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P A Gleeson

Publications and source records attributed to P A Gleeson.

At least 73 records · Page 4Linked to original sources
PubMed1992

Autoimmune gastritis: tolerance and autoimmunity to the gastric H+/K+ ATPase (proton pump).

The alpha and beta subunits of the gastric H+/K(+)-ATPase (proton pump) have been identified as the major molecular targets of parietal cell autoantibodies associated with pernicious anaemia and with murine experimental autoimmune gastritis (EAG) induced by neonatal thymectomy. Recent studies with EAG suggest that the mechanisms of peripheral tolerance and autoimmunity to extrathymic autoantigens are mediated by subsets of "regulator" and "effector" CD4+ T cells, respectively. The persistence of "effector" CD4+ autoreactive T cells in the periphery may be a direct consequence of the delayed developmental expression of the target autoantigen. We hypothesize that cytokines produced by the "regulator" T cells prevent the clonal expansion of the "effector" autoreactive T cells, and that neonatal thymectomy induces organ-specific autoimmunity in genetically susceptible individuals by the reduction of the "regulator" T cell population.

Animals↗
PubMed1992

Diagnostic ELISA for parietal cell autoantibody using tomato lectin-purified gastric H+/K(+)-ATPase (proton pump).

Circulating parietal cell autoantibodies, a useful diagnostic marker for autoimmune gastritis and pernicious anaemia, are currently routinely tested by serum immunofluorescence reactivity with frozen sections of rodent stomach. The major molecular targets of these parietal cell autoantibodies have recently been demonstrated to be the alpha- and the beta-subunits of the gastric H+/K(+)-ATPase (proton pump). We have demonstrated that tomato lectin binds specifically to the beta-subunit of the proton pump and concomittantly co-purifies the alpha-subunit. In the present study, we have exploited the latter observation for the development of a diagnostic ELISA for the detection of parietal cell autoantibodies and compared the performance of this assay with an ELISA using crude gastric membranes. The ELISAs were tested on 72 parietal cell autoantibody-positive sera, 72 parietal cell autoantibody-negative sera and 72 disease-control sera. The ELISA using lectin-purified canine proton pump was superior to that using crude canine gastric membranes in that it was about two-fold more sensitive (82% vs. 43%). With an assay sensitivity of 82% and a specificity of 90%, we propose that the ELISA using the lectin-purified proton pump is a rapid, simple, sensitive and specific diagnostic immunoassay for parietal cell autoantibodies.

Autoantibodies↗
PubMed1991

Reverse immunoaffinity chromatography: application to the purification of the 60- to 90-kDa gastric parietal cell autoantigen associated with autoimmune gastritis.

Previous attempts to purify autoantigens by affinity chromatography using the total IgG from autoantibody-positive sera have not been very successful. We describe here a novel method using parietal cell autoantibodies, isolated on a crude autoantigen immunoabsorbent column, for the purification of the 60- to 90-kDa gastric parietal cell antigen targeted in autoimmune gastritis. The 60- to 90-kDa parietal cell antigen was first enriched 2.5-fold from a 0.5% Triton X-100 extract of total pig gastric mucosal membranes by DEAE-Sepharose 4B chromatography. The fraction containing the 60- to 90-kDa antigen was directly coupled to CNBr-activated Sepharose 4B to produce an immunoadsorbent column for the "reverse" purification of the specific parietal cell autoantibodies from pooled gastric parietal cell autoantibody-positive human sera. A 0.5% Triton X-100 extract of pig gastric mucosal membranes was initially precleared by sequential passage through Protein A-Sepharose 4B and normal human Ig-Sepharose 4B columns. The 60- to 90-kDa gastric parietal cell antigen (approximately 10 micrograms) was then purified from this precleared Triton X-100 membrane extract by affinity chromatography on a 1-ml column of purified autoantibody-Sepharose 4B, followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophonesis. This method, incorporating a "reverse" immunoaffinity chromatography step, has general applicability for the purification of tissue autoantigens using highly specific autoantibodies present in the sera of humans and experimental animals with a variety of other autoimmune diseases.

Animals↗
PubMed1991

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.

