Isolation and characterization of four type-1 ribosome-inactivating proteins, with polynucleotide:adenosine glycosidase activity, from leaves of Phytolacca dioica L.

Abstract

Four type-1 (single-chain) ribosome-inactivating proteins (RIPs), with isoelectric points between 9.5 and 9.7, were isolated from leaves of Phytolacca dioica L. The purification procedure furnished the four proteins with an overall yield of about 16% and separated them from a protein of 29,407 +/- 2 Da, as determined by electrospray mass spectrometry, whose N-terminal amino acid sequence differed from that of pokeweed (Phytolacca americana L.) leaf chitinase (PLC-B) by only one amino acid (R17I). The four RIPs (PD-L1 to PD-L4) inhibited protein synthesis by a rabbit reticulocyte lysate with 50% inhibition at the picomolar level, and produced the beta-fragment, diagnostic of the specific enzymatic action of RIPs, on yeast ribosomes. Comparison of their N-terminal sequences, up to residue 45, showed that PD-L1 is identical to PD-L2 [designated the isoleucine (Ile) form from the N-terminal residue] and PD-L3 is identical to PD-L4 [designated the valine (Val) form from the N-terminal residue] and that there are 35 identical residues between the two forms. Furthermore, the Val form presents the same number of identical residues as PD-S2, an RIP isolated from the seeds of the same plant. With the exception of PD-L4, the purified RIPs gave a positive reaction when stained for sugars on SDS-PAGE gels and, when analyzed by electrospray mass spectrometry, had M(r) values of 32,715 +/- 1 (PD-L1), 31,542 +/- 1 (PD-L2), 30,356 +/- 1 (PD-L3) and 29,185 +/- 1 Da (PD-L4). The 1171 kDa difference in M(r), within the same RIP form, could be due to glycosylation. Like leaf saporins and many other RIPs, the four RIPs released several adenines from poly(A), herring sperm DNA and rRNA 16S + 23S, thus acting as polynucleotide:adenosine glycosidases. This property was less pronounced in PD-L1 and PD-L3 than in PD-L2 and PD-L4, respectively. The proteins PD-L1 and PD-L4 showed 3.7% reactivity with the antiserum anti-dianthin 32 and no reactivity with antisera to PAP-R saporin-S6, momordin 1 and even PD-S2, an RIP isolated from the seeds of the same plant. Protein PD-L4 showed 12.5% cross-reactivity with anti-PD-L1, while the opposite cross-reactivity was 100%.

Purification and enzymatic properties of a peroxidase from leaves of Phytolacca dioica L. (Ombú tree)

“…During the purification of type 1 ribosome-inactivating proteins from P. dioica leaves (19)(20)(21), we obtained different chromatographic profiles between summer (June) and autumn http://bmbreports.org BMB reports Fig. 1.…”

“…The purification procedure has been previously reported (19,21). Leaves (100 g) were homogenized in 500 ml of buffer A by 20 s bursts at full power using a Waring blender (Waring Products; Torrington, USA).…”

Crystal structure of PD‐L1, a ribosome inactivating protein from Phytolacca dioica L. Leaves with the property to induce DNA cleavage

“…30 The protein was concentrated to 10 mg mL 21 using a YM-10 centricon (Amicon Corp.) and stored at 277 K. Preliminary crystallization experiments were performed at 293 K using either the sitting drop or the hanging-drop vapor-diffusion methods and the commercially available sparse-matrix screens (Crystal Screen and Wizard kits I and II from Hampton Research and deCode Genetics, respectively). After optimization of the crystallization conditions by fine-tuning the protein and precipitant concentrations, only tiny needles could be obtained.…”

“…Highly purified PD-L1 and PD-L4 from P. dioica leaves, after Red-Sepharose chromatography, and endowed with adenine polynucleotide glycosylase activity 30,31 were used to carry out DNA cleavage experiments on pBR322. These activities were compared to that of the recombinant (i.e., not glycosylated, manuscript in preparation) PD-L1.…”

“…29 PD-L1-3 isoforms exhibit different degrees of glycosylation, whereas PD-L4 is not glycosylated. [30][31][32] Despite the high sequence identity of these proteins, PD-L1 and PD-L2 are able to cleave supercoiled double-stranded pBR322 DNA, whereas PD-L3 and PD-L4 are not. 7 Here, we report the crystal structure characterization as well as a functional study on DNA cleaving activity of native PD-L1 and its recombinant, non-glycosylated, form.…”