Biomedical subjects
Publications and source records attributed to T W Rademacher.
Glycoconjugate chemistry and glycobiology were rapidly evolving scientific disciplines in the 1980s. Their impact, however, on understanding fundamental biological processes was not immediately forthcoming. Within the past 12-18 months, there has been a resurgence in the field with major discoveries leading to powerful new insights into the complex role of glycoconjugates in biological processes.
OBJECTIVE: This study examined the effect of exogenous dehydroepiandrosterone (DHEA) on the onset, incidence, and severity of collagen-induced arthritis (CIA). METHODS: DHEA was administered subcutaneously prior to arthritis induction in DBA/1 mice, and the severity of the subsequent arthritis was monitored. Serum levels of total IgG and IgG isotype-specific anti-murine type II collagen were measured. RESULTS: Repeated administration of DHEA during arthritis induction delayed the onset and decreased the severity of arthritis in male and female DBA/1 mice. DHEA failed to have an observable effect on established arthritis. IgG isotype autoantibody levels were found to be decreased in the sera of DHEA-treated mice. CONCLUSION: Administration of exogenous DHEA offered protection against the development of CIA. These data support the results of human studies in which low DHEA levels have been identified as a potential risk factor for the development of rheumatoid arthritis. These findings also highlight DHEA as a potential therapy worthy of further investigation.
Extracts of human liver were found to contain activities which copurified and coeluted with the two major subtypes of mediators (type A and type P) isolated from insulin-stimulated rat liver. The putative type A mediator from human liver inhibited cAMP-dependent protein kinase from bovine heart, decreased phosphoenolypyruvate carboxykinase mRNA levels in rat hepatoma cells, and stimulated lipogenesis in rat adipocytes. The putative type P mediator stimulated bovine heart pyruvate dehydrogenase phosphatase. Both fractions were able to stimulate proliferation of EGFR T17 fibroblasts and the type A was able to support growth in organotypic cultures of chicken embryo cochleovestibular ganglia. Both activities were resistant to Pronase treatment and the presence of carbohydrates, phosphate, and free-amino groups were confirmed in the two fractions. These properties are consistent with the structure/ function characteristics of the type A and P inositolphosphoglycans (IPG) previously characterized from rat liver. Further, the ability of the human-derived mediators to interact with rat adipocytes and bovine-derived metabolic enzymes suggests similarity in structure between the mediators purified from different species. Galactose oxidase-susceptible membrane-associated glycosylphosphatidylinositols (GPI) have been proposed to be the precursors of IPG. GPI was purified from human liver membranes followed by treatment with galactose oxidase and reduction with NaB3H4. Serial t.l.c. revealed three radiolabeled bands which comigrated with the putative GPI precursors found in rat liver. These galactose-oxidase-reactive lipidic compounds, however, were only partially susceptible to hydrolysis with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis and were resistant to glycosylphosphatidylinositol-specific phospholipase C from Trypanosoma brucei. These data indicate that IPG molecules with insulin-like biological activities are present in human liver.
This study investigates immobilisation of murine IgG in various ELISA procedures. Monoclonal murine IgG isotypes and polyclonal IgG from sera were studied. Similar binding curves to plastic were found for all four individual murine IgG isotypes. Single isotypes displayed different affinities for both protein A and protein G, in particular IgG1 was poorly and IgG3 strongly bound to both of these proteins. When mixtures of the isotypes were bound to either plastic, protein A or protein G, competition was observed in which IgG3 was dominant. Paradoxically, studies on the binding rates of single isotypes direct to plastic revealed that IgG3 had the slowest binding rate. Heating of bound IgGs resulted in significant but isotypically non-selective losses from the plates. The data demonstrate that despite obtaining equivalent individual IgG isotype binding curves, mixtures of IgG isotypes behave very differently, with competition for binding occurring even on plastic. The IgG isotype levels of murine sera were measured for individual mice, and the capture efficiency of each IgG isotype by protein A determined at different serum dilutions. Comparisons were made between the observed capture levels of IgG isotypes and their known serum levels. At all dilutions tested, greater than expected binding of IgG3, IgG2b and IgG2a was observed. At a serum dilution of 1/100 the binding of these three isotypes was increased 16-, 2.9- and 0.4-fold, respectively. These increases were balanced by a decrease in IgG1 binding which was the most prevalent serum IgG isotype. The results described above suggest that capture techniques are biased and unlikely to provide a coating of IgG isotypes that accurately reflects that of the serum. This bias is derived from the specificity of the individual isotypes for either protein A or protein G, and the errors further compounded by direct competition between isotypes whatever the capture surface. Induced coalescence of IgG3 may explain the latter observations.
