Alciporin, a pore-forming protein as complementary defense mechanism in Millepora alcicornis

Millepora alcicornis (Cnidaria: Hydrozoa), known as fire coral, is a tropical species settled in marine ecosystems of the Canary Islands in the last years. This hydrocoral biosynthesizes toxins involved in chemical defense and prey capture mechanisms. Toxicological studies have shown that the venom contained in the nematocysts of Millepora species is mainly composed of thermolabile proteins that display hemolytic activity, causing skin irritation and burn-like lesions upon contact. As a continuation of a previous study, the chromatographic fractionation of the aqueous extracts of M. alcicornis has confirmed the coexistence of proteins of different nature responsible for the hemolytic effects of red blood cells (RBCs) through two different mechanisms. Aside from the already described phospholipase A2 (PLA2) activity, in this work the presence of alciporin, a pore-forming protein (PFP), has been established for the first time for M. alcicornis. The sequence analysis revealed that alciporin fit an actinoporin with high homology to stichotoxins. The hemolytic effects of alciporin were analyzed and sphingomyelin was identified as its biological target. Also, the evolution of the hemolytic damage produced at the nanoscale has been studied using atomic force microscopy (AFM).This work was funded by projects PID2019–109476RB-C21 (BIOALGRI) (Spanish Ministry of Science), Madrid, Spain; Fundación CajaCanarias–Fundación Bancaria “La Caixa” (2019SP52) and Gobierno de Canarias (ProID 2020010123); PI18/01380 from Instituto de Salud Carlos III, Spain and RICET (RD16/0027/0001 project) from Programa Redes Temáticas de Investigación Cooperativa, FIS (Ministerio Español de Salud, Madrid, Spain) and FEDER. (CIBER) de Enfermedades Infecciosas (CIBERINFEC; CB21/13/00100), Instituto de Salud Carlos III, 28006 Madrid, Spain, Ministerio de Sanidad, Consumo y Bienestar, Spain and Cabildo de Tenerife 21/0587 cofunded by MEDI and FDCAN, and EQC2019-005647-P from the Spanish Ministry of Science. NN thanks the Agustín de Betancourt Programme (Cabildo de Tenerife, TFinnova Programme supported by MEDI and FDCAN funds). AG-O thanks NANOtec, INTec, and ULL for laboratory facilities. The proteomic analysis was performed in the proteomics facility of SCSIE University of Valencia that belongs to Proteored, PRB3 and is supported by grant PT17/0019, of the PE I+D+i 2013-2016, funded by ISCIII and ERDF. Authors acknowledge Estabulario (Dr. M.R. Arnau) and AFM Service of General Research Support Services of University of La Laguna (SEGAI-ULL), and the biological activity service of IPNA-CSIC for the analysis of hemolytic activity.Peer reviewe