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

ITUARTE, SANTIAGO; DREON, MARCOS; BROLA, TABATA; HERAS, HORACIO; CHIUMIENTO, IGNACIO; OTERO, LISANDRO H.

Congreso; XLIX Reunión Anual de la Sociedad Argentina de Biofísica; 2021

Sociedad Argentina de Biofísica

Hemocyanins (HC) are respiratory pigments found in mollusks andarthropods also associated withinnate immunity functions, including phenol oxidase (PO) activity and cleavage intoantimicrobial peptides (AMPs). The aim of this work was to expand the structural knowledgeand the derived physiological implications of gastropod HC within an evolutionarycontext. We focused on the molecular structure of the HC from P. canaliculata (PcH)by Cryo-EM and kinetically characterized its intrinsic and proteolyticallyinduced PO activity. Additionally, we evaluated in silico theproduction of AMPs. Purified PcH wasanalyzed by single-particle Cryo-EM from which we obtained a 5Å resolution densitymap. We constructed an ab initio 3D model by homology modelling using its deducedaminoacid sequence, and the resulting structure was docked and real-space refined in theCryo-EM map, retrieving a cylindrical rearrangement assembled in di-decamers related byD5 symmetry. Each decameric subunit is composed of five antiparallel dimers,each divided into sixteen paralogue functional units showing conserved structuralfeatures among snail HC.We alsocharacterized PcH intrinsic PO activity, using catechol and dopamineas substrates. Likeother 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 limitedproteolysis using trypsin,chymotrypsin and subtilisin. Finally, a bioinformatic analysispredicted the presence of severalAMPs within the PcH sequence, likely to be generated under proteolytictreatment during a microbial infection. As a whole, theseresults reinforce the idea that molluscan HC may be involved ininnate immune system. ThePcH structural model allows a deeper comprehension of these processes, triggeredby still not well-known mechanisms that include structural changes by endogenous and/orexogenous proteases.p { margin-bottom: 0.25cm; line-height: 115%; background: transparent }