Subunit organization of the abalone Haliotis tuberculata hemocyanin type 2(HtH2), and the cDNA sequence encoding its functional units d, e, f, g andh

We have developed a HPLC procedure to isolate the two different hemocyanin types (HtH1 and HtW2) of the European abalone Haliotis tuberculata. On the basis of limited proteolytic cleavage, two-dimensional immunoelectrophoresi s, PAGE, N-terminal protein sequencing and cDNA sequencing, we have identif ied eight different 40-60-kDa functional units (FUs) in HtH2, termed HtH2-a to HtH2-h, and determined their linear arrangement within the elongated 40 0-kDa subunit. From a Haliotis cDNA library, we have isolated and sequenced a cDNA clone which encodes the five C-terminal FUs d, e, f, g and h of HtH 2. As shown by multiple sequence alignments, defg of HtH2 correspond struct urally to defg from Octopus dofleini hemocyanin. HtH2-e is; the first FU of a gastropod hemocyanin to be sequenced. The new Haliotis hemocyanin sequen ces are compared to their counterparts in Octopus, Helix pomatia and HtH1 ( from the latter, the sequences of FU-f, FU-g and FU-h have recently been de termined) and discussed in relation to the recent 2.3 Angstrom X-ray struct ure of FU-g from Octopus hemocyanin and the 15 Angstrom three-dimensional r econstruction of the Megathura crenulata hemocyanin didecamer from electron micrographs. This data allows, for the first time, an insight into the evo lution of the two functionally different hemocyanin isoforms found in marin e gastropods. It appears that they evolved several hundred million years ag o within the Prosobranchia, after separation of the latter from the branch leading to the Pulmonata. Moreover, as a structural explanation for the ine fficiency of the type 1 hemocyanin to form multidecamers in vivo, the addit ional N-glycosylation sites in HtH1 compared to HtH2 are discussed.