RESUMO
The major surface proteins of the parasitic protozoon Leishmania mexicana are anchored to the plasma membrane by glycosylphosphatidylinositol (GPI) anchors. We have cloned the L. mexicana GPI8 gene that encodes the catalytic component of the GPI:protein transamidase complex that adds GPI anchors to nascent cell surface proteins in the endoplasmic reticulum. Mutants lacking GPI8 (DeltaGPI8) do not express detectable levels of GPI-anchored proteins and accumulate two putative protein-anchor precursors. However, the synthesis and cellular levels of other non-protein-linked GPIs, including lipophosphoglycan and a major class of free GPIs, are not affected in the DeltaGPI8 mutant. Significantly, the DeltaGPI8 mutant displays normal growth in liquid culture, is capable of differentiating into replicating amastigotes within macrophages in vitro, and is infective to mice. These data suggest that GPI-anchored surface proteins are not essential to L. mexicana for its entry into and survival within mammalian host cells in vitro or in vivo and provide further support for the notion that free GPIs are essential for parasite growth.
Assuntos
Aciltransferases/genética , Moléculas de Adesão Celular/genética , Glicosilfosfatidilinositóis/metabolismo , Leishmania mexicana/genética , Aciltransferases/isolamento & purificação , Aciltransferases/metabolismo , Sequência de Aminoácidos , Animais , Southern Blotting , Western Blotting , Domínio Catalítico , Moléculas de Adesão Celular/isolamento & purificação , Moléculas de Adesão Celular/metabolismo , Clonagem Molecular , Técnicas In Vitro , Injeções Intraperitoneais , Leishmania mexicana/metabolismo , Leishmania mexicana/patogenicidade , Macrófagos Peritoneais/parasitologia , Proteínas de Membrana/biossíntese , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
A major cysteine proteinase (CPB) of Leishmania mexicana, that is predominantly expressed in the form of the parasite that causes disease in mammals, has been overexpressed in Escherichia coli and purified from inclusion bodies to apparent homogeneity. The CPB enzyme, CPB2.8, was expressed as an inactive pro-form lacking the characteristic C-terminal extension (CPB2.8DeltaCTE). Pro-region processing was initiated during protein refolding and proceeded through several intermediate stages. Maximum enzyme activity accompanied removal of the entire pro-region. This was facilitated by acidification. Purified mature enzyme gave a single band on SDS/PAGE and gelatin SDS/PAGE gels, co-migrated with native enzyme in L. mexicana lysates, and had the same N-terminal sequence as the native enzyme. The procedure yielded >3.5 mg of active enzyme per litre of E. coli culture.