Preliminary results of J-resolved, two-dimensional 1H-NMR studies on glycosphingolipids.

Homonuclear J-resolved two-dimensional spectroscopy has been applied to the simplest glycosphingolipid, glycosylceramide. In comparison with the conventional one-dimensional spectrum, the possible advantages and shortcomings of the two-dimensional method as applied to blood-group-active glycosphingolipids consisting of several saccharide units, have been pr esented. As an example, preliminary results on a more complex pentaosylceramide (IV3Gal-alpha-neolactotetraosylceramide) are discussed.

Analysis of mRNA in hemophilia A patients with undetectable mutations reveals normal splicing in the factor VIII gene.

BACKGROUND: haemophilia A (HA) is characterized by partial or total deficiency of factor VIII (FVIII) protein activity. It is caused by a broad spectrum of mutations in the FVIII gene. Despite tremendous improvements in mutation screening methods, in about 2% of HA patients no DNA change could be found, even after sequencing the whole coding part of the FVIII gene including the flanking splice sites, as well as the promotor and the 3' UTR regions. OBJECTIVES, PATIENTS AND METHODS: In the present study we performed a detailed RNA analysis of three groups of patients. The first included control patients with known splicing defects, the second included two patients with already identified nucleotide changes close to splicing sites, that could potentially alter the normal splicing process, and a third group of 11 unrelated patients whose genomic DNA have already been screened for mutations by DHPLC and direct sequencing with no mutation being identified. RESULTS: Both candidate splice site mutations were shown to result in either skipping or alternative splicing of at least one exon, therefore these DNA changes must be considered as causal for the patients' HA phenotype. In contrast, no abnormalities on the RNA level were observed in any of 11 unrelated patients without mutations in the FVIII gene. CONCLUSIONS: These findings exclude mutations that could be located deep in the introns and affecting either normal splicing or lead to mechanisms causing some unknown rearrangements of the FVIII gene. In fact, our results point to the presence of still unknown factor(s) causing HA, which might be either allelic or in the close proximity of the FVIII gene or non-allelic associated with other genetic loci that are involved in the processing of the FVIII protein.

Structures of fucose-containing ceramide pentasaccharides from the plasma of blood group O Le(a-b-) nonsecretors.

A minor, Lec blood-group inactive ceramide pentasaccharide double band fraction has been isolated from the plasma of blood group O Le(a-b-) nonsecretors. The two purified glycolipids were analysed by NMR spectroscopy, mass spectrometry and combined gas chromatography-mass spectrometry. The following structures could be established: GlcNAc(beta 1----3)Gal(beta 1----4)Glc(beta 1----1)Cer (I); Gal(beta 1----4) [Fuc(alpha 1----3)]GlcNAc(beta 1----3)Gal(beta 1----4)Glc(beta 1----1)Cer (II). It must be concluded that at least part of the secretor gene-independent plasmatic H type 2 blood-group activity can be attributed to glycosphingolipid I, whereas substance II, originally detected in cancerous tissue, also occurs in the plasma of healthy individuals.

Neurologic complications in immune-mediated heparin-induced thrombocytopenia.

OBJECTIVE: To evaluate neurologic complications in patients with immune-mediated heparin-induced thrombocytopenia (HIT) with respect to incidence, clinical characteristics, outcome, and therapy. METHODS: One hundred and twenty consecutive patients with immune-mediated HIT were recruited over a period of 11 years and studied retrospectively for the occurrence of neurologic complications. Diagnosis of HIT was based on established clinical criteria and confirmed by detection of heparin-induced antibodies using functional and immunologic tests. RESULTS: Eleven of the 120 patients (9.2%) presented with neurologic complications; 7 suffered from ischemic cerebrovascular events, 3 from cerebral venous thrombosis, and 1 had a transient confusional state during high-dose heparin administration. Primary intracerebral hemorrhage was not observed. The relative mortality was much higher (Chi-square test, p < 0.01) in HIT patients with neurologic complications (55%) as compared to patients without neurologic complications (11%). The mean platelet count nadir in neurologic patients was 38 +/- 25 x 10(9)/l on average, and was lower in patients with fatal outcome compared to those who survived (21 +/- 13 x 10(9)/l versus 58 +/- 21 x 10(9)/l; p < 0.05, Wilcoxon test). In three patients neurologic complications preceded thrombocytopenia. There was a high coincidence of HIT-associated neurologic complications with other HIT-associated arterial or venous thrombotic manifestations. CONCLUSION: Neurologic complications in HIT are relatively rare, but associated with a high comorbidity and mortality. HIT-associated neurologic complications include cerebrovascular ischemia and cerebral venous thrombosis. They may occur at a normal platelet count.

