[show abstract][hide abstract] ABSTRACT: Fifty years ago, the group of Tony Mathias and Bob Rabin at University College London deduced the first mechanism for catalysis by an enzyme, ribonuclease [Findlay, D., Herries, D. G., Mathias, A. P., Rabin, B. R., and Ross, C. A. (1961) Nature 190, 781-784]. Here, we celebrate this historic accomplishment by surveying knowledge of enzymology and protein science at that time, facts that led to the formulation of the mechanism, criticisms and alternative mechanisms, data that supported the proposed mechanism, and some of the refinements that have since provided a more precise picture of catalysis of RNA cleavage by ribonucleases. The Mathias and Rabin mechanism has appeared in numerous textbooks, monographs, and reviews and continues to have a profound impact on biochemistry.
[show abstract][hide abstract] ABSTRACT: We analyze the effect of ECP on primary cultures of cerebellar granule cells (CGCs) and astrocytes in an effort to understand the role of ECP in the eosinophil-induced neurotoxicity. We have shown that ECP induces dose-dependent cell death in both CGCs and astrocytes. The effect of ECP action on cell morphology is consistent with apoptosis for both cell types. The apoptotic mechanism involves ECP binding on the cell surface and an increase in the free cytosolic Ca(2+) concentration. It is associated with the activation of caspase-3, -8 and -9, processes that are also involved in the apoptosis induced either by stroke or other neurodegenerative conditions. Our results open new insights to clarify the neurotoxic effects associated to ECP in the hypereosinophilic syndrome.
Journal of neuroimmunology 10/2010; 227(1-2):60-70. · 2.84 Impact Factor
[show abstract][hide abstract] ABSTRACT: Analyzing the pattern of oligonucleotide formation induced by HP-RNase cleavage shows that the enzyme does not act randomly and follows a more endonucleolytic pattern when compared to RNase A. The enzyme prefers the binding and cleavage of longer substrate molecules, especially when the phosphodiester bond that is broken is 8-11 nucleotides away from at least one of the ends of the substrate molecule. This more endonucleolytic pattern is more appropriate for an enzyme with a regulatory role. Deleting two positive charges on the N-terminus (Arg4 and Lys6) modifies this pattern of external/internal phosphodiester bond cleavage preference, and produces a more exonucleolytic enzyme. These residues may reinforce the strength of a non-catalytic secondary phosphate binding (p2) or, alternatively, constitute a new non-catalytic phosphate binding subsite (p3).
Archives of Biochemistry and Biophysics 04/2008; 471(2):191-7. · 3.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human eosinophil cationic protein (ECP)/ ribonuclease 3 (RNase 3) is a protein secreted from the secondary granules of activated eosinophils. Specific properties of ECP contribute to its cytotoxic activities associated with defense mechanisms. In this work the ECP cytotoxic activity on eukaryotic cell lines is analyzed. The ECP effects begin with its binding and aggregation to the cell surface, altering the cell membrane permeability and modifying the cell ionic equilibrium. No internalization of the protein is observed. These signals induce cell-specific morphological and biochemical changes such as chromatin condensation, reversion of membrane asymmetry, reactive oxygen species production and activation of caspase-3-like activity and, eventually, cell death. However, the ribonuclease activity component of ECP is not involved in this process as no RNA degradation is observed. In summary, the cytotoxic effect of ECP is attained through a mechanism different from that of other cytotoxic RNases and may be related with the ECP accumulation associated with the inflammatory processes, in which eosinophils are present.
