracted with the non-ionic detergent Triton X-100. The isolated protein antigens were characterized by sodium dodecyl sulphate polyacrylamide gel electrophoresis and immuno-blotting with antisera produced against whole Strep. cremoris cells. When protein profiles of these strains were compared, seven prominent polypeptides were found common to both and were recognized by both antisera. Five of these polypeptides with molecular weights of 70000, 54000, 50000, 47000 and 40000 were identified as cell wall components. The remaining two polypeptides with molecular weights of 42000 and 26000 are being studied further in connection with slime formation for which modified Triton X-100 extraction provides a suitable method for isolation of the surface-associated antigens of lactic streptococci.     

Specific and azurophilic granules from rabbit polymorphonuclear leukocytes. II. Cell surface localization of granule membrane and content proteins before and after degranulation

The compositional relationship between the cell surface of rabbit polymorphonuclear leukocytes (PMNs) and the membranes of PMN cytoplasmic granules has been investigated. Heterophilic PMNs obtained from peritoneal exudates contained 13 cell surface polypeptides ranging in molecular weight from 220,000 to 12,000 daltons as determined by lactoperoxidase-catalyzed protein iodination and gel electrophoresis. Of these, four polypeptides co-migrated with proteins identified as the major constituents of specific (SpG) and azurophilic (AzG) granule membranes. The most notable of these were cell surface proteins of 145,000 and 96,000 daltons that co-migrated with proteins identified as granule content proteins released from PMNs during exocytosis. Extensive washing did not remove these proteins from the cell surface. Iodination of PMNs after the release of SpG and AzG contents by calcium ionophore- induced exocytosis revealed that there was not a dramatic quantitative change in the proteins on the cell surface. Instead, there were large, quantitative increases in the relative amounts of (125)I that were incorporated into several pre-existing cell surface proteins; all of these cell surface proteins co-migrated as a set with those polypeptides identified as either granule membrane or content proteins. Although nearly all of the major polypeptides of SpG and AzG had counterparts on the cell surface of freshly isolated peritoneal exudates PMNs, there were several polypeptides that were unique to the cell surface. Thus, the PMN has at least three membrane compartments with strikingly different protein compositions.     

Parasite specific antibodies in three strains of mice after infection with Trypanosoma cruzi

Three inbred strains of mice (BALB/cJ, C3H/HeJ and NZB/BInJ) were infected with trypomastigotes of Trypanosoma cruzi. Sera were taken at different times after infection and radioimmunoprecipitation assays were used to detect antibodies against individual T. cruzi epimastigote and trypomastigote antigens. The mouse strains differed in regard to the spectrum of antibodies and the time after infection when the various epimastigote specific antibody species appeared. NZB mice had antibodies against at least 25 polypeptides ranging in molecular weight from 20,000 to 90,000 D at 3 wk after infection, and these persisted until at least 10 wk post-infection. C3H and BALB/c had antibodies against fewer than 5 antigens at 3 wk after infection; whereas by week 10, antibodies against at least 25 polypeptides were detected. C3H mice that were most susceptible to infection (but not NZB or BALB/c mice) had antibodies against a 25,000 D molecular weight epimastigote antigen. The antibody response against trypomastigote polypeptides was more uniform. Sera from all mouse strains at 3 wk after infection precipitated the same polypeptides and the radioimmunoprecipitation patterns did not change as a function of time after infection.     

Fractionation of membrane proteins on phenylboronic acid-agarose.

Detergent-solubilized plasma-membrane proteins from lymphocytes of patients with chronic lymphocytic leukaemia were applied to phenylboronic acid-agarose. Some of the polypeptides were bound specifically and subsequently eluted with sorbitol. Immunoprecipitation analyses showed that no surface immunoglobulin M was bound, but that most of the histocompatibility antigens HLA-A, HLA-B, HLA-C and HLA-DR were.     

Surface orientation and antigen properties of Rh and LW polypeptides of the human erythrocyte membrane

Time course digestion of intact human erythrocytes and right side-out vesicles with carboxypeptidase Y altered the Rh polypeptides and removed the 125I label that is normally incorporated by cell-surface radioiodination, but did not affect the RhD, Rhc, or RhE antigens. Under the same conditions, however, the LW antigens were rapidly destroyed. Digestion of inside-out and right side-out vesicles with aminopeptidase M was without any detectable effect on the Rh and LW antigens or polypeptides, although glycophorin A was degraded from right side-out but not from inside-out vesicles. These findings demonstrate that the C-terminal domain of the Rh and LW polypeptides is exposed at the external surface of human erythrocytes and indicate, in addition, that the LW antigens and tyrosine residue(s) of the LW and Rh proteins, respectively, are located close to the C termini of these polypeptides. Further studies using monoclonal and polyclonal antibodies showed that LW antigen expression is inhibited by treatment of red cells with EDTA and is selectively restored by Mg2+, but not by Mn2+ or Ca2+, whereas the Rh antigens were not affected under these conditions. In addition, O- and N-glycanase digestion of the LW glycoprotein removed its sugar chains, but did not alter significantly the epitopes recognized by the monoclonal anti-LW antibody.     

