Plasmodium falciparum: carbohydrates as receptor sites of invasion

Monosaccharides, disaccharides, and trisaccharides were tested as inhibitors of the in vitro growth of Plasmodium falciparum (strain FCB). While certain monosaccharides (N-acetyl-D-glucosamine, D-mannose, and 3-O-methyl-D-glucose) proved to exhibit a toxic or reversibly retarding effect on the intraerythrocytic development of the parasite, the corresponding alpha- or beta-methylglycosides did not. Several methylglycosides, synthetic di- and tri-saccharides, and artificial blood group antigens were further tested for inhibitory effects on invasion of host red blood cells in vitro. The synthetic disaccharides beta DGlcNAc(1----4) alpha DManOMe and beta DGlcNAc(1----4) DGlcNAc (chitobiose) were good inhibitors of invasion at 10 mM concentration, whereas beta DGal(1----4)beta DGlcNAcOMe was negligibly inhibitory. The inhibition rate of N-acetyl-D-glucosamine, beta-glycosidically linked to bovine serum albumin (BSA) by an alipathic spacer, -(CH2)8CO-, was not enhanced, compared to the corresponding hapten, beta DGlcNAcO(CH2)8COOCH3. The inhibition rates of blood group A- and B-trisaccharide haptens, which were inhibitors of invasion, were also not significantly enhanced when coupled to BSA by way of the corresponding amide spacer, -(CH2)2NHCO(CH2)7CO-. A remarkable enhancement of the inhibition rate was, however, observed when beta DGal(1----3) alpha DGalNAcO(CH2)2NHCO(CH2)7COOCH3 (T-hapten) was coupled to BSA. A clear-cut decrease in the inhibition rates of different beta-glycosides of N-acetyl-D-glucosamine, beta DGlcNAcOR, was observed, depending on the nature of the aglycon R(p-nitrophenyl greater than -(CH2)8COOCH3 greater than -(CH2)2NHCO(CH2)2COOCH3 greater than -CH3). Also, p-nitrophenyl-alpha-D-glucopyranoside was a much better inhibitor of invasion than the corresponding methyl glycoside, alpha DGlcOMe, which was not inhibitory. The properties of the aglycon spacer, used for the covalent attachment of the carbohydrate to the carrier protein, may thus be crucial for the outcome of the inhibition rate.     

Phospholipid composition and external labeling of aminophospholipids of human En(a−) erythrocyte membranes which lack the major sialoglycoprotein (glycophorin a)

Erythrocytes of the rare human blood group En(a?) lack the major sialoglycoprotein, glycophorin A, and the cell population heterozygous for the En(a) antigen contain half the normal amount of glycophorin A. With such cells we have studied whether glycophorin A influences the phospholipid composition and the availability of aminophospholipids to external labeling reagents. We here demonstrate that the amounts of all phospholipids are closely similar in normal and variant membranes. However, using the amino-reactive reagent trinitrobenzenesulfonate, we show that phosphatidylethanolamine is more easily labeled in intact En(a?) cells as compared to normal cells, whereas phosphatidylethanolamine shows an intermediate labeling in En(a) heterozygous cells.     

Glycomic mapping of pseudomucinous human ovarian cyst glycoproteins: identification of Lewis and sialyl Lewis glycotopes

Expression of sialyl Lewis x (sLe(x)) and sialyl Lewis a (sLe(a)) on cell-surface glycoproteins endows cells with the ability to adhere to E-, P-, and L-selectins present on endothelia, platelets, or leukocytes. Special arrangements of these glycotopes in cancers are thought to play a key role in metastasis. Previous studies have mostly described membrane-bound sLe(x) and sLe(a) activities. In this report, the major O-glycans of the secreted human ovarian cyst sialoglycoproteins from a Le(a+) nonsecretor individual (human ovarian cyst sample 350) were characterized by MS/MS analyses and immuno-/lectin-chemical assays. The results showed that HOC 350 carries a large number of epitopes for sLe(x), sLe(a), and Le(a) reactive antibodies. Advanced MS/MS sequencing coupled with mild periodate oxidation and exoglycosidase digestions further revealed that the O-glycans from HOC 350 are mostly of core 1 and 2 structures, extended and branched on the 3-arm with both type I and type II chains, complete with variable degrees of terminal sialylation and/or fucosylation to yield the sLe(x) or sLe(a) epitopes. Thus, the underlying core and peripheral backbone structures are similar to that of a previously proposed composite structural model for nonsialylated human ovarian cysts O-glycans, but with some notable distinguishing structural features in addition to sialylation.     

