Monoclonal antibodies specific for the core protein of the beta-subunit of the gastric proton pump (H+/K+ ATPase). An autoantigen targetted in pernicious anaemia.

The gastric H+/K(+)-transporting adenosine triphosphatase (H+/K+ ATPase) (proton pump) consists of a catalytic alpha-subunit and a recently proposed 60-90-kDa glycoprotein beta-subunit. Using dog gastric membranes as the antigen, we have produced two murine monoclonal antibodies, 4F11 (IgG1) and 3A6 (IgA), which are specific for the 60-90-kDa glycoprotein. The monoclonal antibodies (1) specifically stained the cytoplasm of unfixed and formalin-fixed dog gastric parietal cells; (2) specifically reacted by ELISA with gastric tubulovesicular membranes; (3) recognised epitopes located on the luminal face of parietal cell tubulovesicular membranes, the site of the proton pump, by immunogold electron microscopy; (4) immunoblotted a 60-90-kDa molecule from tubulovesicular membranes and a 35-kDa component from peptide N-glycosidase-F-treated membrane extracts; (5) immunoblotted the 60-90-kDa parietal cell autoantigen associated with autoimmune gastritis and pernicious anemia, purified by chromatography on parietal cell autoantibody- or tomato-lectin-Sepharose 4B affinity columns, and the 35-kDa protein core of this autoantigen; this autoantigen has amino acid sequence similarity to the beta-subunit of the related Na+/K(+)-transporting adenosine triphosphatase (Na+/K+ ATPase) [Toh et al. (1990) Proc. Natl Acad. Sci. 87, 6418-6422]; (6) co-precipitated a molecule of 95 kDa with the 60-90-kDa molecule from 125I-labelled detergent extracts of dog tubulovesicular membranes; and (7) co-purified the catalytic alpha-subunit of the H+/K+ ATPase with the 60-90-kDa molecule by immunoaffinity chromatography of tubulovesicular membrane extracts on a monoclonal antibody 3A6-Sepharose 4B column, indicating a physical association between the two molecules. These results provide further evidence that the 60-90-kDa glycoprotein is the beta-subunit of the gastric H+/K+ ATPase. We conclude that the monoclonal antibodies specifically recognise luminal epitopes on the 35-kDa core protein of the 60-90-kDa beta-subunit of the gastric proton pump, a major target molecule in autoimmune gastritis and pernicious anaemia. These monoclonal antibodies will be valuable probes to study the structure and function of this associated beta-subunit, as well as the ontogeny of the gastric proton pump.     

Immunopathology of autoimmune gastritis: lessons from mouse models

Autoimmune gastritis in humans is a chronic inflammatory disease of the stomach accompanied by specific destruction of gastric parietal and zymogenic cells resulting in pernicious anemia. Human gastritis can be accurately reproduced in mice and is characterised by autoantibodies to the alpha- and beta-subunits of the gastric H/K ATPase (the enzyme responsible for gastric acid secretion) and cellular destruction of parietal and zymogenic cells within the gastric gland. Studies with these mouse models have given us our current concepts of the immunopathogenesis of the gastritis. Mouse models have shown that a T cell response is generated to the alpha- and beta-subunits of the H/K ATPase and that an immune response to the beta-subunit seems to be required for disease initiation. Using these models, we have defined key events associated with a damaging autoimmune response to the gastric H/K ATPase. The mechanisms associated with the cellular destruction associated with autoimmune gastritis are not know, but may involve signaling through death inducing pathways such as the Fas/FasL and TNF/TNFR pathways. This knowledge should permit us to develop strategies to prevent and treat the gastritis.     

