A novel adenine-releasing assay for ribosome-inactivating proteins.

Ribosome-inactivating proteins (RIPs) are toxic enzymes that are mostly biosynthesized by plants. RIPs are N-glycosidases that cleave an essential adenine molecule from the 28S rRNA. This is followed by the irreversible inhibition of protein synthesis leading to cell death. By fusing RIPs to cancer cell specific targeting ligands RIPs have been utilized for targeted anti-tumor therapy. The anti-tumoral efficiency of such conjugates depends significantly on the N-glycosidase activity of the RIP domain. Different methods have been developed in order to determine the N-glycosidase activity of RIPs and RIP domain containing anti-tumor toxins. However the existing methods are elaborate and include radioassays, HPLC and enzymatic conversion assays. Here, a simple and cost effective N-glycosidase assay is presented, which is based on the direct determination of the released adenine by thin-layer chromatography (TLC) and TLC-densitometry. An adenine based single stranded oligonucleotide is used as substrate. Following TLC development the released adenine is quantified on silica glass plates by UV absorbance at 260nm.

Transition State Structure of RNA Depurination by Saporin L3.

Saporin L3 from the leaves of the common soapwort is a catalyst for hydrolytic depurination of adenine from RNA. Saporin L3 is a type 1 ribosome inactivating protein (RIP) composed only of a catalytic domain. Other RIPs have been used in immunotoxin cancer therapy, but off-target effects have limited their development. In the current study, we use transition state theory to understand the chemical mechanism and transition state structure of saporin L3. In favorable cases, transition state structures guide the design of transition state analogues as inhibitors. Kinetic isotope effects (KIEs) were determined for an A14C mutant of saporin L3. To permit KIE measurements, small stem-loop RNAs that contain an AGGG tetraloop structure were enzymatically synthesized with the single adenylate bearing specific isotopic substitutions. KIEs were measured and corrected for forward commitment to obtain intrinsic values. A model of the transition state structure for depurination of stem-loop RNA (5'-GGGAGGGCCC-3') by saporin L3 was determined by matching KIE values predicted via quantum chemical calculations to a family of intrinsic KIEs. This model indicates saporin L3 displays a late transition state with the N-ribosidic bond to the adenine nearly cleaved, and the attacking water nucleophile weakly bonded to the ribosyl anomeric carbon. The transition state retains partial ribocation character, a feature common to most N-ribosyl transferases. However, the transition state geometry for saporin L3 is distinct from ricin A-chain, the only other RIP whose transition state is known.

Soapwort Saporin L3 Expression in Yeast, Mutagenesis, and RNA Substrate Specificity.

Saporin L3 from Saponaria officinalis (soapwort) leaves is a type 1 ribosome-inactivating protein. It catalyzes the hydrolysis of oligonucleotide adenylate N-ribosidic bonds to release adenine from rRNA. Depurination sites include both adenines in the GAGA tetraloop of short sarcin-ricin stem-loops and multiple adenines within eukaryotic rRNA, tRNAs, and mRNAs. Multiple Escherichia coli vector designs for saporin L3 expression were attempted but demonstrated high toxicity even during plasmid maintenance and selection in E. coli nonexpression strains. Saporin L3 is >10(3) times more efficient at RNA deadenylation on short GAGA stem-loops than saporin S6, the saporin isoform currently used in immunotoxin clinical trials. We engineered a construct for the His-tagged saporin L3 to test for expression in Pichia pastoris when it is linked to the protein export system for the yeast α-mating factor. DNA encoding saporin L3 was cloned into a pPICZαB expression vector and expressed in P. pastoris under the alcohol dehydrogenase AOX1 promoter. A fusion protein of saporin L3 containing the pre-pro-sequence of the α-mating factor, the c-myc epitope, and the His tag was excreted from the P. pastoris cells and isolated from the culture medium. Autoprocessing of the α-mating factor yielded truncated saporin L3 (amino acids 22-280), the c-myc epitope, and the His tag expressed optimally as a 32 kDa construct following methanol induction. Saporin L3 was also expressed with specific alanines and/or serines mutated to cysteine. Native and Cys mutant saporins are kinetically similar. The recombinant expression of saporin L3 and its mutants permits the production and investigation of this high-activity ribosome-inactivating protein.