Selection and Morphological Alteration of dinoflagellates symbionts by chemical substances in marine invertebrates

海洋无脊椎动物中化学物质对甲藻共生体的选择和形态改变

基本信息

批准号：
15380145
负责人：
KAMIYA Hisao
金额：
$ 8.38万
依托单位：
School of Fisheries Sciences, Kitasato University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

项目摘要

The D-galactose-binding lectin, Sll-2, in the octocoral was effectively purified by affinithy chromatography on D-galactosamine-HiTrap column using Tris-HCl buffer containing 0.5% L-ascorbic acid and 0.1% kojic acid to avoid melanization and insolubilization of SLL-2 during purification. The purified SLL-2 gave many spot in 2D-PAGE with different pI values rangin from 3 to 5. However, after the N-glycopeptidase F treatment, the molecular weight of subunit (16.5kDa) reduced to 9.5kDa with three different pIs. These results suggested that SLL-2 is a mixture of isolectins composed of three glycosilated polypeptides, each equimolar of SLL-2a, b, and c.In frontal affinithy chromatography, SLL-2 showed a high Ka value to Gal α1-3 (Fucα1-2) Gal β1-4 (Fucα1-3) Glc compared to that of Gal α1-3 (Fucα1-2) Gal β1-4 Glc. It also showed high affinity to Forssman antigen which contains GalNAc α1-3 GalNAc structure, and to certain kinds of carbohydrate having a Gal α1-3 Gal structure. These indicate t … More hat SLL-2 binds to carbohydrate constituents derived from glycolipids but not to N-liked carbohydrates.When the protein profiles of coccoid cells induced by the SLL-2 treatment was compared with that by untreated cells on 2-dimentional electrophoresis, the protein profile of SLL-2 treated CS-156 cells was much similar to that of spontaneous coccoid cells than to that of motile cells. Newly expressed proteins, named Bland B2, also confirmed to present in SLL-2-treated cells. B1 showed sequence homology to VDAC1.1 and outer mitochondrial membrane protein porin 1, and B2 to Merozoite surface protein 1 (fragment). In a confocal laser scanning microscopic analysis, we observed the alignment of F-actin and β-tubulin was different between a motile form and coccoid one. The change in the alignment and number of β-tubulin was correlated to the morphological transform from motile to coccoid in Symbiodinium.The D-galactose-binding lectin (TDL) was purified from the hemolymph of a giant clam Tridaca derasa. TDL induced the transform from motile form to coccoid of Symbiodinium sp. However, the effect of TDL on Symbiodinium was species-specic. Interestingly, TDL stimulated the growth of Symbiodinium species tested. D-galactose-bindig lectin from various origins including plants and animals did not stimulate the transform or growth of Symbiodinium.The stony coral Fungia echinata was also confirmed to possess a D-Galactose-binding lectin which alters the motile form to coccoid one to some extent. Some protein fractions were also observed to stimulate significantly the growth of Symbiodinium sp.The factors which showed similar effects on Symbiodinium cells in the sponge Theonella sp. were determined to be depsipeptides, TPId and TP1436. These peptides altered the motile forms to coccoid ones and also depressed cell division completely. The effect was concentration-dependent, and TP-treated CS-156 cells recovered the regular diel alternation between motile and coccoid forms by washing. The similar effect on CS-156 cells was also recognized by the treatment with actin polymerization inhibitors such as Latrunculin A and also with protein kinase inhibitors such as Bisindolylmaleimide I. Present observation strongly suggests that SLL-2 affects the microfilament formulation in Symbiodinium cells and keep these cells in their coccoid stage. Less

八度胶质中的D-半乳糖结合讲座SLL-2通过使用含有0.5％L-抗坏血酸和0.1％Kojic酸的Tris-HCl缓冲液在D-半乳糖胺 - hitrap柱上有效地纯化，以避免纯化后SLL-2的黑色素化和sll-2的溶液化。纯化的SLL-2在2d页中给出了许多斑点，而Pi值不同的rangin从3到5。但是，在N-糖肽酶F处理后，亚基的分子量（16.5KDA）降低至9.5kDa，并使用三种不同的PIS降至9.5kDa。这些结果表明，SLL-2是由三种糖溶解多肽组成的分离蛋白的混合物，每个糖溶性多肽等效于SLL-2A，B和C.in额叶膜片色谱法，SLL-2在GALα1-3（FUCα1-1-2）GALβ1-4（Fucβ1-41-3）GALC的GALCAGALCAGLCα1-3（FUCα1-3）GALCα1-3（FUCα1-3）与该GALC的GALCα1-3（FUCα1-3）相比，SLL-2具有高的Ka值。 GLC。它还显示出对含有galnacα1-3galnac结构的Forssman抗原的高亲和力，并与具有GALα1-3GAL结构的某些类型的碳水化合物。这些适应症……更多的HAT SLL-2与糖脂衍生的碳水合物一致性结合，但不与N类糖碳水化合物结合。当将通过SLL-2处理诱导的球类细胞的蛋白质谱与未经治疗的细胞在二维电泳上进行比较时，SLL-2处理过的CS-156细胞的蛋白质谱与赞助细胞的蛋白质谱相比，而不是在运动细胞的蛋白质。新表达的蛋白质，称为Bland B2，也证实在SLL-2处理的细胞中存在。 B1与VDAC1.1和线粒体外膜蛋白1的序列同源，而B2与Merozoite表面蛋白1（片段）表示。在共聚焦激光扫描显微镜分析中，我们观察到F-肌动蛋白和β-微管蛋白的比对在基序和球类球菌之间有所不同。 β-微管蛋白比对的变化与从基序中的形态变换相关。 D-半乳糖结合讲座（TDL）从巨型蛤Tridaca derasa的血淋巴中纯化。 TDL诱导了Symbiodinium sp。但是，TDL对共生菌的影响是物种特殊的。有趣的是，TDL刺激了测试的共生菌种的生长。来自包括植物和动物在内的各种起源的D-半乳糖 - bindig的讲座没有刺激共生菌的转化或生长。还证实，石质的珊瑚真菌具有D-半乳糖结合的讲座，在某种程度上将基序形式变为球形。还观察到了一些蛋白质级分，以刺激对赞助商Theonella sp中的共生细胞的相似作用的因素。被确定为深度，TPID和TP1436。这些宠物将运动形式改变为球虫的形式，并完全抑制细胞分裂。该作用取决于浓度，而经过TP处理的CS-156细胞通过洗涤恢复了母亲和球类球形之间的常规DIEL替代方案。还通过用肌动蛋白聚合抑制剂（如LATRUNCULIN A）以及诸如Bisindolylylylmaleimide I.的蛋白激酶抑制剂（例如Bisindolylylylmaleimide I.）进行治疗，对CS-156细胞的相似作用也被识别。目前的观察结果表明，SLL-2强烈地表明，SLL-2会影响Symbiodinium细胞和这些细胞中的细胞中的细胞中这些细胞中的细胞。较少的