Structures and fatty acid compositions of neutral glycosphingolipids of human plasma.

Major neutral glycosphingolipids were isolated from human plasma and their structures and fatty acid compositions studied. The four neutral glycosphingolipids of plasma were characterized as Glc beta(1 leads to 1)ceramide, Gal beta(1 leads to 1)- ceramide, Gal beta(1 leads to 4) Glc beta (1 leads to 1)ceramide, Gal alpha(1 leads to 4) Gal beta(1 leads to 4) Glc beta(1 leads to 1)ceramide and GalNAc beta(1 leads to 3) Gal (1 leads to 4) Gal (1 leads to 4) Glc beta(1 leads to 1)-ceramide. The glycosphingolipids contained mostly short chain fatty acids of which most prominent was C16. Erythrocyte glucosylceramide and lactosylceramide exhibited similar fatty acid compositions as their plasma counterparts. Triglycosylceramide and globoside of erythrocytes contained almost exclusively long-chain fatty acids. In lactosylceramide obtained from "p" erythrocytes, an accumulation of long-chain fatty acids was found; this accumulation was not observed, however, in lactosylceramide isolated from "p" plasma. It was concluded that plasma and erythrocyte glycosphingolipids are synthesized at separate sites where short- and long-chain fatty acids, respectively, are available. Plasma and erythrocyte glucosylceramide, and probably a fraction of lactosylceramide, exchange between plasma and erythrocyte pools. The latter conclusion is discussed in the light of the relative roles of carbohydrate and lipid moieties of the glycosphingolipids in maintaining their association with erythrocyte membranes.

Human platelets release alpha-6-L-fucosyltransferase upon activation.

Previously we have shown that human platelets release alpha-6-L-fucosyltransferase (EC 2.4.1.68) during coagulation of blood [(1987) Glycoconjugate J. 4, 43-49]. Here we report that agonists which induce platelet aggregation bring about release of the enzyme. In quantitative terms the release of alpha-6-L-fucosyltransferase by washed, aggregated platelets was very similar to that occurring during blood coagulation.

Chemical modifications of alpha1,6-fucosyltransferase define amino acid residues of catalytic importance.

alpha1,6-Fucosyltransferase (alpha6FucT) of human platelets was subjected to the action of phenylglyoxal (PLG), pyridoxal-5'-phosphate/NaBH(4) (PLP), and diethyl pyrocarbonate (DEPC) the reagents that selectively modify the structure of amino acids arginine, lysine and histidine, respectively, as well as to N-ethylmaleimide (NEM), mersalyl, p-chloromercuribenzoate (pCMB), iodoacetate, iodoacetamide, and methyl iodide that react with sulfhydryl group of cysteine. In addition, we treated the enzyme with beta-mercaptoethanol, a reagent that disrupts disulfide bonds. All reagents except NEM significantly inactivated alpha6FucT. Protection against the action of PLG, PLP and sulfhydryl modifying reagents was offered by GDP-fucose, GDP, and the acceptor substrate, a transferrin-derived biantennary glycopeptide with terminal GlcNAc residues. Neither donor nor acceptor substrate offered, however, any protection against inactivation by DEPC or beta-mercaptoethanol. We conclude that arginine, cysteine and probably lysine residues are present in, or closely by, the donor and acceptor substrate binding domains of the enzyme, whereas histidine may be a part of its catalytic domain. However, the primary structure of alpha6FucT does not show cysteine residues in proximity to the postulated GDP-fucose-binding site and acceptor substrate binding site of the enzyme that contains two neighboring arginine residues and one lysine residue (Glycobiol. 10 (2000) 503). To rationalize our results we postulate that platelet alpha6FucT is folded through disulfide bonds that bring together donor/acceptor-binding- and cysteine- and lysine-rich, presumably acceptor substrate binding sites, thus creating a catalytic center of the enzyme.

Neutrophils promote the release of alpha-6-fucosyltransferase from blood platelets through the action of cathepsin G and elastase.