The glycosylation of tissue plasminogen activator (t-PA) obtained from the Bowes melanoma cell line was re-examined using methods of serial lectin affinity chromatography coupled with Bio-Gel P-4 gel filtration chromatography and exoglycosidase sequencing. This study clarified an earlier discrepancy in the literature and confirmed that the major complex N-linked glycans on Bowes t-PA that carry sialic acid as their sole charged group are bi-antennary, core fucosylated, with terminal N-acetylgalactosamine residues. We also report the characterization of a series of related and previously unidentified sialylated glycans. Further we show that Bowes t-PA expresses glucuronic acid/sulphate containing N-linked glycans and is recognized by anti-carbohydrate L2/HNK-1 monoclonal antibodies. The presence on Bowes t-PA of glycans associated primarily with the nervous system is consistent with its expression in a cell line of neuroectodermal origin.
The galactosylation status of IgG from both control and arthritic DBA-1 mice was determined by exoglycosidase sequencing and an anti-GlcNAc monoclonal based ELISA. Two to three weeks after arthritis onset mice with collagen-induced arthritis (CIA) showed a modest increase in IgG anti-GlcNAc reactivity against controls (0.644 +/- 0.080 and 0.530 +/- 0.087, respectively, mean absorbance +/- SEM n = 4) consistent with previous literature reports. However, the authors were unable to detect any significant changes in the galactosylation of purified IgG from arthritic and control mice using direct sequencing techniques (percentage G0 of 34.5% +/- 1.9 and 37.7% +/- 2.4, respectively, mean +/- SEM n = 4). It has been demonstrated that a correlation exists between percentage G0 and anti-GlcNAc reactivity for purified human IgG and sera samples, respectively; no such correlation was found for murine IgG and sera. The galactosylation of purified polyclonal murine IgG isotypes was analysed on a separate group of arthritic mice selected for their high anti-GlcNAc reactivities. No significant differences were observed between control and arthritic mice. Immunoglobulin G isotype specific differences were found, with IgG1 exhibiting the highest percentage G0 (45-48%) followed by IgG2a (27-37%), IgG3 (20-32%) and IgG2b the lowest (13-17%). The percentage G0 and anti-GlcNAc reactivity of purified IgG1 and IgG2b showed a narrow range of values when compared to those of IgG2a and IgG3 samples. Pooled sera from both arthritic and control mice was used to purify large quantities of IgG3. Which on analysis revealed a fourfold increase in anti-GlcNAc reactivity in the arthritic sample compared to control. Paradoxically these same IgG3 samples contained similar percentage G0 levels as determined by direct sequencing. The results suggest that IgG1 and IgG2b exhibit isotype selective oligosaccharide processing as little sample heterogeneity could be observed. These two purified IgG isotypes displayed a good correlation between percentage G0 and anti-GlcNAc reactivity; this was in contrast to IgG2a and IgG3. Immunoglobulin G3 from arthritic mice may have G0 oligosaccharides selectively paired together with no net increase in percentage G0. This observation is discussed in detail as is the role of agalactosyl IgG in murine type II collagen-induced arthritis.
