INCORPORATION OF [14C]N-ACETYL NEURAMINIC ACID INTO BRAIN GLYCOPROTEINS AND GANGLIOSIDES IN VIVO1

—Intracerebrally administered [¹⁴C]N-acetyl neuraminic acid was incorporated into brain glycoproteins and gangliosides. Incorporation into both classes of compounds was markedly inhibited by acetoxycycloheximide but incorporation into the soluble glycoproteins of the nerve-ending fraction was inhibited least of all. In contrast to glucosamine and fucose, a relatively small proportion of the injected [¹⁴C]NANA was incorporated.     

Syntheses of sulfated glycopolymers and analyses of their BACE-1 inhibitory activity

The glycopolymers for glycosaminoglycan mimic were synthesized, and the inhibitory effects of Alzheimer’s β-secretase (BACE-1) were examined. The regio-selective sulfation was conducted on N-acetyl glucosamine (GlcNAc), and the acrylamide derivatives were synthesized with the consequent sulfated GlcNAc. The glycopolymers were synthesized with acrylamide using radical initiator. The glycopolymer with sulfated GlcNAc showed the strong inhibitory effect on BACE-1, and the inhibitory effects were dependent on the sulfation positions. Especially, glycopolymers carrying 3,4,6-O-sulfo-GlcNAc showed the strong inhibitory effect. The docking simulation suggested that glycopolymers bind to the active site of BACE-1.     

Dynamics of sialyl Lewis(a) in aqueous solution and prediction of the structure of the sialyl Lewis(a)-SelectinE complex

In this article we investigate all possible three-dimensional structures for sialyl Lewis^a (SLe^a) in aqueous solution and we predict without a priori experimental information its conformation when bound to SelectinE by using a combination of long molecular dynamics (MD) simulations. Based on 10 ns MD studies, three structures differing in glycosidic conformations are proposed for SLe^a in aqueous solution. Based on a 4 ns MD study of the SLe^a-SelectinE complex with initial structures derived from our prediction tools, we find that, fucose and N-acetyl neuraminic acid are in close contact with SelectinE and therefore expect interactions of the protein with these two sugar rings to be significantly more important than in the case of galactose and N-acetyl glucosamine. Our predictions indicate that the N-acetyl glucosamine of SLe^a is positioned primarily in the aqueous phase. In order to be able to interact with SLe^a the side chains of amino acid residues Lys99 and Lys111 in SelectinE appear to undergo large conformational changes when contrasted with the positions of these residues in the X-ray crystal structure. Furthermore, amino acid residues Arg97, Glu98 and Lys99 are acting as a holding arm to position the NeuNAc of SLe^a in the binding pocket.     

Plasmodium falciparum: stage-specific lactate production in synchronized cultures

Sequential treatment with gelatin followed by sorbitol-produced Plasmodium falciparum cultures that remained synchronous for ? 144 hr. The schizont stage was associated with more lactate production than the other asexual erythrocytic stages of the parasite (11–21 vs 4–7 neq/hr-10⁶ parasitized red blood cells [PRBC]). Lactate production in asynchronous cultures was relatively constant at 8–11 neq/hr-10⁶ PRBC and was directly proportional to parasitemia (P < 0.001).     

Plasmodium falciparum: Synthesis of glycoprotein by cultured erythrocytic stages

The human malarial parasite, Plasmodium falciparum, incorporated significant radioactivity into glycoconjugates when cultured in the presence of [¹⁴C]- or [³H]glucosamine for 48 to 50 hr. Digestion of the labeled proteins with pronase and subsequent precipitation with absolute ethanol showed that 90 to 95% of the radioactive glucosamine was incorporated into the precipitated material. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the labeled macromolecules revealed eight bands with approximate molecular weights from 19,000 to 90,000 daltons.     

A New Lectin from Meadow Saffron (Colchicum automnale)

A lectin has been isolated from tubers of the meadow saffron (Colchicum autumnale). It is an octameric protein (M_r 100,000) composed of 4A- and 4B-subunits of M_r 15,000 and 10,000, respectively. It is a glycoprotein with 4.4% carbohydrate, the main sugars are (N-acetyl-) glucosamine, mannose, fucose, and xylose. Although the Colchicum autumnale agglutinin (CAA) agglutinates human red blood cells, it has a much higher activity with rabbit erythrocytes. With respect to its carbohydrate-binding specificity CAA behaves rather unusually as it is inhibited by lactose, galactose, N-acetylgalactosamine and related sugars when assayed with human red blood cells but not in assays with rabbit erythrocytes.     

