Cryo-EM structure and biochemical characterization of the hemocyanin of the invasive freshwater snail Pomacea canaliculata: understanding its role in the innate immune system

BROLA, T.R.; CHIUMIENTO, IGNACIO RAFAEL; ITUARTE, S; HERAS, H.; OTERO, L.H.; DREON, M.S.

Congreso; XLIX Reunion Anual de la Sociedad Argentina de Biofisica; 2021

Hemocyanins (HC) are respiratory pigments found in mollusks and arthropods alsoassociated with innate immunity functions, including phenol oxidase (PO) activity andcleavage into antimicrobial peptides (AMPs). The aim of this work was to expand the structural knowledge and the derived physiological implications of gastropod HC within an evolutionary context. We focused on the molecular structure of the HC from P. canaliculata (PcH) by Cryo-EM and kinetically characterized its intrinsic and proteolytically induced PO activity. Additionally, we evaluated in silico the production of AMPs.Purified PcH was analyzed by single-particle Cryo-EM from which we obtained a 5Åresolution density map. We constructed an ab initio 3D model by homology modelling using its deduced aminoacid sequence, and the resulting structure was docked and realspace refined in the Cryo-EM map, retrieving a cylindrical rearrangement assembled in didecamers related by D5 symmetry. Each decameric subunit is composed of five antiparallel dimers, each divided into sixteen paralogue functional units showing conserved structural features among snail HC.We also characterized PcH intrinsic PO activity, using catechol and dopamine assubstrates. Like other gastropod HC, only catechol was catalyzed, showing a KM = 50.5 mM and a Kcat= 0.29.min-1. Furthermore, PO activity was increased by limited proteolysis using trypsin, chymotrypsin and subtilisin. Finally, a bioinformatic analysis predicted the presence of several AMPs within the PcH sequence, likely to be generated under proteolytic treatment during a microbial infection.As a whole, these results reinforce the idea that molluscan HC may be involved in innate immune system. The PcH structural model allows a deeper comprehension of these processes, triggered by still not well-known mechanisms that include structural changes by endogenous and/or exogenous proteases.