Structural analysis of a type 1 ribosome inactivating protein reveals multiple L‑asparagine‑N‑acetyl‑D‑glucosamine monosaccharide modifications: Implications for cytotoxicity

Abstract

Pokeweed antiviral protein (PAP) belongs to the family of type I ribosome‑inactivating proteins (RIPs): Ribotoxins, which function by depurinating the sarcin‑ricin loop of ribosomal RNA. In addition to its antibacterial and antifungal properties, PAP has shown promise in antiviral and targeted tumor therapy owing to its ability to depurinate viral RNA and eukaryotic rRNA. Several PAP genes are differentially expressed across pokeweed tissues, with natively isolated seed forms of PAP exhibiting the greatest cytotoxicity. To help elucidate the molecular basis of increased cytotoxicity of PAP isoenzymes from seeds, the present study used protein sequencing, mass spectroscopy and X-ray crystallography to determine the complete covalent structure and 1.7 Å X‑ray crystal structure of PAP‑S1aci isolated from seeds of Asian pokeweed (Phytolacca acinosa). PAP‑S1aci shares ~95% sequence identity with PAP‑S1 from P. americana and contains the signature catalytic residues of the RIP superfamily, corresponding to Tyr72, Tyr122, Glu175 and Arg178 in PAP‑S1aci. A rare proline substitution (Pro174) was identified in the active site of PAP‑S1aci, which has no effect on catalytic Glu175 positioning or overall active‑site topology, yet appears to come at the expense of strained main‑chain geometry at the pre‑proline residue Val173. Notably, a rare type of N‑glycosylation was detected consisting of N‑acetyl‑D‑glucosamine monosaccharide residues linked to Asn10, Asn44 and Asn255 of PAP‑S1aci. Of note, our modeling studies suggested that the ribosome depurination activity of seed PAPs would be adversely affected by the N‑glycosylation of Asn44 and Asn255 with larger and more typical oligosaccharide chains, as they would shield the rRNA‑binding sites on the protein. These results, coupled with evidence gathered from the literature, suggest that this type of minimal N‑glycosylation in seed PAPs and other type I seed RIPs may serve to enhance cytotoxicity by exploiting receptor‑mediated uptake pathways of seed predators while preserving ribosome affinity and rRNA recognition.

Figure 1

Example of the analysis of glycosylation at Asn10 by tandem MS. MS/MS spectrum of peptide INTITFDAGNATINK with HexNAc attached to Asn10. The two ion series result either from fragmentation of peptide-HexNAc or by initial loss of HexNAc. Accordingly, there are two series of fragment ions in the corresponding parts of the spectrum; those assigned to peptides with the initial loss of HexNAc are marked by apostrophes. Open circles indicate peaks corresponding to loss of water from the indicated fragment ion. P, parent ion. Numerical values for the two series of fragment ions are provided in the accompanying table, with those identified in the spectrum highlighted in boxes. HexNAc, N-acetylhexosamine; MS, mass spectrometry.

Figure 2

X-ray sequencing of PAP-S1_aci at positions 65, 161 and 260. The final model is presented as ball-and-stick and shown in CPK coloring. Water molecules are depicted as red spheres and H-bonds are presented as dashed lines. Simulated-annealing omit maps (F_o-F_c; green; 3σ) are superimposed over the final model. (A) A Leu→Ile correction was applied to residue 65 on the basis of clear electron density features. (B) Electron density and a clear hydrogen-bond network prompted an Ile→Thr correction at position 161. (C) Electron density at position 260 indicated the presence of Thr rather than Ala.

Figure 3

Sequence and structure of PAP-S1_aci. (A) Pairwise sequence alignment between PAP-S1_aci and PAP-S1. The secondary structure of PAP-S1_aci is given above the alignment [α-helices and 3₁₀ (η) helices are presented as squiggles, β-strands as arrows and β-turns as 'TT']. Identical residues are presented on a red background; differences are presented on a yellow background. The three glycosylated Asn residues are framed in magenta. Relative solvent accessibility (acc) for residues of PAP-S1_aci are given below the sequence alignment as a color-coded bar [full accessibility (blue), intermediate (cyan) and buried (white)]. Black stars indicate residues involved in crystal contacts (<4.2 Å from symmetry-related protein atoms) in the PAP-S1_aci crystal form. (B) The 1.7 Å crystal structure of PAP-S1_aci is depicted in the ribbon diagram (purple, helices; orange, β-strands; green, loops). Secondary structure elements are numbered according to the corresponding sequence alignment. The N- and C-termini are labeled and disulfide bonds are shown in yellow (ball-and-stick). Also presented as a ball-and-stick model are the three glycosylated Asn residues (Asn10, Asn45 and Asn255-GlcNAc). GlcNAc, N-acetylglucosamine.

Figure 4

Electron density features of the three N-linked GlcNAc residues in PAP-S1_aci. σ_A-weighted 2F_o-F_c (blue; 1 σ) and F_o-F_c (green; 3 σ) electron density maps are shown, with the final PAP-S1_aci model presented as ball-and-stick. (A) Weak density was observed for an N-linked GlcNAc at Asn10. (B) The Asn44 side-chain and linked sugar are disordered. (C) The Asn255-GlcNAc303 linkage is clearly defined. The GlcNAc moiety is labeled 'NAG'; symmetry-related atoms are labeled 'sym' and distinguished in green. GlcNAc, N-acetylglucosamine.

Figure 5

Active site of PAP-S1_aci. (A) Superposition between PAP-S1_aci (grey) and PAP-S1 (green; Protein Data Bank code 1gik). Active-site residues for PAP-S1_aci are labeled. (B) Electron density features in the vicinity of Val173, a pre-proline Ramachandran outlier according to MolProbity analysis (24). The final model is shown in standard CPK coloring. The final σ_A-weighted 2F_o-F_c electron density map (cyan; 1.5σ) is overlaid. H-bonds are shown as dashed lines, with corresponding distances between non-hydrogen atoms given in Å.