RESUMO
BACKGROUND: Sambucus ebulus is a rich source of ribosome-inactivating proteins (RIPs) and RIP-related lectins generated from multiple genes. These proteins differ in their structure, enzymatic activity and sugar binding specificity. METHODS: We have purified and characterized ebulin-RP from S. ebulus leaves and determined the amino acid sequence by cDNA cloning. Cytotoxicity was studied in a variety of cancer cells and a comparative study of the ability of ebulin-RP to bind sugars using "in vitro" and "in silico" approaches was performed. RESULTS: Ebulin-RP is a novel heterodimeric type 2 RIP present in S. ebulus leaves together with the type 2 RIP ebulin l, which displayed rRNA N-glycosidase activity but unlike ebulin l, lacked functional sugar binding domains. As a consequence of changes in its B-chain, ebulin-RP displayed lower cytotoxicity than ebulin l towards cancer cells and induced apoptosis as the predominant pattern of cell death. CONCLUSIONS: Ebulin-RP is a novel member of the ebulin gene family with low cytotoxicity as a result of deficient sugar binding domains. Type 2 RIP genes from Sambucus have evolved to render proteins with different sugar affinities that may be related to different biological activities and could result in an advantage for the plant. GENERAL SIGNIFICANCE: The ebulin family of RIPs and lectins can serve as a good model for studying the evolutionary process which may have occurred in RIPs. The lack of cytotoxicity of ebulin-RP makes it a good candidate as a toxic moiety in the construction of immunotoxins and conjugates directed against specific targets.
Assuntos
Citotoxinas/isolamento & purificação , Proteínas Inativadoras de Ribossomos Tipo 2/isolamento & purificação , Sambucus/enzimologia , Açúcares/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Sítios de Ligação , Linhagem Celular , Linhagem Celular Tumoral , Sistema Livre de Células , Citotoxinas/química , Citotoxinas/metabolismo , Citotoxinas/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Evolução Molecular , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Modelos Moleculares , Simulação de Acoplamento Molecular , Ácidos Nucleicos/efeitos dos fármacos , Filogenia , Folhas de Planta/enzimologia , Conformação Proteica , Domínios Proteicos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Proteínas Inativadoras de Ribossomos Tipo 2/química , Proteínas Inativadoras de Ribossomos Tipo 2/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 2/farmacologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Ribosome-inactivating proteins (RIPs) are a family of enzymes that trigger the catalytic inactivation of ribosomes. The most known member of the family is the highly poisonous two-chain ricin isolated from Ricinus communis L. Sambucus species contain a number of two-chain RIPs structurally and enzymatically related to ricin which have the noteworthy feature that, having an enzymatic activity on ribosomes, leading to the inhibition of protein synthesis, higher than ricin, they are lacking of the tremendous unspecific toxicity of ricin. Therefore, they have been called non-toxic type 2 RIPs. The most representative and studied members are nigrin b present in the bark of the common (black) elder Sambucus nigra L. and ebulin 1 present in the leaves of the dwarf elder Sambucus ebulus L. The molecular basis for the low unspecific activities of nigrin b and ebulin 1 as compared with ricin seems to be related with single changes of amino acids in the high affinity sugar binding sites of the B chains. These changes determine the intracellular traffic of these proteins and thus the cellular toxicity. Conjugation ofnigrin b or ebulin 1 to either transferrin or monoclonal antibodies provided highly active conjugates targeting cancer. Thus these non-toxic type 2 RIPs are promising tools for cancer therapy.