Human blood platelets release alpha-6-fucosyltransferase during coagulation of blood or after stimulation with thrombin or other agonists that cause platelet activation (Antoniewicz et al., FEBS Lett. 244 (1989) 388-390). However, in the absence of neutrophils the thrombin-stimulated platelets release only a small fraction of alpha-6-fucosyltransferase activity (Koscielak et al., Acta Biochim. Polon. 42 (1995) 35-40). We show that the effect of neutrophils is reproduced by cathepsin G or (less efficiently) by elastase, the two enzymes that are released by neutrophils during coagulation of blood. We have also localized alpha-6-fucosyltransferase to membrane and alpha-granule fractions of platelets that had been disrupted by nitrogen cavitation. It is concluded that thrombin-activated neutrophils release cathepsin G and elastase that promote degranulation of platelets and hence the secretion of alpha-6-fucosyltransferase.

Carbohydrate-deficient glycoprotein syndromes.

Carbohydrate-deficient glycoprotein syndromes are rare, multisystemic diseases, typically with major nervous system impairment, that are caused by hypo- and unglycosylation of N-linked glycoproteins. Hence, a biochemical evidence of this abnormality, like hypoglycosylation of serum transferrin is essential for diagnosis. Clinically and biochemically, six types of the disease have been delineated. Three of them are caused by deficiencies of the enzymes that are required for a proper glycosylation of lipid--(dolichol) linked oligosaccharide (phosphomannomutase or phosphomannose isomerase or alpha-glycosyltransferase), and one results from a deficiency of Golgi resident N-acetylglucosaminyltransferase II. In addition one variant of the disease has been reported as due to a defective biosynthesis of dolichol iself. The diseases are heritable but genetics has been established for only two types. Therapy, based on administration of mannose to patients is currently under investigation. It benefits patients with deficiency of phosphomannose isomerase. Taking into account the complexity of N-linked glycosylation of proteins more of the disease variants is expected to be found.

Diseases of aberrant glycosylation.

Recently a defective glycosylation of glycoconjugates has been implicated in the pathogenesis of a number of heritable or acquired diseases of humans. Herein I discuss them under the name of diseases of aberrant glycosylation. These are: congenital dyserythropoietic anemia type II, carbohydrate-deficient glycoprotein syndrome, I-cell disease, galactosemia in subjects on galactose-free diet, variants of leukocyte adhesion deficiency, and of Ehlers-Danlos syndrome, paroxysmal nocturnal hemoglobinuria, and Tn syndrome. Regarding the present views on the function of glycoconjugates it is probably significant that in most instances defective or missing glycoproteins (or proteoglycans) but not glycosphingolipids, are probably involved in the pathogenesis of these diseases.

Glycophorin A in two patients with congenital dyserythropoietic anemia type I and type II is partly unglycosylated.

Glycophorins A from erythrocyte membranes of two patients with congenital dyserythropoietic anemia type I and type II (CDA type I and II) were analyzed for carbohydrate molar composition employing a modification of the recently published method that allowed simultaneous determination of carbohydrates and protein in electrophoretic bands of glycoproteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Zdebska & Koscielak, 1999, Anal Biochem., 275, 171-179). The modification involved a preliminary extraction of erythrocyte membranes with aqueous phenol, subsequent electrophoresis and analysis of the extracted glycophorins rather than electrophoresis and analysis of the glycophorin from intact erythrocyte membranes. The results showed a large deficit of N-acetylgalactosamine, galactose, and sialic acid residues in glycophorin A from patients with CDA type I and type II amounting to about 45% and 55%, respectively. The results strongly suggest that glycophorin A in these patients is partly unglycosylated with respect to O-linked glycans. In addition, glycophorin A from erythrocytes of a patient with CDA II but not CDA I exhibited a significant deficit of mannose and N-acetylglucosamine suggesting that its N-glycosylation site was also partly unglycosylated.

