A new haemocyanin in cuttlefish (Sepia officinalis) eggs: sequence analysis and relevance during ontogeny

Abstract

BackgroundHaemocyanin is the respiratory protein of most of the Mollusca. In cephalopods and gastropods at least two distinct isoforms are differentially expressed. However, their physiological purpose is unknown. For the common cuttlefish Sepia officinalis, three isoforms are known so far, whereas for only two of them the complete mRNA sequences are available. In this study, we sequenced the complete mRNA of the third haemocyanin isoform and measured the relative expression of all three isoforms during embryogenesis to reveal a potential ontogenetic relevance.ResultsThe cDNA of isoform 3 clearly correlates to the known Sepia officinalis haemocyanin subunits consisting of eight functional units and an internal duplicated functional unit d. Our molecular phylogenetic analyses reveal the third isoform representing a potentially ancestral haemocyanin isoform, and the analyses of the expression of haemocyanin type 3 reveal that haemocyanin type 3 only can be observed within eggs and during early development. Isoforms 1 and 2 are absent at these stages. After hatching, isoform 3 is downregulated, and isoform 1 and 2 are upregulated.ConclusionsOur study clearly shows an embryonic relevance of the third isoform, which will be further discussed in the light of the changes in the physiological function of haemocyanin during ontogeny. Taken together with the fact that it could also be the isoform closest related to the common ancestor of cuttlefish haemocyanin, the phylogeny of cuttlefish haemocyanin may be recapitulated during its ontogeny.

Crystal Structure of the 3.8-MDa Respiratory Supermolecule Hemocyanin at 3.0 Å Resolution

“…The hemocyanin subunit was composed of eight FUs, which share 39%-69% sequence identity ( Figure S2 and Table S1). As previously observed for hemocyanin of cuttlefishes (Thonig et al, 2014), the fourth and fifth FUs show high sequence identity (69%). Hereafter, FUs are designated as FU-a, -b, -c, -d, -d*, -e, -f, and -g.…”

“…Instead of the Cys residues necessary for copper incorporation, all FUs of hemocyanin have two other Cys residues (Cys47 and Cys57) in the vicinity of the Cu 2 O 2 cluster, which form a disulfide bond ( Figure 5C). These Cys residues are completely conserved among hemocyanins (Bergmann et al, 2006;Lieb et al, 2000;Miller et al, 1998;Stoeva et al, 1999Stoeva et al, , 2002Thonig et al, 2014) while no tyrosinase possesses them (Fujieda et al, 2013). In addition, several highly conserved Met and Cys residues are located around them ( Figure 5C).…”

“…Most molluscan hemocyanins are composed of eight FUs (FU-a, -b, -c, -d, -e, -f, -g, and -h), whereas some cephalopod hemocyanins, such as those of octopuses and nautiluses, possess seven FUs (FU-a, -b, -c, -d, -e, -f, and -g), in which FU-h is lost. Hemocyanins from ten-armed cephalopods, such as cuttlefishes and squid, have an additional FU between FU-d and -e (Thonig et al, 2014). Because of the significant sequence similarity with FU-d, this additional FU is designated as FU-d*, which yields the subunit structure of FU-a, -b, -c, -d, -d*, -e, -f, and -g (Markl, 2013;Thonig et al, 2014).…”

“…Hemocyanins from ten-armed cephalopods, such as cuttlefishes and squid, have an additional FU between FU-d and -e (Thonig et al, 2014). Because of the significant sequence similarity with FU-d, this additional FU is designated as FU-d*, which yields the subunit structure of FU-a, -b, -c, -d, -d*, -e, -f, and -g (Markl, 2013;Thonig et al, 2014). Previous cryoelectron microscopic structures have shown that the cylindrical wall consists of FU-a-f, regardless of the FU composition, and remaining FUs assemble to form the inner collar regions (Gatsogiannis et al, 2007(Gatsogiannis et al, , 2015Gatsogiannis and Markl, 2009;Zhang et al, 2013;Zhu et al, 2014).…”

Cross-bracing uncalibrated nodes in molecular dating improves congruence of fossil and molecular age estimates

“…Here, we test this proposed extension of cross-bracing, using as a test case the hemocyanin gene family of chelicerate arthropods. Hemocyanins constitute the oxygen-transporting metalloproteins of various arthropods and mollusks [18][19][20][21][22][23][24][25]. Arthropod hemocyanins are composed of various subunits, each of which contains two copper moieties that reversibly bind oxygen molecules.…”