Removal of blood group determinants from bovine erythrocyte membranes. 2. Degradation of ghosts by butanol and pyridine

Ghosts produced from erythrocytes collected from six different cattle were degraded with butanol and pyridine. Of a total of 38 different antigenic determinants available for investigation among the six cows, F, V, J and L were the only speci-ficties detected in the subtractions resulting from either method of degradation. After butanol degradation V, J and L antigens were found in the soluble protein fraction, while F was found in the insoluble protein. Pyridine digestion resulted in all four determinants being detected in the sialoprotein layer, while J was found in the lipoprotein as well. All antigens were relatively weak, being detected in inhibition strengths of 10.0 to 1.25 mg/ml.     

Removal of blood group determinants from bovine erythrocyte membranes. 1. Action of proteolytic enzymes on ghosts1

Thirty-nine blood group antigens were detected by hemolytic inhibition tests on erythrocyte ghosts prior to enzyme digestion. The ghosts, produced from erythrocytes collected from six different cattle, were digested with the proteolytic enzymes papain, protease, ficin, chymotrypsin and trypsin. Of the 39 antigens, 30 were removed from the membranes and detected in the soluble fraction resulting from the digestions. Some antigens were consistently removed by all enzymes digesting all ghosts possessing them, while the degree to which many other antigenic determinants were removed varied according to the ghosts being digested and the enzymes employed. Of the 9 remaining determinants never removed from the ghosts, some were detected in the insoluble fraction while others were not detected at all. These latter antigens were presumably destroyed by the enzyme digestion.     

Removal of blood group determinants from bovine erythrocyte membranes. 3. Action of proteolytic enzymes on intact cells.

Bovine erythrocyte treatment with chymotrypsin, trypsin, pronase, papain, or ficin eliminated or weakened the reactivity of 18 of the 47 blood group factors which were examined. Thirteen of the affected factors were from the B system, and one each was from the C, FV, L, M, and R'S' systems. Variation attributable to pheno-group (allele) or genotype influences was observed in the effects upon six of the factors. Ficin-treated V/V, but not F/V or F/F, cells were rapidly lysed by normal rabbit serum (complement control). Absorptions with pronase-treated V positive cells indicated that essentially all V antigenicity was removed. However, immunizations with pronase-treated V positive cells elicited V antibody production in one of two recipient cows. The numbers of antigens removed by different enzymes did not appear to be closely related to the amount of protein removed.     

Removal of blood group determinants from bovine erythrocyte membranes. 3. Action of proteolytic enzymes on intact cells

Bovine erythrocyte treatment with chymotrypsin, trypsin, pronase, papain, or ficin eliminated or weakened the reactivity of 18 of the 47 blood group factors which were examined. Thirteen of the affected factors were from the B system, and one each was from the C, FV, L, M, and R'S' systems. Variation attributable to pheno-group (allele) or genotype influences was observed in the effects upon six of the factors. Ficin-treated V/V, but not F/V or F/F, cells were rapidly lysed by normal rabbit serum (complement control). Absorptions with pronase-treated V positive cells indicated that essentially all V antigenicity was removed. However, immunizations with pronase-treated V positive cells elicited V antibody production in one of two recipient cows. The numbers of antigens removed by different enzymes did not appear to be closely related to the amount of protein removed.     

Chromosome mapping of human cell surface molecules: monoclonal anti-human lymphocyte antibodies 4F2, A3D8, and A1G3 define antigens controlled by different regions of chromosome 11

Monoclonal antibodies 4F2, A3D8, and A1G3, directed against cell surface antigens present on subsets of human cells, were used to identify the human chromosome regions that code for the antigenic determinants. Human fibroblasts expressed all three antigens, and no cross-reactivity with Chinese hamster or mouse cells was found. Fourteen rodent X human somatic cell hybrids, derived from six different human donors and from two different Chinese hamster and one mouse cell line, were studied simultaneously for human chromosome content and for antibody binding as detected by indirect immunofluorescence. Concordancy with binding of all three antibodies was observed only for human chromosome 11. All other chromosomes were excluded by three or more discordant hybrid clones. Data from six hybrids containing three different regions of chromosome 11 indicate that it is the long arm of chromosome 11 which is both necessary and sufficient for expression of the human antigen defined by 4F2 while the antigen(s) defined by A3D8 and A1G3 map to short arm.     