Formation of polyprenol-linked mono- and oligosaccharides in Phaseolus aureus

A crude membrane preparation from Phaseolus aureus hypocotyls catalyzes the incorporation of mannose from GDP-[¹⁴C]mannose into a acid labile glycolipid and a methanol insoluble fraction. Addition of dolichyl monophosphate to the incubation mixture stimulated the formation of both the mannolipid and the methanol insoluble endproduct. Thin-layer chromatography of endogenous lipid and of the stimulated lipid fraction revealed that both compounds run identical. Ficaprenyl monophosphate also stimulates the incorporation of mannose; however, the ficaprenyl monophosphate mannose formed is not identical to the endogenous mannolipid. This suggests that the endogenous acceptor has the properties of an α-saturated polyprenyl monophosphate rather than those of the ficaprenyl phosphate type. The same membrane preparation also incorporates N-acetylglucosamine into an acid labile glyolipid as well as into a polymer fraction. Evidence is presented that the N-acetylglucosamine containing lipid consists of a mixture of dolichyl pyrophosphate N-acetylglucosamine and dolichyl pyrophosphate di-N-acetylchitobiose. It seems likely that the two compounds have a precursor-product relationship. Incubation of dolichyl pyrophosphate di-N-acetylchitobiose together with GDP-mannose gives rise to lipid-bound mannosyl-di-N-acetylchitobiose. Radioactivity from either the [¹⁴C]mannolipid or the N-acetyl[¹⁴C]glucosamine containing lipid is incorporated into a methanol insoluble product to 3.4 and 6.3%, respectively; it seems, at least in part, to be a glycoprotein.     

Isolation and properties of N-acetyllactosamine-specific lectins from nine erythrina species

Lectins from seeds of nine species of Erythrina have been purified by affinity chromatography on columns of lactose coupled to Sepharose and their properties compared with those of the lectin from Erythrina cristagalli. All lectins are glycoproteins of M, ca 60 000 composed of two identical or nearly identical subunits. They contain between 3–10% carbohydrates comprised of N-acetylglucosamine, mannose, fucose and xylose. The amino acid composition of all Erythrina lectins is very similar. The N-terminal amino acid is valine, with the exception of the lectin from E. flabelliformis in which it is alanine. To the extent tested, identities or near identities have been found in the N-terminal sequences (up to 15 residues in some cases) of the lectins. Hapten inhibition experiments of agglutination have shown that the lectins are specific for N-acetyllactosamine, this disaccharide being 10–30 times more inhibitory than D-galactose and 10–20 times more than N-acetyl-D-galactosamine. All lectins agglutinate human erythrocytes equally well, irrespective of blood type, at minimal concentrations of 5–20 μg/ml. Six of the lectins are also very effective in agglutinating rabbit erythrocytes and are mitogenic for human peripheral blood lymphocytes, whereas three of them are considerably weaker hemagglutinins for rabbit erythrocytes, and two of these are also very weak mitogens. Our results, while demonstrating striking similarities in the molecular properties and sugar specificity of all Erythrina lectins studied, suggest the existence of differences at or close to the carbohydrate-binding site.     

Schistosoma mansoni: patter of release of secretory products.

Adult Schistosoma mansoni were maintained in vitro for 1 hr with radioactively labeled precursors of protein, glycoprotein, and polysaccharides. The worms were then washed extensively and the supernates analyzed. The precursors N-acetylglucosamine, N-acetylgalactosamine, glucosamine, galactosamine, glucose, leucine, and fucose were incorporated into the worms and both large and small molecular weight products accumulated in the supernatant. For all the precursors except fucose, there was an initial rapid and then slower phase of release for both the large and small molecular weight materials. The amount of label retained by the worms as well as the proportion excreted as large molecular weight material was characteristic for the precursor used. In contrast, the products of fucose were released within 4 to 6 hr and therefore only exhibited the early secretory phase. There was no retention of fucose by the worms. Hydrolysis of large molecular weight products revealed that the N-acetylglucosamine-derived material was incorporated as amino sugars and fucose was incorporated as fucose. Therefore, N-acetylglucosamine and fucose precursors can specifically label secretory glycoproteins of schistosomes in a manner similar to that in mammalian systems.     