Perturbation of gastric mucosa in mice expressing the temperature-sensitive mutant of SV40 large T antigen. Potential for establishment of an immortalised parietal cell line

Gastric parietal cells have a unique secretory membrane system that undergoes a profound transformation when the parietal cell is stimulated to secrete acid. Understanding this process has been hindered by the lack of an immortalised parietal cell line. Here we have explored a strategy for the development of a parietal cell line by the generation of transgenic mice bearing the temperature-sensitive mutant of the SV40 large T antigen (SV40 tsA58) under the control of the regulatory sequences of the gastric H+/K+ ATPase beta-subunit (H/Kbeta-tsA58). Three H/ Kbeta-tsA58 transgenic mouse lines were established, namely 218, 224 and 228, all of which expressed the tsA58 T antigen in the gastric mucosa. Unexpectedly, the gastric mucosae of all lines were hypertrophic indicating that the temperature-sensitive large T antigen was partially active at 37 degrees C. Immunofluorescence together with light and electron microscopic studies revealed that mature parietal and zymogenic cells were absent in H/Kbeta-tsA58 transgenic lines 218 and 224, and small undifferentiated cells were the dominant cell type in the gastric units. On the other hand, a few mature parietal cells were detected in line 228 together with an increased proportion of undifferentiated cells and, normally rare, pre-parietal cells. As line 228 represented a rich source of pre-parietal cells, gastric cells from line 228 were isolated and cultured at 33 degrees C, the permissive temperature for tsA58. Gastric epithelial cells, expressing the T antigen, were maintained in culture for over 6 weeks. Upon a temperature shift to 39 C the cultured gastric cells developed characteristics of differentiated parietal cells, including the presence of a nascent canaliculus and dramatically increased production of the gastric H+/K+ ATPase beta-subunit. Therefore, this system shows the potential to generate an immature parietal cell line that can be induced to differentiate in vitro.     

A convenient model of severe, high incidence autoimmune gastritis caused by polyclonal effector T cells and without perturbation of regulatory T cells

Autoimmune gastritis results from the breakdown of T cell tolerance to the gastric H(+)/K(+) ATPase. The gastric H(+)/K(+) ATPase is responsible for the acidification of gastric juice and consists of an α subunit (H/Kα) and a β subunit (H/Kβ). Here we show that CD4(+) T cells from H/Kα-deficient mice (H/Kα(-/-)) are highly pathogenic and autoimmune gastritis can be induced in sublethally irradiated wildtype mice by adoptive transfer of unfractionated CD4(+) T cells from H/Kα(-/-) mice. All recipient mice consistently developed the most severe form of autoimmune gastritis 8 weeks after the transfer, featuring hypertrophy of the gastric mucosa, complete depletion of the parietal and zymogenic cells, and presence of autoantibodies to H(+)/K(+) ATPase in the serum. Furthermore, we demonstrated that the disease significantly affected stomach weight and stomach pH of recipient mice. Depletion of parietal cells in this disease model required the presence of both H/Kα and H/Kβ since transfer of H/Kα(-/-) CD4(+) T cells did not result in depletion of parietal cells in H/Kα(-/-) or H/Kβ(-/-) recipient mice. The consistency of disease severity, the use of polyclonal T cells and a specific T cell response to the gastric autoantigen make this an ideal disease model for the study of many aspects of organ-specific autoimmunity including prevention and treatment of the disease.     

Sequence motif in control regions of the H+/K+ ATPase alpha and beta subunit genes recognized by gastric specific nuclear protein(s).

A nuclear protein(s) from rat or pig stomach recognized a conserved sequence in the 5'-upstream regions of the rat and human H+/K(+)-ATPase alpha subunit genes. A gel retardation assay suggested that part of the binding site was located in the TAATCAGCTG sequence. No nuclear proteins capable of the binding could be detected in other tissues of rat (liver, brain, kidney, spleen and lung) or pig liver. The sequence motif (GATAGC) located 5'-upstream of the beta-subunit gene also seemed to be recognized by the same protein, because the binding of nuclear protein to the sequence motifs in the alpha and beta subunits was mutually competitive. Considering the sense-strand sequence of the binding motif in the alpha-subunit gene, we conclude that (G/C)PuPu(G/C)NGAT(A/T)PuPy is a core sequence motif for the gastric specific DNA binding protein (PCSF, parietal cell specific factor).     

