Glycosyl-phosphatidylinositol moiety that anchors Trypanosoma brucei variant surface glycoprotein to the membrane.

Two forms of protein-membrane anchor have been described for the externally disposed glycoproteins of eukaryotic plasma membranes; namely, the hydrophobic transmembrane polypeptide and the complex glycosylphosphatidylinositol (G-PI) moiety. The chemical structures of the major species of G-PI anchors found on a single variant surface glycoprotein (VSG) of the parasitic protozoan Trypanosoma brucei were determined by a combination of nuclear magnetic resonance spectroscopy, mass spectrometry, chemical modification, and exoglycosidase digestions. The G-PI anchor was found to be heterogeneous with respect to monosaccharide sequence, and several novel glycosidic linkages were present. The results are pertinent to the mechanism of the biosynthesis of G-PI anchors.

Insight into the role of inositol phosphoglycans in insulin response and the regulation of glucose and lipid metabolism illustrated by the response of adipocytes from two strains of rats.

Differences in biochemical and hormone profiles between two strains of rats provide insights into the relationships between insulin response, inositol phosphoglycans and lipid metabolism in adipose tissue. The results suggest the apparent anomaly of a higher rate of lipogenesis and response to insulin with a lower fat pad weight in the Charles River vs. Harlan Olac group relates to: (i) enzyme pre-programming with IPG-A, (ii) faster turnover of lipid, (iii) effects of leptin and cAMP.

Pre-eclampsia is associated with an increase in trophoblast glycogen content and glycogen synthase activity, similar to that found in hydatidiform moles.

Pre-eclampsia is a placental disorder, but until now, biochemical details of dysfunction have been lacking. During an analysis of the oligosaccharide content of syncytiotrophoblast microvesicles purified from the placental chorionic villi of 10 primigravid women with proteinuric pre-eclampsia, we found an excess of glycogen breakdown products. Further investigation revealed a 10-fold increase in glycogen content (223 +/- 117 micrograms glycogen/mg protein), when compared with controls matched for gestational age at delivery (23 +/- 18 micrograms glycogen/mg protein) (P < 0.01). This was confirmed by examination of electron micrographs of chorionic villous tissue stained for glycogen. The increase in glycogen content was associated with 16 times more glycogen synthase (1,323 +/- 1,013 relative to 83 +/- 96 pmol glucose/mg protein per min) (P < 0.001), and a threefold increase in glycogen phosphorylase activity (2,280 +/- 1,360 relative to 700 +/- 540 pmol glucose/mg protein per min; P < 0.05). Similar changes in glycogen metabolism were found in trophoblast microvesicles derived from hydatidiform moles. Glycogen accumulation in villous syncytiotrophoblast may be a metabolic marker of immaturity of this cell which is unable to divide. The implications of these findings with regard to the pathogenesis of pre-eclampsia are discussed.

P-type inositol phosphoglycans in serum and amniotic fluid in active pre-eclampsia.

OBJECTIVES: Abnormal secretion of P-type inositol phosphoglycans (IPG-P) has been described in maternal urine of pre-eclamptic women. The aim of this study was to determine the origin of production of IPG-P. We examined the IPG-P content of maternal and fetal serum, maternal urine and amniotic fluid in both normal pregnancy and pre-eclampsia. DESIGN: Established extraction and bioactivity assay techniques were used to compare total IPG-P levels in serum samples, and a polyclonal-antibody-based ELISA to assay the amniotic fluid and urine samples in matched pairs of women. SUBJECTS: Eleven women with pre-eclampsia requiring caesarean section (subjects), 11 pregnant women requiring elective caesarean section for reasons other than pre-eclampsia (controls). RESULTS: Our data confirm the abnormal level of IPG-P in maternal urine during pre-eclampsia. Moreover, IPG-P levels were higher in umbilical sera than in maternal sera samples. Amniotic fluid as well as urine ELISA results were significantly higher in the pre-eclamptic group compared with normal controls. Total IPG-P bioactivity in serum did not vary between serum compartments in normal pregnancy. Uterine vein IPG-P levels were lower in pre-eclampsia when compared with normal pregnancy. A possible correlation was observed between urine and amniotic fluid levels in normal women. No correlation was observed between measured blood levels and those in urine and amniotic fluid. CONCLUSIONS: It is hypothesized that steady state equilibrium of IPG-P in serum in normal pregnancy is disrupted in pre-eclampsia. Additionally, an abnormal IPG-P sub-fraction, detectable in urine and amniotic fluid, may be present and involved in the pathophysiology of the syndrome, although sites of production of this abnormal form remain unclear.

