RESUMEN
In mammalian spermiogenesis, sperm mature during epididymal transit to get fertility. The pig sharing many physiological similarities with humans is considered a promising animal model in medicine. We examined the expression profiles of proteins from boar epididymal caput, corpus, and cauda sperm by two-dimensional gel electrophoresis and peptide mass fingerprinting. Our results indicated that protein disulfide isomerase-P5 (PDI-P5) human homolog was down-regulated from the epididymal corpus to cauda sperm, in contrast to the constant expression of protein disulfide isomerase A3 (PDIA3) human homolog. To examine the functions of PDIA3 and PDI-P5, we cloned and sequenced cDNAs of pig PDIA3 and PDI-P5 protein precursors. Each recombinant pig mature PDIA3 and PDI-P5 expressed in Escherichia coli showed thiol-dependent disulfide reductase activities in insulin turbidity assay. Although PDIA3 showed chaperone activity to promote oxidative refolding of reduced denatured lysozyme, PDI-P5 exhibited anti-chaperone activity to inhibit oxidative refolding of lysozyme at an equimolar ratio. SDS-PAGE and Western blotting analysis suggested that disulfide cross-linked and non-productively folded lysozyme was responsible for the anti-chaperone activity of PDI-P5. These results provide a molecular basis and insights into the physiological roles of PDIA3 and PDI-P5 in sperm maturation and fertilization.
Asunto(s)
Disulfuros , Regulación hacia Abajo/fisiología , Precursores Enzimáticos , Muramidasa/química , Proteína Disulfuro Isomerasas , Pliegue de Proteína , Espermatogénesis/fisiología , Espermatozoides/enzimología , Animales , Secuencia de Bases , Clonación Molecular , Disulfuros/química , Disulfuros/metabolismo , Precursores Enzimáticos/biosíntesis , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Epidídimo/enzimología , Fertilización/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Humanos , Masculino , Chaperonas Moleculares/antagonistas & inhibidores , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Muramidasa/metabolismo , Oxidación-Reducción , Desnaturalización Proteica , Proteína Disulfuro Isomerasas/biosíntesis , Proteína Disulfuro Isomerasas/química , Proteína Disulfuro Isomerasas/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidad de la Especie , PorcinosRESUMEN
We have developed a new flexizyme (a flexible de novo tRNA acylation ribozyme) system, a pair of amino-derivatized benzyl thioester (ABT) and amino flexizyme (aFx). ABT bearing the ammonium ion was designed to render the acyl-donor substrates better water solubility. Although the previously reported flexizymes (eFx and dFx) did not show acylation activity for the ABT derivatives, a new flexizyme variant aFx, generated by in vitro selection against an amino acid activated ABT, exhibits high selectivity toward those activated ABT. The flexizymes system including aFx, eFx, and dFx enables us to prepare a wide variety of acyl-tRNAs charged with non-proteinogenic amino acids.