Molecular cloning and characterization of a novel repeat-containing Leishmania major gene, ppg1, that encodes a membrane-associated form of proteophosphoglycan with a putative glycosylphosphatidylinositol anchor.
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Leishmania parasites secrete a variety of proteins that are modified by phosphoglycan chains structurally similar to those of the cell surface glycolipid lipophosphoglycan. These proteins are collectively called proteophosphoglycans. We report here the cloning and sequencing of a novel Leishmania major proteophosphoglycan gene, ppg1. It encodes a large polypeptide of approximately 2300 amino acids. The N-terminal domain of approximately 70 kDa exhibits 11 imperfect amino acid repeats that show some homology to promastigote surface glycoproteins of the psa2/gp46 complex. The large central domain apparently consists exclusively of approximately 100 repetitive peptides of the sequence APSASSSSA(P/S)SSSSS(+/-S). Gene fusion experiments demonstrate that these peptide repeats are the targets of phosphoglycosylation in Leishmania and that they form extended filamentous structures reminiscent of mammalian mucins. The C-terminal domain contains a functional glycosylphosphatidylinositol anchor addition signal sequence, which confers cell surface localization to a normally secreted Leishmania acid phosphatase, when fused to its C terminus. Antibody binding studies show that the ppg1 gene product is phosphoglycosylated by phosphoglycan repeats and cap oligosaccharides. In contrast to previously characterized proteophosphoglycans, the ppg1 gene product is predominantly membrane-associated and it is expressed on the promastigote cell surface. Therefore this membrane-bound proteophosphoglycan may be important for direct host-parasite interactions. (+info)
Glycosphingolipids are not essential for formation of detergent-resistant membrane rafts in melanoma cells. methyl-beta-cyclodextrin does not affect cell surface transport of a GPI-anchored protein.
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Recent data suggest that membrane microdomains or rafts that are rich in sphingolipids and cholesterol are important in signal transduction and membrane trafficking. Two models of raft structure have been proposed. One proposes a unique role for glycosphingolipids (GSL), suggesting that GSL-head-group interactions are essential in raft formation. The other model suggests that close packing of the long saturated acyl chains found on both GSL and sphingomyelin plays a key role and helps these lipids form liquid-ordered phase domains in the presence of cholesterol. To distinguish between these models, we compared rafts in the MEB-4 melanoma cell line and its GSL-deficient derivative, GM-95. Rafts were isolated from cell lysates as detergent-resistant membranes (DRMs). The two cell lines had very similar DRM protein profiles. The yield of DRM protein was 2-fold higher in the parental than the mutant line, possibly reflecting cytoskeletal differences. The same amount of DRM lipid was isolated from both lines, and the lipid composition was similar except for up-regulation of sphingomyelin in the mutant that compensated for the lack of GSL. DRMs from the two lines had similar fluidity as measured by fluorescence polarization of diphenylhexatriene. Methyl-beta-cyclodextrin removed cholesterol from both cell lines with the same kinetics and to the same extent, and both a raft-associated glycosyl phosphatidylinositol-anchored protein and residual cholesterol showed the same distribution between DRMs and the detergent-soluble fraction after cholesterol removal in both cell lines. Finally, a glycosyl phosphatidylinositol-anchored protein was delivered to the cell surface at similar rates in the two lines, even after cholesterol depletion with methyl-beta-cyclodextrin. We conclude that GSL are not essential for the formation of rafts and do not play a major role in determining their properties. (+info)
The glycosphingolipid sulfatide in the islets of Langerhans in rat pancreas is processed through recycling: possible involvement in insulin trafficking.
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In previous studies we have shown that sulfatide (galactosylceramide-3-O-sulfate), in various species, is present in the insulin-producing cells in pancreatic islets of Langerhans. In this study the synthesis of sulfatide in the islets has been investigated by pulse chase labeling at varying glucose levels and in the presence or absence of the glycosphingolipid synthesis inhibitory agents, Brefeldin A, fumonisin B1 and chloroquine and the distribution of sulfatide by immune-electronmicroscopy. The data showed that (1) sulfatide was produced in islets of Langerhans, (2) the main pathway for synthesis was through recycling involving partial degradation in the lysosome, and that (3) high glucose levels, although not primarily reflected in an increased synthesis of sulfatide, lead to an increased expression of mRNA for the UDP-galactose:ceramide galactosyltransferase, producing the immediate precursor of sulfatide. Furthermore, mass spectrometry analyses revealed a high proportion of short chain fatty acids, C16:0 (50%) and no hydroxylated forms and thus special physicochemical properties, indicating important differences between pancreatic and brain/neural sulfatide. Immune electron microscopy revealed an intracellular expression of sulfatide in the secretory granules, the Golgi network and the lysosomes of the islets. These results indicate that sulfatide follows the same intracellular route as insulin and suggest a functional association between these molecules. We have raised the hypothesis that sulfatide possibly plays a role in the trafficking of insulin in the islets of Langerhans in rat pancreas. (+info)
Glycoconjugates in Leishmania infectivity.
