Single amino acid substitutions at the N-terminus of a recombinant cytotoxic ribonuclease markedly influence biochemical and biological properties.

Onconase is a cytotoxic ribonuclease with antitumor properties. A semisynthetic gene encoding the entire protein sequence was constructed by fusing oligonucleotides coding for the first 15 and the last 6 of the 104 amino acids to a genomic clone that encoded the remaining amino acid residues [Newton, D. L., et al. (1997) Protein Eng. 10, 463-470]. The resulting protein product expressed in Escherichia coli exhibited little enzymatic or cytotoxic activity due to the unprocessed N-terminal Met amino acid residue. In this study, we demonstrate that modification of the 5'-region of the gene to encode [Met-(-1)]Ser or [Met-(-1)]Tyr instead of the native pyroglutamate results in recombinant onconase derivatives with restored activities. [Met-(-1)]rOnc(E1S) was more active than [Met-(-1)]rOnc(E1Y) in all assays tested. Consistent with the action of native onconase, [Met-(-1)]rOnc(E1S) was a potent inhibitor of protein synthesis in the cell-free rabbit reticulocyte lysate assay, degrading tRNA at concentrations that correlated with inhibition of protein synthesis. An interesting difference between the recombinant onconase derivatives and the native protein was their susceptibility to inhibition by the major intracellular RNase inhibitor, PRI (onconase is refractory to PRI inhibition). [Met-(-1)]rOnc(E1S) and [Met-(-1)]rOnc(E1Y) inhibited protein synthesis in intact SF539 neuroblastoma cells with IC50's very similar to that of onconase (IC50 3.5, 10, and 10 microg/mL after 1 day and 0.16, 0.35, and 2.5 microg/mL after 5 days for onconase, [Met-(-1)]rOnc(E1S), and [Met-(-1)]rOnc(E1Y), respectively). Similar to that of onconase, cytotoxic activity of the recombinant derivatives was potentiated by monensin, NH4Cl, and retinoic acid. Brefeldin A completely blocked the enhancement of cytotoxicity caused by retinoic acid with all three proteins. Thus, drug-induced alterations of the intracellular trafficking of the recombinant derivatives also resembles that of onconase. Stability studies as assessed in serum-containing medium in the presence or absence of cells at 37 degreesC showed that the recombinant proteins were as stable to temperature and cell culture conditions as the native protein. Therefore, exchanging the Glu amino acid residue at the amino terminus of onconase with an amino acid residue containing a hydroxyl group produces recombinant proteins with ribonuclease and cytotoxic properties similar to native onconase.

Expression and characterization of a cytotoxic human-frog chimeric ribonuclease: potential for cancer therapy.

Onconase is a cytotoxic ribonuclease with antitumor properties. A semisynthetic gene encoding the entire protein sequence was constructed by fusing oligonucleotides coding for the first 15 and last six of the 104 amino acid residues to a genomic clone that encoded the remaining amino acid residues. Additionally, the 15 N-terminal amino acid residues of onconase were replaced with the first 21 amino acid residues of the homologous human RNase, eosinophil-derived neurotoxin, EDN. Two versions of the hybrid EDN-onconase protein were cloned, expressed and purified. The chimera that contained a glycine in lieu of the aspartic acid present in native onconase (position 26 in the chimera) exhibited enzymatic activity more characteristic of EDN than native onconase and was considerably more active with respect to both RNase activity and cellular cytotoxicity than recombinant onconase. In contrast to native or recombinant onconase, the EDN chimera was recognized by anti-EDN polyclonal antibodies, demonstrating that the chimera also shared structural antigenic determinants to the human enzyme. These results demonstrate that a chimeric ribonuclease has cytotoxicity comparable to onconase in two out of four cell lines tested. The implications with regard to cancer therapy are presented.

The structure of MCP-1 in two crystal forms provides a rare example of variable quaternary interactions.

The X-ray crystal structure of recombinant human monocyte chemoattractant protein (MCP-1) has been solved in two crystal forms. One crystal form (P), refined to 1.85 A resolution, contains a dimer in the asymmetric unit, while the other (I) contains a monomer and was refined at 2.4 A. Although both crystal forms grow together in the same droplet, the respective quaternary structures of the protein differ dramatically. In addition, both X-ray structures differ to a similar extent from the solution structure of MCP-1. Such extent of variability of quaternary structures is unprecedented. In the crystal structures, the well-ordered N termini of MCP-1 form 3(10)-helices. Comparison of the three MCP-1 structures revealed a direct correlation between the main-chain conformation of the first two cysteine residues and the quaternary arrangements. These data can be used to explain the structural basis for the assignment of residues responsible for biological activity.

Structure of ribonuclease A derivative II at 2.1-A resolution.

The crystal structure of bovine pancreatic ribonuclease A derivative II, a covalent derivative obtained by reaction of 6-chloropurine 9-beta-D-ribofuranosyl 5'-monophosphate with the alpha-amino group of Lys-1, has been determined and refined at 2.1-A resolution with an agreement factor R = 0.166 for 6254 reflections in the resolution shell 8.0 to 2.1 A. Crystals are orthorhombic and belong to space group C222(1) with unit cell parameters a = 75.73 A, b = 57.85 A, and c = 53.26 A. This crystal packing had never been reported before for pancreatic ribonuclease nor its complexes. The structure found is in accordance with the location of p2, B3, and R3 subsites at the N-terminal region of the protein and provides an explanation of the catalytic behavior observed for this derivative. In particular, differences in kinetic parameters and in the pKa value of His-119 between derivative II and native ribonuclease A can be interpreted on the basis of the position of the phosphate moiety within the derivative structure. Some uncertainty remains on the nucleotide sugar conformation determined.