Adenosine Triphosphatases↗
PubMed1991

The parietal cell autoantigens recognized in neonatal thymectomy-induced murine gastritis are the alpha and beta subunits of the gastric proton pump [corrected].

Murine autoimmune gastritis, induced by neonatal thymectomy, bears a striking similarity in pathology to the human autoimmune disease, pernicious anemia. Autoantibodies to parietal cells are found in both murine and human diseases. Monoclonal immunoglobulin G autoantibodies, obtained from neonatally thymectomized mice, have previously been shown to recognize two groups of gastric parietal cell antigens. In the present study, it is shown that two of these monoclonal autoantibodies, designated 1H9 and 2B6, are directed against the alpha subunit and beta subunit, respectively, of the gastric hydrogen-potassium-stimulated adenosine triphosphatase (H+,K(+)-ATPase; proton pump). Monoclonal antibody 1H9 showed reactivity by immunoblotting with a 95-kilodalton component of dog gastric tubulovesicular membranes and with a fusion protein containing the hydrophilic domain of the alpha subunit of the H+,K(+)-ATPase. Monoclonal antibody 2B6 reacted by immunoblotting with the 60-90-kilodalton glycoprotein (beta subunit) of the tomato lectin-purified dog H+,K(+)-ATPase and with the 60-90-kilodalton autoantigen purified with human parietal cell autoantibodies. Monoclonal antibody 2B6 also reacted with the deglycosylated 35-kilodalton core protein of the tomato lectin-purified 60-90-kilodalton beta subunit and of the purified 60-90-kilodalton autoantigen. Parietal cell autoantibody-positive sera from 20 mice with experimentally induced gastritis showed reactivity predominantly with the alpha and/or beta subunit of the gastric H+,K(+)-ATPase. Therefore, it is concluded that the major molecules targeted by parietal cell autoantibodies from mice with neonatal thymectomy-induced murine autoimmune gastritis and from humans with pernicious anemia are identical.

Adenosine Triphosphatases↗
PubMed1991

Molecular targets in pernicious anaemia.

Autoimmune gastritis, leading to pernicious anaemia, is an organ-specific autoimmune disease characterized by chronic atrophic gastritis and circulating gastric parietal cell autoantibodies. The parietal cell autoantigens have recently been identified as the alpha and beta subunit of the gastric proton pump (H+, K+ ATPase). Here Paul Gleeson and Ban-Hock Toh discuss how the identification of these gastric parietal cell autoantigens and the development of a mouse model of autoimmune gastritis have paved the way for an understanding of the pathogenesis of the gastric lesion.

Anemia, Pernicious↗
PubMed1991

Identification of components of the endoplasmic reticulum and Golgi complex by murine autoreactive monoclonal antibodies.

A number of autoreactive monoclonal antibodies have been produced by the fusion of spleen cells from unprimed BALB/c mice. The specificities of two of these monoclonal autoantibodies, MUI 38 and MUI 100, have been further examined. By indirect immunofluorescence, monoclonal antibody MUI 38 showed discrete perinuclear staining of acetone-fixed murine 3T3 fibroblasts, which was similar to that obtained with the Golgi vital stain, C6-NBD-ceramide, and with rhodamine-labelled wheat germ agglutinin. Furthermore, the staining pattern with antibody MUI 38 in cells treated with either monensin, taxol or nocodazol was altered in a manner consistent with the known effects of these drugs on Golgi morphology. In contrast, monoclonal antibody MUI 100 showed a diffuse cytoplasmic staining pattern, similar to FITC-Con A, indicative of reactivity with the endoplasmic reticulum. At high dilutions antibody MUI 100 showed only a perinuclear staining pattern, indicating that MUI 100 reacted with the Golgi as well as the endoplasmic reticulum. Both monoclonal antibodies are IgM kappa class and both showed reactivity with acetone-fixed fibroblasts from a number of species, indicating that the antigens are highly conserved. By immunoblotting with total membranes of murine 3T3 cells under either reducing or non-reducing conditions, monoclonal antibody MUI 100 reacted with a number of components with apparent molecular weights (MW) from 27,000 to 63,000. This reactivity was abolished when the 3T3 membranes were treated with sodium periodate, indicating antibody MUI 100 may be specific for carbohydrate. In addition, MUI 100, but not MUI 38, possessed rheumatoid factor activity, reacting with IgG from normal sera of a number of different species. Furthermore, monoclonal antibody MUI 100 was shown to be specific for the Fc domain of IgG. Absorption of MUI 100 antibody with normal rabbit IgG-Sepharose reduced the anti-endoplasmic reticulum reactivity, therefore both activities are attributable to the same antibody.