Exchange and stability of HeLa ribosomal proteins in vivo.

The relative stabilities of individual HeLa ribosomal proteins and their capacity for exchange between ribosome-bound and -free states in the cytoplasm were examined. Most ribosomal proteins on cytoplasmic ribosomes were found to have uniform, high stability as measured by comparing the short term (12-hour) to steady state (3-day) labeling ratios determined for each ribosomal protein. This would be expected if the proteins in ribosomes either were all stable or were all degraded as a unit. The data do not rule out the possibility that individual proteins have different stabilities prior to their assembly into ribosomes. Four proteins labeled atypically. One large subunit protein (L5) had a lower than average ratio. We interpret this low ratio as being due to a large free pool of this protein. Three proteins (L10, L28, S2) had higher than average ratios, interpreted as being due to reduced protein stability. Two of these proteins (L10, L28) with high ratios were also found to exchange in vivo. The exchangeable proteins may be subject to increased degradation during the time that they spend in the exchangeable free pool. The third protein (S2) with an atypically high ratio is thought to be degraded or altered while on the ribosome, or slowly lost as ribosomes age, because exchange of this protein was not detected. These interpretations and some alternate interpretations are explained. The exchange of three large subunit proteins (L10, L19, L28) was detected by labeling of protein after ribosome synthesis had been inhibited with actinomycin D. Autoradiography of two-dimensional polyacrylamide gels showed labeling of these spots.     

Murine polypyrimidine tract binding protein. Purification, cloning, and mapping of the RNA binding domain.

A complex of nucleic acid binding proteins (100, 35, and 25 kDa) was purified to apparent homogeneity from nuclear extracts of the murine plasmacytoma J558L. Amino-terminal sequence analysis of the 25-kDa subunit enabled the isolation of a cDNA that encodes a 528-amino acid protein that is highly homologous to the human 62-kDa human polypyrimidine tract binding protein (PTB) (Garcia-Blanco, M. A., Jamison, S. F., and Sharp, P. A. (1989) Genes & Dev. 3, 1874-1886; Gil, A., Sharp, P. A., Jamison, S. F., and Garcia-Blanco, M. A. (1991) Genes & Dev. 5, 1224-1236; Patton, J. G., Mayer, S. A., Tempst, P., and Nadal-Ginard, B. (1991) Genes & Dev. 5, 1237-1251). Sequence comparison programs suggested the presence of domains related to the RNA recognition motif found in other RNA-binding proteins, and deletion analysis revealed that the carboxyl-terminal 195 amino acids of the recombinant PTB was sufficient for specific binding to pre-mRNAs. Cross-linking experiments identified a 25-kDa protein in crude nuclear extracts of J558L cells that possessed the RNA binding properties of PTB, while a approximately 60-kDa protein is detected in other murine cell lines tested. Thus, the 25-kDa protein found in J558L is likely a proteolytic product of the murine polypyrimidine tract binding protein. A probe derived from the PTB cDNA detected a ubiquitous 3.3-kb mRNA in murine cell lines and a 3.6-kb mRNA in human lines. Southern blot analysis revealed three strongly hybridizing DNA fragments and several more weakly hybridizing bands in mouse, human, and yeast DNA. The role of PTB in pre-mRNA splicing is discussed.     

Intranuclear localization of a new snRNP-related antigen.

