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

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 tridecameric 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.

The evolution of hemocyanin genes in Tectipleura: a multitude of conserved introns in highly diverse gastropods

“…The number of internal introns of Tectipleura hemocyanins is greater than that found in hemocyanin genes of Pomacea canaliculata , which comprise 21 to 26 internal introns per gene according to Chiumiento et al . [ 21 ]. The P. canaliculata gene structures are intentionally omitted from Fig.…”

“…Only recently, Chiumiento et al . [ 21 ] showed that exon–intron structures of four hemocyanin genes of the caenogastropod Pomacea canaliculata differ not only from those of Lepetellida but also vary from each other. Furthermore, they contain a much larger number of introns than those known from Vetigastropoda or Cephalopoda (Fig.…”

“…These results are furthermore surprising as they do not correspond with the hemocyanin gene structures of Caenogastropoda elucidated by Chiumiento et al . [ 21 ]. On one hand, their results indicate that accumulation of introns as we have detected in Tectipleura is a common feature of hemocyanin genes of Apogastropoda, but on the other hand, the four hemocyanin genes of P. canaliculata vary in number and positions of introns and do not show such a strong conservation as we have identified for Tectipleura.…”

“…More precisely, introns from all functional units of N. pompilius , Octopodoidea, Lepetellida and Tectipleura are marked together within one model FU-coding sequence. Due to ambiguities of the respective report, exon–intron architectures of hemocyanin genes of P. canaliculata [ 21 ] were not included in this figure.

Fig.

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Pomacea canaliculata Ampullar Proteome: A Nematode-Based Bio-Pesticide Induces Changes in Metabolic and Stress-Related Pathways

“…Rhogocytes have been described in different gastropod molluscs [48,[68][69][70], including P. canaliculata [71], and have been proposed to be involved in several functions, including metabolism and/or detoxification of metal ions [50,[72][73][74][75], cellular defence [68,76,77] and the production of haemocyanin [48,[78][79][80][81][82], which, in its specific multimeric form, was detected in association with rhogocytes by TEM analysis in Lymnaea stagnalis and Haliotis tubercolata [49,78]. TEM observations have also documented the structure of isolated or tissue haemocyanin in P. canaliculata [19,83,84]. In our experimental conditions, we identified haemocyanin by proteomic analysis but not by TEM.…”