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Leishmaniasis is a major health problem to humans and is caused by one of the world's major pathogens, the Leishmania parasite. These protozoa have the remarkable ability to avoid destruction in hostile environments they encounter throughout their life cycle. That Leishmania parasites have adapted to not only survive, but to proliferate largely is due to the protection conferred by unique glycoconjugates that are either on the parasites' cell surface or secreted. Most of these specialized molecules are members of a family of phosphoglycans while others are a family of glycosylinositol phospholipids. Together they have been implicated in a surprisingly large number of functions for the parasites throughout their life cycle and, therefore, are key players in their pathogenesis. This review summarizes the biological roles of these glycoconjugates and how they are believed to contribute to Leishmania survival in destructive surroundings. (+info)
Antigen structure and genetic basis of histo-blood groups A, B and O: their changes associated with human cancer.
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Three areas of research involved in blood group (or histo-blood group) ABO antigens and their genes, developed by our research group, are reviewed: (1) Antigen structures. The structural basis of A and H, A(1) and A(2), i and I antigens expressed in erythrocyte membranes. Major carriers of A and H determinants in erythrocytes are type 2 chain poly-LacNAc, short vs. long and unbranched vs. branched structures termed A(a), A(b), A(c), A(d) and H(1), H(2), H(3), H(4). Regular A (A(1)) and weak A (A(2)) were identified respectively as repetitive A (type 3 chain A) and A-associated H. A(1)- and A(2)-specific type 3 chain A and H, type 1 chain (representing Lewis blood group antigens), and type 4 chain (globo-series antigen; an extremely minor component in erythrocytes) are all glycosphingolipids. A and H determinants in fetal and newborn erythrocytes are carried by unbranched poly-LacNAc, whereas these determinants in adult erythrocytes are carried by branched poly-LacNAc. (2) ABO genes. A few cDNAs encoding A enzyme (UDP-GalNAc: H-a-GalNAc transferase) were cloned based on the amino acid sequence of purified A enzyme and their structures were compared with those of homologous cDNA from blood cells of B and O individuals (genotype BB, OO). Four nucleotide substitutions and four corresponding amino acid sequences essential for expression of A(1) allele and B allele, and differences between A and B enzymes, were identified. Amino acids 266 and 268, i.e. Leu and Gly for A enzyme vs. Met and Ala for B enzyme, were dominant in determining A vs. B activity (presumably recognizing UDP-GalNAc vs. UDP-Gal). The A(2) allele was characterized by deletion of the termination codon, extending nucleotides up to 1128 and thus encoding 21 extra amino acids at the C terminus, which may affect (diminish) the dominant function of amino acids 266 and 268. Typical O allele (O(1)) is characterized by deletion of nucleotide 261 G, causing frame shift and encoding of an entirely different, short polypeptide, due to appearance of early termination codon at nucleotide 354. Structures of other O alleles (O(1 v), O(2)) and other weak A alleles (A(3), A(el)) are also described. The genomic structure of ABO genes consists of seven exons which span approximately 19 kb of genomic DNA on chromosome 9, band q34. Most of the coding sequence is located in exon 7. Analysis of the 5' upstream region revealed the presence of the binding site for transcription factors and enhancer element. (3) Antigens and genes in cancer. A and B phenotypes aberrantly expressed in various types of human cancer, and their genetic basis, have been studied. One widely-occurring change observed in a large variety of human cancers is deletion of A or B epitope, associated with accumulation of their precursor H (Le(y), Le(b)), which causes enhanced malignancy. A less-commonly observed change is expression of incompatible A, identified as real type 1 chain A, in tumors of O or B individuals. A possible molecular genetic mechanism leading to such phenotypic changes is discussed. (+info)
Isolation, characterization and immunolocalization of phosphorylcholine-substituted glycolipids in developmental stages of Caenorhabditis elegans.