In comparison to progenitor platelets, microparticles are deficient in GpIb, GpIb-derived carbohydrates, glycerophospholipids, glycosphingolipids, and ceramides.

Activated blood platelets shed microparticles with procoagulant activity that probably participate in normal hemostasis. We have isolated spontaneously formed microparticles from human blood and analysed them for ultrastructure, antigenic profile, and biochemical composition. In transmission electron microscopy microparticles appeared as regular vesicles with a mean diameter of 300 nm (50-600 nm). In flow cytometry almost all microparticles reacted with fluorescein isothiocyanate (FITC) labeled antibody to platelet glycoprotein complex IIb-IIIa (GpIIb-IIIa) and with FITC-annexin V but only 40-50% of microparticles reacted with FITC-antibody to platelet glycoprotein Ib (GpIb). The latter result was confirmed by double labeling of microparticles with FITC-antibody to GpIIb-IIIa and phycoerythrin (PE) labeled antibody to GpIb. Large microparticles reacted better with anti-GpIb than the small ones. A decreased level of GpIb was also demonstrated by SDS/polyacrylamide gel electrophoresis of microparticles. Compositional studies indicated, that in terms of cholesterol and protein contents, microparticles resembled platelets rather than platelet membranes as previously thought. They are, however, deficient in certain components. Thus, in comparison to platelets, microparticles had reduced contents of sialic acid (by 56.4%), galactosamine (by 48.2%), glucosamine (by 22.4%), galactose by (11.8%) and fucose (by 21.6%). Mannose content was increased by 11.8%. Total phospholipids in microplatelets were lower by 17.8%. Glycerophospholipids only were affected with phosphatidylserine being decreased as much as by 43.2%. Neutral glycosphingolipids, gangliosides and ceramides in microparticles were reduced by half.

The levels of glycosphingolipids, ceramides, sialic acid and glycogen are changed in plasma membranes of rat platelets harvested during recovery from immune-mediated thrombocytopenia.

Plasma membranes of rat platelets produced at normal platelet counts and during early recovery from immune-mediated thrombocytopenia were examined for the contents of carbohydrates, lipids and glycosphingolipids. Glucosylceramide, two monosialo-gangliosides and one disialo-ganglioside were found to be the major glycosphingolipids of platelets. During thrombocytopenia the contents of these glycosphingolipids as well as of ceramides were several fold elevated. Among carbohydrate constituents of platelets and platelet plasma membranes, glycogen content was increased and that of sialic acid decreased. These results are discussed in the light of literature data on relevant biochemical characteristics of megakaryocytes at different stages of maturation and on thrombopoiesis during acute experimental thrombocytopenia.

Activity of platelet alpha-6-fucosyltransferase is inversely related to blood platelet concentration.

The activity of serum alpha-6-fucosyltransferase, a platelet derived enzyme, determined in sera of 22 normal individuals and 86 patients with various disorders was positively correlated with platelet counts. When the enzyme activity in 1 microliters serum was calculated per 1000 of platelets in blood (coefficient F/P) an inverse correlation became evident in that F/P was proportionally the higher the lower was platelet count in blood. The F/P values were in a good agreement with the results of direct assays of enzyme activities in isolated platelets. Neither granulocytes, lymphocytes nor red cells significantly contributed to serum enzyme activity though granulocytes enhanced the thrombin-induced enzyme release from platelets. In platelets separated by centrifugation in density gradients the enzyme was shown to be present in platelets of intermediate and high density but missing from the light ones. It is suggested that alpha-6-fucosyltransferase of platelets may be a marker of the ploidy level of megakaryocytes.

Contents of total and protein-bound carbohydrates are low in leukemic leukocytes from patients with acute myelogenous leukemia.

