The structural integrity exerted by N-terminal pyroglutamate is crucial for the cytotoxicity of frog ribonuclease from Rana pipiens

Abstract

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.

Transfer RNA Cleavages by Onconase Reveal Unusual Cleavage Sites

“…For example, Gly-88 in RNase A responsible for contact with RNase inhibitor is absent in onconase, and this presumably contributes to its inhibitor resistance and cytotoxicity (31). Its N terminus is blocked as pyroglutamic acid and may have implications on its variant catalytic properties as well as cytotoxicity (23,25). Here we show that onconase cleaves predominantly at G-G sites not known for the pancreatic RNase homologues.…”

“…At increasing enzyme concentrations, additional cleavage products, species A, E, and I, were also seen. Species A, I, and E resulted from cleavages 3Ј to pyrimidine as observed for onconase with synthetic substrates (23)(24)(25)(26). Other cleavages seen were less intense.…”

“…Other cleavages were apparent as the enzyme concentration increased. Earlier studies on onconase action on synthetic substrates (23)(24)(25)(26) have suggested a preference for UpG (U binding in the B1 sub-site and G in the B2 sub-site of the substrate-binding site of enzyme). Out of the intense cleavages observed, species A resulted from the cleavage 3Ј to pyrimidine within the dinucleotide U-G. Cleavage at U-G, generating species A, and other cleavages such as those involving residues C-G, are consistent with the base specificity already reported for onconase.…”

“…However, the basis of this specificity toward tRNA has not been understood. When tried with synthetic substrates, onconase predominantly cleaved dinucleotides UpG and CpG (22)(23)(24)(25)(26). Our present studies indicate that the cleavages by onconase in several tRNAs involve selective G-G sites in addition to U-G and C-G dinucleotide bonds.…”

Ranpirnase (Frog RNase) Targeted with a Humanized, Internalizing, Anti–Trop-2 Antibody Has Potent Cytotoxicity against Diverse Epithelial Cancer Cells

“…1A. This design is dictated by the requirement of a pyroglutamate residue at the NH 2 terminus of Rap for the RNase to be fully functional (12).…”