Immunoproteomic analysis of the protective response obtained with subunit and commercial vaccines against Glässer's disease in pigs.

An immunoproteomic analysis of the protective response of subunit and commercial vaccines in colostrum-deprived pigs against Glässer's disease was carried out. A mixture of proteins with affinity to porcine transferrin (PAPT) from Haemophilus parasuis Nagasaki strain (serovar 5) was inoculated intramuscularly (PAPT(M)) and intratracheally (PAPT(Cp)), along with a commercial bacterin. PAPT were separated using 2 dimensional electrophoresis (2DE) gels and with them, 2DE Western blots were carried out. A total of 17 spots were identified as positive with sera of pigs from any of the three vaccinated groups, the highest number of immunoreactive proteins being detected in those having received PAPT(Cp). Among them, six proteins (FKBP-type peptidyl-prolyl cis-trans isomerase, neuraminidase exo-α-sialidase, xanthine-guanine phosphoribosyl transferase, CMP-N-acetylneuraminic acid synthetase, phenylalanyl-tRNA synthetase and glyceraldehyde 3-phosphate dehydrogenase) were found to be novel immunogens in H. parasuis. These proteins showed a high potential as candidates in future subunit vaccines against Glässer's disease. The three experimental groups developed specific systemic total IgG (IgGt), IgG1, IgG2 and IgM antibodies after immunizations. In addition, those receiving PAPT(Cp) yielded a serum IgA response.

Generation of glyco-engineered BY2 cell lines with decreased expression of plant-specific glycoepitopes.

Plants are known to be efficient hosts for the production of mammalian therapeutic proteins. However, plants produce complex N-glycans bearing ß1,2-xylose and core α1,3-fucose residues, which are absent in mammals. The immunogenicity and allergenicity of plant-specific Nglycans is a key concern in mammalian therapy. In this study, we amplified the sequences of 2 plant-specific glycosyltransferases from Nicotiana tabacum L. cv Bright Yellow 2 (BY2), which is a well-established cell line widely used for the expression of therapeutic proteins. The expression of the endogenous xylosyltranferase (XylT) and fucosyltransferase (FucT) was downregulated by using RNA interference (RNAi) strategy. The xylosylated and core fucosylated N-glycans were significantly, but not completely, reduced in the glycoengineered lines. However, these RNAi-treated cell lines were stable and viable and did not exhibit any obvious phenotype. Therefore, this study may provide an effective and promising strategy to produce recombinant glycoproteins in BY2 cells with humanized N-glycoforms to avoid potential immunogenicity.

Human T cell recognition of the blood stage antigen Plasmodium hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT) in acute malaria.

BACKGROUND: The Plasmodium purine salvage enzyme, hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT) can protect mice against Plasmodium yoelii pRBC challenge in a T cell-dependent manner and has, therefore, been proposed as a novel vaccine candidate. It is not known whether natural exposure to Plasmodium falciparum stimulates HGXPRT T cell reactivity in humans. METHODS: PBMC and plasma collected from malaria-exposed Indonesians during infection and 7-28 days after anti-malarial therapy, were assessed for HGXPRT recognition using CFSE proliferation, IFNgamma ELISPOT assay and ELISA. RESULTS: HGXPRT-specific T cell proliferation was found in 44% of patients during acute infection; in 80% of responders both CD4+ and CD8+ T cell subsets proliferated. Antigen-specific T cell proliferation was largely lost within 28 days of parasite clearance. HGXPRT-specific IFN-gamma production was more frequent 28 days after treatment than during acute infection. HGXPRT-specific plasma IgG was undetectable even in individuals exposed to malaria for at least two years. CONCLUSION: The prevalence of acute proliferative and convalescent IFNgamma responses to HGXPRT demonstrates cellular immunogenicity in humans. Further studies to determine minimal HGXPRT epitopes, the specificity of responses for Plasmodia and associations with protection are required. Frequent and robust T cell proliferation, high sequence conservation among Plasmodium species and absent IgG responses distinguish HGXPRT from other malaria antigens.

[Preparation and identification of anti-human polyclonal antibody against xylosyltransferase-I].

AIM: To prepare the rabbit polyclonal antibody against human xylosyltransferase-I (XT-I) protein and to identify its specificity. METHODS: The predominant epitope of XT-I gene was predicted by the DNAssist software. The DNA fragment of this epitope region was synthesized by PCR and cloned into the pGEX-4T-2 vector. The recombinant plasmid was transformed into E.coli ER2566 and the fusion protein GST-XT was induced and isolated. The purified fusion protein was used to immunize New Zealand rabbits. The antibody titer was determined by ELISA. Purified polyclonal antibody was obtained through affinity chromatography column and the specificity of the purified antibody was characterized by Western blot. RESULTS: The amino acid 175-205 of XT-I (QKHQPELAKKPPSRQK-ELLKRKLEQQEKGKG) was selected as an antigen epitope. The synthesized DNA fragment of XT-I was successfully inserted into pGEX-4T-2 vector and the protein GST-XT was expressed. The purified fusion protein GST-XT was used as the immunogen to immunize rabbits and the polyclonal antibody against XT-I protein was obtained. The result of ELISA showed that the antibody titer was 1:640 000. Western blot analysis showed that the antibody had a good specificity. CONCLUSION: The rabbit polyclonal antibody against human XT-I protein has been successfully prepared, which lays the foundation for further study on the biosynthesis of PG by neoplastic myoepithelial cells in salivary tumors.