Leukemic leukocytes from 12 patients with acute myelogenous leukemia (AML) and two patients with chronic myelogenous leukemia (CML) were isolated by centrifugations in Percoll gradients, and examined for total carbohydrates. In leukemic leukocytes from 10 of these patients ceramide-bound carbohydrates were also determined. Protein-bound carbohydrates were calculated by subtraction of ceramide-bound carbohydrates from total carbohydrates. In all samples analysed the contents of total and protein-bound carbohydrates were much lower in leukemic leukocytes than in normal neutrophils, irrespective whether the results were expressed relative to protein, DNA, cell number or dry mass. For immature leukemic cells of M0-M1 phenotype differences up to 10-fold were observed. Contents of ceramide-bound carbohydrates, i.e. those of neutral and acidic glycosphingolipids (GSLs) were also low in leukemic cells. However, when GSL carbohydrates were calculated as percentage of total carbohydrates, GSLs in leukemic leukocytes were elevated in half of the AML patients but depressed in the other half. The results are discussed in the light of the hypothesis on GSL function by one of us (Koscielak J., 1986, Glycoconjugate J. 3, 95-108). According to one element of the hypothesis, during cell differentiation newly synthesized glycoproteins (GPs) that perform specific functions are added to house-keeping GPs that are present in plasma membranes of all types of cells. Thus, during differentiation, the GP content of the cell membrane should increase and that of the so called "membrane packing" glycosphingolipids should decrease.

Enzymatic synthesis of H-active pentaglycosylceramide by human bone marrow fucosyltransferase.

Synthesis of two fucoglycolipides I and II was obtained from GDP-fucose and lacto-N-neotetraosylceramide using either solubilized or particulate enzymic preparation from human bone marrow. Fucoglycolipid I on the basis of its chromatographic mobility and immunoprecipitation with Ulex europeus lectin was identified as H blood group active penta glycosylceramide. Fucoglycolipid II did not react with Ulex europeus and its fucosyl residue was hydrolyzed by alpha (1 leads to 3/4)-L-fucosidase of Trichomonas fetus. Most probably the fucose residue of glycolipid II was linked to N-acetyloglucosamine by 1 leads to 3 linkage.

Structure of a new disialoganglioside GD1c from spontaneous murine thymoma.

A major mono- and a di-sialoganglioside were isolated and purified to homogeneity from a spontaneous thymoma that occurs in AKR mice. Compositional and methylation analyses and the use of exoglycosidases established the monosialoganglioside to be alpha Neu(2----3)beta Gal(1----3)beta GalNAc(1----4)beta Gal(1----4)Glc(1----4)Cer and the disialoganglioside to be alpha NeuAc(2----8)alpha NeuAc(2----3)beta Gal(1----3)beta GalNAc(1----4)beta Gal(1----4)Glc(1----1)Cer (GD1c). A possible pathway for the biosynthesis of this disialoganglioside is presented.

Structure and blood-group I activity of poly(glycosyl)-ceramides.

Nitrous acid deamination of N-deacetylated blood-group O poly(glycosyl)ceramides resulted in a complete degradation of the glycolipids to give O-beta-D-galactopyranosyl-(1 leads to 4)-2,5-anhydro-D-mannose, O-alpha-L-fucopyranosyl-(1 leads to 2)-O-beta-D-galactopyranosyl-(1 leads to 4)-2,5-anhydro-D-mannose, lactosylceramide, and small proportions of free 2,5-anhydro-D-mannose. A series of straight-chain glycolipids containing up to six glycosyl residues and comprising alternating 3-O-substituted D-galactosyl and 4-O-substituted 2-acetamido-2-deoxy-D-glucosyl residues was isolated from partial acid hydrolyzates of poly(glycosyl)ceramides. Branching points of 3,6-di-O-substituted and 6-O-substituted D-galactosyl residues were observed only in fractions containing more than six glycosyl residues. Sequential periodate oxidation of poly(glycosyl)ceramides gradually eliminated the branching points. This elimination was not complete, even after four cycles of degradation. The ability of poly(glycosyl)ceramides to precipitate with anti-I serums disappeared after two cycles of degradation. These results suggest a general structure for poly(glycosyl)ceramides. I blood-group activity of the glycolipids would depend on the periodical arrangement of branched side-chains.