The incorporation of [3H]glucosamine, [3H]fucose and [14C]mannose into protein in the rat anterior pituitary incubated in vitro

Rat anterior hemipituitaries incubated in vitro rapidly take up and incorporate into protein D-[6-³H]-glucosamine · HCl, D-[1-¹⁴C]mannose and L-[G-³H]fucose. The newly labeled protein was only slowly released into a Krebs-Ringer bicarbonate incubation medium. Glucosamine- or mannose-labeled protein was barely detectable in the medium after a 30–60 min incubation whereas about 4% of all fucose-labeled protein had already been released into the incubation medium by 30 min. Puromycin · 2HCl (1 mM) inhibited incorporation of glucosamine or mannose into protein to 40% or less of control values within 30 min; fucose incorporation was not significantly inhibited before 45 min. Acid hydrolysis followed by amino acid analysis of glucosamine-labeled protein yielded significant amounts of label in glucosamine, galactosamine and apparent glucosamine-degradation products but no significant amount of label in any amino acid.     

Catabolism and disposition of exogenous 5-hydroxytryptamine in cockroach,Periplaneta americana,tissues

The concentrations of tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine, N-acetyl 5-hydroxytryptamine and N-acetyl dopamine were determined in the cerebral ganglia, haemolymph and Malpighian tubules of the cockroach Periplaneta americana, using high performance liquid chromatography with electrochemical detection. Injected 5-hydroxytryptamine was rapidly removed from the haemolymph with a concomitant elevation of circulating N-acetyl 5-hydroxytryptamine and little accumulation of 5-hydroxytryptamine in the cerebral ganglia. N-acetyl 5-hydroxytryptamine and N-acetyl dopamine were also rapidly removed from the haemolymph. Incubation of haemolymph from 5-hydroxytryptamine-injected insects and glucosidase or phosphatase, indicated that most of the injected 5-hydroxytryptamine had been converted to a sugar conjugate of N-acetyl 5-hydroxytryptamine. Whole haemolymph did not catabolize 5-hydroxytryptamine or N-acetyl 5-hydroxytryptamine whereas Malpighian tubules N-acetylated both 5-hydroxytryptamine and dopamine and metabolized N-acetyl 5-hydroxytryptamine. Injection of p-chlorophenylalanine (200 and 500 μg/g) had no effect on 5-hydroxytryptamine concentrations in the cockroach cerebral ganglia.     

Plasmodium berghei: The in Vitro immune response

Following primary in vitro Stimulation by Plasmodium berghei, IgM titers by the indirect fluorescent antibody test (IFAT) were negative in in vitro reconstituted syngeneic mouse spleen cultures containing T cells and macrophages, or B cells and macrophages, or macrophages alone, but IgM titers of 1:20 were obtained from cultures containing B cells, T cells, and macrophages. IFAT IgG titers were negative for cultures with T cells and macrophages together, or macrophages alone, but rose to 1:40 with cultures containing B cells and macrophages and 1,80 with cultures of B cells, T cells, and macrophages together. After a second in vitro challenge, IgM and IgG titers were similar to the stimulated cultures from immunized mice; the IgM titer reached only 1:20 but the IgG titer rose to 1:160. Total immunoglobulin was higher in the cultures from immunized mice than from in vitro primed cultures.     

Anticoccidial drugs: Effects on infectivity and survival intracellularly of Eimeria tenella sporozoites

The effects of various anticoccidial drugs on extracellular and intracellular sporozoites were studied in cell culture and in chickens. Treatment of freshly excysted, extracellular sporozoites of Eimeria tenella for 18 hr with monensin, decoquinate, or robenidine at 100 ppm had no effect on oocyst production 7–10 days after the sporozoites were rinsed free of drugs and fed to chickens. Treatment of cultures of E. tenella in chick kidney cell monolayers with monensin (0.001 μg/ml), decoquinate (0.01 μg/ml), zoalene (20.0 μg/ml), or robenidine (0.01 μg/ml) had no effect on intracellular sporozoites at 4 hr following introduction of sporozoites and drugs into the culture. A significant reduction of intracellular parasites occurred at 24 hr in the cultures treated with monensin or zoalene. Remaining intracellular sporozoites in monensin-treated cultures were morphologically abnormal or degenerate, while sporozoites in other cultures appeared normal. The number and condition of sporozoites in the nontreated cultures were unchanged at 24 hr postinoculation. These results indicate that sporozoites undergo changes subsequent to penetration of host cells that render them susceptible to drug action.     