Malaria toxin causes hypoglycemia and induction of tumor necrosis factor. Extracts of parasitized erythrocytes which were coeluted and copurified with one of the two subtypes of mammalian insulin-mimetic inositolphosphoglycans similarly induced fibroblast proliferation in the absence of serum. In addition, induction of tumor necrosis factor in macrophages by malaria toxin and by lipopolysaccharide from Escherichia coli was enhanced by pretreatment of these toxins with alpha-galactosidase. Thus, parasitized erythrocytes contain both soluble inositolphosphoglycan-like insulin second messengers and endotoxin-like lipidic molecules.
The excessive production of tumour necrosis factor (TNF) is associated with the pathology of blood-stage malaria and phosphatidylinositol-containing phospholipid antigens from parasitized erythrocytes stimulate its secretion by macrophages, thus acting as toxins. This brief report describes some properties of an inhibitor present in lysates from erythrocytes infected with malarial parasites that blocked the detection of recombinant TNF in an enzyme-linked immunosorbent assay and diminished or abolished the cytotoxicity of TNF. It was not found in control lysates of normal erythrocytes. Its addition to macrophage cultures stimulated by toxic malarial preparations or by bacterial lipopolysaccharide also blocked the detection of TNF. These findings may explain the contradictory results obtained from different assays for TNF, and emphasize the need for caution when interpreting the results of a single assay system. If released when parasitized erythrocytes rupture in vivo, the inhibitor could help protect both parasite and host from the damaging effects of TNF.
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During pregnancy there is a dramatic reduction in the serum levels of agalactosyl IgG (G0IgG) in both normal women and those with rheumatoid arthritis. In order to determine if a similar reduction in G0IgG were apparent in fetal serum, a comparison of the galactose content of IgG from nine paired samples of umbilical vein or fetal blood and peripheral maternal serum, at gestational ages ranging from 16-41 weeks was performed. The full-term maternal IgG samples were highly galactosylated, so confirming previous observations of reduced G0IgG levels during pregnancy. In addition every paired sample of fetal IgG had a higher level of galactosylation than the corresponding maternal IgG. Therefore, during pregnancy there is both a reduced biosynthesis of the G0IgG glycoform by the mother, and a restriction of its transport across the placenta. The ratio of estimated G0IgG in fetal and maternal serum was found to be related to changes in IgG transport, and in particular the active transport of IgG1 across the placenta during gestation. Our data suggest that the placental IgG transport mechanism is either carbohydrate independent by discriminating for IgG1, or is carbohydrate dependent selecting for highly galactosylated IgG glycoforms. This study emphasizes the need for further investigations on the biological function of G0IgG in normal physiological states, in addition to disease states, such as juvenile and adult rheumatoid arthritis, where elevated G0IgG levels correlate with disease activity.
The glycosylation of IgG results in many different glycoforms. A large body of correlative data (including remission of arthritis during pregnancy) has suggested that IgG molecules lacking galactose were associated with rheumatoid arthritis. We now demonstrate that agalactosyl IgG glycoforms are directly associated with pathogenicity in murine collagen-induced arthritis. We show that passive transfer of an acute synovitis in T-cell-primed mice can be enhanced by using IgG containing autoantibodies to type II collagen when the antibodies are present as the agalactosyl glycoform. Thus, nonpathogenic doses of autoantibodies can be made pathogenic by altering their glycosylation state.
Human serum amyloid P component (SAP) is a normal plasma protein and the precursor of amyloid P component (AP), a universal constituent of the abnormal tissue deposits in amyloidosis, including Alzheimer disease. We show here that its single N-linked biantennary oligosaccharide does not display the microheterogeneity usually characteristic of glycoproteins. The protein and the glycan structures of AP were also invariant, their resistance to degradation suggesting a role in persistence of amyloid deposits. Asialo-SAP was rapidly cleared from the circulation in mice by a mechanism dependent on terminal galactose residues and was catabolized in hepatocytes. However blockade of this pathway did not affect the clearance of native SAP. Rapid hepatic uptake and catabolism of human asialo-SAP in man were also directly demonstrated. The protein and glycan homogeneity of SAP and the integrity of AP suggest that the complete glycoprotein structure is important for the normal and the pathophysiological functions of this molecule.