[Evaluation of immunogenicity and protection efficacy of the recombinant hypoxanthine-guanine-xanthine of plasmodium falciparum in mice].

OBJECTIVE: To investigate immunogenicity and protection efficacy of the recombinant hypoxanthine-guanine-xanthine (HGXPRT) of Plasmodium falciparum expressed in Pichia pastoris. METHODS: 35 BALB/c mice were divided randomly into five groups: HGXPRT+ISA720 experiment group, HGXPRT+Freund experiment group, ISA720 adjuvant control group, Freund adjuvant control group, and blank control group. BALB/c mice were subcutaneously immunized three times with the HGXPRT protein formulated by either Freund or ISA720 adjuvants at a three weeks interval. Mice were bled via tail vein at 2 weeks after each immunization. Specific antibodies were detected by ELISA as well as IFAT using cultured parasites. The immunized mice were challenged with 10(5) P. yoelii 10 days after the third immunization and parasitemia was monitored daily by examining Giemsa-stained thin film. RESULTS: Strong immune response was induced by the HGXPRT antigen formulated with the adjuvant. Antibody titers of more than 1:10(5) were detected after the third immunization while no specific antibody was detected in the mice immunized with adjuvants only. The antibodies against HGXPRT recognized the cultured parasite by IFAT. Four days after mice were challenged with P. yoelii, high parasitemia appeared in the two control groups, which were 24 h earlier than experiment groups. The mean parasitemia of HGXPRT+ISA720 experiment group (29.3%) was significantly lower than that of control groups (70.0%) (P<0.05). The mean parasitemia of HGXPRT+Freund experiment group (51.0%) was significantly lower than that of adjuvant control (60.7%) and blank control(70.0%) (P<0.05). CONCLUSION: HGXPRT of P. falciparum expressed in Pichia pastoris was highly immunogenic in mice. Antibody against the recombinant protein recognizes the cultured parasites, and immunization of mice with the recombinant protein provides partial protection against the challenge of P. yoelii.

Subcellular localization of adenine and xanthine phosphoribosyltransferases in Leishmania donovani.

The subcellular location of a protein is a critical factor in its physiological function and an important consideration in therapeutic paradigms that target the protein. Because Leishmania donovani cannot synthesize purine nucleotides de novo, they rely predominantly upon therapeutically germane phosphoribosyltransferase (PRT) enzymes, hypoxanthine-guanine PRT (HGPRT), adenine PRT (APRT), and xanthine PRT (XPRT), for purine acquisition from the host. Previous studies have shown that the L. donovani HGPRT is localized to the glycosome, a fuel-metabolizing microbody that is unique to kinetoplastid parasites [J. Biol. Chem. 273 (1998) 1534]. The sequences of the other two PRTs indicate that XPRT, but not APRT, possesses a COOH-terminal tripeptide that mediates protein targeting to the glycosome. To determine definitively the intracellular milieu of APRT and XPRT, polyclonal antibodies were raised to each recombinant protein. APRT and XPRT were then shown by immunofluorescence to be localized to the cytosol and glycosome, respectively. The glycosomal milieu for XPRT was also verified by immunoelectron microscopy. Amputation of the glycosomal targeting signal from XPRT resulted in protein mislocalization to the cytosol, but the cytosolic xprt was still functional with respect to purine salvage. These studies establish that APRT is cytosolic and XPRT, like the homologous HGPRT, is glycosomal and demonstrate that a mutant xprt protein that mislocalizes to the cytosol is still functional and supports parasite viability.

First isolation of human UDP-D-xylose: proteoglycan core protein beta-D-xylosyltransferase secreted from cultured JAR choriocarcinoma cells.