Steady-state Inhibitory Kinetic Studies on Almond β-Glucosidase-catalyzed Reaction——Binding Sites of Monosaccharides

Steady-state inhibitory kinetic studies on almond β-glucosidase-catalyzed reactions were done to elucidate the binding subsite of several monosaccharides on this enzyme.

Glucono-1,5-Iactone (a transition-state analog), glucose, 2-deoxy glucose, fucose, and methyl α-glucoside showed mixed-type inhibition, but galactose, galactosamine, mannose, N-acetyl glucosamine, and glucosamine showed pure competitive inhibition on the hydrolysis of P-nitrophenyl β-glucoside.

These results are reasonably accounted for by assuming that the former monosaccharides (the mixed type inhibitors) bind to subsite 1 (the nonreducing-end side subsite to which the nonreducing-end glucose residue of a substrate binds in a productive binding mode), and that the latter (the competitive inhibitors) bind to subsite 2, the adjacent subsite to subsite 1.

The binding affinity ( — ΔG°) of glucono-1,5-lactone (— ΔG° = 6.7 kcal mol ¹ at pH 5.0, 25°C) was significantly greater than those of the others (0.3 ~ 1.6 kcal mol^-1).     

Glycosylation of the Male Genital Ducts and Spermatozeugmata Formation in the Clearnose Skate Raja eglanteria

Genital ducts of three male Raja eglanteria were fixed and embedded in epoxy and methacrylate resin. Epoxy resin sections from the Leydig gland, upper and lower epididymis, ductus deferens and seminal vesicle were stained with 20 labelled lectins to examine their glycosylation. The Leydig gland consisted of columnar epithelial cells expressing N-linked glycans, N-acetyl galactosamine, glucosamine and lactosamine residues and sialic acid. Interspersed were ciliated cells of a different glycotype. The upper epididymis of cuboidal epithelium had a strongly glycosylated, ciliated apical surface and cytoplasmic granules that stained heavily with many lectins, with increased glycosylation compared to the Leydig gland. In the lower epididymis, tall, vacuolated cells showed some differences and a slight reduction in lectin staining. The ductus deferens contained two cell types and showed increased terminal N-acetyl galactosamine. The ciliated cuboidal epithelium of the seminal vesicle had marked differences from the ductus epithelium, with decreased N-acetyl galactosamine and lactosamine expression but increased subterminal N-acetyl lactosamine and galactosamine expression and sialylation. Spermatozoa were suspended in a glycosylated matrix and, in the seminal vesicle, were embedded in solid masses of matrix forming spermatozeugmata. These data show changes in glycan expression along the male genital tract, probably related to the nurture and maturation of the spermatozoa as they travel towards the seminal vesicle.     

Application and limitations of the methyl imidate protection strategy of N-acetylglucosamine for glycosylations at O-4: synthesis of Lewis A and Lewis X trisaccharide analogues

We describe here the synthesis of the allyl Le^a trisaccharide antigen as well as that of an analogue of the Le^x trisaccharide antigen, in which the galactose residue has been replaced by a glucose unit. Although successful fucosylations at O-4 of N-acetylglucosamine acceptors have been reported using perbenzylated thioethyl fucosyl donors under MeOTf activation, such conditions led in our case to the conversion of our acceptor to the corresponding alkyl imidates. Indeed, in this synthesis of the Le^a analogue, we demonstrate that the temporary protection of the N-acetyl group as a methyl imidate is advantageous to fucosylate at O-4. In contrast, we report here that glucosylation at O-4 of an N-acetylglucosamine monosaccharide acceptor using the α-trichloroacetimidate of peracetylated glucopyranose as a donor proceeded in better yields under activation with excess BF₃·OEt₂ than that of the corresponding methyl imidate. Therefore, we conclude that activation of thioglycoside donors by MeOTf to glycosylate at O-4 of a glucosamine acceptor is best accomplished following the temporary protection of the N-acetyl group as a methyl imidate, especially when the donors are highly reactive and prone to degradation. In contrast, if donor and acceptor can withstand multiple equivalents of BF₃·OEt₂, glycosylations at O-4 of a glucosamine acceptor with a trichloroacetimidate donor does not benefit from the temporary protection of the N-acetyl group as a methyl imidate.     

