Quantitative analysis, using MALDI-TOF mass spectrometry, of the N-terminal hydrolysis and cyclization reactions of the activation process of onconase.
Onconase, a member of the ribonuclease superfamily, is a potent cytotoxic agent that is undergoing phase II/III human clinical trials as an antitumor drug. Native onconase from Rana pipiens and its amphibian homologs have an N-terminal pyroglutamyl residue that is essential for obtaining fully active enzymes with their full potential as cytotoxins. When expressed cytosolically in bacteria, Onconase is isolated with an additional methionyl (Met1) residue and glutaminyl instead of a pyroglutamyl residue at position 1 of the N-terminus and is consequently inactivated. The two reactions necessary for generating the pyroglutamyl residue have been monitored by MALDI-TOF MS. Results show that hydrolysis of Met(-1), catalyzed by Aeromonas aminopeptidase, is optimal at a concentration of >or= 3 m guanidinium-chloride, and at pH 8.0. The intramolecular cyclization of glutaminyl that renders the pyroglutamyl residue is not accelerated by increasing the concentration of denaturing agent or by strong acid or basic conditions. However, temperature clearly accelerates the formation of pyroglutamyl. Taken together, these results have allowed the characterization and optimization of the onconase activation process. This procedure may have more general applicability in optimizing the removal of undesirable N-terminal methionyl residues from recombinant proteins overexpressed in bacteria and providing them with biological and catalytic properties identical to those of the natural enzyme.
Ribó
Marc
M
Laboratori d'Enginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Universitat de Girona, Girona, Spain.
Bosch
Montserrat
M
Torrent
Gerard
G
Benito
Antoni
A
Beaumelle
Bruno
B
Vilanova
Maria
M
D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003500:Cyclization; D004789:Enzyme Activation; D019791:Guanidine / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007527:Isoenzymes; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D012260:Ribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013696:Temperature; D013816:Thermodynamics
D000970:Antineoplastic Agents; D007527:Isoenzymes; D011994:Recombinant Proteins; D012260:Ribonucleases; D019791:Guanidine; C058703:ranpirnase