Cellular and Molecular Life Sciences CMLS 02/2008; 65(2):324-37. · 5.62 Impact Factor
[show abstract][hide abstract] ABSTRACT: A general acid-base catalytic mechanism is responsible for the cleavage of the phosphodiester bonds of the RNA by ribonuclease A (RNase A). The main active site is formed by the amino acid residues His12, His119, and Lys41, and the process follows an endonucleolytic pattern that depends on the existence of a noncatalytic phosphate-binding subsite adjacent, on the 3'-side, to the active site; in this region the phosphate group of the substrate establishes electrostatic interactions through the side chains of Lys7 and Arg10. We have obtained, by means of site-directed mutagenesis, RNase A variants with His residues both at positions 7 and 10. These mutations have been introduced with the aim of transforming a noncatalytic binding subsite into a putative new catalytic active site. The RNase activity of these variants was determined by the zymogram technique and steady-state kinetic parameters were obtained by spectrophotometric methods. The variants showed a catalytic efficiency in the same order of magnitude as the wild-type enzyme. However, we have demonstrated in these variants important effects on the substrate's cleavage pattern. The quadruple mutant K7H/R10H/H12K/H119Q shows a clear increase of the exonucleolytic activity; in this case the original native active site has been suppressed, and, as consequence, its activity can only be associated to the new active site. In addition, the mutant K7H/R10H, with two putative active sites, also shows an increase in the exonucleolytic preference with respect to the wild type, a fact that may be correlated with the contribution of the new active site.
Protein Science 02/2007; 16(1):99-109. · 2.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Eosinophil cationic protein (ECP)/ribonuclease 3 is a member of the RNase A superfamily involved in inflammatory processes mediated by eosinophils. ECP is bactericidal, helminthotoxic, and cytotoxic to tracheal epithelium cells and to several mammalian cell lines although its RNase activity is low. We studied the thermal stability of ECP by fourth-derivative UV absorbance spectra, circular dichroism, differential scanning calorimetry, and Fourier transform infrared spectroscopy. The T (1/2) values obtained with the different techniques were in very good agreement (T (1/2) approximately 72 degrees C), and the stability was maintained in the pH range between 5 and 7. The ECP calorimetric melting curve showed, in addition to the main transition, a pretransitional conformational change with a T (1/2) of 44 degrees C. Both calorimetric transitions disappeared after successive re-heatings, and the ratio DeltaH versus DeltaH (vH) of 2.2 indicated a significant deviation from the two-state model. It was observed that the thermal unfolding was irreversible. The unfolding process gives rise to changes in the environment of aromatic amino acids that are partially maintained in the refolded protein with the loss of secondary structure and the formation of oligomers. From the thermodynamic analysis of ECP variants, the contribution of specific amino acids, such as Trp10 and the region 115-122, to thermal stability was also determined. The high thermal stability of ECP may contribute to its resistance to degradation when the protein is secreted to the extracellular medium during the immune response.
Protein Science 01/2007; 15(12):2816-27. · 2.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Background. Capan-1 is a human pancreatic adeno-carcinoma cell line of presumed ductal origin. This is based on the histologic appearance of the tumor from which it arose. Yet considerable controversy exists regarding the actual cell of origin for these exocrine carcinomas. Two acinar antigens, ribonuclease and trypsin, were analyzed in cells growing in synthetic serum.Methods. Capan-1 cells were adapted to grow in basal medium supplemented with synthetic serum, because fetal bovine serum (FBS) normally used to culture cells contains bovine ribonuclease, which can interfere with measurements of the ribonuclease secretion. These cells were also adapted to grow in different serum-free media, allowing us to determine its minimal growth requirements. The presence of ribonuclease in Capan-1 and PANC-1 conditioned media was monitored by activity. Other acinar and ductal markers were monitored using Northern blot analysis.Results. Capan-1, PANC-1, IBF-CP3, and MDA Amp-7 cell lines were successfully adapted to grow in synthetic serum by means of the adaptation protocol reported here. The adaptation of Capan-1 to serum-free media showed that the cells are capable of growing in a medium containing insulin, transferrin, selenium, a nonprotein carrier, and lipoic and linoleic acids. Northern blot analysis showed the expression of carbonic anhydrase II, cytokeratin 18, ribonuclease, and trypsin in Capan-1 cells growing in FBS and synthetic serum. No changes in morphology, karyotype, or gene expression were observed in these cells as a result of the adaptation process.Conclusions. The cell line Capan-1 is expressing some ductal as well as acinar products despite its supposed ductal origin. The expression of trypsin at the mRNA level and ribonuclease at mRNA and protein levels is shown in Capan-1 cells. The protein expression will be further investigated as the cell line has been adapted to grow in synthetic serum and serum-free media with no apparent changes with respect to their growth in FBS.