Transfer of N, N'-diacetylchitobiose from dolichyl diphosphate into a gray matter membrane glycoprotein

Gray matter and white matter membranes catalyze the transfer of label from UDP-N-acetyl-[${}^{14}\text{C}$] glucosamine into N-acetyl[${}^{14}\text{C}$]glucosaminyl-pyrophosphoryl-dolichol, N,N′-diacetyl [${}^{14}\text{C}$]chitobiosyl-pyrophosphoryl-dolichol, and N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycoprotein. Gel filtration of the Pronase digests of gray matter N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycoprotein reveals two N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycopeptide fractions. One fraction (A) contains approximately eight glycose units. All of the radioactivity is at nonreducing termini and can be released by treatment with an exo-β-N-acetylglucosaminidase. A smaller N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycopeptide (B) is recovered in the elution volume expected for an asparaginyl disaccharide. Structural studies show that the labeled saccharide unit in glycopeptide B is N,N′-diacetyl[${}^{14}\text{C}$]chitobiose. The linkage between the ${}^{14}\text{C}$-labeled disaccharide and the polypeptide has the properties of an N-glycosidic attachment to asparagine. Only the larger N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycopeptide (A) is found in Pronase digests of white matter membrane N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycoprotein after incubation with UDP-N-acetyl[${}^{14}\text{C}$]glucosamine. When gray matter membranes are incubated with UDP-N-acetyl[${}^{14}\text{C}$]glucosamine in the presence of tunicamycin or UMP, the labeling of glycolipid and the asparaginyl disaccharide is inhibited. UMP and tunicamycin have no effect on the transfer of N-acetyl[${}^{14}\text{C}$]glucosamine to external acceptor sites of the larger glycopeptide (A). The transfer of N,N′-diacetyl[${}^{14}\text{C}$]-chitobiose from carrier lipid to protein is observed when extensively washed membranes containing endogenous, prelabeled ${}^{14}\text{C}$-labeled glycolipids are incubated in the presence or absence of unlabeled GDP-mannose. UMP treatment of the prelabeled membranes selectively discharged over 80% of the label from N-acetyl[${}^{14}\text{C}$]glucosaminyl-pyrophosphoryl-dolichol, but had no effect on the transfer of the ${}^{14}\text{C}$-labeled disaccharide to protein. All of these results are concordant with transfer of N,N′-diacetylchitobiose from dolichyl diphosphate to gray matter glycoprotein. The major membrane glycoprotein labeled by the lipid-mediated [${}^{14}\text{C}$]disaccharide transfer reaction has an apparent molecular weight of 24,000. Tunicamycin prevents the enzymatic labeling of the gray matter glycoprotein having an apparent molecular weight of 24,000.     

Structural and Functional Analysis of Fucose-Processing Enzymes from Streptococcus pneumoniae

Fucose metabolism pathways are present in many bacterial species and typically contain the central fucose-processing enzymes fucose isomerase (FcsI), fuculose kinase (FcsK), and fuculose-1-phosphate aldolase (FcsA). Fucose initially undergoes isomerization by FcsI producing fuculose, which is then phosphorylated by FcsK. FcsA cleaves the fuculose-1-phosphate product into lactaldehyde and dihydroxyacetone phosphate, which can be incorporated into central metabolism allowing the bacterium to use fucose as an energy source. Streptococcus pneumoniae has fucose-processing operons containing homologs of FcsI, FcsK, and FcsA; however, this bacterium appears unable to utilize fucose as an energy source. To investigate this contradiction, we performed biochemical and structural studies of the S. pneumoniae fucose-processing enzymes SpFcsI, SpFcsK, and SpFcsA. These enzymes are demonstrated to act in a sequential manner to ultimately produce dihydroxyacetone phosphate and have structural features entirely consistent with their observed biochemical activities. Analogous to the regulation of the Escherichia coli fucose utilization operon, fuculose-1-phosphate appears to act as an inducing molecule for activation of the S. pneumoniae fucose operon. Despite our evidence that S. pneumoniae appears to have the appropriate regulatory and biochemical machinery for fucose metabolism, we confirmed the inability of the S. pneumoniae TIGR4 strain to grow on fucose or on the H-disaccharide, which is the probable substrate of the transporter for the pathway. On the basis of these observations, we postulate that the S. pneumoniae fucose-processing pathway has a non-metabolic role in the interaction of this bacterium with its human host.