Animals↗
PubMed1990

Expression of human immunodeficiency virus 1 (HIV-1) envelope gene products transcribed from a heterologous promoter. Kinetics of HIV-1 envelope processing in transfected cells.

The expression of human immunodeficiency virus 1 (HIV-1) envelope glycoprotein products was studied in cells transfected with env gene constructs transcribed from an SV40 promoter. Gene constructs possessing the complete tat, rev (tat+ rev+) and env genes were transiently expressed in COS-1 cells as precursor SU-TM (gp160), SU.TM (gp120 x 41), and nucleolar rev protein. In addition, envelope glycoprotein was detected on the surface of those transfected COS-1 cells expressing abundant levels of env protein. Transfected constructs possessing a mutated tat translational initiation codon (tat-rev+) were expressed in COS-1 cells with at least a 10-fold increase in the level of envelope glycoprotein expression compared to the analogous constructs with an intact tat AUG codon (tat+ rev+). Mutation of the rev initiation codon (tat+ rev-) and (tat-rev-) resulted in no detectable expression of env products but expression of these proteins could be rescued by co-transfection of a cDNA encoding the rev gene. Subgenomic tat/rev transcripts were detected following transfection of all of the gene constructs indicating splicing of the env mRNAs transcribed from a heterologous promoter. Unspliced env transcripts were only detected in the cytoplasm of cells transfected with (rev+) constructs or with the (tat- rev-) construct in the presence of the rev cDNA supplied in trans. In contrast, unspliced transcripts were detected in the nuclear and total cellular RNA of all transfected cells. Expression of rev protein was localized to the nucleolus of transfected COS-1 cells. These results indicate that the export of unspliced env mRNA to the cytoplasm is facilitated by the expression of rev. Following env synthesis, the conversion of SU-TM (gp160) to SU.TM (gp120 x 41) was not quantitative. After a 20-h pulse-chase, only 40% of the SU-TM (gp160) present at the start of the chase period was subsequently accountable as mature SU (gp120), and approximately 30% of the detectable SU (gp120) was found in the culture medium of transfected COS-1 cells. The findings indicate that the surface expression of SU.TM (gp120 x 41) derived from heterologous gene transcripts is modulated (i) the co-expression of rev, (ii) the efficiency of proteolytic processing of SU-TM (gp160), and (iii) the degree of SU (gp120) shedding and/or secretion from the cell.

Animals↗
PubMed1990

N-linked oligosaccharides of the murine transferrin receptor from a plasmacytoma cell line. Comparison with total cellular N-glycans.

The N-linked oligosaccharides synthesised by the murine plasmacytoma cell line NS-1 have been analysed by lectin affinity chromatography on columns of immobilised concanavalin A (Con A), Lens culinaris (lentil), Ricinus communis agglutinin (RCA) and leuko-phytohemagglutinin (L-PHA). The majority of complex N-glycans in this transformed cell line were branched structures with only a low level of biantennary complex chains detected. The analysis showed the major complex N-glycan fraction consisted of a minimum sialylated triantennary structure. [3H]Mannose-labelled transferrin receptor was isolated from NS-1 cells by immunoprecipitation followed by electroelution from SDS polyacrylamide gels. The isolated receptor was digested with Pronase and the 3H-labelled glycopeptides analysed by lectin affinity chromatography. Analysis by Con A-Sepharose indicated that approx. 50% of the labelled glycopeptides were branched complex N-glycans (unbound fraction) while the remainder were oligomannose structures (strongly bound). The presence of tri and/or tetraantennary structures in the Con A unbound fraction was further suggested by the interaction of 61% of the fraction with L-PHA. The lectin profiles obtained for the complex N-glycans of the transferrin receptor glycopeptides were similar to those for the total cellular glycopeptides of NS-1 cells. Reverse-phase HPLC analysis of tryptic glycopeptides of the isolated [3H]mannose-labelled transferrin receptor gave three 3H-labelled peaks, indicating that all three potential N-glycosylation sites on the receptor are utilised. The Con A-Sepharose profiles of the three fractions indicated the presence of branched complex N-glycans and high mannose chains at each site. The profiles of two of the tryptic glycopeptide fractions were very similar, while the third had a higher content of oligomannose oligosaccharides.