[Possibility of using antibodies to lactosylsphingosine in the diagnosis of neoplasms]. / Mozliwosc wykorzystania przeciwcial reagujacych z laktozylosfingozyna w diagnostyce chorób nowotworowych.

A radioimmunoassay was evolved for detection of antibodies reacting with lactosylsphingosine in the serum. Positive results were obtained in cases of digestive tract neoplasms, cervical carcinoma and burns.

Mobilities of complex glycosphingolipids in SDS gel electrophoresis.

Poly/glycosyl/ceramides, highly complex and blood-group active glycosphingolipids of human erythrocyte membranes were subjected to SDS polyacrylamide gel electrophoresis. The substances exhibited similar electrophoretic mobilities as membrane sialoglycoproteins.

[Erythrocyte freezing in the presence of hydroxyethyl starch]. / Badania nad zamrazaniem erythrocytów w obecnosci hydroksyetylowanej skrobi

Biochemical properties of erythrocytes frozen in the presence of hydroxyethyl starch (HES) as preserving substance produced in this country were assessed. Erythrocytes were frozen adding HES in 4 different concentrations. The most favourable results were obtained with erythrocytes subjected to the action of HES in 30% and 35% concentrations. The erythrocyte 2,3-DPG level during freezing with 35% HES was within normal limits while it decreased by 10% after defreezing when 30% HES had been used. The ATP concentration in erythrocytes fell by 50% in relation to the initial value. A slight fall of P50 was observed and erythrocyte loss after defreezing did not exceed 10%. The 35% HES solution in a 0.15 mol/l sodium chloride solution was most favourable for freezing erythrocytes in liquid nitrogen. A simple method of preparation of defrozen erythrocytes for transfusion was elaborated.

[Fate of C-14 labeled hydroxyethyl starch in mice]. / Utrzymywanie sie [14C]-znakowanej hydroksyetylowanej skrobi w tkankach i narzadach myszy.

Three preparations of hydroxyethyl starch having MS 0.55, 0.65 and 0.8 respectively and labelled with 14C in hydroxyethyl residue were administered to mice. After 1, 5, 10, 20 and 60 days the mice were sacrificed and radioactivities determined in the following organs and tissues: blood, spleen, liver, kidneys, heart, lungs and muscles. Most of the initial dose of radioactivity was eliminated from mice within 24 h. The remaining portion was eliminated slowly and even after 60 days 0 07--1.2% of the initial dose could be detected in different organs. The rate of clearance of radioactivity was fastest for hydroxyethyl starch with MS = 0.55 and slowest for the preparation with MS = 0.8. The retention of radioactivity was most conspicous in the muscles.

[RNA in surface antigen of hepatitis B virus (HBsAg)]. / RNA w antygenie powierzchniowym wirusa zóltaczki B (HBsAg).

RNA was isolated from HBsAg particles obtained by the method of preparatory immunoprecipitation with anti-HBs antibodies. RNA electrophoresis in polyacrylamide gel showed presence of one fraction with molecular weight about 28 000. The index of RNA sedimentation determined by the method of ultracentrifugation was about 4 S. RNA from HBsAg had no informative features of nucleic acids (m RNA) such as presence of methyl group 5' of the end sequence m7 GpppN ("cap"), translatory activity. It had also no features of tRNA since it was not aminoacylated in the presence of aminoacylo-tRNA synthetases.

[Case of heterozygotic SC hemoglobinopathy with high titre of indirect immunofluorescence reaction with Plasmodium falciparum antigen]. / Przypadek heterozygotycznej hemoglobinopatii SC z wysokim mianem odczynu immunofluorescencji posredniej (IFP) z antygenem Plasmodium falciparum.

The authors report the first in the Polish literature case of combined heterozygotic SC haemoglobinopathy in a Nigerian aged 20 years. The diagnosis was based on the results of haemoglobin electrophoresis and clinical signs. The titre of IFP with Plasmodium falciparum was very high and malarial pigment was found in lymph nodes.