Integral membrane proteins specific to the inner nuclear membrane and associated with the nuclear lamina

We obtained a monoclonal antibody (RL13) that identifies three integral membrane proteins specific to the nuclear envelope of rat liver, a major 75-kD polypeptide and two more minor components of 68 and 55 kD. Immunogold labeling of isolated nuclear envelopes demonstrates that these antigens are localized specifically to the inner nuclear membrane, and that the RL13 epitope occurs on the inner membrane's nucleoplasmic surface where the nuclear lamina is found. When nuclear envelopes are extracted with solutions containing nonionic detergent and high salt to solubilize nuclear membranes and pore complexes, most of these integral proteins remain associated with the insoluble lamina. Since the polypeptides recognized by RL13 are relatively abundant, they may function as lamina attachment sites in the inner nuclear membrane. Major cross-reacting antigens are found by immunoblotting and immunofluorescence microscopy in all rat cells examined. Therefore, these integral proteins are biochemical markers for the inner nuclear membrane and will be useful models for studying nuclear membrane biogenesis.     

Trypanosoma cruzi: shedding of surface antigens as membrane vesicles

Tissue culture-derived trypomastigotes from Trypanosoma cruzi spontaneously shed surface antigens into the culture medium. The shedding is a temperature- and time-dependent phenomenon and is independent of the presence of proteins or immune serum in the medium. The analysis of this process in four strains (Y, YuYu, CA1, and RA) showed differences in the amounts of polypeptides released. However, for all strains the liberation of the entire set of surface polypeptides ranging in molecular mass from 70 to 150 kDa was observed. Biochemical and electron microscopic data strongly suggest that most of the surface antigens are released as plasma membrane vesicles, ranging from 20 to 80 nm in diameter.     

Characterization of the T, L, I, S, M and P cell surface (immobilization) antigens of Tetrahymena thermophila: molecular weights, isoforms, and cross-reactivity of antisera.

In the ciliate protist Tetrahymena thermophila the L, H, T, I, S, M and P cell surface proteins (immobilization antigens) are expressed under different conditions of temperature (L, H, T), culture media (I, S), and mutant genotype (M, P). Immunoblot and autoradiographic studies using antisera to purified protein show that the molecular weights of these proteins range from 25,000 to 59,000. The H, T, S, M and P antigens are recognized as single polypeptides, whereas L, I, and one allelic form of T each appear to consist of a family of polypeptides. Although antisera are specific in immobilization and immunofluorescence assays of surface protein in living cells, cross-reactivity is seen with denatured protein on immunoblots. It is hypothesized that the surface protein genes are organized into families of evolutionarily related isoloci.     

Antigenic analysis of purified surface antigens of filarial nematode Setaria digitata.

The surface antigens of S. digitata were isolated by treatment with Triton X-100. In non SDS-PAGE the surface antigen preparation resolved into more than 6 protein bands. Electroelution of gel slices corresponding to the protein bands with relative mobilities 0.09, 0.32, 0.41, 0.53, 0.61 and 0.76 gave 6 purified surface antigen fractions (SAF). Analysis of SAFs by SDS-PAGE showed that the proteins with molecular weights 17, 29 and 36 KD were the three major polypeptides and different combination of these gave rise to the 6 native surface proteins. The 29 KD protein existed as a monomer and as cross-linked with the 17 and 36 KD proteins. All surface antigen fractions showed antigenicity, where as 29 KD protein remained as a high avidity surface antigen.     

Characterization of the T, L, I, S, M and P Cell Surface (Immobilization) Antigens of Tetrahymena thermophila: Molecular Weights, Isoforms, and Cross-Reactivity of Antisera

ABSTRACT. In the ciliate protist Tetrahymena thermophila the L, H, T, I, S, M and P cell surface proteins (immobilization antigens) are expressed under different conditions of temperature (L, H, T), culture media (I, S), and mutant genotype (M, P). Immunoblot and autoradiographic studies using antisera to purified protein show that the molecular weights of these proteins range from 25,000 to 59,000. The H, T, S, M and P antigens are recognized as single polypeptides, whereas L, I, and one allelic form of T each appear to consist of a family of polypeptides. Although antisera are specific in immobilization and immunofluorescence assays of surface protein in living cells, cross-reactivity is seen with denatured protein on immunoblots. It is hypothesized that the surface protein genes are organized into families of evolutionarily related isoloci.     

