Person:
Lacadena García-Gallo, Francisco Javier

First Name

Francisco Javier

Last Name

Lacadena García-Gallo

URI

https://hdl.handle.net/20.500.14352/76233

Affiliation

Universidad Complutense de Madrid

Faculty / Institute

Ciencias Químicas

Department

Bioquímica y Biología Molecular

Area

Bioquímica y Biología Molecular

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Now showing 1 - 10 of 22

Der p 1‑based immunotoxin as potential tool for the treatment of dust mite respiratory allergy
(Scientific Reports, 2020) Lázaro‑Gorines, Rodrigo; López Rodríguez, Juan Carlos; Benedé Pérez, Sara; González, Miguel; Mayorga, Cristobalina; Vogel, Lothar; Martínez Del Pozo, Álvaro; Lacadena García-Gallo, Francisco Javier; Villalba Díaz, María Teresa
Immunotoxins appear as promising therapeutic molecules, alternative to allergen-specifcimmunotherapy. In this work, we achieved the development of a protein chimera able to promote specifc cell death on efector cells involved in the allergic reaction. Der p 1 allergen was chosen as cell-targeting domain and the powerful ribotoxin α-sarcin as the toxic moiety. The resultant construction, named proDerp1αS, was produced and purifed from the yeast Pichia pastoris. Der p 1-protease activity and α-sarcin ribonucleolytic action were efectively conserved in proDerp1αS. Immunotoxin impact was assayed by using efector cells sensitized with house dust mite-allergic sera. Cell degranulation and death, triggered by proDerp1αS, was exclusively observed on Der p 1 sera sensitized-humRBL-2H3 cells, but not when treated with non-allergic sera. Most notably, equivalent IgE-binding and degranulation were observed with both proDerp1αS construct and native Der p 1 when using purifed basophils from sensitized patients. However, proDerp1αS did not cause any cytotoxic efect on these cells, apparently due to its lack of internalization after their surface IgEbinding, showing the complex in vivo panorama governing allergic reactions. In conclusion, herein we present proDerp1αS as a proof of concept for a potential and alternative new designs of therapeutic tools for allergies. Development of new, and more specifc, second-generation of immunotoxins following proDerp1αS, is further discussed
Role of histidine-50, glutamic acid-96 and histidine-137 in the ribonucleolytic mechanism of the ribotoxin alpha-sarcin
(Proteins: Structure, Function and Genetics, 1999) Lacadena García-Gallo, Francisco Javier; Martínez Del Pozo, Álvaro; Martínez Ruiz, Antonio; Pérez-Cañadillas, José Manuel; Bruix, Marta; Mancheño Gómez, José Miguel; Gavilanes Franco, José Gregorio; Oñaderra Sánchez, Mercedes
alpha-Sarcin is a ribotoxin secreted by the mold Aspergillus giganteus that degrades the ribosomal RNA by acting as a cyclizing ribonuclease. Three residues potentially involved in the mechanism of catalysis--histidine-50, glutamic acid-96, and histidine-137--were changed to glutamine. Three different single mutation variants (H50Q, E96Q, H137Q) as well as a double variant (H50/137Q) and a triple variant (H50/137Q/E96Q) were prepared and isolated to homogeneity. These variants were spectroscopically (circular dichroism, fluorescence emission, and proton nuclear magnetic resonance) characterized. According to these results, the three-dimensional structure of these variants of alpha-sarcin was preserved; only very minor local changes were detected. All the variants were inactive when assayed against either intact ribosomes or poly(A). The effect of pH on the ribonucleolytic activity of alpha-sarcin was evaluated against the ApA dinucleotide. This assay revealed that only the H50Q variant still retained its ability to cleave a phosphodiester bond, but it did so to a lesser extent than did wild-type alpha-sarcin. The results obtained are interpreted in terms of His137 and Glu96 as essential residues for the catalytic activity of alpha-sarcin (His137 as the general acid and Glu96 as the general base) and His50 stabilizing the transition state of the reaction catalyzed by alpha-sarcin.
Secretion of Recombinant Pro- and Mature Fungal α-Sarcin Ribotoxin by the Methylotrophic YeastPichia pastoris:The Lys–Arg Motif Is Required for Maturation
(Protein Expression and Purification, 1998) Martínez Ruiz, Antonio; Martínez Del Pozo, Álvaro; Lacadena García-Gallo, Francisco Javier; Mancheño Gómez, José Miguel; Oñaderra Sánchez, Mercedes; Carlos López-Otı́n; Gavilanes Franco, José Gregorio
