Double domain swapping in bovine seminal RNase: formation of distinct N- and C-swapped tetramers and multimers with increasing biological activities.
Bovine seminal (BS) RNase, the unique natively dimeric member of the RNase super-family, represents a special case not only for its additional biological actions but also for the singular features of 3D domain swapping. The native enzyme is indeed a mixture of two isoforms: M = M, a dimer held together by two inter-subunit disulfide bonds, and MxM, 70% of the total, which, besides the two mentioned disulfides, is additionally stabilized by the swapping of its N-termini.When lyophilized from 40% acetic acid, BS-RNase oligomerizes as the super-family proto-type RNase A does. In this paper, we induced BS-RNase self-association and analyzed the multimers by size-exclusion chromatography, cross-linking, electrophoresis, mutagenesis, dynamic light scattering, molecular modelling. Finally, we evaluated their enzymatic and cytotoxic activities.Several BS-RNase domain-swapped oligomers were detected, including two tetramers, one exchanging only the N-termini, the other being either N- or C-swapped. The C-swapping event, confirmed by results on a BS-K113N mutant, has been firstly seen in BS-RNase here, and probably stabilizes also multimers larger than tetramers.Interestingly, all BS-RNase oligomers are more enzymatically active than the native dimer and, above all, they display a cytotoxic activity that definitely increases with the molecular weight of the multimers. This latter feature, to date unknown for BS-RNase, suggests again that the self-association of RNases strongly modulates their biological and potentially therapeutic properties.
Dipartimento di Scienze della Vita e della Riproduzione, Sezione di Chimica Biologica, Università degli Studi di Verona, Verona, Italy. giovanni.gotte@univr.it
Giovanni
G
Gotte
Alexander
A
Mahmoud Helmy
Carmine
C
Ercole
Roberta
R
Spadaccini
Douglas V
DV
Laurents
Massimo
M
Donadelli
Delia
D
Picone
D019342:Acetic Acid / Q000737:chemistry; D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D002417:Cattle; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000737:chemistry; D004139:Dinitrofluorobenzene / Q000031:analogs & derivatives / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D007700:Kinetics; D008297:Male; D008958:Models, Molecular; D009154:Mutation; D011063:Poly A-U / Q000378:metabolism; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D013696:Temperature
D000970:Antineoplastic Agents; D003432:Cross-Linking Reagents; D004220:Disulfides; D011063:Poly A-U; D004139:Dinitrofluorobenzene; D004722:Endoribonucleases; C032523:ribonuclease SPL; C002041:1,5-difluoro-2,4-dinitrobenzene; D019342:Acetic Acid