Rhogocytes (pore cells) are specific molluscan cell types that are scattered throughout the connective tissues of diverse body parts. We have identified rhogocytes in large numbers in tissue taken from mantle, foot and midgut gland of the abalone Haliotis tuberculata (Vetigastropoda). Within cisternae of the endoplasmic reticulum, particles are visible that resemble, in shape and size, hemocyanin molecules, the respiratory protein of many molluscs. Immunohistochemical experiments using hemocyanin-specific antibodies demonstrated that these cells contain hemocyanin. In situ hybridization with a cDNA probe specific for Haliotis hemocyanin showed that hemocyanin-specific mRNA is present in rhogocytes, which confirmed that they are the site of hemocyanin biosynthesis in this gastropod. A possible path of hemocyanin release into the hemolymph is discussed. Also in the vetigastropod Megathura crenulata, many rhogocytes could be detected. However, they lacked hemocyanin molecules which, together with published data, indicates a seasonal expression of hemocyanin in this animal.
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.
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