α-Sarcin is a ribosome-inactivating protein from the moldAspergillus giganteus.The methylotrophic yeastPichia pastorishas been transformed with two plasmids (pHILD2preαS and pHILS1preαS), which contain the complete α-sarcin cDNA, including its original fungal leader peptide, under the control of yeast alcohol oxidase promoter. The second one is indeed fused to the signal sequence ofP. pastorisacid phosphatase. The transformed yeasts secreted both mature and pro-α-sarcin. The presence of this pro-α-sarcin in the yeast extracellular medium is due to an inefficient recognition of the pro-sequence by a putative Kex2p-like endopeptidase. A third plasmid accounting for a single mutation of the α-sarcin leader peptide was designed to produce a more efficient Kex2p recognition motif. This approach resulted in the extracellular production of only the mature protein, suggesting the existence of a two-step mechanism for processing its leader peptide. This recombinant α-sarcin is identical to the original fungal protein, according to activity and spectroscopic criteria. In addition, pro-α-sarcin, which has been characterized for the first time, also exhibits ribonucleolytic activity as the mature protein does. Therefore, protection of the producing cells against this kind of ribotoxins may depend on an efficient recognition of the signal sequence followed by translocation of the nascent polypeptide to the endoplasmic reticulum.
Characterization of a natural larger form of the antifungal protein (AFP) from Aspergillus giganteus
(Biochim Biophys Acta, 1997) Martínez Ruiz, Antonio; Martínez Del Pozo, Álvaro; Lacadena García-Gallo, Francisco Javier; Mancheño Gómez, José Miguel; Oñaderra Sánchez, Mercedes; Gavilanes Franco, José Gregorio
Two major proteins, a-sarcin and an antifungal polypeptide AFP , are secreted by the mould Ž . Aspergillus giganteus MDH 18894 when it is cultured for 70–80 h. A third major protein is also found in the extracellular medium at 48–60 h, but it disappears as the culture proceeds. This protein has been isolated and characterized in terms of apparent molecular mass, electrophoretic and chromatographic behaviour, NH -terminal primary structure, amino acid content, spectroscopical 2 features, reactivity against anti-AFP antibodies, and antifungal activity. Based on the obtained results it would be an extracellular inactive precursor form of AFP, designated as the large form of AFP lf-AFP . Its amino acid composition is Ž . identical to that of AFP but containing six extra residues. NH -terminal sequence analysis of the first eight amino acid 2 residues of this polypeptide revealed that the extra residues can be perfectly accommodated within the DNA-deduced sequence of the precursor form of AFP. Its alignment with precursor sequences of different proteins, secreted by a variety of Aspergillus spp., reveals the existence of a common tetrapeptide at the carboxy-terminal end of their leader peptides. This sequence would be IlerLeu-Xaa-Yaa-Arg, being mostly Xaa and Yaa an acid residue Asp Ž . rGlu and alanine, respectively. The presence of lf-AFP as an extracellular protein would be in perfect agreement with the existence of this tetrapeptide motif, that can be involved in the protein secretion mechanisms of filamentous fungi.
The cytotoxin α‐sarcin behaves as a cyclizing ribonuclease
(FEBS Letters, 1998) Lacadena García-Gallo, Francisco Javier; Martínez Del Pozo, Álvaro; Valle Lacadena; Martínez Ruiz, Antonio; Mancheño Gómez, José Miguel; Oñaderra Sánchez, Mercedes; Gavilanes Franco, José Gregorio
The hydrolysis of adenylyl(3PC5P)adenosine (ApA) and guanylyl(3PC5P)adenosine (GpA) dinucleotides by the cytotoxic protein K-sarcin has been studied. Quantitative analysis of the reaction has been performed through reversephase chromatographic (HPLC) separation of the resulting products. The hydrolysis of the 3P-5P phosphodiester bond of these substrates yields the 2P-3P cyclic mononucleotide; this intermediate is converted into the corresponding 3P-monophosphate derivative as the final product of the reaction. The values of the apparent Michaelis constant (KM), kcat and kcat/ KM have also been calculated. The obtained results fit into a twostep mechanism for the enzymatic activity of K-sarcin and allow to consider this protein as a cyclizing RNase. z 1998 Federation of European Biochemical Societies.
