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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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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.
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f1-mmr-12-04-5737: 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.

Mentions: It was difficult to identify amino-acid residues at three positions within the PAP-S1aci sequence (residues 10, 44 and 255). These positions were characterized by low yields of phenylthiohydantoin (PTH)-amino acids with a similar elution profile to PTH-Asp. Although this may be indicative of any post-translational modification, they could be identified as Asn-GlcNAc linkages - an extremely rare type of N-glycosylation. Large carbohydrates attached to Asn residues may prevent extraction of the corresponding PTH-Asn-oligosaccharide from the cartridge (genuine 'empty' cycle), however, PTH-Asn-GlcNAc is extractable (albeit at a lower yield), and would possibly elute earlier than PTH-Asn in reversed-phase HPLC. Definitive evidence for this assignment at each position in the sequence was provided by tandem MS. An example of the MS/MS spectrum for the N-terminal glycosylated peptide having the structure INTITFDAG [N-HexNAc (L-asparagine-N-acetyl-hexosamine)]ATINK is shown in Fig. 1, where extensive sequence coverage by fragments generated following the initial loss of HexNAc as well as those containing the modification is evident.


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)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1-mmr-12-04-5737: 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.
Mentions: It was difficult to identify amino-acid residues at three positions within the PAP-S1aci sequence (residues 10, 44 and 255). These positions were characterized by low yields of phenylthiohydantoin (PTH)-amino acids with a similar elution profile to PTH-Asp. Although this may be indicative of any post-translational modification, they could be identified as Asn-GlcNAc linkages - an extremely rare type of N-glycosylation. Large carbohydrates attached to Asn residues may prevent extraction of the corresponding PTH-Asn-oligosaccharide from the cartridge (genuine 'empty' cycle), however, PTH-Asn-GlcNAc is extractable (albeit at a lower yield), and would possibly elute earlier than PTH-Asn in reversed-phase HPLC. Definitive evidence for this assignment at each position in the sequence was provided by tandem MS. An example of the MS/MS spectrum for the N-terminal glycosylated peptide having the structure INTITFDAG [N-HexNAc (L-asparagine-N-acetyl-hexosamine)]ATINK is shown in Fig. 1, where extensive sequence coverage by fragments generated following the initial loss of HexNAc as well as those containing the modification is evident.

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