RESUMEN
The removal of N-terminal translation initiator Met by methionine aminopeptidase (MetAP) is often crucial for the function and stability of proteins. On the basis of crystal structure and sequence alignment of MetAPs, we have engineered Escherichia coli MetAP by the mutation of three residues, Y168G, M206T, Q233G, in the substrate-binding pocket. Our engineered MetAPs are able to remove the Met from bulky or acidic penultimate residues, such as Met, His, Asp, Asn, Glu, Gln, Leu, Ile, Tyr, and Trp, as well as from small residues. The penultimate residue, the second residue after Met, was further removed if the antepenultimate residue, the third residue after Met, was small. By the coexpression of engineered MetAP in E. coli through the same or a separate vector, we have successfully produced recombinant proteins possessing an innate N terminus, such as onconase, an antitumor ribonuclease from the frog Rana pipiens. The N-terminal pyroglutamate of recombinant onconase is critical for its structural integrity, catalytic activity, and cyto-toxicity. On the basis of N-terminal sequence information in the protein database, 85%-90% of recombinant proteins should be produced in authentic form by our engineered MetAPs.
Asunto(s)
Aminopeptidasas/metabolismo , Escherichia coli/enzimología , Metionina/metabolismo , Mutación , Rana pipiens/genética , Ribonucleasas/metabolismo , Sustitución de Aminoácidos/genética , Aminoácidos/genética , Aminoácidos/metabolismo , Aminopeptidasas/genética , Animales , Sitios de Unión/genética , Escherichia coli/genética , Expresión Génica , Metionil Aminopeptidasas , Mutagénesis Sitio-Dirigida , Ingeniería de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleasas/genética , Especificidad por Sustrato/genéticaRESUMEN
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
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.
Asunto(s)
Rana catesbeiana/genética , Ribonucleasas/química , Secuencia de Aminoácidos , Animales , Catálisis , Supervivencia Celular , Dicroismo Circular , Cristalografía por Rayos X , ADN Complementario/metabolismo , Vectores Genéticos , Ácido Glutámico/química , Guanina/metabolismo , Humanos , Hidrógeno/metabolismo , Cinética , Lisina/química , Espectrometría de Masas , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Mutación , Estructura Terciaria de Proteína , ARN/metabolismo , Proteínas Recombinantes/metabolismo , Ribonucleasa Pancreática/metabolismo , Ribonucleasas/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Treonina/químicaRESUMEN
Multiple ribonucleases are widely found in living organisms, but the function and regulation of individual ribonucleases are still not clear. In the present study, we found that one oocytic ribonuclease, RC-RNase, is developmentally expressed in the liver and stored in the oocyte of the bullfrog, while another ribonuclease, RC-RNase L1, is constitutively expressed and retained in the liver at all stages. In females, the expression of RC-RNase increased with the degree of maturity and the concentration of plasma estradiol during oogenesis. In males, the RC-RNase gene was activated in the liver and the newly synthesized protein was secreted into plasma if estradiol was administered. To investigate the mechanism of estrogen-mediated activation of ribonuclease expression, we cloned the RC-RNase promoter and analyzed the putative transcription factor binding sites, e.g. TATA box, ERE, AP1 and CAAT box. Using luciferase as a reporter gene, we found that an estrogen response element in the promoter of RC-RNase was essential for both basic transcription and estradiol-mediated gene activation in estrogen receptor-positive MCF7 cells. These results support the hypothesis that RC-RNase is synthesized in the liver upon stimulation by estradiol during oogenesis, then secreted into the bloodstream and stored in oocytes for embryonic development.