Allelic discrimination of factor V Leiden using a 5' nuclease assay.

The G1691A (Leiden) mutation of the factor V gene is the most prevalent identified cause of venous thrombosis. Therefore, we developed a new genetic test using the TaqMan system. With this assay which combines PCR amplification and detection reaction in one closed tube, a cohort of 234 patients with a history of thrombosis was screened. In parallel, amplification products of the same patients were screened with a previously described test using endonuclease digestion of PCR products followed by gel electrophoresis. Identical results were obtained by both methods. Among cases, 122 (52%) individuals were homozygous normal, 99 (42%) were heterozygous affected and 13 (5.5%) showed homozygous pattern for the Factor V Leiden mutation. Thus, it could be demonstrated that the new TaqMan assay is a robust, rapid and automated method for high throughput application which avoids time consuming and difficult post-PCR steps.

Characterization of B and H blood-group active glycosphingolopids from human B erythrocyte membranes.

Two blood group B active glycosphingolipids (B-I and B-II) previously isolated and highly purified from human B erythrocytes [21] were analysed first by degradation with alpha-D-galactosidase from coffee beans, alpha-L-fucosidase from bovine kidney and with 0,1 N trichloracetic acid; the native B-glycolipids as well as their degradation products were then investigated by methylation analysis with combined gas chromatography-mass spectromety, by thin layer chromatography, two dimensional immunodiffusion and by the hemagglutination inhibition technique. Together with the results obtained by mass spectrometry of permethylated glycolipids [26] the following structures were elucidated: alpha-D-gakactpurampsu;-(1 leads to 3) [alpha-L-fucopyranosyl-(1 leads to 2)]-D-galactopyranosyl-(1 leads to 4)-N-acetyl-D-glucosaminosyl-(1 leads to 3)-D-galactopyranosyl-(1 leads to 4)-D-glucopyranosyl-(1 leads to 1)-ceramide for the B-I glycosphingolipid and alpha-D-galactopyransosyl-(1 leads to 3)-[alpha-L-fucopyranosyl-(1 leads to 2)]-D-galactopyranosyl-(1 leads to 4)-N-acetyl-D-glucosaminosyl-(1 leads to 3)-D-galactopyranosyl-(1 leads to 4)-N-acetyl-D-glucosaminosyl-(1 leads to 3)-D-galactopyranosyl-(1 leads to 4)-D-glucopyranosyl-(1 leads to 1)-ceramide for the B-II glycophingolipid. AH active glycolipid fraction from B erythrocytes further purified by thin layer chromatography was also investigated by methylation analysis. The pattern of its partially methylated alditol acetates was essentially the same as that of the alpha-galactosidase treated and permethylated B-I glycoliped. It is also exhibited strongly precipitating and hemagglutination inhibiting H properties as well as the two alpha-galactosidase treated B-I and B-II glycosphingolipids. Based upon these data the following tentative structure was proposed: alpha-L-fucopyranosyl-(1 leads to 2)-D-galactopyranosyl-(1 leads to 4)-N-acetyl-D-glucosaminosyl-(1 leads to 3)-D-Galactopyranosyl-(1 leads to 4)-D-glucopyranosyl-( 1 leads to 1)-ceramide. Gas chromatographic analysis revealed sphingosine and lignoceric, nervonic and behenic acids to be the main components of the ceramide residues of the three glycophingolipids. From the data presented the H active substance very probably can be regarded as the immediate precursor of the B-I gly cosphingolipid from human B erythrocyte membranes.

