Abalone ( Haliotis tuberculata ) hemocyanin type 1 (HtH1)

Abstract

We have identified two separate hemocyanin types (HtH1 and HtH2) in the European abalone Haliotis tuberculata . HtH1/HtH2 hybrid molecules were not found. By selective dissociation of HtH2 we isolated HtH1 which, as revealed by electron microscopy and SDS/PAGE, is present as didecamers of a ≈ 400 kDa subunit. Immunologically, HtH1 and HtH2 correspond to keyhole limpet hemocyanin (KLH)1 and KLH2, respectively, the two well‐studied hemocyanin types of the closely related marine gastropod Megathura crenulata . On the basis of limited proteolytic cleavage, two‐dimensional immunoelectrophoresis, SDS/PAGE and N‐terminal sequencing, we identified eight different 40–60 kDa functional units in HtH1, termed HtH1‐ a to HtH1‐ h , and determined their linear arrangement within the elongated subunit. From Haliotis mantle tissue, rich in hemocyanin‐producing pore cells, we isolated mRNA and constructed a cDNA library. By expression screening with HtH‐specific rabbit antibodies, a cDNA clone was isolated and sequenced which codes for the three C‐terminal functional units f , g and h of HtH1. Their sequences were aligned to those available from other molluscs, notably to functional unit f and functional unit g from the cephalopod Octopus dofleini . HtH1‐ f , which is the first sequenced functional unit of type f from a gastropod hemocyanin, corresponds to functional unit f from Octopus . Also functional unit g from Haliotis and Octopus correspond to each other. HtH1‐h is a gastropod hemocyanin functional unit type which is absent in cephalopods and has not been sequenced previously. It exhibits a unique tail extension of ≈ 95 amino acids, which is lacking in functional units a to g and aligns with a published peptide sequence of 48 amino acids from functional unit h of Helix pomatia hemocyanin. The new Haliotis sequences are discussed with respect to their counterparts in Octopus , the 15 Å three‐dimensional reconstruction of the KLH1 didecamer from electron micrographs, and the recent 2.3 Å X‐ray structure of functional unit g from Octopus hemocyanin.