Hemocyanin of the caenogastropod Pomacea canaliculata exhibits evolutionary differences among gastropod clades

Structural knowledge of gastropod hemocyanins is scarce. To better understand their evolution and diversity we studied the hemocyanin of a caenogastropod, Pomacea canaliculata (PcH). Through a proteomic and genomic approach, we identified 4 PcH subunit isoforms, in contrast with other gastropods that usually have 2 or 3. Each isoform has the typical Keyhole limpet-type hemocyanin architecture, comprising a string of eight globular functional units (FUs). Correspondingly, genes are organized in eight FUs coding regions. All FUs in the 4 genes are encoded by more than one exon, a feature not found in non- caenogastropods. Transmission electron microscopy images of PcH showed a cylindrical structure organized in di, tri and tetra-decamers with an internal collar structure, being the di and tri-decameric cylinders the most abundant ones. PcH is N-glycosylated with high mannose and hybrid-type structures, and complex-type N-linked glycans, with absence of sialic acid. Terminal β-N-GlcNAc residues and nonreducing terminal α-GalNAc are also present. The molecule lacks O-linked glycosylation but presents the T-antigen (Gal-β1,3-GalNAc). Using an anti-PcH polyclonal antibody, no cross-immunoreactivity was observed against other gastropod hemocyanins, highlighting the presence of clade-specific structural differences among gastropod hemocyanins. This is, to the best of our knowledge, the first gene structure study of a Caenogastropoda hemocyanin.