Animals↗
PubMed1990

The 60- to 90-kDa parietal cell autoantigen associated with autoimmune gastritis is a beta subunit of the gastric H+/K(+)-ATPase (proton pump).

Autoantibodies in the sera of patients with pernicious anemia recognize, in addition to the alpha subunit of the gastric H+/(+)-ATPase, an abundant gastric microsomal glycoprotein of apparent Mr 60,000-90,000. Herein we have colocalized the glycoprotein and the alpha subunit of the gastric H+/K(+)-ATPase to the tubulovesicular membranes of the parietal cell by immunogold electron microscopy. Moreover, the glycoprotein and the alpha subunit were coimmunoprecipitated, and copurified by immunoaffinity chromatography, with an anti-glycoprotein monoclonal antibody. The pig glycoprotein was purified by chromatography on tomato lectin-Sepharose, and five tryptic peptides from the purified glycoprotein were partially sequenced. The complete amino acid sequence, deduced from the nucleotide sequence of overlapping cDNA clones, showed 33% similarity to the sequence of the beta subunit of the pig kidney Na+/K(+)-ATPase. We therefore propose that the 60- to 90-kDa glycoprotein autoantigen is the beta subunit of the gastric H+/K(+)-ATPase and that the alpha and beta subunits of the proton pump are major targets for autoimmunization in autoimmune gastritis.

Adenosine Triphosphatases↗
PubMed1990

Poly-N-acetyllactosamine-specific tomato lectin interacts with gastric parietal cells. Identification of a tomato-lectin binding 60-90 X 10(3) Mr membrane glycoprotein of tubulovesicles.

The cytoplasmic tubulovesicular and canalicular membranes of gastric parietal cells are intimately involved in hydrochloric acid secretion. To characterise the glycoproteins of these membranes, we examined a panel of lectins for reactivity with parietal cells in paraffin sections of rat, dog and pig stomach. The poly-N-acetyllactosamine-specific lectin from Lycopersicon esculentum (tomato) and from Solanum tuberosum (potato), and the galactose-specific lectin Ricinus communis agglutinin (RCA120), showed strong cytoplasmic binding of parietal cells of all three species, with a pattern indicative of an intracellular membrane network. Binding to parietal cells was confirmed by double-labelling studies with parietal cell auto-antibodies from patients with autoimmune gastritis. Mucous cells and mucin also bound these lectins strongly. Other gastric cell types did not stain with either tomato or potato lectin, but stained weakly with RCA120. Electron-microscopic examination of lectin binding sites using biotinylated tomato lectin or RCA120 and streptavidin-gold, revealed specific binding to the luminal face of parietal cell tubulovesicular and canalicular membranes as well as the contents of mucous cell secretory granules. Tomato lectin and RCA120 reacted by lectin blotting with a major species of apparent molecular weight 60-90 X 10(3) Mr from rat, dog and pig gastric membranes. A tubulovesicular membrane fraction, enriched 10-fold for K(+)-dependent phosphatase activity, was also enriched three-fold for tomato lectin binding as assessed by a solid-phase lectin assay. The 60-90K (K = 10(3) Mr) component, in 125I-labelled detergent extracts of dog tubulovesicular membranes, bound to an affinity support of tomato lectin-Sepharose and was specifically eluted with N,N',N'-triacetylchitotriose. Digestion with N-glycanase collapsed the 60-90K component into a sharp 35K band. We conclude that: (1) a 60-90K membrane glycoprotein localised on the luminal face of tubulovesicles and canaliculi of parietal cells interacts strongly with tomato lectin and RCA120; and (2) the glycoprotein is composed of a 35K core protein glycosylated with N-glycans probably containing poly-N-acetyllactosamine sequences with terminal galactosyl residues. The properties of this 60-90K glycoprotein are identical to a major parietal cell autoantigen recognised by sera of patients with autoimmune gastritis.(ABSTRACT TRUNCATED AT 400 WORDS)

4-Nitrophenylphosphatase↗
PubMed1989

Effect of glycosylation inhibitors on the structure and function of the murine transferrin receptor.