Structure and expression of the human glycosylphosphatidylinositol phospholipase D1 (GPLD1) gene.

Here we report the structure of the human glycosylphosphatidylinositol phospholipase D1 gene, which covers at least 80 kb on chromosome 6p22. The gene comprises 25 exons and encodes a 5.8 kb mRNA, which was detected only in the liver. Southern blot analysis shows that the human genome contains only one GPLD gene and we could only detect one of the two previously reported cDNAs.

The use of two-dimensional correlated spectroscopy to obtain new assignments in the high-resolution 1H nuclear magnetic resonance spectrum of the biantennary complex oligosaccharide isolated from human serum transferrin by hydrazinolysis.

The asialo biantennary complex type oligosaccharide from human serum transferrin was isolated by hydrazinolysis, a method which results in the quantitative release of the intact oligosaccharide free of all amino acids. The 1H-NMR chemical shifts of the previously assigned anomeric and H-2 protons from the peripheral residues of the glycopeptide are identical to the corresponding values for the reduced oligosaccharide. The chemical shift of GlcNAc-1 H-1 proton in the reduced oligosaccharide was assigned by selective deuteration. Proton J connectivities were determined using two-dimensional 1H-1H correlated high resolution NMR spectroscopy. Twelve new assignments were made within the central envelope of the NMR spectrum and a further six were tentatively proposed. The ability to assign proton resonances in this way should allow further conformational studies of the oligosaccharide using nuclear Overhauser effects between the relevant assigned protons on different saccharide residues (Homans, S.W., Dwek, R.A., Fernandes, D.L. and Rademacher, T.W. (1982) FEBS Lett. 150, 503-506).

The analysis of coupling networks in a complex oligosaccharide mixture derived from the Fc region of rabbit immunoglobulin G using 1H-1H correlated NMR spectroscopy combined with double quantum NMR spectroscopy.

In glycoproteins, even for those containing a single glycosylation site, diversity is manifest in the occurrence of a family of structurally-related yet distinct oligosaccharides. To date this 'microheterogeneity' is universal in mammalian glycoproteins. A method is described, using 1H-1H correlated and double quantum nuclear magnetic resonance NMR spectroscopy, for the assignment of proton resonances within a mixture of complex-type oligosaccharides derived from the Fc region of rabbit immunoglobulin G. The ability to assign resonances in heterogeneous populations will be of importance in the chemical shift analysis of the 1H-NMR spectra of glycopeptides since these cannot generally be separated on the basis of their carbohydrate sequence. The resulting assignments will be necessary before conformational studies on glycopeptides using nuclear Overhauser effects can be made.

Characterisation of oligosaccharides from Drosophila melanogaster glycoproteins.

An analysis of the released oligosaccharides from a membrane glycoprotein preparation of third instar larvae (3rdIL), and purified larval serum protein 2 (LSP2) from Drosophila melanogaster was performed. Sequential exoglycosidase digestion in combination with high-resolution gel permeation chromatography and partial acetolysis indicated the presence of two series of oligomannosides; one of these series was unusual and characterized by the presence of a core alpha 1-6 linked fucose, the other was a typical mammalian oligomannose series containing the following isomers -D1, -D2, -D12, -D123 and -CD123 as well as the unprocessed Man9GlcNAc2 structure. Conventional oligomannose could only be detected in the LSP2 sample. This study opens the way to use powerful molecular and classical genetic techniques to analyse the control and functional significance of glycosylation in higher organisms.

Two-domain structure of the native and reactive centre cleaved forms of C1 inhibitor of human complement by neutron scattering.

