Refinement of the structure of bovine seminal ribonuclease

Capasso, Sante; Giordano, Federico; Mattia, Carlo Andrea; Mazzarella, L.; Zagari, Adriana

doi:10.1002/bip.360220142

Cited by 69 publications

(41 citation statements)

References 12 publications

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“…The initial scFv-BSRNase fusion protein construct consisted of a straightforward linkage of the C-terminus of the scFv V K region to the native BSRNase N-terminus. This site was chosen as examination of the three-dimensional structure indicated that the C-terminus was less accessible (Capasso et al, 1983). The amplification oligonucleotides were designed to produce a BSRNase cassette with an N-terminal XhoI site followed by a IleLys-Arg sequence to replace the same sequence found at the end of the scFv ( Figure IA).…”

Section: Enzyme-linked Immunosorbent Assays and Western Blotsmentioning

confidence: 99%

Design, characterization and anti-tumour cytotoxicity of a panel of recombinant, mammalian ribonuclease-based immunotoxins

Deonarain

Epenetos²

1998

Br J Cancer

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Summary Bovine seminal ribonuclease (BSRNase) is an unusual member of the ribonuclease superfamily, because of its remarkable antitumour and immunosuppressive properties. We describe here the construction, expression, purification and characterization of a panel of six immunotoxins based upon this enzyme and show that we can increase its anti-tumour activity by over 2 x 1 04-fold. This is achieved by improving tumour cell targeting using a single-chain Fv (scFv) directed against the oncofetal antigen placental alkaline phosphatase. As well as the simple scFv-BSRNase fusion protein, we have constructed five other derivatives with additional peptides designed to improve folding and intracellular trafficking and delivery. We find that the molecule most cytotoxic to antigen (PLAP)-positive cells in vitro is one that contains a C-terminal 'KDEL' endoplasmic reticulum retention signal and a peptide sequence derived from diphtheria toxin. All these molecules are produced in Escherichia cofi (E. coli) as insoluble inclusion bodies and require extensive in vitro processing to recover antigen binding and ribonuclease activity. Despite incomplete ribonuclease activity and quaternary assembly, these molecules are promising reagents for specific chemotherapy of cancer and are potentially less harmful and immunogenic than current immunotoxins.

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Section: Enzyme-linked Immunosorbent Assays and Western Blotsmentioning

confidence: 99%

Design, characterization and anti-tumour cytotoxicity of a panel of recombinant, mammalian ribonuclease-based immunotoxins

Deonarain

Epenetos²

1998

Br J Cancer

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“…The presence of Leu 28 in BS-RNase, rather than a glutamine residue in RNase A, makes this region highly hydrophobic and thus facilitates the contact between the two subunits (Capasso et al, 1983). Lys 34 of BS-RNase is responsible for the high reactivity of Cys 31 and Cys 32 toward reducing agents (Parente et al, 1985).…”

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confidence: 99%

Mechanism of Ribonuclease Cytotoxicity

Kim

Souček

Matoušek

et al. 1995

Journal of Biological Chemistry

104

115

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Bovine seminal ribonuclease (BS-RNase), a dimeric homolog of bovine pancreatic ribonuclease A (RNase A), is toxic to mammalian cells. In contrast to dimeric BSRNase, monomeric BS-RNase and RNase A are not cytotoxic and are bound tightly by cytosolic ribonuclease inhibitor. To elucidate the mechanism of ribonuclease cytotoxicity, we constructed a series of hybrid and semisynthetic enzymes and examined their properties. In five hybrid enzymes, divergent residues in BS-RNase were replaced with the analogous residues of RNase A so as to diminish an interaction with a putative cellular receptor. In a semisynthetic enzyme, the disulfide bonds that cross-link the monomeric subunits of dimeric BSRNase were replaced with thioether bonds, which can withstand the reducing environment of the cytosol. Each hybrid and semisynthetic enzyme had ribonucleolytic and cytotoxic activities comparable with those of wild-type BS-RNase. These results suggest that dimeric BS-RNase (pI ‫؍‬ 10.3) enters cells by adsorptive rather than receptor-mediated endocytosis and then evades cytosolic ribonuclease inhibitor so as to degrade cellular RNA. This mechanism accounts for the need for a cytosolic ribonuclease inhibitor and for the cytotoxicity of other homologs of RNase A.Bovine seminal ribonuclease (EC 3.1.27.5, BS-RNase) 1 is a cytotoxic protein. In various assays, this cytotoxicity can manifest itself as an immunosuppressive, antitumor, embryotoxic, and aspermatogenic activity (Dostá l and Motoušek, 1973; Souček et al., 1986;Tamburrini et al., 1990;Matoušek and D'Alessio, 1991;Laccetti et al., 1992), each of which has potential therapeutic value Deonarain and Epenetos, 1994). Among these cytotoxic activities, the immunosuppressive activity is most likely to be physiologically significant, since this activity may be required to suppress the female immune response against components of bull seminal fluid (James and Hargreave, 1984).The cytotoxicity of BS-RNase is related to its quaternary structure. BS-RNase is isolated as a dimer that is cross-linked by two disulfide bonds. RNase A and monomeric BS-RNase are not cytotoxic (Vescia et al., 1980;Tamburrini et al., 1990;Kim et al., 1995b). In contrast, artificially dimerized RNase A is cytotoxic, but to a lesser extent than is BS-RNase (Bartholeyns and Baudhuin, 1976;Bartholeyns and Zenebergh, 1979;Vescia et al., 1980;Di Donato et al., 1994). At equilibrium, dimeric BS-RNase is a mixture of two distinct quaternary forms, MϭM and MxM . The conversion of MϭM to MxM entails the exchange of N-terminal ␣-helices between subunits, as occurs during the artificial dimerization of RNase A. Recently, we and others demonstrated that the MxM form is responsible for the cytotoxicity of BS-RNase Cafaro et al., 1995;Kim et al., 1995b). We also demonstrated that the ribonucleolytic activity of BS-RNase is necessary for its cytotoxic activity (Kim et al., 1995a).The cytosol of cells contains a ribonuclease inhibitor (RI) (Roth, 1967;Blackburn and Moore, 1982;Lee and Vallee, 1993;Hofsteenge, 1994).2 This pro...

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“…Recent results (unpublished) indicate that the amide group in question is that of Asne,. This residue is far away from the subunit active site and also from the intersubunit region, as shown by the tri-dimensional structure of the protein [21]. Thus it would seem very unlikely that the side chain of this residue could affect the catalytic act of the reaction, without significantly affecting the K,, i.e., the binding of substrate.…”

Section: Discussionmentioning

confidence: 97%