The murine transferrin receptor is a disulphide-linked dimer with three N-glycosylation sites. We have investigated the structural and functional properties of the transferrin receptor from murine plasmacytoma cells (NS-1 cells) treated with the glycosylation inhibitor, tunicamycin and the glycosylation-processing inhibitors, swainsonine and castanospermine. 1. Tunicamycin (1 microgram/ml) inhibited mannose incorporation in NS-1 cells by greater than 90%, but also inhibited methionine incorporation by up to 50%. Both swainsonine (1 microgram/ml) and castanospermine (50 micrograms/ml) resulted in mannose incorporation greater than 100% of untreated cells and neither drug affected methionine incorporation. 2. Incubation of NS-1 cells with tunicamycin resulted in a shift in the apparent molecular mass of the transferrin receptor from 96 kDa and 94 kDa to approximately 82 kDa. 3. Peptide N-glycosidase F digestion of the receptor from untreated cells resulted in the fully deglycosylated 82 kDa component as well as an 87 kDa component which represents partially deglycosylated receptor resistant to peptide N-glycosidase F digestion. 4. The receptor from swainsonine-treated cells was equally sensitive to peptide N-glycosidase F and endo-beta-N-acetylglucosaminidase H (endo H; resulting in both 87-kDa and 82-kDa components), whereas the receptor from castanospermine-treated cells was only partially sensitive to endo H. 5. Analysis of mannose- and fucose-labelled cellular glycopeptides by concanavalin-A--Sepharose chromatography showed that swainsonine (1 microgram/ml) treatment resulted in approximately 90% inhibition of the synthesis of complex N-glycans and an accumulation of fucosylated hybrid structures. In contrast, castanospermine (100 micrograms/ml) treatment resulted in only partial inhibition (60%) of the synthesis of complex N-glycans. 6. Analysis of the receptor from tunicamycin, swainsonine and castanospermine treated cells under nonreducing conditions showed a single component corresponding to the dimer, indicating that dimerisation of newly synthesised murine receptor is independent of carbohydrate. 7. The non-glycosylated receptor from tunicamycin-treated cells appears to bind transferrin as demonstrated by interaction with transferrin-Sepharose. 8. Surface expression of the receptor was not significantly altered in the presence of either swainsonine or castanospermine as judged by flow cytometry.

Alkaloids↗
PubMed1989

Characterization of the hydroxyproline-rich protein core of an arabinogalactan-protein secreted from suspension-cultured Lolium multiflorum (Italian ryegrass) endosperm cells.

An arabinogalactan-protein (AGP) purified from the filtrate of liquid-suspension-cultured Italian-ryegrass (Lolium multiflorum) endosperm cells by affinity chromatography on myeloma protein J539-Sepharose was deglycosylated with trifluoromethanesulphonic acid to remove polysaccharide chains that are covalently associated with hydroxyproline residues in the peptide component of the proteoglycan. The protein core, which accounts for less than 10% (w/w) of the intact proteoglycan, was purified by h.p.l.c. It has an apparent Mr of 35,000, but reacts very poorly with both Coomassie Brilliant Blue R and silver stains. Amino-acid-sequence analysis of the N-terminus of the h.p.l.c.-purified protein core and of tryptic peptides generated from the unpurified protein reveals a high content of hydroxyproline and alanine. These are sometimes arranged in short (Ala-Hyp) repeat sequences of up to six residues. Polyclonal antibodies raised against the protein core do not cross-react with native AGP, the synthetic peptide (Ala-Hyp)4, poly-L-hydroxyproline or poly-L-proline. The results suggest that the polysaccharide chains in the native AGP render the protein core of the proteoglycan inaccessible to the antibodies and that the immunodominant epitopes include domains of the protein other than those rich in Ala-Hyp repeating units.