The C1 inhibitor component of human complement is a member of the serpin superfamily, and controls C1 activation. Carbohydrate analyses showed that there are seven O-linked oligosaccharides in C1 inhibitor. Together with six N-linked complex-type oligosaccharides, the carbohydrate content is therefore 26% by weight and the molecular weight (Mr) is calculated as 71,100. Neutron scattering gives an Mr of 76,000 (+/- 4000) and a matchpoint of 41.8 to 42.3% 2H2O, in agreement with this carbohydrate and amino acid composition. Guinier plots to determine the radius of gyration RG were biphasic. Neutron contrast variation of C1 inhibitor in H2O-2H2O mixtures gave an overall radius of gyration RG at infinite contrast of 4.85 nm, from analyses at low Q, and a cross-sectional RG of 1.43 nm. The reactive centre cleaved form of C1 inhibitor has the same Mr and structure as the native molecule. The length of C1 inhibitor, 16 to 19 nm, is far greater than that of the putative serpin domain. This is attributed to an elongated structure for the carbohydrate-rich 113-residue N-terminal domain. The radial inhomogeneity of scattering density, alpha, is large at 59 x 10(-5) from the RG data and 28 x 10(-5) from the cross-sectional analysis, and this is accounted for by the high oligosaccharide content of C1 inhibitor. The scattering data were modelled using small spheres. A two-domain structure of length 18 nm based on two distinct scattering densities accounted for all the contrast variation data. One domain is based on the crystal structure of alpha 1 antitrypsin (7 nm x 3 nm x 3 nm). The other corresponds to an extended heavily glycosylated N-terminal domain of length 15 nm, whose long axis is close to the longest axis of the serpin domain. Calculation of the sedimentation coefficient s0(20),w for C1 inhibitor using the hydrodynamic sphere approach showed that a two-domain head-and-tail structure with an Mr of 71,000 and longest axis of 16 to 19 nm successfully reproduced the s0(20),w of 3.7 S. Possible roles of the N-terminal domain in the function of C1 inhibitor are discussed.

Recent advances in glycoconjugate analysis and glycobiology.

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.

Effector functions of a monoclonal aglycosylated mouse IgG2a: binding and activation of complement component C1 and interaction with human monocyte Fc receptor.

Aglycosylated monoclonal anti-DNP mouse IgG2a produced in the presence of tunicamycin was compared with the native monoclonal IgG2a with respect to its ability to interact with the first component of complement, C1, and to compete with human IgG for binding to human monocyte Fc receptors. The aglycosylated IgG2a was found to bind subcomponent C1q with an equivalent capacity to the native IgG2a, but the dissociation constant was found to be increased three-fold. When activation of C1 by the glycosylated and aglycosylated IgG2a was compared, the rate of C1 activation by the aglycosylated IgG2a was reduced approximately three-fold. In contrast aglycosylation was accompanied by a large decrease (greater than or equal to 50-fold) in the apparent binding constant of monomeric IgG2a to human monocytes. The data suggest that the aglycosylated IgG2a has a structure which differs in the CH2 domain from the native IgG2a, and that the heterogeneous N-linked oligosaccharides of this monoclonal IgG2a which occur at a conserved position in the CH2 domain play a role in maintaining the integrity of its monocyte-binding site. This lack of monocyte binding may result either from a localized conformational change occurring in a single CH2 domain or from an alteration in the CH2-CH2 cross-domain architecture which is normally structured by a pair of opposing and interacting oligosaccharides. The minimal changes in C1q binding and C1 activation suggest that the oligosaccharides are, at most, indirectly involved in these events.

Solution conformation of the biantennary N-linked oligosaccharide of human serotransferrin using 1H NMR nuclear Overhauser effect measurements.

The conformation in solution of the biantennary complex type oligosaccharide unit derived from human serotransferrin has been investigated using 1H-1H Nuclear Overhauser Effect (NOE) measurements at 300 MHz. From quantitation of the NOE, the alpha(1-3) antenna is shown to exist in a preferred solution conformation with respect to the mannosyl-chitobiose core. The flexibility of the alpha(1-6) arm, together with the absence of NOE data between this arm and the core, indicates that, in contrast to the alpha(1-3) arm the alpha(1-6) arm has no preferred conformation with respect to the core.

Bias in murine IgG isotype immobilisation. Implications for IgG glycoform analysis ELISA procedures.

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.

Analysis of glycosylation changes in IgG using lectins.