The primary structures of the N-linked oligosaccharides from normal human serum IgA1 were determined by a combination of sequential exoglycosidase digestion, Bio-Gel P-4 chromatography, anion-exchange chromatography and one-dimensional n.m.r. spectroscopy. Three major N-linked disialylated biantennary-complex-type structures were found (55%). The remaining N-linked oligosaccharides consisted of at least nine further structures, some of which (7%) were of the triantennary type and included disialylated triantennary oligosaccharides with outer-arm fucose substitution [Fuc alpha 1-3(4)]. Compared with IgG, the N-glycan structures on IgA are more completely processed: the outer arms have a higher proportion of galactose and sialic acid, and only trace levels of incompletely galactosylated oligosaccharides, commonly found on IgG, were detected. Analysis of the sialylated O-glycans revealed that 64% were [NeuAc2 alpha 3(6)]2Gal beta 3GalNAc and 9% were [NeuAc2 alpha 3(6)]-Gal beta 4GlcNAc beta 6[NeuAc2 alpha 3(6)Gal beta 3]GalNAc, and 27% were monosialylated. The N-linked glycosylation of both serum IgA1 and IgG isolated from a group of six normal individuals was compared with that from ten patients with rheumatoid arthritis (RA). In contrast with the hypogalactosylation found in IgG from diseased sera, there was no evidence of an equivalent decrease in the galactosylation of the IgA1 oligosaccharides. In addition, the N-glycosylation of IgA1 was remarkably consistent within the group of normal individuals. These data suggest that incomplete galactosylation of N-linked glycans and its augmentation in RA does not extend to IgA1 and that the RA-associated galactosyltransferase deficiency may be restricted to cells producing gamma-chain.
Immunosuppressive factors isolated from trophoblast are known to block both innate and major histocompatibility complex (MHC)-dependent cell-mediated immune responses in vitro and, in some cases, in vivo. We investigated the biochemical nature of these factors, which is presently unknown. Immunosuppressive activity, assessed by inhibition of two-way MLR, was extracted from term syncytiotrophoblast microvilli using 3 M KCl. The activity resisted both extensive pronase digestion and heating to 90 degrees C for 1 h, demonstrating that intact membrane proteins were not required. Although purified protein-linked oligosaccharides released by hydrazinolysis from the syncytiotrophoblast membrane were themselves inactive, they blocked the immunosuppressive activity of the KCl extract. After pronase digestion, the activity could be fractionated by TSK 55S gel filtration, followed by C18 reverse-phase chromatography. Sequential exoglycosidase digestion of hydrazine-released sugars of the active fraction demonstrated that it contained neutral N-linked oligomannose and hybrid oligosaccharides, which normally make up < 3% of the total syncytiotrophoblast-derived protein glycan library. These glycopeptides of the active fraction were associated with membrane phospholipid micelles. The possible mechanism by which incompletely processed N-linked oligosaccharides expressed by a variety of syncytiotrophoblast membrane glycoproteins may block allogeneic reactivity when presented as polyvalent sugar groups is discussed.
The clinical significance of the percentage agalactosyl IgG oligosaccharides [%G(O)] was investigated in serum of a well-characterized cohort of 127 female RA patients who were followed for a mean duration of 6 yr. The %G(O) was determined in the first available serum sample which was obtained at a mean of 3.4 yr after symptom onset. It could be shown that patients with a %G(O) more than 2 S.D. above the mean level of controls (n = 34), had significantly more erosions, disease activity, and were treated with more second-line drugs, than patients without an increased %G(O) (n = 93), both at the time the serum sample was obtained, and during follow-up. These findings suggest that G(O) may serve as an indicator for the disease course in patients with RA.