Amino Acid Sequence↗
PubMed1989

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.

Amidohydrolases↗
PubMed1985

Structures of N-glycans of a ricin-resistant mutant of baby hamster kidney cells. Synthesis of high-mannose and hybrid N-glycans.

The asparagine-linked glycopeptides (N-glycans) of a ricin-resistant mutant of baby hamster kidney (BHK) cells, RicR21, have been isolated and fractionated from a Pronase digest of disrupted cells by concanavalin A (Con A)-Sepharose chromatography, ion-exchange chromatography, and lentil lectin chromatography. The structures of all the major N-glycans have been determined by 500-MHz H NMR spectroscopy. RicR21 synthesizes only hybrid and high-mannose N-glycans. All the hybrid structures contain only three mannose residues. The major hybrid glycopeptide has the following structure: (Formula: see text). There is also about 15% of the nonfucosylated species present. Only a small amount (less than or equal to 5%) of the asialo hybrid is produced. Branched hybrid N-glycans are also present in RicR21 cells, containing two complex antenna linked beta 1----2 and beta 1----4 to the Man alpha 1----3 arm; about 70% of this species is core fucosylated. Man6GlcNAc2 glycopeptide is the most abundant (about 70%) of the high-mannose N-glycans. These studies account for the very poor ricin binding property of this mutant, as the sialic acid residues of the major hybrid N-glycan are exclusively linked alpha 2----3 to galactose and ricin is unable to bind to alpha 2----3-substituted galactosyl residues [Baenziger, J. U., & Fiete, D. (1979) J. Biol. Chem. 254, 9795-9799].

Animals↗
PubMed1985

Binding and uptake of the toxic lectin modeccin by baby hamster kidney (BHK) cells. Isolation of mutants defective in the internalization of modeccin.

The effects of the toxic lectins, ricin and modeccin, on baby hamster kidney (BHK) cells have been compared. Modeccin is about 20-50 times more toxic to BHK cells than ricin. Binding studies showed that there are 10(5) to 2 X 10(5) modeccin binding sites/cell compared with approximately 10(7) binding sites for ricin. Inhibition studies with galactosides indicate that both N- and O-glycans with terminal galactosyl residues are effective inhibitors of modeccin binding. Surface-bound modeccin, at 0 degrees C, was found to be very rapidly endocytosed (75-80% in 10 min) when cells were warmed to 37 degrees C. Two modeccin-resistant BHK cell lines have been isolated and characterized. These mutants bind normal levels of modeccin and retain sensitivity to the toxic action of ricin. Both cell lines were shown to be defective in their ability to internalize modeccin. Also, these mutants are morphologically different from parental cells as they align and elongate very prominently at low cell densities. These mutants will be very useful in understanding the mechanism of uptake and transport of modeccin.

Animals↗
PubMed1984

Galactosyl transferases of baby hamster kidney (BHK) cells. Characterization of two oligosaccharide products synthesised using bovine asialo submaxillary-gland mucin as acceptor.

Extracts of BHK (baby hamster kidney) cells catalyse incorporation of galactose from UDP-galactose into asialo bovine submaxillary gland mucin. The galactosylated oligosaccharide products were released by alkaline-borohydride treatment and purified by Bio-Gel P2 chromatography and high-performance liquid chromatography. The structures of the oligosaccharide sequences synthesised have been identified unequivocally by high resolution 500 MHz 1H-NMR as galactosyl-(beta 1----3) N-acetylgalactosamine and galactosyl (beta 1----4) N-acetylglucosaminyl (beta 1----3)-N-acetylgalactosamine. Characterization of the latter sequence shows the presence in bovine mucin of the type III core sequence N-acetylglucosamine-(beta 1----3) N-acetylgalactosamine. Fractionation of BHK cell extracts on alpha-lactalbumin-Agarose has shown that the (beta 1----4)-galactosyl transferase responsible for synthesis of the trisaccharide binds to alpha-lactalbumin, a modulator of the (beta 1----4)-galactosyl transferase involved in N-glycan assembly. The evidence that the same transferase activity may be responsible for galactose transfer to both O-glycans and N-glycans is discussed.

Animals↗