Abalone (Haliotis tuberculata) hemocyanin type 1 (HtH1) -: Organization of the ≈400 kDa subunit, and amino acid sequence of its functional units f, g and h
Abalone (Haliotis tuberculata) hemocyanin type 1 (HtH1) -: Organization of the ≈400 kDa subunit, and amino acid sequence of its functional units f, g and h
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DOI：
10.1046/j.1432-1327.1999.00564.x
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发表时间：
1999-08-01
期刊：
影响因子：
--
通讯作者：
Markl, J
中科院分区：
文献类型：
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作者：
Keller, H;Lieb, B;Markl, J
我们已经确定了两种不同的血青素类型（HtH1和HtH2）在欧洲鲍鱼结核。未发现HtH1/HtH2杂化分子。通过HtH2的选择性解离，我们分离出HtH1，通过电子显微镜和SDS/PAGE显示，HtH1以大约400 kDa亚基的二十聚体存在。在免疫学上，HtH1和HtH2分别对应于keyhole帽贝血青素（KLH）1和KLH2，这两种血青素类型是密切相关的海洋腹足动物Megathura crenulata的两种研究得很好的血青素类型。基于有限的蛋白水解裂解、二维免疫电泳、SDS/PAGE和n端测序，我们在HtH1中鉴定了8个不同的40-60 kDa的功能单元，称为HtH1-a至HtH1-h，并确定了它们在细长亚基中的线性排列。从富含产血青素孔细胞的盘藻套膜组织中分离出mRNA并构建cDNA文库。通过兔hth特异性抗体的表达筛选，分离出一个编码HtH1 c端3个功能单元f、g和h的cDNA克隆并测序。它们的序列与其他软体动物的序列一致，特别是与头足类章鱼的功能单元f和功能单元g一致。HtH1-f是腹足类血青素中第一个被测序的f型功能单位，与章鱼的f型功能单位相对应。此外，海螺和章鱼的功能单位g也相互对应。htl -h是腹足类血青素功能单位类型，在头足类中不存在，以前没有测序。它显示出一个独特的尾部延伸，大约95个氨基酸，缺乏功能单位a到g，与已发表的Helix pomatia hemocyanin功能单位h的48个氨基酸序列一致。本文讨论了新的Haliotis序列与章鱼的对应序列、KLH1二十聚体的15埃三维重建和章鱼血青素功能单元g的2.3埃x射线结构。
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 approximate to 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 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 hemocyanin functional unit type which is absent in cephalopods and has not been sequenced 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 Helix pomatia hemocyanin. The new Haliotis sequences are discussed with respect to their counterparts in Octopus, the 15 Angstrom three-dimensional reconstruction of the KLH1 didecamer from electron micrographs, and the recent 2.3 Angstrom X-ray structure of functional unit g from Octopus hemocyanin.