A simple rapid assay based on the ability of lectins to bind carbohydrates has been developed to analyse changes in the oligosaccharide chains of IgG. Bandeiraea simplicifolia lectin and Ricinus communis agglutinin have been used to detect terminal N-acetylglucosamine and galactose moieties respectively in IgG using immunodot-blotting. IgG samples (approximately 1 micrograms) were dot-blotted onto nitrocellulose followed by boiling of the blots to expose the carbohydrate moieties. The blots were then treated with biotinylated lectins followed by either streptavidin-biotin-hydrogen peroxidase conjugate or 125I-labelled streptavidin. The colour was developed using chloronaphthol and the blots read on a densitometer. The labelled blots were cut and read on a gamma counter. The use of a monoclonal antibody to N-acetylglucosamine is also discussed. The results obtained using this method are comparable to those obtained by structural analysis.

Syncytiotrophoblast membrane protein glycosylation patterns in normal human pregnancy and changes with gestational age and parturition.

The fetally derived syncytiotrophoblast in the placenta form the major interface with the maternal circulation. Cell surface N-linked oligosaccharides are known to influence cell-cell interactions in a variety of ways. The N-linked oligosaccharide component of the human syncytiotrophoblast membrane has been purified from term placentae, and its biochemical structure analysed. Ninety-five per cent of structures were complex N-linked oligosaccharides, with the remaining 5 per cent being of the oligomannose type. Seventy-two per cent of oligosaccharides were sialylated; 50 per cent having two or more sialic acid residues. Such a population of N-linked oligosaccharides would be expected to provide a surface which inhibits interactions between trophoblast and maternal leukocytes. The temporal changes in syncytiotrophoblast N-linked oligosaccharides from the end of the second, and through the third trimester (25-41 weeks) were analysed, as were the changes which occur during parturition. There was no change in the degree of sialylation of these structures. The only significant change was a 37 per cent decrease in core fucosylation of complex N-linked sugars during gestation (P less than 0.005). Women delivered by caesarean section at term, had significantly higher levels of fucosylation (equivalent to women with a gestational age of 31-36 weeks), than those who laboured at term. Present knowledge of core fucosylation of N-linked oligosaccharides is discussed in relation to trophoblast functioning.

Short communication: selective placental transport of maternal IgG to the fetus.

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.

Correlation between IgG anti-type II collagen levels and arthritic severity in murine arthritis.

This study examines the relationship between the serum levels of IgG antibodies to heterologous and homologous type II collagen and the subsequent arthritic severity in a collagen induced arthritis model. Arthritis was induced in DBA-1 mice using intradermal injections of heterologous type II collagen in Freunds complete adjuvant, the time of arthritis onset was noted and the severity was monitored regularly. Serum samples were taken and IgG levels of anti-heterologous and homologous type II collagen were analyzed both pre and post arthritis onset. We observed that post induction/pre arthritic serum IgG anti-heterologous and homologous type II collagen levels showed a significant correlation (both p < 0.01) with the severity of the arthritis that subsequently developed. Mice with early arthritis showed a highly significant correlation (p < 0.002) between sera IgG anti-homologous type II collagen levels and arthritic severity, a lesser correlation was also apparent between anti-heterologous type II collagen titres and arthritic severity (p < 0.05). The high levels of correlation observed in this study between anti-type II collagen titres and arthritic severity before actual onset of arthritis, clearly suggest that the magnitude of the initial humoral response to type II collagen plays a crucial role in determining the resultant arthritic severity. This observation is only apparent due to the use of an arthritis susceptible inbred mouse strain, which removes variables such as H-2 restriction, antigen processing/presentation and possible complement deficiencies, and the early time scale of the analysed sera samples.

Placental GPI-PLD is of maternal origin and its GPI substrate is absent from placentae of pregnancies associated with pre-eclampsia.