Human UDP-d-xylose:proteoglycan core protein beta-d-xylosyltransferase (EC, XT) initiates the biosynthesis of glycosaminoglycan lateral chains in proteoglycans by transfer of xylose from UDP-xylose to specific serine residues of the core protein. In this study, we report the first isolation of the XT and present the first partial amino acid sequence of this enzyme. We purified XT 4,700-fold with 1% yield from serum-free JAR choriocarcinoma cell culture supernatant. The isolation procedure included a combination of ammonium sulfate precipitation, heparin affinity chromatography, ion exchange chromatography, and protamine affinity chromatography. Among other proteins an unknown protein was detected by matrix-assisted laser desorption ionization mass spectrometry-time of flight analysis in the purified sample. The molecular mass of this protein was determined as 120 kDa by SDS-polyacrylamide gel electrophoresis. The isolated protein was enzymatically cleaved by trypsin and endoproteinase Lys-C. Eleven peptide fragments were sequenced by Edman degradation. Searches with the amino acid sequences in protein and EST data bases showed no homology to known sequences. XT was enriched by immunoaffinity chromatography with an immobilized antibody against a synthetic peptide deduced from the sequenced peptide fragments and was specifically eluted with the antigen. In addition, XT was purified alternatively with an aprotinin affinity chromatography and was detected by Western blot analysis in the enzyme-containing fraction.

Toxoid of Pseudomonas aeruginosa exotoxin A generated by deletion of an active-site residue.

Glutamic acid-553 of Pseudomonas aeruginosa exotoxin A (ETA), identified previously as an active-site residue, was deleted by oligonucleotide-directed mutagenesis of the cloned toxin gene in Escherichia coli. The purified mutant toxin was stable, fully immunoreactive, and capable of blocking toxin receptors. ADP-ribosyltransferase and cytotoxic activities were at least 10(6)-fold lower than those of wild-type ETA, and injection of mice with 50 micrograms (equivalent to 400 lethal doses of ETA) produced no ill effects. The mutant toxin elicited high levels of neutralizing anti-ETA antibodies in mice, which protected against a challenge with 100 micrograms of authentic ETA (greater than 600 lethal doses). The mutant protein has the attributes of a toxoid and may be useful as a component of vaccines for individuals at risk for infection by P. aeruginosa.

MHC-restricted recognition of immunogenic T cell epitopes of pertussis toxin reveals determinants in man distinct from the ADP-ribosylase active site.

The S1 subunit of Pertussis toxin (PT) is responsible for the reactogenicity and in part the immunogenicity of Bordetella pertussis vaccine. The critical residues associated with the immunomodulatory effects of PT were located around Glu140 in the S1 subunit. In man, T cell responses to PT are directed at S1 peptides distinct from Glu140. Two such epitopes, p64-75 and p151-161, are immunogenic in a panel of individuals covering a wide range of HLA genotypes. The response to PT peptides is HLA class II restricted. The response to p64-75 is blocked by an anti-HLA-DQ mAb, while that to p151-161 is blocked by an anti-HLA-DR mAb. These findings may allow for the development of a B. pertussis vaccine free from reactogenicity.

Use of synthetic peptides to map antigenic sites of Bordetella pertussis toxin subunit S1.

We synthesized six peptides (15-22 amino acids) from the amino acid sequence of the S1 subunit of pertussis toxin. The antigenicity of the polypeptides was investigated by enzyme-linked immunosorbent assays, in which the polypeptides coupled to bovine serum albumin were used as coating antigens. The polypeptides were examined as antigens for reaction with pre- and postvaccination sera from infants receiving either a Bordetella pertussis whole-cell vaccine or a vaccine containing pertussis toxoid. An elevated serum titer was noted upon vaccination with both types of vaccines when bovine serum albumin conjugates of five synthetic peptides were used as antigens. Similarly, mice immunized with whole-cell pertussis vaccine showed an elevated titer against all five peptides that were reactive with human sera. Thus, we identified five peptide sequences of S1 against which, in two species, antibodies are formed upon exposure of these species to whole cells of B. pertussis.

Functional modification of a 21-kilodalton G protein when ADP-ribosylated by exoenzyme C3 of Clostridium botulinum.

Exoenzyme C3 from Clostridium botulinum types C and D specifically ADP-ribosylated a 21-kilodalton cellular protein, p21.bot. Guanyl nucleotides protected the substrate against denaturation, which implies that p21.bot is a G protein. When introduced into the interior of cells, purified exoenzyme C3 ADP-ribosylated intracellular p21.bot and changed its function. NIH 3T3, PC12, and other cells rapidly underwent temporary morphological alterations that were in certain respects similar to those seen after microinjection of cloned ras proteins. When injected into Xenopus oocytes, C3 induced migration of germinal vesicles and potentiated the cholera toxin-sensitive augmentation of germinal vesicle breakdown by progesterone, also as caused by ras proteins. Nevertheless, p21.bot was immunologically distinct from p21ras.

Botulinum ADP-ribosyltransferase C3 but not botulinum neurotoxins C1 and D ADP-ribosylates low molecular mass GTP-binding proteins.