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Caenorhabditis elegans displays three neutral glycosphingolipids with structural homology to glycosphingolipids from the porcine nematode parasite, Ascaris suum. The present findings extend the degree of structural conservation between the two nematode species to glycosphingolipids with a phosphodiester substitution. Using a combination of hydrofluoric acid pretreatment, immunochemical characterization and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, three zwitterionic, phosphorylcholine-substituted glycosphingolipids could be identified in the neutral glycolipid fraction of C. elegans. The components were isolated as their zwitterionic, phosphorylcholine-substituted, pyridylaminated oligosaccharides by HPLC. Structural analysis was performed using hydrofluoric acid treatment, partial acid hydrolysis, methylation analysis, gas chromatography-mass spectrometry, cleavage with exoglycosidases and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Their chemical structures are proposed as: component Nz1, GalNAc(beta1-4)[phosphorylcholine]GlcNAc(beta1-3)Man(beta1-4)Glc-cera mide; component Nz2, Gal(alpha1-3)GalNAc(beta1-4)[phosphorylcholine]-GlcNAc(beta1-3)Man(be ta1-4)Glc-ceramide; and component Nz3, Gal(beta1-3)- Gal(alpha1-3)GalNAc(beta1-4)[phosphorylcholine]GlcNAc(beta1-3)Man(bet a1-4)Glc-ceramide. The oligosaccharide core is characteristic of the biosynthetic arthro-carbohydrate series of protostomial glycosphingolipids. The ceramide moiety was specified by a d17 : 1 sphingoid-base with iso-branching and anteiso-branching, and 2-hydroxy, saturated fatty acids as represented by docosanoic and tetracosanoic acids. Analysis of the spatial and temporal expression of the phosphorylcholine epitope, during embryonic and postembryonic development, showed it to be localized predominantly in seam cells and basement membranes, respectively. In early embryonic ontogenesis the phosphorylcholine epitope was only lipid bound, while in late embryonic and postembryonic development this epitope was both lipid bound and protein bound. (+info)
Extracellular signal-related kinase (ERK) and p38 mitogen-activated protein (MAP) kinases differentially regulate the lipopolysaccharide-mediated induction of inducible nitric oxide synthase and IL-12 in macrophages: Leishmania phosphoglycans subvert macrophage IL-12 production by targeting ERK MAP kinase.
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Macrophage activation by cytokines or microbial products such as LPS results in the induction and release of several key immune effector molecules including NO and IL-12. These have been shown to play crucial roles in the development of immunity to intracellular pathogens such as Leishmania. The molecular mechanisms underlying the induction of these effector molecules are not fully understood. We now show that the extracellular signal-related kinase (ERK) and p38 mitogen-activated protein (MAP) kinases play differential roles in the regulation of LPS-stimulated inducible NO synthase and IL-12 gene expression. In macrophages, LPS stimulates the simultaneous activation of all three classes of MAP kinases, ERK, c-jun N-terminal kinase, and p38, albeit with differential activation kinetics. However, studies using inhibitors selective for ERK (PD98059) and p38 (SB203580) show that while p38 plays an essential role in the induction of inducible NO synthase, ERK MAP kinases play only a minor role in promoting NO generation. In contrast, while p38 promotes induction of IL-12 (p40) mRNA, ERK activation suppresses LPS-mediated IL-12 transcription. The biological relevance of these regulatory signals is demonstrated by our finding that Leishmania lipophosphoglycans, which promote parasite survival, act by stimulating ERK MAP kinase to inhibit macrophage IL-12 production. Thus, as ERK and p38 MAP kinases differentially regulate the induction of the macrophage effector molecules, inducible NO synthase and IL-12, these kinases are potential targets not only for the development of novel strategies to combat intracellular pathogens but also for therapeutic immunomodulation. (+info)
Novel plasmalogalactosylalkylglycerol from equine brain.
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A novel galactosylalkylglycerol modified with a long-chain cyclic acetal at the sugar moiety, 3-O-(4'6'-plasmalogalactosyl) 1-O-alkylglycerol, was isolated from equine brain. The presence of cyclic acetal linkage, its linked position, and the length of the acetal chain of the natural plasmalo lipid were determined by proton NMR spectroscopy and fast-atom bombardment;-mass spectrometry, as well as gas chromatography;-mass spectrometry and gas;-liquid chromatography. To identify the isomeric stereostructure of the natural product, the plasmalo derivative was chemically synthesized from 3-O-galactosyl 2-O-acyl 1-O-alkyl glyceride through acetalization after deacylation. As a result, the direction and position of the acetal chain of the natural plasmalo lipid were characterized as an "endo"-type 4',6'-O-acetal derivative linked to galactoside by comparison with the NMR data of the synthesized product. The chain lengths of alkyl and acetal groups were C(14) for the former and C(16) and C(18) for the latter, and those for the latter group were mostly similar to those of plasmalogalactosyl ceramide, which was previously isolated from equine brain. (+info)