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

Hogg T, Mendel JT, Lavezo JL - Mol Med Rep (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Education, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, TX 79905, USA.

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.

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Related in: MedlinePlus

X-ray sequencing of PAP-S1aci 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 (Fo-Fc; 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.
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f2-mmr-12-04-5737: X-ray sequencing of PAP-S1aci 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 (Fo-Fc; 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.

Mentions: The high-quality X-ray data allowed us to identify three positions in the sequence where the Edman/MS data were evidently erroneous (residues 65, 161 and 260). On the basis of clear electron density and/or H-bonding features, the original assignments of Leu65 (derived by Edman and MS/MS), Ile161 (Edman) and Ala260 (Edman) were corrected to Ile65, Thr161 and Thr260. Electron density at these positions is presented in Fig. 2, illustrating the utility of high-quality X-ray data as a complementary sequencing method, particularly for direct visualization of isomeric (Ile/Leu) or isobaric (Gln/Lys) residues, which are frequently misannotated by other protein sequencing methods.


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

Hogg T, Mendel JT, Lavezo JL - Mol Med Rep (2015)

X-ray sequencing of PAP-S1aci 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 (Fo-Fc; 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581812&req=5

f2-mmr-12-04-5737: X-ray sequencing of PAP-S1aci 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 (Fo-Fc; 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.
Mentions: The high-quality X-ray data allowed us to identify three positions in the sequence where the Edman/MS data were evidently erroneous (residues 65, 161 and 260). On the basis of clear electron density and/or H-bonding features, the original assignments of Leu65 (derived by Edman and MS/MS), Ile161 (Edman) and Ala260 (Edman) were corrected to Ile65, Thr161 and Thr260. Electron density at these positions is presented in Fig. 2, illustrating the utility of high-quality X-ray data as a complementary sequencing method, particularly for direct visualization of isomeric (Ile/Leu) or isobaric (Gln/Lys) residues, which are frequently misannotated by other protein sequencing methods.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Education, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, TX 79905, USA.

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.

Show MeSH
Related in: MedlinePlus