Giuseppe D'Alessio

Seminal RNase: a unique member of the ribonuclease superfamily

The RNase found in bull semen, although a member of the mammalian superfamily of ribonucleases, possesses some unusual properties. Besides its unique structure and enzymic properties, it displays antispermatogenic, antitumor and immunosuppressive activities. Seminal RNase belongs to an interesting group of RNases, the RISBASES (RIbonucleases with Special, i.e. non catalytic, Biological Actions) other members of which include angiogenin, selectively neurotoxic RNases, a lectin and the self-incompatibility factors from a flowering plant.

THE ANTITUMOR ACTION OF SEMINAL RIBONUCLEASE AND ITS QUATERNARY CONFORMATIONS

It has been previously shown that the antitumor action of bovine seminal ribonuclease (BS‐RNase) is dependent on its dimeric structure. However, two distinct quaternary structures, each in equilibrium with the other, have been described for the enzyme: one in which the two subunits exchange their N‐terminal ends, the other with no exchange. Antitumor activity assays, carried out on homogeneous quaternary forms of the enzyme, as well as on dimeric mutants of bovine pancreatic RNase A, reveal that another structural determinant of the antitumor activity of BS‐RNase is the exchange of N‐terminal ends between subunits.

A Fully Human Antitumor ImmunoRNase Selective for ErbB-2-Positive Carcinomas

We report the preparation and characterization of a novel, fully human antitumor immunoRNase (IR). The IR, a human RNase and fusion protein made up of a human single chain variable fragment (scFv), is directed to the ErbB-2 receptor and overexpressed in many carcinomas. The anti-ErbB-2 IR, named hERB-hRNase, retains the enzymatic activity of the wild-type enzyme (human pancreatic RNase) and specifically binds to ErbB-2-positive cells with the high affinity (Kd = 4.5 nm) of the parental scFv. hERB-hRNase behaves as an immunoprotoxin and on internalization by target cells becomes selectively cytotoxic in a dose-dependent manner at nanomolar concentrations. Administered in five doses of 1.5 mg/kg to mice bearing an ErbB-2-positive tumor, hERB-hRNase induced a dramatic reduction in tumor volume. hERB-hRNase is the first fully human antitumor IR produced thus far, with a high potential as a poorly immunogenic human drug devoid of nonspecific toxicity, directed against ErbB-2-positive malignancies.

Seminal Ribonuclease: The Importance of Diversity

Publisher Summary This chapter provides an overview of the seminal ribonuclease. Bovine seminal RNase (BS-RNase) is a diverse RNase “different” from the historic prototype RNase A—and from all other RNases of the vertebrate superfamily—for its dimeric structure, for its non-Michaelian kinetics, and for its special, noncatalytic, biological actions. BS-RNase is also a diverse RNase because it exists in a multiplicity of structural forms, and is endowed with a multiplicity of biological actions. Two quaternary conformations and three isoenzymatic subunit compositions are known for BS-RNase. BS-RNase performs a surprising array of biological actions: aspermatogenic, antitumor, immunosuppressive, and antiviral. BS-RNase may not be the only seminal RNase: an RNase has been purified from human semen and low levels of RNase activity have been detected in the semen of several mammals, including mouse, rabbit, and sheep. This chapter discusses isolation and production of seminal RNase. It explains preparation of seminal ribonuclease from natural sources. Production of recombinant BS-RNase is discussed. The chapter elaborates covalent structure, three-dimensional structure, and folding pathway of seminal RNase. The chapter also outlines the functions of seminal ribonuclease.

Interchain disulfide bridges in ribonuclease BS-1

Abstract RNAase BS-1, a dimeric ribonuclease isolated from bovine seminal plasma, is made up of two identical subunits whose amino acid sequence is homologous to the sequence of bovine pancreatic RNAase A. The dimeric structure, resistant to denaturating agents, is sensitive to thiol reagents even in the absence of denaturants. The isolation and characterization of a cystine peptide containing two adjacent 1 2 cystine residues is reported. As the peptide molecular weight is halved after reductive cleavage with dithiothreitol, a structure based on two interchain disulfide bonds between the two adjacent 1 2 cystine of each subunit is proposed. The singularity of such a structure for a small enzymatic protein is discussed.

