Double Domain Swapping in Bovine Seminal RNase: Formation of Distinct N- and C-swapped Tetramers and Multimers with Increasing Biological Activities

Citation metadata

From: PLoS ONE(Vol. 7, Issue 10)
Publisher: Public Library of Science
Document Type: Report
Length: 9,875 words
Lexile Measure: 1560L

Document controls

Main content

Article Preview :

Author(s): Giovanni Gotte 1 , * , Alexander Mahmoud Helmy 1 , Carmine Ercole 2 , Roberta Spadaccini 3 , Douglas V. Laurents 4 , Massimo Donadelli 1 , Delia Picone 2


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, also by the three dimensional (3D) swapping [5] of its N-terminal domains (residues 1-15) [6]. BS-RNase is endowed with special biological actions, especially a potentially therapeutic antitumor activity [7]. Notably, only MxM is selectively [8] cytotoxic against malignant cells [9], because it maintains the dimeric structure necessary to evade the RNase inhibitor (RI) even under the reducing cytosolic environment that breaks the inter-subunits disulfide bonds and causes the disassociation of the unswapped M = M form [9], [10].

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]. Later, Mazzarella and colleagues hypothesized the existence of more than one tetrameric isoform (dimer+dimer) ascribable to different induced orientations of the BS-RNase N-termini [13]. Nevertheless, despite many studies focused on the propensity to natively swap its N-termini [14]-[18] no additional investigations on BS-RNase oligomers have been performed. However, 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].

It is noteworthy that the 3D domain swapping mechanism is shared by several fibril-forming proteins, such as cystatin C [22], [23], human prion protein [24], [25], T7-Endonuclase I [26], [beta]-2 microglobulin [27], [28], but also by proteins that are not fibrillogenic, such as cytochrome c [29], and RNase A [20]. By the way, RNase A is considered a model for the formation of amyloid or amyloid-like fibrils through domain swapping [19], although only mutants containing poly-Q- or poly-G-expanded loops were shown to produce native-like fibrils [30], [31]. Contrarily, no conditions yielded fibrils from wt RNase A [31], [32], even if this enzyme displays more than one cross-[beta]-spine-prone sequence [33], and although more recently another pancreatic-type [2] RNase, the eosinophil cationic protein (ECP), was shown to form fibrils [34]. Anyway, RNase A can form several N-or C-domain-swapped oligomers [20], [21] if it is lyophilized from 40% HAc solutions [11], or if highly concentrated protein solutions are subjected to a...

Source Citation

Source Citation   

Gale Document Number: GALE|A498257192