Mass spectrometric analysis of permethylated glycosphingolipids I. Sequence analysis of two blood-group B active glycosphingolipids from human B erythrocyte membranes.

Two blood group B active glycosphingolipids (B-I and B-II) formerly isolated and purified from human B erythrocytes (16) were investigated by mass spectrometry after permethylation. B-I yielded fragments up to m/e 1266 and B-II up to m/e 1495, showing the sequence of six and seven carbohydrate residues respectively. In combination with additional experimental evidence (18) the glycosphingolipids are demonstrated to be a gal-[ fuc ]-gal-glcNAc-gal-glc-ceramide (B-I) and a gal-[ fuc ]-gal-glcNAc-gal-glcNAc-gal-glc-ceramide (B-II). Mass spectrometric evidence for the ceramide residues are also obtained indicating besides spingosine C24-,C24:1-, and C22-fatty acids as main constituents.

Mass spectrometric analysis of permethylated glycosphingolipids II. Comparative studies on different blood-group active and related erythrocyte membrane glycosphingolipids.

Three isomeric ceramide tetrasaccharides--P blood-group active globoside, lacto-N-neotetraosyl ceramide as ABH blood-group precursor, both isolated from human erythrocytes and "asialo ganglioside" from human brain as reference standard--and two ceramide pentasaccharides--H blood-group active glycosphingolipid, obtained from blood-group B active ceramide hexasaccharide of human B erythrocytes after alpha-galactosidase treatment and ceramide pentasaccharide from rabbit erythrocytes with B-like blood-group activity--were investigated by mass spectrometry after permethylation. The carbohydrate moiety exhibits differences not only concerning the sugar sequence but also with regard to the position of some glycosidic linkages: Oligosaccharides containing N-acetylhexosamine substituted at position 4 produce spectra that are distinctly different from those containing C-3 substituted N-acetylhexosamines, thus allowing the differentation between type 1 and type 2 carbohydrate chains. Moreover, oligosaccharide ions with a hexose at the cleavage site exhibit a fragmentation pattern different from those with a N-acetylhexosamine at the "reducing terminal". The intensity ratio between parent ion and parent ion -32 mass units is Q greater than or equal to 3 in the first case, whereas in the latter case Q is less than 1. The Q-values are given for 14 oligosaccharide ions. Differences in the composition of the ceramide residues can also be deduced from the mass spectra.

High resolution nuclear magnetic resonance spectroscopy of glycosphingolipids. I: 360 MHz 1H and 90.5 MHz 13C NMR analysis of galactosylceramides.

The high resolution 1H and 13C nuclear magnetic resonance (NMR) spectra of galactosylceramides containing n-fatty acids and alpha-hydroxy fatty acids were recorded in dimethylsulfoxide solution with and without addition of D2O. From the coupling constants of the sugar ring protons, a 4C1 conformation can be deduced. In contrast to the conformation in aqueous solution, the C6 hydroxymethylene group is freely rotating around the C6--C5 bond. In the ceramide residue all signals produced by protons linked to carbons bearing electronegative substituents could be attributed. The large difference in coupling constants of the methylene protons of C1' to the C2' methine proton of the sphingosine indicates a restricted rotation around the C1'--C2' bond. The assignments of the hydroxy and amino protons follow from the decoupling of the corresponding methine protons.

Isolation and purification of Lea blood-group active and related glycolipids from human plasma of blood-group A Lea individuals.