Melittin inhibition of the gastric (H+ + K+) ATPase and photoaffinity labeling with [125I]azidosalicylyl melittin

Melittin is a 26-amino acid amphipathic polypeptide toxin from bee venom which forms anion-selective ion channels in bilayers and biological membranes under the influence of membrane potential. Melittin has been shown to interact with a number of membrane proteins. We found that melittin inhibited K+-stimulated ATP hydrolysis by the (H+ + K+) ATPase in parietal cell apical membrane vesicles derived from histamine-stimulated rabbit gastric mucosa with a KIapp of 0.5 micron. Melittin also inhibited K+-stimulated p-nitrophenyl hydrolysis activity which is associated with the gastric (H+ + K+) ATPase in a dose-dependent manner with a KIapp of 0.95 micron. ATP-driven, K+-dependent H+ transport was inhibited over this same concentration range, even in the absence of a membrane potential. Melittin did not appear to increase the H+ leak from vesicle with preformed H+ gradients when the H+ pump was arrested by Mg2+ chelation, but all possible membrane perturbation effects were difficult to rule out. However, the data suggest that melittin exerts its inhibitory effect through interaction with the (H+ + K+) ATPase. In order to determine whether direct interactions between the (H+ + K+) ATPase and melittin occurred, a radioactive derivative of melittin, [125I]azidosalicylyl melittin, was prepared and photoreacted with sealed rabbit gastric membranes and highly purified hog gastric membrane containing the (H+ + K+) ATPase. In the purified hog preparation only a 95,000-Da band, the (H+ + K+) ATPase was labeled, while in the rabbit preparation a 95,000-Da band and one other membrane protein of 70,000 Da were labeled with this reagent. Label incorporation into the (H+ + K+) ATPase and the 70,000-Da band was greatly reduced by addition of excess unlabeled melittin, suggesting specificity of the interaction. Label incorporation occurred in the absence of ATP or added salts and was not reduced by SCH28080 (a K+ site inhibitor) suggesting that the melittin binding site was distinct from the luminal K+ site of action of SCH28080.     

Immunopurification of gastric parietal cell tubulovesicles.

1. The tubulovesicles of hog and rabbit gastric parietal cells were immunopurified from microsomes using monoclonal antibodies against the (H+, K+)-ATPase. 2. The best yields of immunoprecipitation were obtained with an ATPase/mAb molar ratio of 0.3: the immunoprecipitate contained 79 and 90% of the hog and rabbit microsomal PNPPase activity respectively and K(+)-stimulated ATPase specific activity was 221 +/- 29 mumoles Pi per hr and per mg of membrane protein. 3. The immunoprecipitate contained vesicles that were 85% cytoplasmic-side out, like tubulovesicles in vivo, demonstrating that the epitopes were cytoplasmic. 4. The alpha-beta protomer of (H+, K+)-ATPase accounted for 80 +/- 12% of the immunopurified proteins. 5. The major other proteins ran at 80, 75, 69, 57, 47, 44, 39, 34 and 32 kDa on the SDS-PAGE. 6. Comparative analysis between sucrose-gradient purified fractions and immunopurified tubulovesicles demonstrated that carbonic anhydrase and actin were contaminants and that the 53 kDa and presumably the 50 kDa bands of the gradient fraction were alpha and beta subunits of F1 ATPase.     

cDNA cloning of the beta-subunit of the human gastric H,K-ATPase.

A full-length cDNA clone encoding the human gastric H,K-ATPase (EC 3.6.1.36)beta-subunit was isolated from a human gastric mucosal lambda gt10 library using oligonucleotide probes which were based on the cDNA sequence from rat and rabbit H,K-ATPase beta-subunits. The insert was 1407 bp in length and encoded a polypeptide of 291 amino acids with a MW = 33,367 Da. It exhibited 84.2%, 85.6% and 81.3% identity to the H,K-ATPase beta-subunits of rabbit, pig and rat, respectively.     

