RESUMEN
Onconase, a cytotoxic ribonuclease from Rana pipiens, possesses pyroglutamate (Pyr) at the N-terminus and has a substrate preference for uridine-guanine (UG). To identify residues responsible for onconase's cytotoxicity, we cloned the rpr gene from genomic DNA and expressed it in Escherichia coli BL21(DE3). The recombinant onconase with Met at the N-terminus had reduced thermostability, catalytic activity and antigenicity. Therefore, we developed two methods to produce onconase without Met. One relied on the endogeneous E.coli methionine aminopeptidase and the other relied on the cleavage of a pelB signal peptide. The Pyr1 substitutional variants maintained similar secondary structures to wild-type onconase, but with less thermostability and specific catalytic activity for the innate substrate UG. However, the non-specific catalytic activity for total RNAs varied depending on the relaxation of base specificity. Pyr1 promoted the structural integrity by forming a hydrogen bond network through Lys9 in alpha1 and Val96 in beta6, and participated in catalytic activity by hydrogen bonds to Lys9 and P(1) catalytic phosphate. Residues Thr35 and Asp67 determined B(1) base specificity, and Glu91 determined B(2) base specificity. The cytotoxicity of onconase is largely determined by structural integrity and specific catalytic activity for UG through Pyr1, rather than non-specific activity for total RNAs.
Asunto(s)
Ácido Pirrolidona Carboxílico/metabolismo , Rana pipiens/genética , Ribonucleasas/metabolismo , Animales , Catálisis , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Dicroismo Circular , Clonación Molecular , ADN/química , ADN/genética , Electroforesis en Gel de Poliacrilamida , Epítopos/química , Epítopos/genética , Epítopos/inmunología , Escherichia coli/genética , Regulación Enzimológica de la Expresión Génica , Células HeLa , Humanos , Concentración 50 Inhibidora , Células K562 , Cinética , Espectrometría de Masas , Datos de Secuencia Molecular , Mutación , Ácido Pirrolidona Carboxílico/química , Ácido Pirrolidona Carboxílico/farmacología , Rana pipiens/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Ribonucleasas/genética , Ribonucleasas/farmacología , Análisis de Secuencia de ADN , Especificidad por SustratoRESUMEN
Cytotoxic ribonucleases with antitumor activity are mainly found in the oocytes and early embryos of frogs. Native RC-RNase 4 (RNase 4), consisting of 106 residues linked with four disulfide bridges, is a cytotoxic ribonuclease isolated from oocytes of bullfrog Rana catesbeiana. RNase 4 belongs to the bovine pancreatic ribonuclease (RNase A) superfamily. Recombinant RC-RNase 4 (rRNase 4), which contains an additional Met residue and glutamine instead of pyroglutamate at the N terminus, was found to possess less catalytic and cytotoxic activities than RNase 4. Equilibrium thermal and guanidine-HCl denaturation CD measurements revealed that RNase 4 is more thermally and chemically stable than rRNase 4. However, CD and NMR data showed that there is no gross conformational change between native and recombinant RNase 4. The NMR solution structure of rRNase 4 was determined to comprise three alpha-helices and two sets of antiparallel beta-sheets. Superimposition of each structure with the mean structure yielded an average root mean square deviation (RMSD) of 0.72(+/-0.14)A for the backbone atoms, and 1.42(+/-0.19)A for the heavy atoms in residues 3-105. A comparison of the 3D structure of rRNase 4 with the structurally and functionally related cytotoxic ribonuclease, onconase (ONC), showed that the two H-bonds in the N-terminal pyroglutamate of ONC were not present at the corresponding glutamine residue of rRNase 4. We suggest that the loss of these two H-bonds is one of the key factors responsible for the reductions of the conformational stability, catalytic and cytotoxic activities in rRNase 4. Furthermore, the differences of side-chain conformations of subsite residues among RNase A, ONC and rRNase 4 are related to their distinct catalytic activities and base preferences.
Asunto(s)
Proteínas del Huevo/química , Endorribonucleasas/química , Oocitos/enzimología , Estructura Secundaria de Proteína , Rana catesbeiana/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Dicroismo Circular , Cristalografía por Rayos X , Proteínas del Huevo/metabolismo , Endorribonucleasas/metabolismo , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
The Rana catesbeiana (bullfrog) ribonucleases, which belong to the RNase A superfamily, exert cytotoxicity toward tumor cells. RC-RNase, the most active among frog ribonucleases, has a unique base preference for pyrimidine-guanine rather than pyrimidine-adenine in RNase A. Residues of RC-RNase involved in base specificity and catalytic activity were determined by site-directed mutagenesis, k(cat)/K(m) analysis toward dinucleotides, and cleavage site analysis of RNA substrate. The results show that Pyr-1 (N-terminal pyroglutamate), Lys-9, and Asn-38 along with His-10, Lys-35, and His-103 are involved in catalytic activity, whereas Pyr-1, Thr-39, Thr-70, Lys-95, and Glu-97 are involved in base specificity. The cytotoxicity of RC-RNase is correlated, but not proportional to, its catalytic activity. The crystal structure of the RC-RNase.d(ACGA) complex was determined at 1.80 A resolution. Residues Lys-9, His-10, Lys-35, and His-103 interacted directly with catalytic phosphate at the P(1) site, and Lys-9 was stabilized by hydrogen bonds contributed by Pyr-1, Tyr-28, and Asn-38. Thr-70 acts as a hydrogen bond donor for cytosine through Thr-39 and determines B(1) base specificity. Interestingly, Pyr-1 along with Lys-95 and Glu-97 form four hydrogen bonds with guanine at B(2) site and determine B(2) base specificity.