The intranuclear distribution of a new antigen (F78) associated with U snRNPs (small nuclear RNA-protein complexes) was compared with that of the RNP and Sm protein antigens previously identified on individual snRNP particles. Human and rat cells were double stained with human autoantisera and mouse monoclonal antibodies. The binding of the human and mouse antibodies was detected with secondary antibodies conjugated with fluorescein and rhodamine, respectively. The resulting immunofluorescence patterns were compared by digital image analysis. The F78, RNP, and Sm antigens show speckled fluorescence patterns which overlap to a great extent. The F78 pattern, however, also contains two classes of structural elements not present in the RNP pattern. Furthermore, during mitosis expression of the F78 antigen is completely suppressed from early prophase to telophase, while the RNP and Sm antigens are found evenly distributed throughout the cytoplasm of the dividing cells.     

Induction of Epstein-Barr virus nuclear antigens.

Lymphocytes were infected with the QIMR-WIL strain of Epstein-Barr virus, and the induction of Epstein-Barr virus-associated nuclear antigens was determined by using the protein immunoblot. There was a temporal increase in six antigens, with Epstein-Barr nuclear antigen 2 being detected 1 day after infection. The appearance of these antigens was shown to be independent of cellular DNA synthesis.     

Biochemical characterization of polypeptide components involved in neurite fasciculation and elongation

Polypeptide components and carbohydrate linkage types of F11 antigen and G4 antigen, two chick cell-surface glycoproteins implicated in neurite fasciculation and elongation [Rathjen, F.G., Wolff, J.M., Bonhoeffer, F. and Rutishauser, U. (1987) J. Cell Biol. 104, 343-353], have been studied in comparison to mouse L1 antigen. Tryptic fingerprint analysis does not reveal any relation of the 130-kDa components of G4 or F11 antigens to each other or to neural cell-adhesion molecules. The 180/190-kDa component of G4 antigen comprises parts of the 130-kDa and 80/65-kDa components and shares a sequence corresponding to the amino terminus of the G4 130-kDa component as shown serologically with anti-peptide sera. This closely parallels the relationship found for mouse L1 antigen components. In contrast, the F11 170-kDa component is different from the F11 130-kDa component, as shown serologically and by fingerprint analysis. A combination of chemical and enzymatic deglycosylation methods reveals that while O-glycosylation cannot be detected F11 130-kDa, G4 130-kDa and L1 140-kDa components contain N-linked carbohydrates. Endoglycosidase H treatment shows that the oligosaccharides present in the G4 130-kDa component and mouse L1 are mostly of the complex type, while the F11 130-kDa component consists of two populations, one containing mainly complex-type carbohydrates and a second containing high-mannose/hybrid-type carbohydrates.     

Expression of the two major envelope proteins of equine arteritis virus as a heterodimer is necessary for induction of neutralizing antibodies in mice immunized with recombinant Venezuelan equine encephalitis virus replicon particles

RNA replicon particles derived from a vaccine strain of Venezuelan equine encephalitis virus (VEE) were used as a vector for expression of the major envelope proteins (G(L) and M) of equine arteritis virus (EAV), both individually and in heterodimer form (G(L)/M). Open reading frame 5 (ORF5) encodes the G(L) protein, which expresses the known neutralizing determinants of EAV (U. B. R. Balasuriya, J. F. Patton, P. V. Rossitto, P. J. Timoney, W. H. McCollum, and N. J. MacLachlan, Virology 232:114-128, 1997). ORF5 and ORF6 (which encodes the M protein) of EAV were cloned into two different VEE replicon vectors that contained either one or two 26S subgenomic mRNA promoters. These replicon RNAs were packaged into VEE replicon particles by VEE capsid protein and glycoproteins supplied in trans in cells that were coelectroporated with replicon and helper RNAs. The immunogenicity of individual replicon particle preparations (pVR21-G(L), pVR21-M, and pVR100-G(L)/M) in BALB/c mice was determined. All mice developed antibodies against the recombinant proteins with which they were immunized, but only the mice inoculated with replicon particles expressing the G(L)/M heterodimer developed antibodies that neutralize EAV. The data further confirmed that authentic posttranslational modification and conformational maturation of the recombinant G(L) protein occur only in the presence of the M protein and that this interaction is necessary for induction of neutralizing antibodies.     

Identification of a tumor-associated antigen of the guinea pig L2C leukemia by using syngeneic antisera.

