RESUMEN
Axonal regeneration requires changes in the lipid dynamics of the axon membrane for growth and extension. Here, we examined the expression of genes associated with lipid transport after nerve injury. The expression of ATP-binding cassette transporter-A1 (ABCA1), which participates in the transport of cholesterol from the plasma membrane, was markedly upregulated in motor and sensory neurons after nerve injury. Stimulation of PC12 cells with the nerve growth factor induced neurite extension and ABCA1 expression predominantly in regions proximal to the neurite tip. To clarify the functional role of ABCA1 in neurite elongation, we examined the morphology of neurons cultured from conditionally-injured dorsal root ganglia from ABCA1-deficient mice. We found a significant increase in neurite branch formation in these neurons. In addition, the neurite tips of ABCA1-deficient neurons appeared excessively ruffled, and the direction of neurite elongation was unsteady. In contrast, the neurite tips of wild-type neurons were not excessively ruffled, and the neurites elongated rapidly in a stable directionally-oriented manner. Together, these findings suggest that ABCA1 plays an important role in regulating the membrane lipid composition of injured neurons and in axonal regeneration following nerve injury.
Asunto(s)
Neuritas , Enfermedades del Sistema Nervioso Periférico , Ratas , Animales , Ratones , Neuritas/fisiología , Células Cultivadas , Ganglios Espinales , Colesterol , Células Receptoras Sensoriales , Células PC12 , Regeneración Nerviosa/fisiologíaRESUMEN
Within the endoplasmic reticulum (ER), mannoses and glucoses, donated from dolichol-phosphate-mannose and -glucose, are transferred to N-glycan and GPI-anchor precursors, and serine/threonine residues in many proteins. Glycosyltransferases that mediate these reactions are ER-resident multitransmembrane proteins with common characteristics, forming a superfamily of >10 enzymes. Here, we report an essential component of glycosylphosphatidylinositol-mannosyltransferase I (GPI-MT-I), which transfers the first of the four mannoses in the GPI-anchor precursors. We isolated a Chinese hamster ovary (CHO) cell mutant defective in GPI-MT-I but not its catalytic component PIG-M. The mutant gene, termed phosphatidylinositolglycan-class X (PIG-X), encoded a 252-amino acid ER-resident type I transmembrane protein with a large lumenal domain. PIG-X and PIG-M formed a complex, and PIG-M expression was <10% in the absence of PIG-X, indicating that PIG-X stabilizes PIG-M. We found that Saccharomyces cerevisiae Pbn1p/YCL052Cp, which was previously reported to be involved in autoprocessing of proproteinase B, is the functional homologue of PIG-X; Pbn1p is critical for Gpi14p/YJR013Wp function, the yeast homologue of PIG-M. This is the first report of an essential subcomponent of glycosyltransferases using dolichol-phosphate-monosaccharide.
Asunto(s)
Manosiltransferasas/química , Manosiltransferasas/fisiología , Glicoproteínas de Membrana/fisiología , Proteínas de la Membrana/química , Proteínas de Saccharomyces cerevisiae/fisiología , Animales , Antiinfecciosos/farmacología , Secuencia de Bases , Western Blotting , Antígenos CD59/biosíntesis , Células CHO , Catálisis , Línea Celular , Separación Celular , Supervivencia Celular , Clonación Molecular , Codón Iniciador , Cricetinae , Dolicoles/química , Retículo Endoplásmico/metabolismo , Citometría de Flujo , Glucosa/química , Glicosiltransferasas/química , Humanos , Lípidos/química , Manosiltransferasas/metabolismo , Ratones , Datos de Secuencia Molecular , Monosacáridos/química , Mutación , Oligosacáridos/química , Fenotipo , Fosfatos/química , Plásmidos/metabolismo , Unión Proteica , Ratas , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/química , TransfecciónRESUMEN
Many eukaryotic cell surface proteins are anchored to the membrane via glycosylphosphatidylinositol (GPI). GPI is synthesized from phosphatidylinositol by stepwise reactions and attached en bloc to nascent proteins. In mammalian cells, the major GPI species transferred to proteins is termed H7. By attachment of an additional ethanolamine phosphate (EtNP) to the second mannose, H7 can be converted to H8, which acts as a minor type of protein-linked GPI and also exists as a free GPI on the cell surface. Yeast GPI7 is involved in the transfer of EtNP to the second mannose, but the corresponding mammalian enzyme has not yet been clarified. Here, we report that the human homolog of Gpi7p (hGPI7) forms a protein complex with PIG-F and is involved in the H7-to-H8 conversion. We knocked down hGPI7 by RNA interference and found that H7 accumulated with little production of H8. Immunoprecipitation experiments revealed that hGPI7 was associated with and stabilized by PIG-F, which is known to bind to and stabilize PIG-O, a protein homologous to hGPI7. PIG-O is a transferase that adds EtNP to the third mannose, rendering GPI capable of attaching to proteins. We further found that the overexpression of hGPI7 decreased the level of PIG-O and, therefore, decreased the level of EtNP transferred to the third mannose. Finally, we propose a mechanism for the regulation of GPI biosynthesis through competition between the two independent enzymes, PIG-O and hGPI7, for the common stabilizer, PIG-F.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Proteínas Portadoras/química , Clonación Molecular , Secuencia Conservada , Cricetinae , ADN Complementario/metabolismo , Glicoproteínas/química , Humanos , Células KB , Proteínas de la Membrana , Proteínas de Transporte de Membrana , Datos de Secuencia Molecular , Plásmidos , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Glycosylphosphatidylinositol (GPI) is a glycolipid that anchors many proteins to the eukaryotic cell surface. The biosynthetic pathway of GPI is mediated by sequential additions of sugars and other components to phosphatidylinositol. Four mannoses in the GPI are transferred from dolichol-phosphate-mannose (Dol-P-Man) and are linked through different glycosidic linkages. Therefore, four Dol-P-Man-dependent mannosyltransferases, GPI-MT-I, -MT-II, -MT-III, and -MT-IV for the first, second, third, and fourth mannoses, respectively, are required for generation of GPI. GPI-MT-I (PIG-M), GPI-MT-III (PIG-B), and GPI-MT-IV (SMP3) were previously reported, but GPI-MT-II remains to be identified. Here we report the cloning of PIG-V involved in transferring the second mannose in the GPI anchor. Human PIG-V encodes a 493-amino acid, endoplasmic reticulum (ER) resident protein with eight putative transmembrane regions. Saccharomyces cerevisiae protein encoded in open reading frame YBR004c, which we termed GPI18, has 25% amino acid identity to human PIG-V. Viability of the yeast gpi18 deletion mutant was restored by human PIG-V cDNA. PIG-V has two functionally important conserved regions facing the ER lumen. Taken together, we suggest that PIG-V is the second mannosyltransferase in GPI anchor biosynthesis.