Exposure of thyroxine residues in human thyroglobulin. Two-site binding studies

Human thyroglobulin (Tg) could be adsorbed through one of its thyroxine (T4) residues by either of two T4-binding antibodies which had been covalently attached to Sepharose- CL4B . The antibodies used were (i) a purified human autoantibody specific for a T4-containing epitope in human Tg, or (ii) a rabbit antibody raised against T4 conjugated to bovine albumin side chains. Tg adsorbed by either immobilized antibody could then itself adsorb either type of antibody free in solution on to a further T4 residue. At least two T4 residues in human Tg are therefore sufficiently exposed to interact with T4-binding antibodies. Furthermore, these T4 residues are sufficiently far apart to allow the binding of two immunoglobulin molecules simultaneously. Previous observations of a marked preference by human autoantibodies for one of the T4-containing epitopes in Tg therefore reflect a higher binding energy with that epitope rather than an inability to interact with others. The T4-containing epitope which preferentially reacts with human Tg autoantibodies must therefore have a distinctive topography.     

Human insulin autoantibody fine specificity and H and L chain use

Fine specificity and H and L chain isotypes of insulin autoantibodies in sera from 11 subjects were examined. None of these 11 subjects was treated with exogenous insulin. Two patterns of fine specificity were found. In one, the autoantibodies were specific for human insulin, with a requirement for threonine at B30. The conservative substitution in pork insulin (threonine to alanine) abrogated IgG binding by these sera. Insulin autoantibodies in other sera cross-reacted with beef, pork, and human insulin; not requiring threonine at B30. Reciprocal competitive inhibition experiments showed that epitopes recognized by the human specific insulin autoantibodies were exclusively on the B chain, whereas the cross-reactive sera contain autoantibodies that recognize both the B chain and combinatorial (A and B chain) epitopes. The fine specificity of cross-reactive insulin autoantibodies are thus similar to insulin antibodies from insulin-treated subjects. When IgG subclasses and L chains of insulin autoantibodies were examined, however, restricted C region usage was found. The hierarchy was IgG3 greater than G1 greater than G2 greater than G4; with one subclass dominant in each serum, although others were used. L chain use was similarly restricted. There was no correlation between isotype and fine specificity or between H and L chain type. It is concluded that heterogeneity of insulin autoantibodies is restricted. The response is probably more oligo- or pauciclonal than insulin antibody from insulin-treated subjects.     

Localization of LewisX, sialyl-LewisX and alpha-galactosyl epitopes on glycosphingolipids in lens tissues

Mammalian lens contains several neutral and acidic glycosphingolipids, the core structures of which are ganglio-, neolacto-, globo-, and isoglobo-series sugar chains. Old World monkey lens shows glycosphingolipid compositions similar to those of human cataractous lens, in particular the presence of Lewisxand sialyl-Lewisxepitopes and the absence of alpha-galactosyl epitope. Dog and pig lenses contain globotriaosylceramide and the sialyl-Lewisxcontaining neolactotetraosylceramide, respectively, which were found in primate lens, together with the alpha-galactosyl epitope containing neolactotetraosylceramide. Thin-layer chromatography immunostaining revealed the enrichment of some neolacto-series glycosphingolipids in the cortical and nuclear fibers, but not in lens epithelia, of dog, pig, and Japanese monkey lenses. Immunohistochemical studies confirmed the expression of Lewisx, sialyl-Lewisx, and alpha-galactosyl epitopes in the inner cortical and nuclear fibers, in association with the differentiation and maturation of lens epithelial cells to lens fibers. Glycobiological approaches thus suggested that some neolacto-series glycosphingolipids are involved in lens fiber development, in which the physiological roles of the alpha-galactosyl epitope are evolutionarily replaced by the Lewisxand sialyl-Lewisxepitopes in Old World monkeys and humans.      

Gastric parietal cell antigens of 60-90, 92, and 100-120 kDa associated with autoimmune gastritis and pernicious anemia. Role of N-glycans in the structure and antigenicity of the 60-90-kDa component