Inbred strain 2 guinea pigs immunized with L2C leukemia cells produced antibodies to L2C cells detected by 125I-protein A assay. L2C-associated tumor antigens were reacted with syngeneic antisera and analyzed by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. These sera recognized idiotypic determinants on surface IgM molecules of L2C cells but did not recognize any determinants on normal strain 2 spleen cells. Thus, determinants on IgM molecular act as tumor-associated antigens in the L2C system and can be detected by syngeneic sera.     

Monoclonal antibodies specific for Leishmania tropica. I. Characterization of antigens associated with stage- and species-specific determinants

Four monoclonal antibodies (T-1 through T-4), which were produced to membrane-enriched preparations of Leishmania tropica major promastigotes, reacted specifically with the members of L. tropica complex. The antibodies T-1 and T-4 react exclusively with L. t. major and L. t. minor. The remaining two monoclonal antibodies bind, in addition, to L. t. aethiopica and weakly to L. mexicana amazonensis. No significant cross-reactivity was observed with L. donovani, L. braziliensis braziliensis, and Trypanosoma cruzi. Antibodies T-1, T-2, and T-4 were found to be specific for the promastigote stage (insect) of L. tropica. Antibody T-3 reacts with both the amastigote and promastigote stages of the parasite. All of the monoclonal antibodies react with cell surface components on intact promastigotes. The protein antigens containing the species-specific determinants recognized by each of the four antibodies were identified by radioimmunoprecipitation of solubilized 125I-labeled L. t. major promastigotes. A single 50 kilodalton protein is recognized by clone T-4. T-1 recognized two high m.w. proteins (100 and 200 kilodaltons). These two antigens plus an additional protein of lower m.w. (70 kilodaltons) are also immunoprecipitated by the antibodies T-2 and T-3, demonstrating that species-specific determinants are present on several different cell surface proteins of L. t. major.     

Isolation of immunologically active uterine luminal proteins associated with follicular and luteal phases of the ovary in buffalo (Bubalus bubalus)

Uterine luminal proteins (ULP) collected from the genital tract of buffalo during the follicular (Group F) and luteal (Group L) phases of the estrous cycle were chromatographed using sephacryl S-200 gel. Five peaks were detected in each group. Different protein concentrations (10 to 200 microg) from Peaks I and V in each group were examined for immunological activity on polymorph nuclear leukocytic cells (PMNL) in vitro. All concentrations except 10 microg of ULP Peak I (< or = 250 kDa) in Group F enhanced phagocytic activity of PMNL. Peak V (56 kDa) in the same group enhanced phagocytic activity of PMNL only at low protein concentrations (10, 20 and 40 microg protein), while at greater concentrations (80, 150 and 200 microg protein) PMNL activity was suppressed. On the other hand, all protein concentrations from Peak 1 (> or = 250 kDa) in Group L suppressed PMNL activity in a dose-dependent manner. Proteins from Peak V (31 kDa) in Group L suppressed PMNL phagocytic activity at all concentrations but not to the same extent as in Peak I. Electrophoretic analysis of Peaks I and V in both groups revealed only 3 detectable protein bands (subunits) in Peak I and 1 detectable subunit in Peak V. Several additional proteins were probably not detected. The molecular weights of the detected subunits in Peaks I and V in Group F were greater than those in Group L as indicated by SDS-PAGE analysis. The results of this study show that ULP collected from buffalo possessed proteins that modulated phagocytic activity of PMNL in vitro. Proteins collected during the follicular phase, especially Peak I, enhanced phagocytic activity of the PMNL, whereas those collected during the luteal phase (Peaks I and V) suppressed activity. Changes in the molecular weights of ULP detected in this experiment may be related to the changes in phagocytic activity of PMNL tested in vitro.     

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.     

Structure and organization of plasmid genes required to produce the translation inhibitor microcin C7.