Antibody profiles by EITB and ELISA of cattle and sheep infected with Fasciola hepatica

In evaluating potential mechanisms of immunity in fascioliasis we compared the time-course analysis of the antibody responses between a resistant (cattle) and a susceptible model (sheep). Sera from sheep and cows experimentally infected with F. hepatica were reacted with both somatic (FhWWE) and excretory-secretory (ES) antigens in order to evaluate the antibody repertoires in the 2 different hosts. Analysis of these sera by ELISA showed a significant increase in antibody levels by 2 wk in most infected cattle using both somatic and ES antigens, whereas with most infected sheep antibodies are not clearly detected until week 4. By EITB, both infected sheep and cows recognize major somatic polypeptides in a molecular weight range of 30-38 kDa by 8 wk. Cattle recognized 3 additional major antigens of 56, 64, and 69 kDa as early as 6 wk. Various polypeptides of 20-25 kDa are prominently detected by most sheep and very faintly, if at all, by the cow sera. The sera from both sheep and cows also identify ES polypeptides of 20-28 kDa. The patterns of polypeptides recognized by sheep infected with S. mansoni and challenged with F. hepatica in EITB are almost identical to those with a simple F. hepatica primary infection. No significant differences were detected in the antibody kinetics in ELISA between these 2 groups. Differences and similarities between these models could eventually help determine which antibodies may be predictive of resistance or susceptibility in fascioliasis.     

Identification of human sperm surface glycoproteins recognized by autoantisera from immune infertile men, women, and vasectomized men

To identify the surface antigens of human sperm recognized by antisera from immune infertility patients and vasectomized men, we labeled sperm surface proteins with 125I- and used patient antisera for immunoprecipitation. Sera were studied from 27 infertile males, 18 infertile females, and 4 vasectomized males, each possessing anti-sperm antibodies detected by immunobead binding. Sera from different infertile males, different infertile females, and vasectomized males were remarkably similar in their surface antigen recognition. The different sera specifically immunoprecipitated the same small group of 125I-labeled surface proteins, which included polypeptides in the region 90 kDa, 40-45 kDa, and 26 kDa. Treatment with N-glycanase showed that the proteins of 90 kDa, 40-45 kDa, and 26 kDa were glycoproteins with N-linked carbohydrate. The immunoprecipitated 125I-labeled proteins and the total extract of 125I-labeled surface proteins were compared on two-dimensional (2D) gels. The results show the 90 kDa polypeptide is a major sperm surface component, whereas 40-45 kDa and 26 kDa polypeptides are minor components. The 2D gel comparison also indicates that 90 kDa, 40-45 kDa, and 26 kDa are a small subset of the total ensemble of sperm surface proteins. Clinical data suggest antibodies to these few proteins interfere with sperm function.     

Antigens of human cytomegalovirus

In considering HCMV antigens one must take into consideration not only structural proteins of virus particles but also HCMV specific proteins associated with the infected cell, for all of these proteins may play a part in eliciting an humoral and/or cell-mediated immune response in the infected individual. The virion is composed of some 35 polypeptides ranging in molecular weight from 12 000 to more than 200 000 daltons (Table I). Viral polypeptide synthesis at the level of the infected cell occurs in three waves, the immediate early, the early and the late (Table II). During the immediate early and early phases a dozen polypeptides appear. Two glycoproteins appear during the early period but these are poorly represented in the virion. Many antigens have been described both in the cytoplasm and nucleus during these periods (Table II). Viral DNA synthesis marks the beginning of the late phase of virus replication. Many new proteins and glycoproteins appear but not all of them will become part of the virus particle (Table II). It is interesting to compare the kinetics of appearance of antibodies as detected by different serodiagnostic techniques, at the time of primary infection, with the location of the antigens which these antibodies detect in the infected cells (Table III). CMV-IgM, the first antibodies to be detected, react with late appearing intracellular nuclear inclusion antigens. This contrasts with the relatively long time required for the development of neutralizing antibodies which react with antigens accessible not only on the viral envelope and at the infected cell membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell-substrate adhesion during Trypanosoma cruzi differentiation

