Molecular characterization and Cryo-EM structure of the hemocyanin of the invasive freshwater snail Pomacea canaliculata.

BROLA, TABATA ROMINA; QIU, JIAN-WEN; DREON, MARCOS SEBASTIÁN; CHIUMIENTO, IGNACIO RAFAEL; SUN, JIN; HERAS, HORACIO; ITUARTE, SANTIAGO; OTERO, LISANDRO HORACIO

San Luis

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Biofísica; 2019

Hemocyanins (HC) are the respiratory proteins found in most mollusks and arthropods. Probably due to their huge size and glycan moieties molluscan HC can be used as immunostimulants. Nevertheless, structural knowledge of these proteins is scarce. In this work we studied the molecular assembly, glycosylation pattern and gene structure of the HC from the gastropod P. canaliculata (PcH). Through a proteomic and genomic approach, we identified 4 gene sequences of PcH subunits. Each subunit has the typical architecture of a gastropod HC, which is a string of eight globular functional units (FUs) of about 50 kDa each. Correspondingly, genes are organized in eight structurally related coding regions, with linker introns of variable length. Of special relevance for evolutionary considerations are introns interrupting regions that encode a discrete FU, a feature not found in other mollusks. Although the positioning of the FUs linker introns is highly conserved among mollusks, introns within FUs show no relationship neither in location nor phase. A preliminary low-resolution model of its structure was achieved by single-particle cryo-electron microscopy. PcH structure shows a cylindrical rearrangement assembled by di, tri, and tetra-decamers with an internal collar structure. PcH is N-glycosylated with oligo- or high mannose and hybrid-type structures, and complex-type N-linked glycans, without sialic acid. It is also decorated by terminal β-N-GlcNAc residues and nonreducing terminal α-GalNAc. This glycosylation profile, with an abundance of N-linked glycans, is similar to that of Megathura crenulata HC. In addition, polyclonal antibodies against PcH do not cross-react with other gastropod HCs, thus highlighting structural differences among them and opening a research avenue on the immunostimulant activity of PcH and the role that carbohydrates play in it.