Ricin A-Chain Substrate Specificity in RNA, DNA, and Hybrid Stem−Loop Structures

Abstract

Ricin toxin A-chain (RTA) is the catalytic subunit of ricin, a heterodimeric toxin from castor beans. Its ribosomal inactivating activity arises from depurination of a single adenine from position A(4324) in a GAGA tetraloop from 28S ribosomal RNA. Minimal substrate requirements are the GAGA tetraloop and stem of two or more base pairs. Depurination activity also occurs on stem-loop DNA with the same sequence, but with the k(cat) reduced 200-fold. Systematic variation of RNA 5'-G(1)C(2)G(3)C(4)[G(5)A(6)G(7)A(8)]G(9)C(10)G(11)C(12)-3' 12mers via replacement of each nucleotide in the tetraloop with a deoxynucleotide showed a 16-fold increase in k(cat) for A(6) --> dA(6) but reduced k(cat) up to 300-fold for the other sites. Methylation of individual 2'-hydroxyls in a similar experiment reduced k(cat) by as much as 3 x 10(-3)-fold. In stem-loop DNA, replacement of d[G(5)A(6)G(7)A(8)] with individual ribonucleotides resulted in small kinetic changes, except for the dA(6) --> A(6) replacement for which k(cat) decreased 6-fold. Insertion of d[G(5)A(6)G(7)A(8)] into an RNA stem-loop or G(5)A(6)G(7)A(8) into a DNA stem-loop reduced k(cat) by 30- and 5-fold, respectively. Multiple substitutions of deoxyribonucleotides into RNA stem-loops in one case (dG(5),dG(7)) decreased k(cat)/K(m) by 10(5)-fold, while a second change (dG(5),dA(8)) decreased k(cat) by 100-fold. Mapping these interactions on the structure of GAGA stem-loop RNA suggests that all the loop 2'-hydroxyl groups play a significant role in the action of ricin A-chain. Improved binding of RNA-DNA stem-loop hybrids provides a scaffold for inhibitor design. Replacing the adenosine of the RTA depurination site with deoxyadenosine in a small RNA stem-loop increased k(cat) 20-fold to 1660 min(-1), a value similar to RTA's k(cat) on intact ribosomes.

An activity-dependent assay for ricin and related RNA N-glycosidases based on electrochemiluminescence

“…If the opposite was true, false negative signals would be possible with high concentrations of ricin in test samples. The high apparent specificity of ricin toward the dA target residue is consistent with previous findings [17,18]. Indeed, these rate differences may account for the undetectable activity with ricin and RNA GAGA as tested in our study (Fig.…”

“…reagents used in the assay were similarly optimized (not shown). Previous research showed that RNA substrates for ricin A chain containing a GdAGA loop exhibited large rate enhancements relative to substrates with a GAGA loop [17,18]. Therefore, we designed a synthetic biotinylated RNA substrate containing a 2 0 -deoxyadenosine (dA) residue within the four-residue loop, GdAGA, in a hairpin structure (RNA GdAGA; Table 1).…”

“…Thus, commercially prepared substrate is sufficiently pure to achieve the required sensitivity. Amukele and Schramm [17] showed that dA undergoes abiotic hydrolysis (deadenylation) much faster than A residues at pH 4. Longer incubations (6 h; not shown) did not improve the sensitivity due to a higher background signal, which we attributed primarily to abiotic hydrolysis of RNA GdAGA.…”

“…However, the DNA substrate allows a (minimal) detectable ricin concentration that is about two orders of magnitude higher than the concentration detectable with RNA GdAGA (data not shown). Similarly, the DNA substrate ''dA12'' was reported to have a much lower turnover number (k cat ) for ricin A chain than an RNA substrate with a GdAGA loop [17].…”

Ricin

“…In addition to the highly specific action on ribosomes, ricin and related RIPs have a less specific action in vitro on super-coiled double-stranded DNA (dsDNA), dsDNA, single-stranded DNA (ssDNA), and RNA substrates releasing multiple adenine residues and, in some instances, guanine residues (Wang and Tumer, 1999). RTA also catalyzes the hydrolysis of synthetic oligonucleotides as short as 6 base pairs, provided a GAGA tetra loop is present (Amukele and Schramm, 2004). Other reported RIP activities are lipase, chitinase, and superoxide dismutase (Xu et al, 2008).…”