The transformation of Trypanosoma cruzi epimastigotes to the mammal infective metacyclic trypomastigotes (metacyclogenesis) can be performed in vitro under chemically defined conditions. Under these conditions, differentiating epimastigotes adhere to a surface before their transformation into metacyclic trypomastigotes. Scanning and transmission electron microscopy of adhered and non-adhered parasites during the metacyclogenesis process show that only epimastigotes and few transition forms are found in the first population, whereas metacyclic trypomastigotes are exclusively found in the cell culture supernatant. PAGE analysis of the [35S]methionine metabolic labeling products of adhered and non-adhered parasites shows that although most of the polypeptides are conserved, adhered parasites express specifically four polypeptides in the range of 45-50 kD with an isoelectric point of 4.8. These proteins might be involved in the adhesion process and are recognized by an antiserum against total adhered parasite proteins. This antiserum also recognized a group of 45-50 kD in the iodine-radiolabeled surface proteins of differentiating cells, providing direct evidence that these components are indeed surface antigens. The results suggest that epimastigotes must adhere to a substrate before their transformation to metacyclic trypomastigotes, being released to the medium as the metacyclogenesis process is accomplished. This could correspond to the process naturally occurring within the triatomine invertebrate host.     

Electrophoretic analysis of proteins from Mycoplasma hominis strains detected by SDS-PAGE, two-dimensional gel electrophoresis and immunoblotting

The proteins of 14 strains of Mycoplasma hominis were compared by SDS-PAGE in gradient gels, by two-dimensional (2D) gel electrophoresis of extracts of 35S-labelled cells and by immunoblot analysis of cell proteins. The strains examined included the M. hominis type strain PG21 and 13 others isolated variously from genital tract, mouth, blood, upper urinary tract and a wound. These 14 strains shared 76-99% of proteins in SDS-gradient gel analysis and 41-72% in the 2D gels. As expected, the immunoblot analysis likewise revealed the existence of an extensive common protein pattern in M. hominis, in addition to a number of antigens shared only by some strains.     

Sequential detection of different antigens induced by Epstein-Barr virus and herpes simplex virus in the same Western blot by using dual antibody probes

A dual antibody probing technique that permitted a color-coded identification of polypeptides representing different classes of Epstein-Barr virus (EBV) antigens as well as differentiation of the polypeptides induced by different herpesviruses in the same Western blot was developed. When the nitrocellulose sheet was probed first with monoclonal antibody against EBV early antigen diffuse component (EA-D) and then stained with 4-chloro-1-naphthol, four polypeptides specific for EA-D were identified by purple bands. Subsequently, the same nitrocellulose sheet was reprobed with human serum containing antibodies against EBV early antigen, viral capsid antigen, and nuclear antigen and stained with 3,3'-diaminobenzidine. Several brown bands corresponding to early, viral capsid, and nuclear antigen polypeptides were detected. The dual antibody probing technique was used in an analysis to differentiate polypeptides resulting from either EBV or herpes simplex virus infection, either in cells infected by individual virus or in a cell line dually infected by both viruses. On the basis of different colored bands in different lanes of the same gel, 20 polypeptides with molecular weights ranging from 31,000 to 165,000 were identified as herpes simplex virus-specific proteins. These results suggested that the dual antibody probing technique may be applicable in clinical diagnosis for detecting antigens and antibodies derived from different pathogens.     

Isolation and characterisation of two sperm membrane proteins recognised by sperm-associated antibodies in infertile men

Antisperm antibodies eluted from the surface of spermatozoa obtained from infertile men recognised several common epitopes. We tested whether these epitopes were relevant to fertility by isolating the immunodominant 37/36 and 19/18 protein zones. These protein zones were cut out of preparative slab gels and electro-eluted. The isolated proteins, P36 and P18, were used for biochemical characterisation and to produce specific antibodies in rabbits. The specific reactivity of P36 and P18 with WGA and AAL lectins, respectively, indicated the presence of lactosaminyl structures with sialic acid termini in P36 and of fucosylated residues in P18. Isoelectric focusing showed that the two proteins consist of several polypeptides. Some of these polypeptides were recognised by both human and rabbit antibodies: the pl of these epitopes was around 5.5 for P36 and 8.3-10.3 for P18. Rabbit antibodies detected the corresponding proteins on the sperm heads of methanol-fixed and of live acrosome-reacted spermatozoa. Anti-P36 antibodies bound mainly to the equatorial segment. They reduced the binding and, consequently, the penetration of zona-free hamster oocytes by human spermatozoa. Anti-P18 antibodies gave more diffuse staining of the acrosomal and post-acrosomal regions and reduced sperm-oocyte penetration without a significant effect on sperm binding. These results suggest that P36 and P18 antigens located in different compartments of the sperm head may participate in the sperm-oolemma interaction. We are currently investigating the physiological role of these antigens by sequencing the proteins isolated from the gels.