RESUMEN
Alpha-sarcin and ricin represent two structurally and mechanistically distinct families of site-specific enzymes that block translation by irreversibly modifying the sarcin/ricin loop (SRL) of 23S-28S rRNA. alpha-Sarcin family enzymes are designated as ribotoxins and act as endonucleases. Ricin family enzymes are designated as ribosome inactivating proteins (RIP) and act as N-glycosidases. Recently, we demonstrated that basic surface residues of the ribotoxin restrictocin promote rapid and specific ribosome targeting by this endonuclease. Here, we report that three RIP: ricin A, saporin, and gypsophilin depurinate the ribosome with strong salt sensitivity and achieve unusually fast kcat/Km approximately 10(9)-10(10) M(-1) s(-1), implying that RIP share with ribotoxins a common mechanism of electrostatically facilitated ribosome targeting. Bioinformatics analysis of RIP revealed that surface charge properties correlate with the presence of the transport chain in the RIP molecule, suggesting a second role for the surface charge in RIP transport. These findings put forward surface electrostatics as an important determinant of RIP activity.
Asunto(s)
Endorribonucleasas/química , Proteínas Fúngicas/química , Familia de Multigenes/fisiología , N-Glicosil Hidrolasas/química , N-Glicosil Hidrolasas/fisiología , Proteínas de Plantas/química , Proteínas de Plantas/fisiología , Inhibidores de la Síntesis de la Proteína/química , Ribosomas/metabolismo , Ricina/química , Ésteres del Ácido Sulfúrico/química , Triterpenos/química , Endorribonucleasas/fisiología , Proteínas Fúngicas/fisiología , N-Glicosil Hidrolasas/clasificación , Proteínas de Plantas/clasificación , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Inactivadoras de Ribosomas Tipo 1 , Ribosomas/química , Ricina/clasificación , Ricina/farmacología , Saporinas , Electricidad Estática , Ésteres del Ácido Sulfúrico/clasificación , Ésteres del Ácido Sulfúrico/farmacología , Propiedades de Superficie , Triterpenos/clasificación , Triterpenos/farmacologíaAsunto(s)
Síndrome de Fuga Capilar/tratamiento farmacológico , Endotelio/efectos de los fármacos , Inmunotoxinas/genética , Ingeniería de Proteínas/métodos , Ricina/administración & dosificación , Animales , Síndrome de Fuga Capilar/inducido químicamente , Inmunotoxinas/administración & dosificación , Inmunotoxinas/química , Inmunotoxinas/toxicidad , Pulmón/irrigación sanguínea , Pulmón/efectos de los fármacos , Linfoma/tratamiento farmacológico , Ratones , Ratones SCID , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/toxicidad , Ricina/química , Ricina/clasificación , Ricina/genéticaRESUMEN
The lectin isolated from the phytopathogenic basidiomycete Rhizoctonia solani (RSA) is a homodimer of two noncovalently associated monomers of 15.5 kDa. RSA is a basic protein (pI > 9) which consists mainly of beta-sheets. A presumed relationship with ricin-B is supported by the sequence similarity between the N-terminus of RSA and the N-terminal subdomain of ricin-B. Hydrophobic cluster analysis confirms that the N-terminus of both proteins has a comparable folding. RSA exhibits specificity towards Gal/GalNAc whereby the hydroxyls at the C3', C4', and C6' positions of the pyranose ring play a key role in the interaction with simple sugars. The carbohydrate-binding site of RSA apparently accommodates only a single sugar unit. Our results demonstrate an obvious evolutionary relationship between some fungal and plant lectins, but also provide evidence for the occurrence of a lectin consisting of subunits corresponding to a single subdomain of ricin-B.