Identification, chemical synthesis, structure, and function of a new KV1 channel blocking peptide from Oulactis sp.

Punnepalli Sunanda, Bankala Krishnarjuna, Steve Peigneur, Michela L. Mitchell, Rosendo Estrada, Jessica Villegas-Moreno, Michael W. Pennington, Jan Tytgat, Raymond S. Norton

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Rapid progress in transcriptomic and proteomic studies of sea anemones has led to the identification of a large number of new peptide sequences. Some of these peptides have high sequence similarity and identical cysteine frameworks to those of previously reported sequences. One such peptide we have identified from a transcriptomic study of Oulactis sp is OspTx2a, which has a cysteine framework similar to that of ShK (from Stichodactyla helianthus) and BgK (from Bunodosoma granulifera). This peptide was made using solid-phase peptide synthesis, but, upon oxidative folding, it generated two peptides with identical masses (OspTx2a-p1 and OspTx2a-p2) that were distinguishable by high-performance liquid chromatography. The structures of OspTx2a-p1 and OspTx2a-p2 were determined using nuclear magnetic resonance spectroscopy, and voltage-clamp electrophysiology assays were performed in order to assess the activity against a range of potassium channels. The structures of the two peptides were very similar to each other and to BgK, with the same disulfide bond connectivities, and both had an all-trans backbone conformation. In functional assays, both OspTx2a-p1 and OspTx2a-p2 inhibited K V 1.2 and K V 1.6 channel currents at low µM concentrations, with similar but not identical IC 50 values. Peptides containing a C-terminal Cys residue are particularly sensitive to racemization at this residue, and the two products obtained for OspTx2a could be a consequence of racemization of the Cys residue at its C-terminus during synthesis. NMR chemical shift differences between the two products and their structural preferences for a d-Cys residue were consistent with this interpretation.

Original languageEnglish
Article numbere24073
Number of pages10
JournalPeptide Science
Volume110
Issue number4
DOIs
Publication statusPublished - 1 Jul 2018

Keywords

  • cysteine-rich peptide
  • isomers
  • K channel
  • NMR spectroscopy
  • OspTx2a
  • sea anemone
  • structure

Cite this

Sunanda, P., Krishnarjuna, B., Peigneur, S., Mitchell, M. L., Estrada, R., Villegas-Moreno, J., ... Norton, R. S. (2018). Identification, chemical synthesis, structure, and function of a new KV1 channel blocking peptide from Oulactis sp. Peptide Science, 110(4), [e24073]. https://doi.org/10.1002/pep2.24073
Sunanda, Punnepalli ; Krishnarjuna, Bankala ; Peigneur, Steve ; Mitchell, Michela L. ; Estrada, Rosendo ; Villegas-Moreno, Jessica ; Pennington, Michael W. ; Tytgat, Jan ; Norton, Raymond S. / Identification, chemical synthesis, structure, and function of a new KV1 channel blocking peptide from Oulactis sp. In: Peptide Science. 2018 ; Vol. 110, No. 4.
@article{3f1db34eb9764c18972e733ac2755306,
title = "Identification, chemical synthesis, structure, and function of a new KV1 channel blocking peptide from Oulactis sp.",
abstract = "Rapid progress in transcriptomic and proteomic studies of sea anemones has led to the identification of a large number of new peptide sequences. Some of these peptides have high sequence similarity and identical cysteine frameworks to those of previously reported sequences. One such peptide we have identified from a transcriptomic study of Oulactis sp is OspTx2a, which has a cysteine framework similar to that of ShK (from Stichodactyla helianthus) and BgK (from Bunodosoma granulifera). This peptide was made using solid-phase peptide synthesis, but, upon oxidative folding, it generated two peptides with identical masses (OspTx2a-p1 and OspTx2a-p2) that were distinguishable by high-performance liquid chromatography. The structures of OspTx2a-p1 and OspTx2a-p2 were determined using nuclear magnetic resonance spectroscopy, and voltage-clamp electrophysiology assays were performed in order to assess the activity against a range of potassium channels. The structures of the two peptides were very similar to each other and to BgK, with the same disulfide bond connectivities, and both had an all-trans backbone conformation. In functional assays, both OspTx2a-p1 and OspTx2a-p2 inhibited K V 1.2 and K V 1.6 channel currents at low µM concentrations, with similar but not identical IC 50 values. Peptides containing a C-terminal Cys residue are particularly sensitive to racemization at this residue, and the two products obtained for OspTx2a could be a consequence of racemization of the Cys residue at its C-terminus during synthesis. NMR chemical shift differences between the two products and their structural preferences for a d-Cys residue were consistent with this interpretation.",
keywords = "cysteine-rich peptide, isomers, K channel, NMR spectroscopy, OspTx2a, sea anemone, structure",
author = "Punnepalli Sunanda and Bankala Krishnarjuna and Steve Peigneur and Mitchell, {Michela L.} and Rosendo Estrada and Jessica Villegas-Moreno and Pennington, {Michael W.} and Jan Tytgat and Norton, {Raymond S.}",
year = "2018",
month = "7",
day = "1",
doi = "10.1002/pep2.24073",
language = "English",
volume = "110",
journal = "Peptide Science",
issn = "2475-8817",
publisher = "American Peptide Society",
number = "4",