Pre-eclampsia (PE) is a disorder affecting 5-10% of all pregnancies and is characterised by abnormal trophoblast invasion, maternal endothelial cell dysfunction and a systemic maternal response. A unifying factor responsible for eliciting these effects remains unknown. However, levels of the autocrine insulin mediators, inositolphosphoglycans (IPG), are elevated 3-fold in pre-eclamptic placentae compared with controls and are also elevated 3-fold in maternal urine of pre-eclamptic women, suggesting an abnormal paracrine role of the mediator in the systemic maternal response. At the placental level, IPGs are metabolic second messengers capable of eliciting some of the characteristic features of PE, such as the 10-fold increase in glycogen synthesis and 16-fold increase in the activity of the IPG-dependent enzyme glycogen synthase. IPGs are derived from their lipidic precursors, the glycosylphosphatidylinositols (GPI), in membrane associated caveolae by the action of a GPI-specific phospholipase D whose activity is regulated by its membrane microenvironment. We show that the lipidic GPI precursor was detected in total placental membrane and microvillous membrane from normal placentae. The presence of GPI could not be detected in PE placentae, suggesting that the GPI/IPG signalling system is dysregulated in this disorder. Equivalent amounts of a proteolytically-cleaved 50 kDa GPI-PLD protein is detected in both normal and PE placentae. However, GPI-PLD mRNA is absent, suggesting a mechanism of uptake from maternal serum. Since GPI-PLD, whose presence is required for hydrolysis of GPI and release of free IPG, is detectable with equal activity in both normal and PE placentae, we postulate that dysregulation of the tubular caveolar structure of the microvilli in pre-eclamptic placentae provides an environment which promotes the unregulated hydrolysis of GPI in this disorder.

Possible involvement of inositol phosphoglycan-P in human parturition.

Preterm labour is a major cause of neonatal morbidity and mortality but the pathophysiology that underlies preterm labour is unknown. Inositolphosphoglycans (IPGs) comprise a ubiquitous family of putative carbohydrate second messengers and they have been linked to the pathogenesis of various conditions, including diabetes and pre-eclampsia. Studying IPG-P levels in normal and pre-eclamptic pregnancies, we noticed a constant rise of urinary IPG-P levels in all women at the time of delivery. A prospective pilot study of urinary IPG-P levels in 23 non-labouring and labouring women with uncomplicated pregnancies has, therefore, been performed. Levels of urinary IPG-P were significantly higher in labour than in the non-labouring group (P<0.0001). These higher levels have been found in both spontaneous and induced labour. The clinical significance of this observation with particular reference to the onset of labour itself is discussed.

Improvement of glucose homeostasis in obese diabetic db/db mice given Plasmodium yoelii glycosylphosphatidylinositols.

We have previously reported that infection with Plasmodium yoelii, Plasmodium chabaudi, or injection of extracts from malaria-parasitized red blood cells induces hypoglycemia in normal mice and normalizes the hyperglycemia in streptozotocin (STZ)-diabetic mice. P yoelii glycosylphosphatidylinositols (GPIs) were extracted in chloroform:methanol:water (CMW) (10:10:3), purified by high-performance thin layer chromatography (HPTLC) and tested for their insulin-mimetic activities. The effects of P yoelii GPIs on blood glucose were investigated in insulin-resistant C57BL/ks-db/db diabetic mice. A single intravenous injection of GPIs (9 and 30 nmol/mouse) induced a significant dose-related decrease in blood glucose (P < .001), but insignificantly increased plasma insulin concentrations. A single oral dose of 2.7 micromol GPIs per db/db mouse significantly lowered blood glucose (P < .01). P yoelii GPIs in vitro (0.062 to 1 micromol/L) significantly stimulated lipogenesis in rat adipocytes in a dose-dependent manner both in the presence and absence of 10(-8) mol/L insulin (P < .01). P yoelii GPIs stimulated pyruvate dehydrogenase phosphatase (PDH-Pase) and inhibited both cyclic adenosine monophosphate (cAMP)-dependent protein kinase A and glucose-6-phosphatase (G6Pase). P yoelii GPIs had no effect on the activity of the gluconeogenic enzymes fructose-1,6-bisphosphatase (FBPase) and phosphoenolpyruvate carboxykinase (PEPCK). This is the first report of the hypoglycemic effect of P yoelii GPIs in murine models of type 2 diabetes. In conclusion, P yoelii GPIs demonstrated acute antidiabetic effects in db/db mice and in vitro. We suggest that P yoelii GPIs, when fully characterized, may provide structural information for the synthesis of new drugs for the management of diabetes.