Changes in the Expression of a Neuronal Surface Protein During Development of Cerebellar Neurones In Vivo and in Culture

The expression of the neurone-specific D2 protein changes both quantitatively and qualitatively during development in vivo and in cultures of cerebellar nerve cells. The total D2 content per unit protein shows a two-fold increase in vivo from birth to postnatal day 6, after which it declines progressively to about 50% of the maximal value. This increase can be accounted for by an immature form of the protein anodic D2 being preferentially expressed at the early stages of cerebellar development. After postnatal day 9 this form gradually switches to a mature form cathodic D2. This switch can be mimicked by neuraminidase treatment, suggesting a developmental loss of sialic acid from the D2 protein. In freshly isolated cells the total D2 content per unit protein is only 30% of that in the corresponding intact tissue from 8-day-old cerebella, but it increases rapidly during the first 8 days of culture to levels similar to those of the equivalent age in vivo. The switch from anodic D2 to cathodic D2 also occurs at a faster rate in culture, probably reflecting the culture conditions that favour differentiation. The changes in the expression of D2 during development of cerebellar nerve cells in culture suggest that anodic D2 is preferentially expressed on nerve cells that are proliferating, migrating, or in the initial stages of differentiation, whereas cathodic D2 is associated with differentiated neurones. The transition between the two forms appears to occur during the formation of interneuronal contacts.     

Changes in expression of a major sialoglycoprotein associated with ascites forms of a mammary adenocarcinoma

Glycoproteins of a cultured form (MR) of the 13762 rat mammary adenocarcinoma and its variants have been studied by analyses for peanut agglutinin receptors, [³H]glucosamine labeling, lactoperoxidase labeling and CsCl density gradient centrifugation. The 13762 MR cells, derived from 13762 MAT-B ascites cells, do not contain detectable ASGP-1, the predominant cell surface sialoglycoprotein of the ascites forms of the 13762 tumor.Transplantation and continued passage as ascites cells of MR cells or clonal lines derived from MR results in abrupt expression of ASGP-1 at about passage 16; it is absent in early passages of the ascites tumor. When these ascites cells are transferred to culture, ASGP-1 is again lost. No ASGP-1 is found in solid tumors derived from subcutaneous transplantation of the 13762 MR cells. The results suggest modulation of ASGP-1 content of the 13762 tumor cells.     

Glycoprotein Properties of the Solubilized Atrial Muscarinic Acetylcholine Receptor

Abstract: The muscarinic acetylcholine receptor from porcine atria exhibits sialoglycoprotein characteristics based on its sensitivity to neuraminidase digestion and its ability to interact specifically with lectin affinity resins when solubilized with a digitonin/cholate mixed detergent system. Differential lectin binding properties of the neuraminidase-treated and untreated receptor suggest that high-affinity binding to immobilized wheat germ agglutinin is accomplished through the presence of both terminal sialic acid and internal N -acetylglucosamine or its β(1→4)-linked oligomers.     

Surface glycoprotein of human natural killer cells recognized by wheat germ agglutinin

We analyzed surface glycoproteins of human natural killer (NK) cells by utilizing lectins. Among the lectins tested, wheat germ agglutinin (WGA) was found to bind preferentially to CD16(Leu11)-positive lymphocytes as determined by two-colour flow cytometry. Analysis of glycoproteins in the lysate prepared from NK cells with sodium dodecyl sulfate (SDS) gel electrophoresis followed by Western blotting and¹²⁵I labeled WGA staining revealed that a glycoprotein with anM _r of 65 kDa was strongly bound to the lectin, but no corresponding glycoprotein was detected in the lysate of T lymphocytes. This glycoprotein (GP65) gave several spots in the pI range 4.1–4.6 on 2-dimensional gel electrophoresis. Sialidase treatment of GP65 resulted in a single spot on the 2-dimensional gel, suggesting that GP65 is heterogeneous in the degree of sialylation. GP65 was shown to be exposed on the cell surface, since it was radiolabeled with¹²⁵I by the lactoperoxidase-catalyzed method. We next isolated GP65 from human peripheral blood lymphocytes by a combination of chromatography on a cation-exchange column and a WGA-agarose column and preparative SDS gel electrophoresis. It is suggested that GP65 is a novel surface glycoprotein on human NK cells.