The structural integrity exerted by N-terminal pyroglutamate is crucial for the cytotoxicity of frog ribonuclease from Rana pipiens.
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.
Liao
You-Di
YD
Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan. ydliao@ibms.sinica.edu.tw
Wang
Sui-Chi
SC
Leu
Ying-Jen
YJ
Wang
Chiu-Feng
CF
Chang
Shu-Ting
ST
Hong
Yu-Ting
YT
Pan
Yun-Ru
YR
Chen
Chinpan
C
D000818:Animals; D002384:Catalysis; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D006367:HeLa Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D020014:K562 Cells; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009154:Mutation; D011761:Pyrrolidonecarboxylic Acid / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011894:Rana pipiens / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012260:Ribonucleases / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D000939:Epitopes; D011994:Recombinant Proteins; D004247:DNA; D012260:Ribonucleases; D011761:Pyrrolidonecarboxylic Acid; C058703:ranpirnase