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.

The Sequence of a Gastropod Hemocyanin (HtH1 from Haliotis tuberculata)

“…This is very close to the value of ϳ400 kDa measured in SDSpolyacrylamide gel electrophoresis for both HtH1 and KLH1 (12,24). From the multitude of conserved and variable structural features visible in Fig.…”

“…5). The recent biochemical characterization of Haliotis hemocyanin (12,13), in combination with the complete amino acid sequence presented here, now establishes HtH1 as the gastropod reference hemocyanin. Together with the partial sequence of HtH2 (13), which will soon be completed, questions of the biological significance and regulation of the two physicochemically and apparently physiologically distinct hemocyanin isoforms found in prosobranch species (see Refs.…”

“…Functional unit HtH1-h has two sugar sites in quite unusual positions ( Fig. 2 and 3), which has already been discussed in detail elsewhere (12,13). Significantly, Haliotis hemocyanin isoform HtH1 shows 13 potential N-glycosylation sites per subunit, and Octopus hemocyanin shows only seven (excluding the NPS/T sites).…”

“…However, when we started our sequencing work on the two isoforms of Haliotis tuberculata hemocyanin, HtH1 and HtH2, the primary structure of gastropod hemocyanin was only partially known, namely from FU-d and FU-g of Helix pomatia and FU-a of Rapana thomasiana hemocyanin (9 -11). Recently, we published the cDNA sequence coding for HtH1-fgh (12) and for HtH2-defgh (13). The present study was designed to complete the entire cDNA sequence coding for HtH1 and thereby provide the first complete primary structure of a gastropod hemocyanin.…”

Structures of two molluscan hemocyanin genes: Significance for gene evolution

“…Therefore, it seemed a logical next step to follow the cDNA sequencing of HtH and OdH with determination of the sequences of their genes. In sequencing the cDNA corresponding to the HtH, the two distinct isoforms HtH1 and HtH2 previously described at the protein level were found (14,15); counterparts termed KLH1 and KLH2 have been studied in detail in another gastropod, the keyhole limpet Megathura crenulata (16), and shown to exhibit differential regulation. The hemocyanin isoforms HtH1 and HtH2 share only Ϸ65% sequence identity (4), and therefore their genes could be easily distinguished.…”