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Non-specific electrostatic interactions and the conversion of horseshoe crab haemocyanin into a phenoloxidase

Christopher Coates, Jacqueline Nairn

Developmental & Comparative Immunology, Volume: 169, Start page: 105401

Swansea University Author: Christopher Coates

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DOI (Published version): 10.1016/j.dci.2025.105401

Abstract

Haemocyanin is a haemolymph (blood)-based protein and the functional equivalent to haemoglobin - supplying tissues with oxygen in decapod crustaceans, chelicerates, and shelled molluscs. In addition to oxygen transport, haemocyanin plays several roles in innate immunity, wound healing, and ecdysis....

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Published in: Developmental & Comparative Immunology
ISSN: 0145-305X 1879-0089
Published: Elsevier Ltd 2025
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URI: https://cronfa.swan.ac.uk/Record/cronfa69780
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last_indexed 2025-07-22T05:04:02Z
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spelling 2025-07-21T11:08:11.8362669 v2 69780 2025-06-20 Non-specific electrostatic interactions and the conversion of horseshoe crab haemocyanin into a phenoloxidase af160934b75bea5b8ba83d68b3d1a003 Christopher Coates Christopher Coates true false 2025-06-20 BGPS Haemocyanin is a haemolymph (blood)-based protein and the functional equivalent to haemoglobin - supplying tissues with oxygen in decapod crustaceans, chelicerates, and shelled molluscs. In addition to oxygen transport, haemocyanin plays several roles in innate immunity, wound healing, and ecdysis. Under certain conditions in vitro and in vivo, horseshoe crab (Limulus polyphemus) haemocyanin is converted into a phenoloxidase-like enzyme, yet the protein-ligand interactions associated with this conversion remain unclear. Negatively charged ligands, such as phosphatidylserine and sodium dodecyl sulphate, represent effective endogenous and exogenous activators, respectively. Herein, we explored the nature of the interaction between haemocyanin and phosphatidylserine. We used several spectroscopic techniques and phenoloxidase assays to follow the electrostatic interactions. Manipulating the ionic strength of the assay resulted in less enzyme activity, and reversed haemocyanin conformational changes associated with phosphatidylserine binding (confirmed by fluorescence emission spectra). The addition of wild type and rearranged peptides - mimicking the P181 to K196 region close to the active site of haemocyanin subunit II - to phenoloxidase assays resulted in less product (dopachrome) formation. We propose that non-specific electrostatic interactions between haemocyanin and endogenous activators such as phosphatidylserine facilitate the switch to a phenoloxidase-like enzyme. Journal Article Developmental & Comparative Immunology 169 105401 Elsevier Ltd 0145-305X 1879-0089 Limulus polyphemus; Innate immunity; Melanogenesis; Protein-phospholipid interactions; Peptide competition assays 1 8 2025 2025-08-01 10.1016/j.dci.2025.105401 COLLEGE NANME Biosciences Geography and Physics School COLLEGE CODE BGPS Swansea University Another institution paid the OA fee Some of the initial experimental work was financed by the University of Stirling and supplemented by Swansea University. 2025-07-21T11:08:11.8362669 2025-06-20T13:20:49.6110080 Faculty of Science and Engineering School of Biosciences, Geography and Physics - Biosciences Christopher Coates 1 Jacqueline Nairn 2 69780__34534__ea6b39b9b2a14158b8369674092df038.pdf 69780.VOR.pdf 2025-06-20T13:24:52.3014802 Output 3172991 application/pdf Version of Record true © 2025 The Authors. This is an open access article under the CC BY license. true eng http://creativecommons.org/licenses/by/4.0/
title Non-specific electrostatic interactions and the conversion of horseshoe crab haemocyanin into a phenoloxidase
spellingShingle Non-specific electrostatic interactions and the conversion of horseshoe crab haemocyanin into a phenoloxidase
Christopher Coates
title_short Non-specific electrostatic interactions and the conversion of horseshoe crab haemocyanin into a phenoloxidase
title_full Non-specific electrostatic interactions and the conversion of horseshoe crab haemocyanin into a phenoloxidase
title_fullStr Non-specific electrostatic interactions and the conversion of horseshoe crab haemocyanin into a phenoloxidase
title_full_unstemmed Non-specific electrostatic interactions and the conversion of horseshoe crab haemocyanin into a phenoloxidase
title_sort Non-specific electrostatic interactions and the conversion of horseshoe crab haemocyanin into a phenoloxidase
author_id_str_mv af160934b75bea5b8ba83d68b3d1a003
author_id_fullname_str_mv af160934b75bea5b8ba83d68b3d1a003_***_Christopher Coates
author Christopher Coates
author2 Christopher Coates
Jacqueline Nairn
format Journal article
container_title Developmental & Comparative Immunology
container_volume 169
container_start_page 105401
publishDate 2025
institution Swansea University
issn 0145-305X
1879-0089
doi_str_mv 10.1016/j.dci.2025.105401
publisher Elsevier Ltd
college_str Faculty of Science and Engineering
hierarchytype
hierarchy_top_id facultyofscienceandengineering
hierarchy_top_title Faculty of Science and Engineering
hierarchy_parent_id facultyofscienceandengineering
hierarchy_parent_title Faculty of Science and Engineering
department_str School of Biosciences, Geography and Physics - Biosciences{{{_:::_}}}Faculty of Science and Engineering{{{_:::_}}}School of Biosciences, Geography and Physics - Biosciences
document_store_str 1
active_str 0
description Haemocyanin is a haemolymph (blood)-based protein and the functional equivalent to haemoglobin - supplying tissues with oxygen in decapod crustaceans, chelicerates, and shelled molluscs. In addition to oxygen transport, haemocyanin plays several roles in innate immunity, wound healing, and ecdysis. Under certain conditions in vitro and in vivo, horseshoe crab (Limulus polyphemus) haemocyanin is converted into a phenoloxidase-like enzyme, yet the protein-ligand interactions associated with this conversion remain unclear. Negatively charged ligands, such as phosphatidylserine and sodium dodecyl sulphate, represent effective endogenous and exogenous activators, respectively. Herein, we explored the nature of the interaction between haemocyanin and phosphatidylserine. We used several spectroscopic techniques and phenoloxidase assays to follow the electrostatic interactions. Manipulating the ionic strength of the assay resulted in less enzyme activity, and reversed haemocyanin conformational changes associated with phosphatidylserine binding (confirmed by fluorescence emission spectra). The addition of wild type and rearranged peptides - mimicking the P181 to K196 region close to the active site of haemocyanin subunit II - to phenoloxidase assays resulted in less product (dopachrome) formation. We propose that non-specific electrostatic interactions between haemocyanin and endogenous activators such as phosphatidylserine facilitate the switch to a phenoloxidase-like enzyme.
published_date 2025-08-01T05:31:01Z
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