}

Sunanda, P, Krishnarjuna, B, Peigneur, S, Mitchell, ML, Estrada, R, Villegas-Moreno, J, Pennington, MW, Tytgat, J & Norton, RS 2018, 'Identification, chemical synthesis, structure, and function of a new KV1 channel blocking peptide from Oulactis sp.' Peptide Science, vol. 110, no. 4, e24073. https://doi.org/10.1002/pep2.24073

Identification, chemical synthesis, structure, and function of a new KV1 channel blocking peptide from Oulactis sp. / Sunanda, Punnepalli; Krishnarjuna, Bankala; Peigneur, Steve; Mitchell, Michela L.; Estrada, Rosendo; Villegas-Moreno, Jessica; Pennington, Michael W.; Tytgat, Jan; Norton, Raymond S.

In: Peptide Science, Vol. 110, No. 4, e24073, 01.07.2018.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Identification, chemical synthesis, structure, and function of a new KV1 channel blocking peptide from Oulactis sp.

AU - Sunanda, Punnepalli

AU - Krishnarjuna, Bankala

AU - Peigneur, Steve

AU - Mitchell, Michela L.

AU - Estrada, Rosendo

AU - Villegas-Moreno, Jessica

AU - Pennington, Michael W.

AU - Tytgat, Jan

AU - Norton, Raymond S.

PY - 2018/7/1

Y1 - 2018/7/1

N2 - Rapid progress in transcriptomic and proteomic studies of sea anemones has led to the identification of a large number of new peptide sequences. Some of these peptides have high sequence similarity and identical cysteine frameworks to those of previously reported sequences. One such peptide we have identified from a transcriptomic study of Oulactis sp is OspTx2a, which has a cysteine framework similar to that of ShK (from Stichodactyla helianthus) and BgK (from Bunodosoma granulifera). This peptide was made using solid-phase peptide synthesis, but, upon oxidative folding, it generated two peptides with identical masses (OspTx2a-p1 and OspTx2a-p2) that were distinguishable by high-performance liquid chromatography. The structures of OspTx2a-p1 and OspTx2a-p2 were determined using nuclear magnetic resonance spectroscopy, and voltage-clamp electrophysiology assays were performed in order to assess the activity against a range of potassium channels. The structures of the two peptides were very similar to each other and to BgK, with the same disulfide bond connectivities, and both had an all-trans backbone conformation. In functional assays, both OspTx2a-p1 and OspTx2a-p2 inhibited K V 1.2 and K V 1.6 channel currents at low µM concentrations, with similar but not identical IC 50 values. Peptides containing a C-terminal Cys residue are particularly sensitive to racemization at this residue, and the two products obtained for OspTx2a could be a consequence of racemization of the Cys residue at its C-terminus during synthesis. NMR chemical shift differences between the two products and their structural preferences for a d-Cys residue were consistent with this interpretation.

AB - Rapid progress in transcriptomic and proteomic studies of sea anemones has led to the identification of a large number of new peptide sequences. Some of these peptides have high sequence similarity and identical cysteine frameworks to those of previously reported sequences. One such peptide we have identified from a transcriptomic study of Oulactis sp is OspTx2a, which has a cysteine framework similar to that of ShK (from Stichodactyla helianthus) and BgK (from Bunodosoma granulifera). This peptide was made using solid-phase peptide synthesis, but, upon oxidative folding, it generated two peptides with identical masses (OspTx2a-p1 and OspTx2a-p2) that were distinguishable by high-performance liquid chromatography. The structures of OspTx2a-p1 and OspTx2a-p2 were determined using nuclear magnetic resonance spectroscopy, and voltage-clamp electrophysiology assays were performed in order to assess the activity against a range of potassium channels. The structures of the two peptides were very similar to each other and to BgK, with the same disulfide bond connectivities, and both had an all-trans backbone conformation. In functional assays, both OspTx2a-p1 and OspTx2a-p2 inhibited K V 1.2 and K V 1.6 channel currents at low µM concentrations, with similar but not identical IC 50 values. Peptides containing a C-terminal Cys residue are particularly sensitive to racemization at this residue, and the two products obtained for OspTx2a could be a consequence of racemization of the Cys residue at its C-terminus during synthesis. NMR chemical shift differences between the two products and their structural preferences for a d-Cys residue were consistent with this interpretation.

KW - cysteine-rich peptide

KW - isomers

KW - K channel

KW - NMR spectroscopy

KW - OspTx2a

KW - sea anemone

KW - structure

UR - http://www.scopus.com/inward/record.url?scp=85047100105&partnerID=8YFLogxK

U2 - 10.1002/pep2.24073

DO - 10.1002/pep2.24073

M3 - Article

VL - 110

JO - Peptide Science

JF - Peptide Science

SN - 2475-8817

IS - 4

M1 - e24073

ER -

Sunanda P, Krishnarjuna B, Peigneur S, Mitchell ML, Estrada R, Villegas-Moreno J et al. Identification, chemical synthesis, structure, and function of a new KV1 channel blocking peptide from Oulactis sp. Peptide Science. 2018 Jul 1;110(4). e24073. https://doi.org/10.1002/pep2.24073