Thirty-four human sera containing parietal cell autoantibodies (PCA) specifically immunoprecipitated two antigens, with apparent molecular masses of 60-90 kDa and 100-120 kDa under nonreducing conditions and 60-90 kDa and 120-150 kDa under reducing conditions, from porcine gastric membrane extracts. A third antigen of 92 kDa was only observed in immunoprecipitates analyzed under reducing conditions. By immunoblotting, 24 of the 34 PCA-positive sera reacted with only the 60-90-kDa antigen, three reacted with a broad 60-120-kDa smear, one reacted only with a 92-kDa antigen and six did not react. Reactivity with the 60-90-kDa antigen was observed with gastric membranes from dog, pig, rat, and rabbit. Twenty PCA-negative sera did not react with these components by immunoprecipitation or immunoblotting. PCA reactivity with the 60-90-kDa antigen was abolished when the gastric membranes were (a) digested with Pronase, (b) reduced with 100 mM dithiothreitol, (c) treated with sodium periodate, or (d) digested with N-glycanase. The 60-90-kDa and 100-120-kDa components were insensitive to neuraminidase treatment. N-glycanase digestion of 125I-labeled antigens purified by immunoprecipitation and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis collapsed the 60-90-kDa antigen to a sharp 34-kDa band; the 100-120-kDa component was unaffected. These observations suggest that (i) parietal cell antigens comprise three components of 60-90, 92, and 100-120 kDa; (ii) the epitopes differ in conformational sensitivity; (iii) the 60-90-kDa antigen is a conserved molecule comprising a 34-kDa core protein extensively glycosylated with N-linked oligosaccharides; (iv) sialic acid residues are not present in the 60-90- and 100-120-kDa molecules, and (v) the carbohydrate and protein moieties of the 60-90-kDa molecule are required for antibody binding.     

Identification of ribosomal protein autoantigens

Approximately 20% of patients with systemic lupus erythematosus and with anti-Sm autoantibodies synthesize autoantibodies, called anti-rRNP, to components of the ribosome. We found that anti-rRNP sera reacted predominantly with three ribosomal phosphoproteins of approximate Mr = 38,000, 16,000 and 15,000, both by immunoprecipitation and by immunoblotting. The human autoantibodies cross-reacted with similar antigens present in rodent, brine shrimp, and yeast cells but reacted weakly if at all with proteins of bacteria. Thus the human autoantibodies recognize epitopes that are widely conserved in evolution. Purified ribosomal proteins together with specific rabbit antisera were used to identify the two smaller rRNP antigens as the acidic phosphoproteins of the large ribosomal subunit, designated P1/P2(L40/L41) (rat), eL7/eL12 (Artemia, brine shrimp), and A1/A2 (yeast). These proteins function in the elongation step of protein synthesis in an analogous fashion to the L7/L12 ribosomal proteins of E. coli. The 38,000-dalton rRNP antigen corresponds to a nonacidic protein also associated with the large ribosomal subunit. The human autoantibodies appear to have a specificity similar to that of a previously described mouse monoclonal antibody obtained from mice injected with heterologous (chick) ribosomes, suggesting that both the human polyclonal autoantibodies and the mouse monoclonal recognize a class of epitope(s) that is common in all three ribosomal proteins. In addition, we found that many of the anti-ribosomal sera contained a further class of autoantibodies reactive with naked RNA. These may be similar to the anti-RNA antibodies previously described in both humans and mice with autoimmune disease.     

Identification of Immunodominant Neutralizing Epitopes on the Hemagglutinin Protein of Rinderpest Virus

The immunodominant epitopes on the hemagglutinin protein of rinderpest virus (RPV-H) were determined by analyzing selected monoclonal antibody (MAb)-resistant mutants and estimating the level of antibody against each epitope in five RPV-infected rabbits with the competitive enzyme-linked immunosorbent assay (c-ELISA). Six neutralizing epitopes were identified, at residues 474 (epitope A), 243 (B), 548 to 551 (D), 587 to 592 (E), 310 to 313 (G), and 383 to 387 (H), from the data on the amino acid substitutions of hemagglutinin protein of MAb-resistant mutants and the reactivities of MAbs against RPV-H to the other morbilliviruses. The epitopes identified in this study are all positioned on the loop of the propeller-like structure in a hypothetical three-dimensional model of RPV-H (J. P. M. Langedijk et al., J. Virol. 71:6155-6167, 1997). Polyclonal sera obtained from five rabbits infected experimentally with RPV were examined by c-ELISA using a biotinylated MAb against each epitope as a competitor. Although these rabbit sera hardly blocked binding of each MAb to epitopes A and B, they moderately blocked binding of each MAb to epitopes G and D and strongly blocked binding of each MAb to epitopes E and H. These results suggest that epitopes at residues 383 to 387 and 587 to 592 may be immunodominant in humoral immunity to RPV infection.     