Dimeric structure of seminal ribonuclease

RNAase BSl., the major component of ribonuclease activity in bull seminal plasma, is a basic protein with a molecular weight of 29,000 [ 11. Its catalytic properties are very similar to those of bovine pancreatic RNAase A [2] , except for a lower kcat and the ability to degrade also double-stranded RNA under conditions in which RNAase A is only slightly active [3]. We wish to report on the subunit structure of this enzyme, the first instance, to our knowledge, of a dimeric ribonuclease. This conclusion rests on several lines of evidence: i) peptide mapping of the protein; ii) quantitation of end groups; iii) estimation of the subunit molecular weight by electrophoresis on polyacrylamide gels in sodium dodecylsulphate; iv) estimation of the number of covalently linked species obtained by amidination of the protein with a crosslinking reagent. The peptide map of RNAase BS-1 was obtained by tryptic digestion of the protein oxidized with performic acid [4]. A total of 19 f 2 spots was obtained in several experiments (fig. la). From the ammo acid composition (28 lysine and 8 arginine residues per molecule), one would expect a number of about 37 peptides for a protein consisting of a single polypeptide chain. The observed value can therefore only be explained if one assumes that the protein is made up of two identical, or very similar, subunits. On the other hand, the possibility of RNAase BS1 being a dimer of RNAase A is ruled out by a comparison of the peptide maps of the two proteins (fig. la and b). Several peptides appear to be different, and this is in

Ribonuclease/angiogenin inhibitor 1 regulates stress-induced subcellular localization of angiogenin to control growth and survival.

Summary Angiogenin (ANG) promotes cell growth and survival. Under growth conditions, ANG undergoes nuclear translocation and accumulates in the nucleolus where it stimulates rRNA transcription. When cells are stressed, ANG mediates the production of tRNA-derived stress-induced small RNA (tiRNA), which reprograms protein translation into a survival mechanism. The ribonucleolytic activity of ANG is essential for both processes but how this activity is regulated is unknown. We report here that ribonuclease/angiogenin inhibitor 1 (RNH1) controls both the localization and activity of ANG. Under growth conditions, ANG is located in the nucleus and is not associated with RNH1 so that the ribonucleolytic activity is retained to ensure rRNA transcription. Cytoplasmic ANG is associated with and inhibited by RNH1 so that random cleavage of cellular RNA is prevented. Under stress conditions, ANG is localized to the cytoplasm and is concentrated in stress granules where it is not associated with RNH1 and thus remains enzymatically active for tiRNA production. By contrast, nuclear ANG is associated with RNH1 in stressed cells to ensure that the enzymatic activity is inhibited and no unnecessary rRNA is produced to save anabolic energy. Knockdown of RNH1 abolished stress-induced relocalization of ANG and decreased cell growth and survival.

Effective expression and purification of recombinant onconase, an antitumor protein

Several members of the RNase A superfamily are endowed with antitumor activity, showing selective cytotoxicity toward several tumor cell lines. One of these is onconase, the smallest member of the RNase A superfamily, which is at present undergoing phase III clinical trials. We report here the expression of recombinant onconase in Escherichia coli inclusion bodies, the correct processing of the protein, followed by its purification in high yields. The recombinant protein has biological and catalytic properties identical to those of the natural enzyme.

A human, compact, fully functional anti-ErbB2 antibody as a novel antitumour agent.

A new human, compact antibody was engineered by fusion of a human, antitumour ErbB2-directed scFv with a human IgG1 Fc domain. Overexpression of the ErbB2 receptor is related to tumour aggressiveness and poor prognosis. This new immunoagent meets all criteria for a potential anticancer drug: it is human, hence poorly or not immunogenic; it binds selectively and with high affinity to target cells, on which it exerts an effective and selective antiproliferative action, including both antibody-dependent and complement-dependent cytotoxicity; it effectively inhibits tumour growth in vivo. Its compact molecular size should provide for an efficient tissue penetration, yet suitable to a prolonged serum half-life.

Essential stations in the intracellular pathway of cytotoxic bovine seminal ribonuclease

Bovine seminal RNase (BS-RNase) is a dimeric RNase selectively cytotoxic for malignant cells. No information is available on its pathway from the extracellular matrix through the cytosol, where it degrades rRNA. An investigation of this pathway is reported here, carried out by immunofluorescence studies, by assessing the effects on BS-RNase cytotoxicity of drugs that affect specific intracellular compartments and by assaying the behaviour of a protein variant, BS-RNase-KDEL (BS-RNase in which a Lys-Asp-Glu-Leu peptide segment is inserted at the C-terminal ends of the subunit chains), endowed with a consensus sequence that directs proteins to the endoplasmic reticulum. BS-RNase was found to bind both normal and malignant cells and to be internalized by both cell types in endosome vesicles. Non-cytotoxic RNases, such as RNase A and a monomeric derivative of BS-RNase, did not bind to the cell surface and were not internalized. However, an engineered, dimeric and cytotoxic variant of RNase A bound effectively and permeated cells. The results of immunofluorescence studies, the effects of nigericin, monensin and brefeldin A on the cytotoxic action of seminal RNase, and the behaviour of the BS-RNase-KDEL variant, led to the conclusion that the pathway of BS-RNase in malignant cells from the extracellular matrix to the cytosol has two essential intracellular stations: endosomes and the trans-Golgi network. In normal cells, however, the protein does not progress from the endosomal compartment to the Golgi complex.