Expansion of Hydra actinoporin-like toxin (HALT) gene family: Expression divergence and functional convergence evolved through gene duplication. (December 2019)

• Record Type: Journal Article
• Authors: Yap, Wei Yuen; Tan, Katrina Joan Shu Xian; Hwang, Jung Shan
• Is Part Of: Toxicon. Volume 170(2019)
• Page Start: 10
• Page End: 20
• Publication Date: 2019-12
• Subjects: Hydra actinoporin-like toxin -- Pore-forming toxin -- Sulfatide -- Protein-lipid interaction -- Gene duplication; Arg Arginine -- BCIP 5-bromo-4-chloro-3′-indolyphosphate p-toluidine -- BLAST Basic Local Alignment Search Tool -- BSA Bovine serum albumin -- CC50 50% cytotoxic concentration -- CHOL Cholesterol -- CTX Cobra cardiotoxin -- Dab2 Disabled homolog 2 DMSO, Dimethyl sulfoxide -- ELISA Enzyme-linked immunosorbent assay -- Eqt Equinatoxin -- EST Expressed sequence tag -- FraC Fragaceatoxin -- GPI Glycosylphosphatidylinositol -- GST Glutathione S-transferase -- HALT Hydra actinoporin like toxin -- IPTG Isopropyl β-D-1-thiogalactopyranoside -- LPA Lysophosphatidic acid Lys, Lysine mRNA Messenger RNA -- MTT 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide -- NBT Nitro-blue tetrazolium chloride -- NEP Nematocyte expressed proteins -- NOWA Nematocyst outer wall antigen -- PBS Phosphate buffered saline -- PCNA Proliferating cell nuclear antigen -- PFT Pore-forming toxin -- Phe Phenylalanine -- POC phosphocholine -- PMSF Phenylmethylsulfonyl fluoride -- pNPP para-Nitrophenylphosphate -- Pro Proline -- rHALT recombinant HALT -- RNA Ribonucleic acid -- RNA-Seq RNA Sequencing -- RT-PCR Reverse transcription – polymerase chain reaction -- S1P Sphingosine-1-phosphate -- SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis -- SFT Sulfatide -- SILAC Stable Isotope Labeling by/with Amino acids in Cell culture -- SM Sphingomyelin -- St Sticholysin -- STb Heat-stable enterotoxin b -- TBS Tris buffered saline -- TBS-T 1 X TBS containing 0.1% Tween 20 -- Trp Tryptophan -- t-SNE t-distributed Stochastic Neighbour Embedding -- Tyr Tyrosine; Toxins -- Periodicals; Venom -- Periodicals; 615.9
• DOI: 10.1016/j.toxicon.2019.09.007
• Languages: English
• ISSNs: 0041-0101

Abstract

Abstract: Hydra actinoporin-like toxin 1 (HALT-1) was previously shown to cause cytolysis and haemolysis in a number of human cells and has similar functional properties to the actinoporins equinatoxin and sticholysin. In addition to HALT-1, five other HALTs (HALTs 2, 3, 4, 6 and 7) were also isolated from Hydra magnipapillata and expressed as recombinant proteins in this study. We demonstrated that recombinant HALTs have cytolytic activity on HeLa cells but each exhibited a different range of toxicity. All six recombinant HALTs bound to sulfatide, while rHALT-1 and rHALT-3 bound to two additional sphingolipids, lysophosphatidic acid and sphingosine-1-phosphate as indicated by the protein-lipid overlay assay. When either tryptophan 133 or tyrosine 129 of HALT-1 was mutated, the mutant protein lost binding to sulfatide, lysophosphatidic acid and sphingosine-1-phosphate. As further verification of HALTs' binding to sulfatide, we performed ELISA for each HALT. To determine the cell-type specific gene expression of seven HALTs in Hydra, we searched for individual HALT expression in the single-cell RNA-seq data set of Single Cell Portal. The results showed that HALT-1, 4 and 7 were expressed in differentiating stenoteles. HALT-1 and HALT-6 were expressed in the female germline during oogenesis. HALT-2 was strongly expressed in the gland and mucous cells in the endoderm. Information on HALT-3 and HALT-5 could not be found in the single-cell data set. Our findings show that subfunctionalisation of gene expression following duplication enabled HALTs to become specialized in various cell types of the interstitial cell lineage. Highlights: All expressed HALTs were cytolytic towards HeLa cells. Sulfatide is a common lipid-binding site for all HALTs. Subfunctionalisation of actinoporins following gene duplication enabled specialisation of HALTs in the interstitial cell lineage.