RESUMO
Alectina ligante de manose (MBL) e uma proteina com importantepapel na primeira linha de defesa do sistema imune inato, cujos valores sericos sao determinados geneticamente. A MBL ativa a via da lectina do complemento, alem de mediar a opsonisaçao e fagocitose de microorganismos. A hanseniase e uma doença infecciosa cronica causada pelo Mycobacterium leprae, bacteria intracelular obrigatoria, que infecta fagocitos mononucleares do ospedeiro...
Assuntos
Humanos , Hanseníase/fisiopatologia , Hanseníase/imunologia , Hanseníase/microbiologia , Lectinas/fisiologia , Lectinas/imunologia , Mycobacterium leprae/citologia , Mycobacterium leprae/fisiologia , Mycobacterium leprae/imunologia , Dolicol Monofosfato Manose , Dolicol Monofosfato Manose/imunologiaRESUMO
A membrane fraction obtained from the filamentous form of Sporothrix schenckii was able to transfer mannose from GDP-Mannose into dolichol phosphate mannose and from this inTermediate into mannoproteins in coupled reactions catalyzed by dolichol phosphate mannose synthase and protein mannosyl transferase(s), respectively. Although the transfer reaction depended on exogenous dolichol monophosphate, membranes failed to use exogenous dolichol phosphate mannose for protein mannosylation to a substantial extent. Over 95% of the sugar was transferred to proteins via dolichol phosphate mannose and the reaction was stimulated several fold by Mg2+ and Mn2+. Incubation of membranes with detergents such as Brij 35 and Lubrol PX released soluble fractions that transferred the sugar from GDP-Mannose mostly into mannoproteins, which were separated by affinity chromatography on Concanavilin A-Sepharose 4B into lectin-reacting and non-reacting fractions. All proteins mannosylated in vitro eluted with the lectin-reacting proteins and analytical electrophoresis of this fraction revealed the presence of at least nine putative mannoproteins with molecular masses in the range of 26-112 kDa. The experimental approach described here can be used to identify and isolate specific glycoproteins mannosylated in vitro in studies of O-glycosylation.
Assuntos
Dolicol Monofosfato Manose/metabolismo , Proteínas Fúngicas/biossíntese , Glicoproteínas/biossíntese , Manosiltransferases/metabolismo , Glicoproteínas de Membrana/biossíntese , Proteínas de Membrana/metabolismo , Sporothrix/metabolismo , Coenzimas/farmacologia , Detergentes/farmacologia , Eletroforese em Gel de Poliacrilamida , Humanos , Magnésio/farmacologia , Manganês/farmacologia , Manosiltransferases/isolamento & purificação , Glicoproteínas de Membrana/isolamento & purificação , Peso Molecular , Polidocanol , Polietilenoglicóis/farmacologiaRESUMO
Leishmania parasites synthesize an abundance of mannose (Man)-containing glycoconjugates thought to be essential for virulence to the mammalian host and for viability. These glycoconjugates include lipophosphoglycan (LPG), proteophosphoglycans (PPGs), glycosylphosphatidylinositol (GPI)-anchored proteins, glycoinositolphospholipids (GIPLs), and N-glycans. A prerequisite for their biosynthesis is an ample supply of the Man donors GDP-Man and dolicholphosphate-Man. We have cloned from Leishmania mexicana the gene encoding the enzyme phosphomannomutase (PMM) and the previously described dolicholphosphate-Man synthase gene (DPMS) that are involved in Man activation. Surprisingly, gene deletion experiments resulted in viable parasite lines lacking the respective open reading frames (DeltaPMM and DeltaDPMS), a result against expectation and in contrast to the lethal phenotype observed in gene deletion experiments with fungi. L. mexicana DeltaDPMS exhibits a selective defect in LPG, protein GPI anchor, and GIPL biosynthesis, but despite the absence of these structures, which have been implicated in parasite virulence and viability, the mutant remains infectious to macrophages and mice. By contrast, L. mexicana DeltaPMM are largely devoid of all known Man-containing glycoconjugates and are unable to establish an infection in mouse macrophages or the living animal. Our results define Man activation leading to GDP-Man as a virulence pathway in Leishmania.
Assuntos
Leishmania mexicana/enzimologia , Leishmania mexicana/patogenicidade , Manosiltransferases/genética , Fosfotransferases (Fosfomutases)/genética , Virulência/genética , Sequência de Aminoácidos , Animais , Sequência de Carboidratos , Clonagem Molecular , Dolicol Monofosfato Manose/metabolismo , Regulação para Baixo , Citometria de Fluxo , Deleção de Genes , Marcação de Genes , Glicoconjugados/metabolismo , Glicosilação , Guanosina Difosfato Manose/metabolismo , Leishmania mexicana/genética , Macrófagos/parasitologia , Camundongos , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutação , Fenótipo , Homologia de Sequência de AminoácidosRESUMO
The cell surface of the parasitic protozoan Leishmania mexicana is coated by glycosylphosphatidylinositol (GPI)-anchored glycoproteins, a GPI-anchored lipophosphoglycan and a class of free GPI glycolipids. To investigate whether the anchor or free GPIs are required for parasite growth we cloned the L.mexicana gene for dolichol-phosphate-mannose synthase (DPMS) and attempted to create DPMS knockout mutants by targeted gene deletion. DPMS catalyzes the formation of dolichol-phosphate mannose, the sugar donor for all mannose additions in the biosynthesis of both the anchor and free GPIs, except for a alpha1-3-linked mannose residue that is added exclusively to the free GPIs and lipophosphoglycan anchor precursors. The requirement for dolichol-phosphate-mannose in other glycosylation pathways in L.mexicana is minimal. Deletion of both alleles of the DPMS gene (lmdpms) consistently resulted in amplification of the lmdpms chromosomal locus unless the promastigotes were first transfected with an episomal copy of lmdpms, indicating that lmdpms, and possibly GPI biosynthesis, is essential for parasite growth. As evidence presented in this and previous studies indicates that neither GPI-anchored glycoproteins nor lipophosphoglycan are required for growth of cultured parasites, it is possible that the abundant and functionally uncharacterized free GPIs are essential membrane components.