Hemocyanin of the caenogastropod Pomacea canaliculata exhibitsevolutionary differences among gastropod clades.

Ignacio Rafael Chiumiento,Marcos Sebastián Dreon,Jian Wen Qiu,Jin Sun,Santiago Ituarte,Horacio Heras,Maria Gasset

doi:10.1371/journal.pone.0228325

Abstract

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.

Highlights

Molluscs transport oxygen by copper-containing respiratory proteins called hemocyanins (Hc)
The purity of the PcH preparation was checked by sodium dodecyl sulfate (SDS)-PAGE and the protein content was determined spectrophotometrically recording the absorbance of the samples at 280 nm and employing an averaged theoretical extinction coefficient obtained from the deduced PcH subunit sequences (ε280 = 5.37 M-1 x cm-1)
After hemolymph ultracentrifugation on NaBr δ = 1.28 g/mL fractions containing PcH were identified by absorbance at 350 nm (Fig 1A)

Summary

Introduction

Molluscs transport oxygen by copper-containing respiratory proteins called hemocyanins (Hc). Molluscan Hc may play crucial roles in the physiological adaptations of these animals to their habitats, influencing their lifestyles. They may allow a better adaptation to oxygen-poor and low-temperature environments than hemoglobins [1].

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