Cancer 06/2006; 73(9):2285 - 2295. · 5.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: Eosinophil cationic protein (ECP) is a ribonuclease secreted from activated eosinophils that may cause tissue injure as a result of eosinophilic inflammation. ECP possesses bactericidal, antiviral and helminthotoxic activity and inhibits mammalian cell growth. The mechanism by which ECP exerts its toxicity is not known but it has been related to the ability of the protein to destabilise lipid bilayers. We have assessed the involvement of some cationic and aromatic surface exposed residues of ECP in the inhibition of proliferation of mammalian cell lines. We have constructed ECP mutants for the selected residues and assessed their ability to prevent cell growth. Trp10 and Trp35 together with the adjacent stacking residue are critical for the damaging effect of ECP on mammalian cell lines. These residues are also crucial for the membrane disruption activity of ECP. Other exposed aromatic residues packed against arginines (Arg75-Phe76 and Arg121-Tyr122) and specific cationic amino acids (Arg101 and Arg104) of ECP play a secondary role in the cell growth inhibition. This may be related to the ability of the protein to bind carbohydrates such as those found on the surface of mammalian cells.
Molecular and Cellular Biochemistry 05/2005; 272(1-2):1-7. · 2.33 Impact Factor
[show abstract][hide abstract] ABSTRACT: To characterize eosinophil granule-derived proteins in cats.
Eosinophils collected via peritoneal lavage from 2 cats.
The cats were infested orally with Toxocara canis eggs and subsequently challenge-exposed with T. canis antigen injected IP to induce peritoneal eosinophilia; eosinophils were collected via peritoneal lavage. Eosinophil granule proteins were acid-extracted, separated by gel-filtration chromatography, and examined for their peroxidase, ribonuclease, and bactericidal activities; the N-terminal sequence of some of these proteins was determined and compared with homologue proteins from other species.
3 protein peaks were separated in the chromatogram. The first peak had both peroxidase and bactericidal activities. The second peak had ribonuclease and bactericidal activities, and the N-terminal sequence of the major protein was homologous with that of proteins of the ribonuclease A superfamily, including eosinophil ribonucleases from humans and other animal species. The third protein peak had bactericidal activity, and the N-terminal sequence of the major protein was homologous with that of human and murine major basic proteins.
Results indicated that feline eosinophil granules contain major basic protein and eosinophil-associated ribonuclease and the granule proteins have peroxidase, ribonuclease, and bactericidal activities. In cats, characterization of eosinophil granule proteins may be useful in elucidation of the mechanism of tissue damage in eosinophil-associated diseases and development of new treatment options for those diseases. In addition, the identification of conserved structure and function of eosinophil granule proteins in cats is relevant from an evolutionary viewpoint.
American Journal of Veterinary Research 08/2004; 65(7):957-63. · 1.35 Impact Factor
[show abstract][hide abstract] ABSTRACT: Eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN) are proteins of the ribonuclease A (RNase A) superfamily that have developed biological properties related to the function of eosinophils. ECP is a potent cytotoxic molecule, and although the mechanism is still unknown this cytotoxic activity has been associated with its highly cationic character. Using liposome vesicles as a model, we have demonstrated that ECP tends to disrupt preferentially acidic membranes. On the basis of structure analysis, ECP variants modified at basic and hydrophobic residues have been constructed. Changes in the leakage of liposome vesicles by these ECP variants have indicated the role of both aromatic and basic specific amino acids in cellular membrane disruption. This is the case with the two tryptophans at positions 10 and 35, but not phenylalanine 76, and the two arginines 101 and 104. The bactericidal activity of both native ECP and point-mutated variants, tested against Escherichia coli and Staphylococcus aureus, suggests that basic amino acids play, in addition to the effect on the disruption of the cellular membrane, other roles such as specific binding on the surface of the bacteria cell.