Hirsutellin A: A Paradigmatic Example of the Insecticidal Function of Fungal Ribotoxins
(Insects, 2013) Herrero Galán, Elías; García Ortega, Lucía; Lacadena García-Gallo, Francisco Javier; Olombrada Sacristán, Miriam; Martínez Del Pozo, Álvaro; Gavilanes Franco, José Gregorio; Oñaderra Sánchez, Mercedes
The fungal pathogen Hirsutella thompsonii produces an insecticidal protein named hirsutellin A (HtA), which has been described to be toxic to several species of mites, insect larvae, and cells. On the other hand, on the basis of an extensive biochemical and structural characterization, HtA has been considered to be a member of the ribotoxins family. Ribotoxins are fungal extracellular ribonucleases, which inactivate ribosomes by specifically cleaving a single phosphodiester bond located at the large rRNA. Although ribotoxins were brought to light in the 1960s as antitumor agents, their biological function has remained elusive. Thus, the consideration of hirsutellin A, an insecticidal protein, as a singular ribotoxin recalled the idea of the biological activity of these toxins as insecticidal agents. Further studies have demonstrated that the most representative member of the ribotoxin family, α-sarcin, also shows strong toxic action against insect cells. The determination of high resolution structures, the characterization of a large number of mutants, and the toxicity assays against different cell lines have been the tools used for the study of the mechanism of action of ribotoxins at the molecular level. The aim of this review is to serve as a compilation of the facts that allow identification of HtA as a paradigmatic example of the insecticidal function of fungal ribotoxins.
A novel Carcinoembryonic Antigen (CEA)-Targeted Trimeric Immunotoxin shows signifcantly enhanced Antitumor Activity in Human Colorectal Cancer Xenografts
(Scientific Reports, 2019) Lázaro Gorines, R.; Ruiz de la Herrán, J.; Navarro, R.; Sanz, L.; Alvarez Vallina, L.; Martínez Del Pozo, Álvaro; Gavilanes, José G.; Lacadena García-Gallo, Francisco Javier
Immunotoxins are chimeric molecules, which combine antibody specifcity to recognize and bind with high-afnity tumor-associated antigens (TAA) with the potency of the enzymatic activity of a toxin, in order to induce the death of target cells. Current immunotoxins present some limitations for cancer therapy, driving the need to develop new prototypes with optimized properties. Herein we describe the production, purifcation and characterization of two new immunotoxins based on the gene fusion of the anti-carcinoembryonic antigen (CEA) single-chain variable fragment (scFv) antibody MFE23 to α-sarcin, a potent fungal ribotoxin. One construct corresponds to a conventional monomeric single-chain immunotoxin design (IMTXCEAαS), while the other one takes advantage of the trimerbody technology and exhibits a novel trimeric format (IMTXTRICEAαS) with enhanced properties compared with their monomeric counterparts, including size, functional afnity and biodistribution, which endow them with an improved tumor targeting capacity. Our results show the highly specifc cytotoxic activity of both immunotoxins in vitro, which was enhanced in the trimeric format compared to the monomeric version. Moreover, the trimeric immunotoxin also exhibited superior antitumor activity in vivo in mice bearing human colorectal cancer xenografts. Therefore, trimeric immunotoxins represent a further step in the development of next-generation therapeutic immunotoxins.
Involvement of loops 2 and 3 of alpha-sarcin on its ribotoxic activity
(Toxicon, 2015) Castaño Rodríguez, Carlos; Olombrada Sacristán, Miriam; Partida Hanon, Angélica; Lacadena García-Gallo, Francisco Javier; Oñaderra Sánchez, Mercedes; Gavilanes, José G.; García Ortega, Lucía; Martínez Del Pozo, Álvaro