Isolation and characterization of gastric microsomal glycoproteins. Evidence for a glycosylated beta-subunit of the H+/K(+)-ATPase.

Detergent-solubilization of hog gastric microsomal membrane proteins followed by affinity chromatography using wheat germ agglutinin or Ricinus communis I agglutinin resulted in the isolation of five glycoproteins with the apparent molecular masses on sodium dodecyl sulfate polyacrylamide gels of (in kDa): 60-80 (two glycoproteins sharing this molecular mass); 125-150; and 190-210. In the nonionic detergent Nonidet P-40 (NP-40), the 94 kDa H+/K(+)-ATPase was recovered exclusively in the lectin-binding fraction; however, in the cationic detergent dodecyltrimethylammonium bromide, most of the ATPase was recovered in the nonbinding fraction. Detection of glycoproteins either by periodic acid-dansyl hydrazine staining of carbohydrate in polyacrylamide gels or by Western blots probed with lectins indicated that the majority of the ATPase molecules are not glycosylated. In addition, in the absence of microsomal glycoproteins, the NP-40-solubilized ATPase does not bind to a lectin column. Taken together, these results suggest that the recovery of NP-40-solubilized ATPase in the lectin-binding fraction is due to its noncovalent interaction with a gastric microsomal glycoprotein. Immunoprecipitation of the ATPase from NP-40-solubilized microsomal membrane proteins resulted in the co-precipitation of a single 60-80 kDa glycoprotein. Characterization of the 60-80 kDa glycoprotein associated with the ATPase revealed that: it is a transmembrane protein; it has an apparent core molecular mass of 32 kDa; and, it has five asparagine-linked oligosaccharide chains. Given its similarity to the glycosylated beta-subunit of the Na+/K(+)-ATPase, this 60-80 kDa gastric microsomal glycoprotein is suggested to be a beta-subunit of the H+/K(+)-ATPase.     

Location of the cytoplasmic epitope for a K(+)-competitive antibody of the (H+,K+)-ATPase.

The monoclonal antibody (mAb) 95-111 binds the alpha subunit of (H+,K+)-ATPase and inhibits the K(+)-ATPase activity. To map the epitope, all of the partial sequences of the alpha subunit were expressed in Escherichia coli HB101 using rabbit alpha subunit cDNA restriction fragments ligated into PuEx vector. Bacterial recombinant lysates were separated by sodium dodecyl sulfate-gel electrophoresis, and the epitope was detected by Western blotting. The antibody site was mapped between Cys529 and Glu561. This is close to the Lys517 that binds fluorescein isothiocyanate (FITC) and is considered to be between M4 and M5 close to the ATP binding domain. However, the mAb inhibition of ATPase is not ATP-competitive but is K(+)-competitive with a KI of 2 x 10(-9) M. The mAb also inhibits K+ quench of FITC fluorescence competitively with a KI of 8 x 10(-9) M. The K+ activation of ATPase activity and quench of FITC fluorescence are dependent on K+ binding to an E2 form of the enzyme from the extracytoplasmic surface. The mAb epitope is cytoplasmic since the K(+)-ATPase activity of ion-tight gastric vesicles is inhibited. The 125I-mAb 95-111 binds to a single class of sites with an apparent KD of 2.3 +/- 0.8 x 10(-9) M and K+ does not displace bound mAb. Hence, antibody binding to a cytoplasmic Cys529-Glu561 epitope allosterically competes with K(+)-dependent reactions at extracytoplasmic sites.     

Epitopes of the p70 and p80 (Ku) lupus autoantigens.