From 81 of human plasma of blood-group A Lea nonsecretors three different Lea blood-group active ceramide pentasaccharides (a total of 4.65 mg) have been isolated, all revealing glucose, galactose, N-acetylglucosamine and fucose in molar ratios of 1 : 2 : 1 : 1 as determined by gas liquid chromatography. A fourth blood-group active fraction (0.72 mg) represents a mixture of a Lea active ceramide pentasaccharide and an A active ceramide hexasaccharide (molar ration 7.7 : 2.3 as calculated from the content of different aminosugars). Additionally, two different globosides, two different hematosides and a new N-acetylglucosamine containing ceramide tetrasaccharide were obtained. All 9 glycolipid fractions demonstrated homogeneity in analytical high performance thin layer chromatography (HPTLC) using 4 different solvent systems. 0.2 microgram of each Lea active glycolipid completely inhibited the agglutination of O Le(a+b-) erythrocytes by 50 microliter of 4 hemagglutinating units of caprine anti Lea serum. At least 0.04 microgram of each Lea antigen are sufficient for incubation to convert 9 x 10(7) O Le(a-b-) erythrocytes into Lea-positive cells. Mainly due to the relatively low content of the blood-group A glycolipid in plasma (0.17 mg/81), previously negative erythrocytes readily become agglutinable by anti Lea sera and not by anti A sera after incubation with appropriate plasma.

A multiplicity of erythrocyte glycolipids of the neolacto series revealed by immuno-thin-layer chromatography with monoclonal anti-I and anti-i antibodies.

The thin-layer-chromatography immunostaining procedure was applied to human erythrocyte glycolipids using monoclonal anti-i and anti-I antibodies which are directed against epitopes on linear and branched carbohydrate chains of the neolacto (poly-N-acetyllactosamine) series. An examination of native and mild-acid-treated glycolipids from normal adult (I(adult) antigen type), neonatal (i(cord)), and I-antigen-deficient adult (i(adult)) erythrocytes enabled certain structural inferences to be made as follows: (a) cells of both I and i phenotypes contain a multiplicity of glycolipids of the neolacto series whose backbones consist of 8 or more sugar residues; (b) the octasaccharide backbones are predominantly linear in cells of i phenotype and branched in those of I type; and (c) more complex glycolipids having decasaccharide and larger backbones with both linear and branched sequences occur in erythrocytes of both phenotypes.

Structure elucidation of blood group B-like and I-active ceramide eicosa- and pentacosasaccharides from rabbit erythrocyte membranes by combined gas chromatography-mass spectrometry; electron-impact and fast-atom-bombardment mass spectrometry; and two-dimensional correlated, relayed-coherence transfer, and nuclear Overhauser effect 500-MHz 1H-n.m.r. spectroscopy.

The structures of two glycosphingolipids, a ceramide eicosasaccharide BIrab-3 and a ceramide pentacosasaccharide BIrab-4 with "B-like" and distinct I blood-group activity, isolated in high yield from rabbit erythrocyte membranes, were investigated. The determination of their general structure, alpha-D-Galp-(1----3)-beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)- [alpha-D-Galp-(1----3)-beta-D-Galp-(1----4)-beta-D-Glcp-NAc-(1----6)]-be ta- D-Galp-(1----n4)-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-beta-D-Gl cp- (1----1)-ceramide, with n = 3 for BIrab-3 and n = 4 for BIrab-4, was based on the results of methylation analysis, fast-atom-bombardment and electron-impact mass spectrometry, 1D and 2D COSY, RCT, and n.O.e. 1H-N.m.r. spectra, and specific enzymic and chemical degradation.

Studies of the binding specificity of the soluble 14,000-dalton bovine heart muscle lectin using immobilised glycolipids and neoglycolipids.