Human monoclonal antibodies to the Rh D blood group antigen were produced by EBV-transformed B cell lines grown in serum-free medium in low density (LD) static cultures or high density (HD) hollow fiber bioreactors. Glycosylation analysis of the purified IgG was determined by the binding of anti-GlcNAc monoclonal antibody (GN7) and by analysis of oligosaccharides released by hydrazinolysis. The LD MAbs had only trace levels of agalactosyl oligosaccharides (G0), the major species (> 70%) being digalactosyl structures (G2). The HD MAbs, by contrast, contained about 10% G0 and relatively high levels (over 50%) of monogalactosyl (G1) oligosaccharides. beta 1-4 galactosyltransferase activity in the LD cell lines was similar to that found previously for other EBV-transformed B cell lines. The predominant oligosaccharides of an IgG3 anti-D, BRAD-3, contained bisecting N-acetylglucosamine. In functional assays with Fc receptor (Fc gamma R) positive effector cells, the highly galactosylated LD form of BRAD-3 was more active than the HD form in monocyte (Fc gamma RI) and K cell (Fc gamma RIII) mediated lysis of erythrocytes in ADCC assays, although these preparations showed no difference in Fc gamma RI-mediated rosette formation with U937 cells. One MAb, JAC10, was over 10-fold less active than two other IgG1 MAbs, 2B6 and BRAD-5, at mediating lysis of erythrocytes by Fc gamma RIII+ K cells; differences in sialylation may have contributed to this heterogeneity.(ABSTRACT TRUNCATED AT 250 WORDS)
The mechanisms by which cellular receptors can elicit different biological responses in a maturation state-dependent manner is one of the central problems in cell differentiation which remains to be resolved. The signals generated are likely to be due to additional (as yet unknown) transmembrane signalling pathways. In addition, the recent observation that a single growth factor receptor can activate a whole family of different putative second messengers and that the combinatorial interactions and stoichiometric ratios between the different messengers determine the resulting biological activities has opened up a whole new area of cell biology. It has been proposed that membrane GPI-anchors may function in signal transduction. We have recently confirmed the presence of a family of inositolphosphoglycan second messengers. Partial structural data suggests that these second messengers are not derived from known GPI membrane anchors and may thus constitute a novel class of non-protein-conjugated GPI.
Recently we have describe a simple efficient chemical method of generating an asparagine side-chain linker with beta-stereochemistry at the anomeric position of neutral oligosaccharides. We now report the 1-N-glycyl beta-derivatization of sialylated saccharides. Several neoglycoconjugates formed using these N-linked inter-mediates were investigated for their usefulness in probing carbohydrate-protein interactions. First, biotinyl derivatives of two xylose/fucose class plant-type oligosaccharides purified from horseradish peroxidase were effective in demonstrating the carbohydrate specificity of polyclonal anti-(horseradish peroxidase) antibodies. Secondly, a fluorescein-labelled asialo- and digalactosylated biantennary complex sugar was synthesized and shown to bind to a Ricinus communis agglutinin column. This galactose-specific recognition was abolished by treating this fluorescein-labelled oligosaccharide with jack bean beta-galactosidase. Finally, two 1-N-glycyl beta-saccharide derivatives were modified with thiophosgene to form their corresponding isothiocyanate derivatives. Coupling of these isothiocyanate derivatives of sugars to BSA, amino-derivatized polystyrene plates and glass-fibre discs resulted in multiple sugar presentation. The binding of an anti-N-acetylglucosamine monoclonal antibody to N,N'-diacetylchitobiose residues presented on BSA and solid supports was shown by e.l.i.s.a. Similarly the binding of concanavalin A to asialo-, agalactosylated biantennary complex oligosaccharide residues attached to BSA was demonstrated by a competitive e.l.i.s.a. Our results demonstrate that N-linked neoglycoconjugates could be made readily available and they are valuable tools for the detailed analyses of carbohydrates and carbohydrate-binding proteins.