Ribotoxins are a family of fungal ribosome-inactivating proteins displaying highly specific ribonucleolytic activity against the sarcin/ricin loop (SRL) of the larger rRNA, with a-sarcin as its best-characterized member. Their toxicity arises from the combination of this activity with their ability to cross cell membranes. The involvement of a-sarcin's loops 2 and 3 in SRL and ribosomal proteins recognition, as well as in the ribotoxin-lipid interactions involving cell penetration, has been suggested some time ago. In the work presented now different mutants have been prepared in order to study the role of these loops in their ribonucleolytic and lipid-interacting properties. The results obtained confirm that loop 3 residues Lys 111, 112, and 114 are key actors of the specific recognition of the SRL. In addition, it is also shown that Lys 114 and Tyr 48 conform a network of interactions which is essential for the catalysis. Lipid-interaction studies show that this Lys-rich region is indeed involved in the phospholipids recognition needed to cross cell membranes. Loop 2 is shown to be responsible for the conformational change which exposes the region establishing hydrophobic interactions with the membrane inner leaflets and eases penetration of ribotoxins target cells.
Efficient in vivo antitumor effect of an immunotoxin based on ribotoxin α-sarcin in nude mice bearing human colorectal cancer xenografts
(Springerplus, 2015) Tomé Amat, Jaime; Olombrada Sacristán, Miriam; Ruiz de la Herrán, Javier; Pérez Gómez, Eduardo; Andradas Arias, Clara; Sánchez García, Cristina; Martínez, Leopoldo; Martínez Del Pozo, Álvaro; Gavilanes, José G.; Lacadena García-Gallo, Francisco Javier
Tagging of RNases, such as the ribotoxin α-sarcin, with the variable domains of antibodies directed to surface antigens that are selectively expressed on tumor cells endows cellular specificity to their cytotoxic action. A recombinant single-chain immunotoxin based on the ribotoxin α-sarcin (IMTXA33αS), produced in the generally regarded as safe (GRAS) yeast Pichia pastoris, has been recently described as a promising candidate for the treatment of colorectal cancer cells expressing the glycoprotein A33 (GPA33) antigen, due to its high specific and effective cytotoxic effect on in vitro assays against targeted cells. Here we report the in vivo antitumor effectiveness of this immunotoxin on nude mice bearing GPA33-positive human colon cancer xenografts. Two sets of independent assays were performed, including three experimental groups: control (PBS) and treatment with two different doses of immunotoxin (50 or 100 μg/ injection) (n = 8). Intraperitoneal administration of IMTXA33αS resulted in significant dose-dependent tumor growth inhibition. In addition, the remaining tumors excised from immunotoxin-treated mice showed absence of the GPA33 antigen and a clear inhibition of angiogenesis and proliferative capacity. No signs of immunotoxin-induced pathological changes were observed from specimens tissues.Overall these results show efficient and selective cytotoxic action on tumor xenografts, combined with the lack of severe side effects, suggesting that IMTXA33αS is a potential therapeutic agent against colorectal cancer.
Fungal ribotoxins: structure, function and evolution
(Microbial Toxins: Current Research and Future Trends, 2009) Herrero Galán, Elías; Álvarez García, Elisa; Carreras Sangrà, Nelson; Lacadena García-Gallo, Francisco Javier; Alegre Cebollada, Jorge; Martínez Del Pozo, Álvaro; Oñaderra, Mercedes; Gavilanes, José G.; Proft, Thomas
Ribotoxins are a family of fungal extracellular ribonucleases which inactivate ribosomes by specifically cleaving a single phosphodiester bond located at the universally conserved sarcin/ricin loop of the large rRNA. The subsequent inhibition of protein biosynthesis is followed by cell death via apoptosis. Ribotoxins are also able to interact with membranes containing acid phospholipids, their cytotoxicity being preferentially directed towards cells showing altered membrane permeability, e.g. transformed or virus infected cells. Many features of their cytotoxic action and their ribonucleolytic mechanism have been elucidated by comparison with other extracellular non toxic fungal RNases, best represented by RNase T1. The study of structure-function relationships in ribotoxins is of particular interest, since they are postulated as potential therapeutic agents against different human pathologies. The production of hypoallergenic variants with application in several Aspergillus-related allergic syndromes and the construction of immunotoxins against different carcinomas are promising examples of such potential therapeutic utilisation.