ABSTRACT
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.
Subject(s)
Endoribonucleases/chemistry , Fungal Proteins/chemistry , Multigene Family/physiology , N-Glycosyl Hydrolases/chemistry , N-Glycosyl Hydrolases/physiology , Plant Proteins/chemistry , Plant Proteins/physiology , Protein Synthesis Inhibitors/chemistry , Ribosomes/metabolism , Ricin/chemistry , Sulfuric Acid Esters/chemistry , Triterpenes/chemistry , Endoribonucleases/physiology , Fungal Proteins/physiology , N-Glycosyl Hydrolases/classification , Plant Proteins/classification , Protein Synthesis Inhibitors/pharmacology , Ribosome Inactivating Proteins, Type 1 , Ribosomes/chemistry , Ricin/classification , Ricin/pharmacology , Saporins , Static Electricity , Sulfuric Acid Esters/classification , Sulfuric Acid Esters/pharmacology , Surface Properties , Triterpenes/classification , Triterpenes/pharmacologySubject(s)
Capillary Leak Syndrome/drug therapy , Endothelium/drug effects , Immunotoxins/genetics , Protein Engineering/methods , Ricin/administration & dosage , Animals , Capillary Leak Syndrome/chemically induced , Immunotoxins/administration & dosage , Immunotoxins/chemistry , Immunotoxins/toxicity , Lung/blood supply , Lung/drug effects , Lymphoma/drug therapy , Mice , Mice, SCID , Mutagenesis, Site-Directed , Recombinant Proteins/administration & dosage , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/toxicity , Ricin/chemistry , Ricin/classification , Ricin/geneticsABSTRACT
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.