Botulinum ADP-ribosyltransferase C3 modified 21-24 kDa proteins in a guanine nucleotide-dependent manner similar to that described for botulinum neurotoxin C1 and D. Whereas GTP and GTP gamma S stimulated C3-catalyzed ADP-ribosylation in the absence of Mg2+, in the presence of added Mg2+ ADP-ribosylation was impaired by GTP gamma S. C3 was about 1000-fold more potent than botulinum C1 neurotoxin in ADP-ribosylation of the 21-24 kDa protein(s) in human platelet membranes. Antibodies raised against C3 blocked ADP-ribosylation of the 21-24 kDa protein by C3 and neurotoxin C1 but neither cross reacted with neurotoxin C1 immunoblots nor neutralized the toxicity of neurotoxin C1 in mice. The data indicate that the ADP-ribosylation of low molecular mass GTP-binding proteins in various eukaryotic cells is not caused by botulinum neurotoxins but is due to the action of botulinum ADP-ribosyltransferase C3. The weak enzymatic activities described for botulinum neurotoxins appear to be due to the contamination of C1 and D preparations with ADP-ribosyltransferase C3.

Potential for ambiguity in the interpretation of immunochemical studies on multienzymic proteins. The case of uridylate synthase.

Rabbit antibodies directed against homogeneous uridylate synthase multienzyme from mouse Ehrlich ascites carcinoma precipitate both the orotidine-5'-monophosphate decarboxylase (EC 4.1.1.23) and orotate phosphoribosyltransferase (EC 2.4.2.10) activities of mouse and human erythrocyte uridylate synthase. When the partially purified human enzyme is used as antigen the two activities coprecipitate with the same apparent titer; however, when the mouse carcinoma protein was studied under the same conditions the decarboxylase activity immunoprecipitated with significantly higher avidity than did the transferase activity. Since the mouse multienzyme has been shown to be a single polypeptide that contains both activities (McClard, R.W., Black, M.J., Livingstone, L.R. and Jones, M.E. (1980) Biochemistry 19, 4699-4706), these results were, at face value, surprising. However, when the mouse orotate phosphoribosyltransferase activity (which is largely lost upon dilution into the immunoassay medium) was stabilized with 5-phosphoribosyl 1-pyrophosphate, both enzyme activities displayed the same apparent antibody titer. The immunochemical studies indicate that the antibodies, as a population, preferentially bind to a form or forms of the enzyme which contain(s) denatured transferase domains. A calculation based on a simple model yields a value of approximately 100 for the relative selectivity of the antibody for the denatured form of uridylate synthase. These results illustrate an ambiguity that is inherent in the interpretation of immunochemical studies on such multienzymic proteins; that is, it is possible to conclude incorrectly that two enzyme activities are not functionally associated if one of the catalytic domains is particularly unstable and thereby displays greater immunoreactivity for the specific antiserum.

Purine nucleoside phosphorylase: immunodetection and characterization of the human enzyme.

An antibody prepared against human placental purine nucleoside phosphorylase (PNP) has been used to characterize the subunit structure of the normal enzyme by two-dimensional gel electrophoresis. The antibody against the placental enzyme reacts with PNP present in human lymphocytes, fibroblasts, erythrocytes and placenta. The enzyme can be detected in intact cells by immunofluorescence with the same antibody preparation. The techniques allow the identification of cross-reacting material in a small number of lymphocytes and could therefore be used to detect abnormal enzyme protein in immunodeficiency.

Composition of the first enzyme of histidine biosynthesis isolated from wild-type and mutant operator strains of Salmonella typhimurium.

The first enzyme of histidine biosynthesis in Salmonella typhimurium, adenosine triphosphate phosphoribosyltransferase (EC 2.4.2.17), has been purified from two bacterial strains containing histidine operator deletions and compared to the eenzyme from a strain that has a normal operator. The enzymes isolated in different ways also are compared. Evidence as to the separateness of the operator and first structural gene or covalent modification of the first enzyme was sought. Specific activity, histidine feedback inhibition, amino acid analysis, discontinuous-gel electrophoresis, and gel filtration of the native enzyme, and Ouchterlony double-immunodiffusion tests were carried out. The purified enzyme contains little phosphorous and has five cysteine residues per subunit, which all are readily titratable. No evidence for differences in the enzyme preparations was obtained. Thus, no evidence for overlap of the histidine operator with the first structural gene was obtained.

Purification and characterization of mouse hypoxanthine-guanine phosphoribosyltransferase.

Hypoxanthine-guanine phosphoribosyltransferase (HGPR transferase) (EC 2.4.2.8) has been purified approximately 4500-fold to apparent homogeneity from mouse liver. The procedure involves the use of affinity chromatography and was designed to be readily adaptable to small scale isolations. The enzyme appears to be composed of 3 subunits of identical molecular weight (27,000 per subunit). The subunit molecular weight has also been determined by the analysis of radioactively labeled HGPR transferase immunoprecipitated from wild type and mutant (HGPR transferase) mouse tissue culture cell lines.