High titer autoantibodies to the Ku Ag, a DNA-protein complex containing 70- and approximately 80-kDa protein subunits (p70 and p80, respectively), are found in sera of certain patients with systemic lupus erythematosus and related disorders. Autoepitopes of the Ku Ag were identified and partially characterized by expressing fragments of the p70 and p80 cDNA as fusion proteins in bacteria. Systemic lupus erythematosus sera reacted on immunoblots with at least three epitopes of p70 (amino acids 560-609, 506-535, and 115-467), and three epitopes of p80 (amino acids 682-732, 558-681, and 1-374). These six antigenic regions had distinct amino acid sequences, and were also immunologically distinct, as determined by using immunoaffinity-purified auto-antibodies to particular epitopes. Detailed mapping of the strongly antigenic region near the C terminus of p70 revealed a complex conformational or discontinuous epitope, the antigenicity of which was abolished by deleting either amino acids 560-571 or 601-609. The C terminus of p80 may also contain a discontinuous or conformational epitope(s). Although only some sera reacted with p70 or p80 on immunoblots, all sera that immunoprecipitated the native Ku complex reacted with native Ku by ELISA, and inhibited the binding of mAb directed at epitopes of native Ku. Taken together, these studies indicate that anti-Ku autoantibodies target a diversity of independent epitopes located on p70, p80, and the intact Ku complex, and that a significant portion of the autoantibodies in most patients' sera is directed against conformational/discontinuous epitopes.     

The K(+) affinity of gastric H(+),K(+)-ATPase is affected by both lipid composition and the beta-subunit

It is generally assumed that negatively charged residues present in the alpha-subunit of gastric H(+),K(+)-ATPase are involved in K(+) binding and transport. Despite the fact that there is no difference between various species regarding these negatively charged residues, it was observed that the apparent K(+) affinity of the pig enzyme was much lower than that of the rat H(+),K(+)-ATPase. By determining the K(+)-stimulated dephosphorylation reaction of the phosphorylated intermediate K(0.5) values for K(+) of 0.12+/-0.01 and 1.73+/-0.03 mM were obtained (ratio 14.4) for the rat and the pig enzyme, respectively. To investigate the reason for the observed difference in K(+) sensitivity, both enzymes originating from the gastric mucosa were either reconstituted in a similar lipid environment or expressed in Sf9 cells. After reconstitution in K(+)-permeable phosphatidylcholine/cholesterol liposomes K(0.5) values for K(+) of 0.16+/-0.01 and 0.35+/-0.05 mM for the rat and pig enzyme respectively were measured (ratio 2.2). After expression in Sf9 cells the pig gastric H(+),K(+)-ATPase still showed a 4.1 times lower K(+) sensitivity than that of the rat enzyme. This means that the difference in K(+) sensitivity of the rat and pig gastric H(+), K(+)-ATPase is not only due to a different lipid composition but also to the structure of either the alpha- or beta-subunit. Expression of hybrid enzymes in Sf9 cells showed that the difference in K(+) sensitivity between the rat and pig gastric H(+),K(+)-ATPase is primarily due to differences in the beta-subunit.     

Molecular cloning of endo-beta -galactosidase C and its application in removing alpha -galactosyl xenoantigen from blood vessels in the pig kidney

Galalpha1-3Gal is the major xenoantigenic epitope responsible for hyperacute rejection upon pig to human xenotransplantation. Endo-beta-galactosidase C from Clostridium perfringens destroys the antigenic epitope by cleaving the beta-galactosidic linkage in the Galalpha1-3Galbeta1-4GlcNAc structure. Based on partial peptide sequences of the enzyme, we molecularly cloned the enzyme gene, which encodes a protein with a predicted molecular mass of about 93 kDa. The deduced protein sequence of the enzyme has limited homology in the C-terminal half with endo-beta-galactosidase from Flavobacterium keratolyticus and beta-1,3-glucanases. The enzyme expressed in Escherichia coli removed the alpha-galactosyl epitope nearly completely from pig erythrocytes and from pig aortic endothelial cells. The enzyme-treated endothelial cells in culture were greatly reduced in cell surface antigens, which were recognized by IgM, IgG, or IgA in human sera, and became much less susceptible to complement-mediated cytotoxicity caused by human sera. When the pig kidney was perfused with the enzyme, the vascular endothelial cells became virtually devoid of the alpha-galactosyl epitope, with concomitant decrease in binding to IgM in human plasma. These results demonstrated that the recombinant endo-beta-galactosidase C is a valuable aid in xenotransplantation.