The translation inhibitor microcin C7 (MccC7) is a linear heptapeptide whose N terminus has been replaced by an N-formyl group and whose C terminus has been replaced by the phosphodiester of 5'-adenylic acid and n-aminopropanol (J. I. Guijarro, J. E. González-Pastor, F. Baleux, J. L. San Millán, M. A. Castilla, M. Rico, F. Moreno, and M. Delepierre, J. Biol. Chem. 270:23520-23532, 1995). MccC7 production and immunity determinants lie on a 6.2-kb region of the Escherichia coli plasmid pMccC7. This region was entirely sequenced. It contains six open reading frames, which were shown to be true genes by different complementary approaches. Five genes, mccABCDE, which are transcribed in the same direction, are required to produce mature extracellular microcin. The sixth gene, mccF, adjacent to mccE, is transcribed in the opposite direction and encodes specific self-immunity. Genes mccA to -E constitute an operon transcribed from a promoter (mccp) located upstream of mccA. mccA is 21 nucleotides long and encodes the unmodified heptapeptide (J. E. González-Pastor, J. L. San Millán, and F. Moreno, Nature [London] 369:281, 1994). A comparison of predicted gene polypeptide products with those included in databases shows that an 81-amino-acid stretch of MccB is strikingly homologous to fragments of the same length of proteins ThiF and ChlN from E. coli, HesA from Anabaena sp. strain PCC7120, and UBA1, the ubiquitin-activating enzyme from different eukaryotic species. MccC displays several hydrophobic domains, suggesting a transmembrane location. The carboxyl end of MccE displays 41.2% identity with RimL, a protein required to acetylate the ribosome protein L12 from E. coli. In the absence of the other mcc genes, mccA impairs the growth of host cells, suggesting that unmodified MccA has antibiotic activity. A model for MccC7 biosynthesis, export, and immunity is proposed.     

Comparative analysis of the immune response of a rabbit to antigens to live and killed Francisella species bacteria]

Serum antibodies were analyzed in rabbits immunized with live and formalin-killed Francisella (F. tularensis, F. novicida, F. novicida-like, and F. philomiragia). Passive hemagglutination test with erythrocytes sensitized by these bacteria' LPS showed much higher titers of species-specific antibodies in all sera to live microorganisms than sera to killed bacteria. The results of immunoblotting with purified LPS and bacterial lysates indicate that sera to live bacteria contained mainly immunoglobulins to species-specific antigenic epitopes of LPS O-polysaccharide chain and few antibodies to the protein component of the cell. By contrast, killed bacterial cells induced weak production of antibodies to S-LPS and a pronounced antibody response to protein antigens. Besides the quantitative differences, live and killed bacteria differed by the qualitative spectrum of immunodominant proteins. Serum to live F. tularensis 15/10 contained antibodies to at least 3 immunodominant antigens of the cell, while serum to killed bacteria contained antibodies to only two of these. Immunoglobulins to protein antigens, absent in homologous sera to live bacteria, were detected in the sera to killed F. novicida and F. novicida-like bacteria. Both sera to F. philomiragia had antibodies reacting with LPS epitopes and immunodominant complex containing protein. In contrast to other Francisella, F. philomiragia was found to synthesize an uncommon LPS representing two major lipooligosaccharides with different molecular weights and antigenic specificity. Therefore, immune response of the host to live and killed Francisella is different: live cells more effectively induce the production of antibodies to S-LPS epitopes, while killed ones to protein antigens.     

Metabolism of phosphatidylglycerol and bis(monoacylglycero)-phosphate in macrophage subcellular fractions