[show abstract][hide abstract] ABSTRACT: Eosinophil cationic protein (ECP) is a component of the eosinophil granule matrix. It shows marked toxicity against helminth parasites, bacteria single-stranded RNA viruses, and host epithelial cells. Secretion of human ECP is related to eosinophil-associated allergic, asthmatic, and inflammatory diseases. ECP belongs to the pancreatic ribonuclease superfamily of proteins, and the crystal structure of ECP in the unliganded form (determined previously) exhibited a conserved RNase A fold [Boix, E., et al. (1999) Biochemistry 38, 16794-16801]. We have now determined a high-resolution (2.0 A) crystal structure of ECP in complex with adenosine 2',5'-diphosphate (2',5'-ADP) which has revealed the details of the ribonucleolytic active site. Residues Gln-14, His-15, and Lys-38 make hydrogen bond interactions with the phosphate at the P(1) site, while His-128 interacts with the purine ring at the B(2) site. A new phosphate binding site, P(-)(1), has been identified which involves Arg-34. This study is the first detailed structural analysis of the nucleotide recognition site in ECP and provides a starting point for the understanding of its substrate specificity and low catalytic efficiency compared with that of the eosinophil-derived neurotoxin (EDN), a close homologue.
[show abstract][hide abstract] ABSTRACT: The cleavage pattern of oligocytidylic acid substrates by bovine pancreatic ribonuclease A (RNase A) was studied by means of reversed-phase HPLC. Oligocytidylic acids, ranging from dinucleotides to heptanucleotides, were obtained by RNase A digestion of poly(C). They were identified by MALDI-TOF mass spectrometry; it was confirmed that all of them corresponded to the general structure (Cp)(n)C>p, in which C>p indicates a 2',3'-cyclic phosphate. This is a confirmation of the proposed mechanism for RNase A, wherein the so-called hydrolytic (or second) step is in fact a special case of the reverse of transphosphorylation (first step). The patterns of cleavage for the oligonucleotide substrates show that the native enzyme has no special preference for endonucleolytic or exonucleolytic cleavage, whereas a mutant of the enzyme (K7Q/R10Q-RNase A) lacking p(2) (a phosphate binding subsite adjacent, on the 3' side, to the main phosphate binding site p(1)) shows a clear exonucleolytic pattern; a mutant (K66Q-RNase A) lacking p(0) (a phosphate binding subsite adjacent, on the 5' side, to the main phosphate binding site p(1)) shows a more endonucleolytic pattern. This indicates the important role played by the subsites on the preference for the bond cleaved. Molecular modeling shows that, in the case of the p(2) mutant, the amide group of glutamine can form a hydrogen bond with the 2',3'-cyclic terminal phosphate, whereas the distance to a 3',5'-phosphodiester bond is too long to form such a hydrogen bond. This could explain the preference for exonucleolytic cleavage shown by the p(2) mutant.
Protein Science 01/2002; 11(1):117-28. · 2.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: The eosinophil cationic protein (ECP) is a basic secretion protein involved in the immune response system. ECP levels in biological fluids are an indicator of eosinophil-specific activation and degranulation and are currently used for the clinical monitoring and diagnosis of inflammatory disorders. A polyclonal epitope-specific antibody has been obtained by immunizing rabbits with a conjugated synthetic peptide. A sequence corresponding to a large exposed loop in the human ECP three-dimensional structure (D115-Y122) was selected as a putative antigenic epitope. The antibody was purified on an affinity column using recombinant ECP (rECP) as antigen. The antibody (D112-P123 Ab) specifically recognizes rECP and its native glycosylated and nonglycosylated forms in plasma, granulocytes, and sputum. The antibody detects as little as 1 ng of rECP, can be used both in reducing and nonreducing conditions, and does not cross-react with the highly homologous eosinophil-derived neurotoxin or other proteins of the pancreatic ribonuclease superfamily.
Journal of Leukocyte Biology 07/2001; 69(6):1027-35. · 4.57 Impact Factor
[show abstract][hide abstract] ABSTRACT: Nucleic-acid cleavage can be processive, with the enzyme moving from one site to the next in the polymer before dissociating
from the substrate, or distributive, with partially cleaved substrates released to the medium after the initial reaction.