The aim of the present study has been to investigate the binding specificity of the soluble 14,000-dalton lectin of bovine heart muscle towards immobilised oligosaccharides in clustered form. To this end, chromatogram overlay assays and quantitative plastic-microwell-binding assays have been performed using several natural glycolipids and neoglycolipids containing one or more of the disaccharide units, beta-D-Galp-(1----4 or 3)-D-GlcNAc or beta-D-Galp-(1----4)-D-Glc and related structures. The microwell assay gave the most consistent results. It was observed that for binding by the soluble lectin the optimal sequence, which is beta-D-Galp-(1----4 or 3)-D-GlcNAc, must occur at the nonreducing end of longer oligosaccharides when linked to lipid. These oligosaccharides may be of poly(N-acetyllactosamine) type or they may be mono- or multi-antennary, complex-type chains in which the disaccharide is joined directly to a trimannosyl core. The lectin bound to such immobilised lipid-linked oligosaccharides on which the terminal D-galactosyl groups are substituted with alpha-L-Fucp-(1----2), alpha-D-Galp-(1----3), or alpha-NeuAc-(2----3) groups. However, no binding was detected if the terminal D-galactosyl groups were substituted with an alpha-NeuAc-(2----6) group or the subterminal N-acetylglucosamine units with an alpha-L-Fucp-(1----3 or -4) group. Internally located N-acetyllactosamine units where the D-galactose units are disubstituted by beta-D-GlcNacp-(1----3) and -(1----6) units, as in branched poly(N-acetyllactosamine) backbones were not bound by the bovine lectin. These results are in accord with previous observations on the bovine lectin and the corresponding human and rat lectins, using structurally defined oligosaccharides as inhibitors of binding. The results of comparative binding experiments using paragloboside and ceramide hexasaccharide which contain one and two N-acetyllactosamine units, respectively, joined in linear sequence to the lactosylceramide core, were equivocal with respect to the availability of the internal N-acetyllactosamine units for binding by the bovine lectin.

Structure analysis of glycosphingolipids using fast atom bombardment (FAB) techniques.

The results presented show, that with the aid of negative ion FAB MS native glycosphingolipids and especially gangliosides are amenable to sequence analysis. The preferred formation of pseudomolecular ions M-1 and of sialic acid containing fragments gives conclusive information on the number of sialic acids present and the sites of their attachment to the oligosaccharide backbone. Positive ion FAB MS of branched permethylated glycosphingolipids with up to 25 sugar units yielded pseudomolecular ions [M+Na]+ in excess of 6000 daltons, that allowed an exact calculation of carbohydrate constituents. Furthermore highly (Formula: see text) specific fragmentation patterns furnished information on number and positions of branching points as well as on the ceramide moiety. It can be anticipated, that FAB MS will be very useful in the analysis of more complex gangliosides carrying additional fucose or acyl residues and of even larger molecules with molecular weights up to 15,000 daltons.

[Characterization of factor VIII antibody epitopes from haemophilia A patients using cellulose bound FVIII peptide libraries]. / Charakterisierung von Faktor-VIII-Antikörperepitopen mit Faktor-VIII-Peptid-Bibliotheken.

Approximately 30% of patients suffering from severe haemophilia A develop antibodies against factor VIII (FVIII) neutralizing the effect of the pro-coagulant activity of intravenously injected FVIII as a complication of replacement therapy. Generally, various epitopes on the FVIII molecule are bound by these antibodies. The detailed structure of such epitopes is unknown. In this study epitopes on the FVIII molecule are identified using solid phase bound peptide arrays carrying the whole amino acid sequence of FVIII as small oligopeptides. The binding of FVIII antibodies by specific peptide sequences on the array indicates potential epitopes. FVIII antibodies of inhibitor patients and healthy blood donors are currently investigated by this method. Identified epitopes may lead to new concepts in therapy aiming at avoidance of inhibitor formation or improvement of inhibitor eradication. As participant of the 'haemophilia A' consortium dealing with genotype/phenotype correlation in haemophilia A we investigate, if the site or type of the mutation correlates with the epitopes, and if there is any relation between epitopes and clinical course. Furthermore, the influence of epitopes on therapeutical effects and the outcome of immune tolerance induction is under scrutiny.