Bis(monoacylglycero)phosphate (BMP) is synthesized from exogenous phosphatidylglycerol (PG) by macrophages (Cochran, F. R., Roddick, V. L., Connor, J. R., Thornburg, J. T., and Waite, M. (1987) J. Immunol. 138, 1877-1883). Previous work from our laboratory showed that arachidonic acid in BMP was released by the macrophages upon challenge of the cells with PMA (Cochran, F. R., Connor, J. R., Roddick, V. L., and Waite, M. (1985) Biochem. Biophys. Res. Commun. 130, 800-806). Here we extend those studies using a model cultured cell line of macrophages, RAW 264.7. When PG labeled with 32P- and [3H]glycerol in both moieties was added to the culture medium, 32P/[3H]BMP was synthesized in a time-dependent manner. Fractionation of cell homogenates on a discontinuous sucrose gradient in which the light membranes were floated from dense sucrose showed an enrichment of [3H]BMP in light membrane fractions. The precursor [3H]PG was also found in the light fractions but, relative to the [3H]BMP, was more abundant in the denser membrane fractions. The appearance of [3H]PG and [3H]BMP in the light membrane fraction was time-dependent which suggested that the initial uptake and metabolism of [3H]PG was into the denser membranes. Incubation of the light membranes under conditions that are optimal for the lysosomal phospholipase A1 led to significant metabolism of [3H]PG. Both degradation of [3H]PG to water-soluble compounds and its conversion to acylphosphatidylglycerol occurred while no lyso-PG was detected. On the other hand, little BMP was found to be degraded. From these studies we postulate that in lysosomes acylphosphatidylglycerol is a precursor of BMP and that the previously reported turnover of arachidonic acid by BMP may occur via transacylation rather than hydrolysis.     

Mutant alpha-galactosidase A enzymes identified in Fabry disease patients with residual enzyme activity: biochemical characterization and restoration of normal intracellular processing by 1-deoxygalactonojirimycin

Fabry disease is a lysosomal storage disorder caused by the deficiency of alpha-Gal A (alpha-galactosidase A) activity. In order to understand the molecular mechanism underlying alpha-Gal A deficiency in Fabry disease patients with residual enzyme activity, enzymes with different missense mutations were purified from transfected COS-7 cells and the biochemical properties were characterized. The mutant enzymes detected in variant patients (A20P, E66Q, M72V, I91T, R112H, F113L, N215S, Q279E, M296I, M296V and R301Q), and those found mostly in mild classic patients (A97V, A156V, L166V and R356W) appeared to have normal K(m) and V(max) values. The degradation of all mutants (except E59K) was partially inhibited by treatment with kifunensine, a selective inhibitor of ER (endoplasmic reticulum) alpha-mannosidase I. Metabolic labelling and subcellular fractionation studies in COS-7 cells expressing the L166V and R301Q alpha-Gal A mutants indicated that the mutant protein was retained in the ER and degraded without processing. Addition of DGJ (1-deoxygalactonojirimycin) to the culture medium of COS-7 cells transfected with a large set of missense mutant alpha-Gal A cDNAs effectively increased both enzyme activity and protein yield. DGJ was capable of normalizing intracellular processing of mutant alpha-Gal A found in both classic (L166V) and variant (R301Q) Fabry disease patients. In addition, the residual enzyme activity in fibroblasts or lymphoblasts from both classic and variant hemizygous Fabry disease patients carrying a variety of missense mutations could be substantially increased by cultivation of the cells with DGJ. These results indicate that a large proportion of mutant enzymes in patients with residual enzyme activity are kinetically active. Excessive degradation in the ER could be responsible for the deficiency of enzyme activity in vivo, and the DGJ approach may be broadly applicable to Fabry disease patients with missense mutations.     

Antigenic variation of influenza A virus nucleoprotein detected with monoclonal antibodies.

Monoclonal antibodies were used to study antigenic variation in the nucleoprotein of influenza A viruses. We found that the nucleoprotein molecule of the WSN/33 strain possesses at least five different determinants. Viruses of other influenza A virus subtypes showed antigenic variation in these nucleoprotein determinants, although changes in only one determinant were detected in H0N1 and animal strains. The nucleoprotein of human strains isolated from 1933 through 1979 could be divided into six groups, based on their reactivities with monoclonal antibodies; these groups did not correlate with any particular hemagglutinin or neuraminidase subtype. Our results indicate that antigenic variation in the nucleoproteins of influenza A viruses proceeds independently of changes in the viral surface antigens and suggest that point mutations and genetic reassortment may account for nucleoprotein variability.