Sapovaccarin-S1 and -S2, Two Type I RIP Isoforms from the Seeds of Saponaria vaccaria L.

Abstract

Type I ribosome-inactivating proteins (RIPs) are plant toxins that inhibit protein synthesis by exerting rRNA N-glycosylase activity (EC 3.2.2.22). Due to the lack of a cell-binding domain, type I RIPs are not target cell-specific. However once linked to antibodies, so called immunotoxins, they are promising candidates for targeted anti-cancer therapy. In this study, sapovaccarin-S1 and -S2, two newly identified type I RIP isoforms differing in only one amino acid, were isolated from the seeds of Saponaria vaccaria L. Sapovaccarin-S1 and -S2 were purified using ammonium sulfate precipitation and subsequent cation exchange chromatography. The determined molecular masses of 28,763 Da and 28,793 Da are in the mass range typical for type I RIPs and the identified amino acid sequences are homologous to known type I RIPs such as dianthin 30 and saporin-S6 (79% sequence identity each). Sapovaccarin-S1 and -S2 showed adenine-releasing activity and induced cell death in Huh-7 cells. In comparison to other type I RIPs, sapovaccarin-S1 and -S2 exhibited a higher thermostability as shown by nano-differential scanning calorimetry. These results suggest that sapovaccarin-S1 and -S2 would be optimal candidates for targeted anti-cancer therapy.

Mutational Analysis of RIP Type I Dianthin-30 Suggests a Role for Arg24 in Endocytosis

“…Arginine at position 24 is not only preserved in dianthin-30 but also in saporin-S6, as previously shown [55]. In addition, the alignment of dianthin-30 with common type I and type II RIPs indicates that Arg24 is also conserved in sapovaccarin-S1, gelonin, gypsophilin-S, agrostin, bouganin, abrin A chain and ricin A chain [34,46,[68][69][70][71][72][73]. Thus, Arg24 is even found in type I RIPs like agrostin, which have an amino acid sequence that is unusual for the latter ones.…”

Biological Activities of Ribosome-Inactivating Proteins

“…Four examples of these type 1 RIPs are described in this Special Issue: the curcins from the euphorbiaceous Jatropha curcas L. [ 11 ], the sodins from the amaranthaceous Salsola soda L. [ 12 ], the sapovaccarins from the caryophyllaceous Vaccaria hispanica (Mill.) Rauschert (= Saponaria vaccaria L.) [ 13 ], and the PD-Ls from the phytolaccaceaous Phytolacca dioica L. [ 14 ].…”

“…Schlaak et al isolated a new type 1 RIP from S. vaccaria seeds homologous to type 1 RIPs from other caryophyllaceous that, similar to dianthin 30 and saporin-S6, are used for the construction of immunotoxins [ 13 ]. Compared to other type 1 RIPs, they exhibited greater thermostability, suggesting that they would be optimal candidates for targeted cancer therapy.…”