Limits...
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.

Show MeSH

Related in: MedlinePlus

Sequence and structure of PAP-S1aci. (A) Pairwise sequence alignment between PAP-S1aci and PAP-S1. The secondary structure of PAP-S1aci 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-S1aci 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-S1aci crystal form. (B) The 1.7 Å crystal structure of PAP-S1aci 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4581812&req=5

f3-mmr-12-04-5737: Sequence and structure of PAP-S1aci. (A) Pairwise sequence alignment between PAP-S1aci and PAP-S1. The secondary structure of PAP-S1aci 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-S1aci 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-S1aci crystal form. (B) The 1.7 Å crystal structure of PAP-S1aci 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.

Mentions: The sequence and X-ray structure of PAP-S1aci are presented in Fig. 3. The figure was generated with DSSP (25), Multalin (31) and ESPript 2.2 (32). PAP-S1aci contains invariant active-site residues of the RIP superfamily (corresponding to Tyr72, Tyr122, Glu175 and Arg178 in PAP-S1aci), which are critical for catalysis (15,33). Four conserved Cys residues forming two intramolecular disulfide bonds (Cys34-Cys258 and Cys84-Cys105) are also present. A protein-protein BLAST search (34) was performed to determine the closest known sequence relative, identifying P. americana PAP-S1 (30) with 96% sequence identity (250 of 261 residues) to PAP-S1aci (Fig. 3A). The 1.8 Å crystal structure of PAP-S1 from P. americana has been elucidated and includes a fully resolved carbohydrate structure consisting of three GlcNAc monosaccharide linkages at Asn10, Asn44 and Asn255 (17). The current results demonstrated that the same three N-glycosylation sequons are also found in PAP-S1aci and that each site is similarly modified with a single GlcNAc residue. In our crystal form there is clear electron density for the sugar at Asn255 due to its involvement in crystal packing (18,35), however, Asn10 and Asn45 are freely exposed to solvent and the corresponding electron density for the linked GlcNAc residues is weak (Fig. 4).


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)

Sequence and structure of PAP-S1aci. (A) Pairwise sequence alignment between PAP-S1aci and PAP-S1. The secondary structure of PAP-S1aci 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-S1aci 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-S1aci crystal form. (B) The 1.7 Å crystal structure of PAP-S1aci 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3-mmr-12-04-5737: Sequence and structure of PAP-S1aci. (A) Pairwise sequence alignment between PAP-S1aci and PAP-S1. The secondary structure of PAP-S1aci 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-S1aci 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-S1aci crystal form. (B) The 1.7 Å crystal structure of PAP-S1aci 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.
Mentions: The sequence and X-ray structure of PAP-S1aci are presented in Fig. 3. The figure was generated with DSSP (25), Multalin (31) and ESPript 2.2 (32). PAP-S1aci contains invariant active-site residues of the RIP superfamily (corresponding to Tyr72, Tyr122, Glu175 and Arg178 in PAP-S1aci), which are critical for catalysis (15,33). Four conserved Cys residues forming two intramolecular disulfide bonds (Cys34-Cys258 and Cys84-Cys105) are also present. A protein-protein BLAST search (34) was performed to determine the closest known sequence relative, identifying P. americana PAP-S1 (30) with 96% sequence identity (250 of 261 residues) to PAP-S1aci (Fig. 3A). The 1.8 Å crystal structure of PAP-S1 from P. americana has been elucidated and includes a fully resolved carbohydrate structure consisting of three GlcNAc monosaccharide linkages at Asn10, Asn44 and Asn255 (17). The current results demonstrated that the same three N-glycosylation sequons are also found in PAP-S1aci and that each site is similarly modified with a single GlcNAc residue. In our crystal form there is clear electron density for the sugar at Asn255 due to its involvement in crystal packing (18,35), however, Asn10 and Asn45 are freely exposed to solvent and the corresponding electron density for the linked GlcNAc residues is weak (Fig. 4).

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