Kinetics indicate that either mechanism can be seen in reactions with polymerases, helicases, or nucleases (1). However, many factors affect these kinetics, including the strength of the enzyme-substrate binding, which can be altered
by the salt concentration of the buffer, and intermediates in the formation of the complex or the release of the products
that may be poorly characterized. The distinction between processivity and distributivity is based primarily on the ratio
of rate constants for cleavage and dissociation. Unlike the distinction between endo- and exo-nucleases (i.e., those cleaving
within or from one end of the nucleic acid), which will not change with reaction conditions, although in some cases enzymes
show only a preference from one or the other activity, assay conditions can blur the distinction between processivity and
Methods in molecular biology (Clifton, N.J.) 02/2001; 160:15-24.
[show abstract][hide abstract] ABSTRACT: Eosinophil cationic protein (ECP) is located in the matrix of the eosinophil's large specific granule and has marked toxicity for a variety of helminth parasites, hemoflagellates, bacteria, single-stranded RNA virus, and mammalian cells and tissues. It belongs to the bovine pancreatic ribonuclease A (RNase A) family and exhibits ribonucleolytic activity which is about 100-fold lower than that of a related eosinophil ribonuclease, the eosinophil-derived neurotoxin (EDN). The crystal structure of human ECP, determined at 2.4 A, is similar to that of RNase A and EDN. It reveals that residues Gln-14, His-15, Lys-38, Thr-42, and His-128 at the active site are conserved as in all other RNase A homologues. Nevertheless, evidence for considerable divergence of ECP is also implicit in the structure. Amino acid residues Arg-7, Trp-10, Asn-39, His-64, and His-82 appear to play a key part in the substrate specificity and low catalytic activity of ECP. The structure also shows how the cationic residues are distributed on the surface of the ECP molecule that may have implications for an understanding of the cytotoxicity of this enzyme.
[show abstract][hide abstract] ABSTRACT: With the use of a high yield prokaryotic expression system, large amounts of human eosinophil cationic protein (ECP) have been obtained. This has allowed a thorough kinetic study of the ribonuclease activity of this protein. The catalytic efficiencies for oligouridylic acids of the type (Up)nU>p, mononucleotides U>p and C>p, and dinucleoside monophosphates CpA, UpA, and UpG have been interpreted by the specific subsites distribution in ECP. The distribution of products derived from digestion of high molecular mass substrates, such as poly(U) and poly(C), by ECP was compared with that of RNase A. The characteristic cleavage pattern of polynucleotides by ECP suggests that an exonuclease-like mechanism is predominantly favored in comparison to the endonuclease catalytic mechanism of RNase A. Comparative molecular modeling with bovine pancreatic RNase A-substrate analog crystal complexes revealed important differences in the subsite structure, whereas the secondary phosphate-binding site (p2) is lacking, the secondary base subsite (B2) is severely impaired, and there are new interactions at the po, Bo, and p-1 sites, located upstream of the P-O-5' cleavable phosphodiester bond, that are not found in RNase A. The differences in the multisubsites structure could explain the reduced catalytic efficiency of ECP and the shift from an endonuclease to an exonuclease-type mechanism.
Journal of Biological Chemistry 05/1999; 274(22):15605-14. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Pancreatic ribonuclease A (RNase A) is a endonuclease that catalyzes depolymerization of ribonucleic acid (RNA) releasing oligonucleotides. In the process of binding enzyme with substrate are involved several non-catalytic phosphate binding subsites, one of them is p2, additional to main catalytic site p1. RNaza A prefers binding and cleavage of longer substrate molecules, and 3',5'-phosphodiester bond should be some six-seven residues apart from the end of molecules of the chain of RNA. In this work is analysed endonuclease activity of recombinant pancreatic RNase A (K7H), that in position seven instead of a lysine there is a histidine, amino acid residue that participates in main catalytic site p1. Mutant enzyme is obtained by site-directed mutagenesis by Kunkel. Results of this investigation have shown that substitution of lysine by histidine in position seven of RNase A has produced total deletion of p2 subsite, and K7H has lost endonuclease activity, and has become exonuclease. These results confirm central role of Lys-7 in establishing p2 subsite and endonuclease activity of pancreatic RNase A.