Abstract
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.
Highlights
Bovine seminal ribonuclease (BS-RNase) is the sole natively dimeric [1] member of the pancreatic-type RNase super-family [2], and it is a mixture of two isoforms
Preliminary results obtained through size-exclusion chromatography (SEC) with a Sephadex G-100 column showed the presence of BS-RNase tetramers (TT), hexamers (H), and larger oligomers (L.O.) (Figure 1A)
We attempted to refine the separation using two different cation-exchange columns, but, contrarily to RNase A [20], no conditions allowed us to improve the quality of the purification previously obtained with SEC
Summary
Bovine seminal ribonuclease (BS-RNase) is the sole natively dimeric [1] member of the pancreatic-type RNase super-family [2], and it is a mixture of two isoforms. The first, called M = M, accounts for about the 30% of the total, and it is dimeric due to two anti-parallel disulfide bonds linking the Cys-31 and 32 of the two subunits [3,4]; the second isoform, called MxM, (70% of the total) is stabilized, in addition to the mentioned disulfides, by the three dimensional (3D) swapping [5] of its N-terminal domains (residues 1–15) [6]. Libonati first showed that, when dissolved in 40–50% acetic acid (HAc) and subjected to lyophilization [11], BS-RNase forms a mixture of meta-stable oligomeric aggregates [12], whose stability increases in sodium phosphate buffers (NaPi), as occurs to bovine pancreatic ribonuclease (RNase A), the monomeric proto-type of the super-family [11]. The high sequence identity (about 82%) existing between BS-RNase and RNase A [4], and the similar chromatographic behavior of the two proteins after their multimerization [12,13], has lead us to hypothesize that BS-RNase could oligomerize through the same mechanism of its pancreatic monomeric counterpart, i.e. the double domain swapping [19] of both N- and/or C-termini [20,21]
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