Abalone (Haliotis tuberculata) hemocyanin type 1 (HtH1) - Organization of the approximate to 400 kDa subunit, and amino acid sequence of its functional units f, g and h

We have identified two separate hemocyanin types (HtH1 and HtH2) in the Eur opean abalone Haliotis tuberculata. HtH1/HtH2 hybrid molecules were not fou nd. By selective dissociation of HtH2 we isolated HtH1 which, as revealed b y electron microscopy and SDS/PAGE, is present as didecamers of a approxima te to 400 kDa subunit. Immunologically, HtH1 and HtH2 correspond to keyhole limpet hemocyanin (KLH)1 and KLH2, respectively, the two well-studied hemo cyanin types of the closely related marine gastropod Megathura crenulata. O n the basis of limited proteolytic cleavage, two-dimensional immunoelectrop horesis, 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 mantl e tissue, rich in hemocyanin-producing pore cells, we isolated mRNA and con structed a cDNA library. By expression screening with HtH-specific rabbit a ntibodies, a cDNA clone was isolated and sequenced which codes for the thre e C-terminal functional units f, g and h of HtH1. Their sequences were alig ned to those available from other molluscs, notably to functional unit f an d functional unit g from the cephalopod Octoyus 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. HtHL-h is a gastropod hemocy anin functional unit type which is absent in cephalopods and has not been s equenced previously. It exhibits a unique tail extension of approximate to 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 H elix pomatia hemocyanin. The new Haliotis sequences are discussed with resp ect to their counterparts in Octopus, the 15 Angstrom three-dimensional rec onstruction of the KLH1 didecamer from electron micrographs, and the recent 2.3 Angstrom X-ray structure of functional unit g from Octopus hemocyanin.