Identification, characterization, and complete amino acid sequence of the conjugation-inducing glycoprotein (blepharmone) in the ciliate Blepharisma japonicum
Identification, characterization, and complete amino acid sequence of the conjugation-inducing glycoprotein (blepharmone) in the ciliate Blepharisma japonicum
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DOI：
10.1073/pnas.221457698
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发表时间：
2001-12-04
影响因子：
11.1
通讯作者：
Harumoto, T
中科院分区：
文献类型：
--
作者：
Sugiura, M;Harumoto, T
Blepharisma japonicum中的接合是由互补交配型I和II之间的相互作用诱导的，它们分别分泌blepharmone（配子体1）和blepharismone（配子体2）。配子1转化II型细胞，使它们能够结合，配子2类似地转化I型细胞。此外，每个配子体促进另一配子体的产生。伽马酮2已被鉴定为3-（2 '-甲酰氨基-5'-羟基-苯甲酰基）乳酸钙，并已被化学合成。Gamone 1被分离并表征为20-30 kDa的糖蛋白，含有175个氨基酸和6个糖。然而，这种配子中的氨基酸序列和糖的排列仍然未知。为了确定gamone 1的部分氨基酸序列，我们建立了一种分离方法，该方法的基础上发现，这种糖蛋白可以被ConA亲和柱浓缩。Gamone 1非常不稳定，一旦吸附到我们测试的任何柱上，就会失去其生物活性。通过使用ConA亲和柱和非变性聚丙烯酰胺凝胶电泳，我们检测到一个30 kDa的蛋白对应于gamone 1的活性，并确定了部分氨基酸序列的四个肽。为了分离配子1 cDNA，我们从合成配子2刺激的交配型I细胞中分离mRNA，然后通过使用基因特异性引物和克隆配子1的DNA进行cDNA末端程序的快速扩增。该cDNA序列含有一个305个氨基酸的开放阅读框，编码一种可能是新的蛋白质。我们还通过比较对各种凝集素柱的亲和力来估计糖的排列。
Conjugation in Blepharisma japonicum is induced by interaction between complementary mating-types I and II, which excrete blepharmone (gamone 1) and blepharismone (gamone 2), respectively. Gamone 1 transforms type II cells such that they can unite, and gamone 2 similarly transforms type I cells. Moreover, each gamone promotes the production of the other gamone. Gamone 2 has been identified as calcium-3-(2'-formylamino-5'-hydroxy-benzoyl) lactate and has been synthesized chemically. Gamone 1 was isolated and characterized as a glycoprotein of 20-30 kDa containing 175 amino acids and 6 sugars. However, the amino acid sequence and arrangement of sugars in this gamone are still unknown. To determine partial amino acid sequences of gamone 1, we established a method of isolation based on the finding that this glycoprotein can be concentrated by a Con A affinity column. Gamone 1 is extremely unstable and loses its biological activity once adsorbed to any of the columns that we tested. By using a Con A affinity column and native PAGE, we detected a 30-kDa protein corresponding to gamone 1 activity and determined the partial amino acid sequences of the four peptides. To isolate gamone 1 cDNA, we isolated mRNA from mating-type I cells stimulated by synthetic gamone 2 and then performed rapid amplification of cDNA ends procedures by using gene-specific primers and cloned DNA of gamone 1. The cDNA sequence contains an ORF of 305 amino acids and codes a possibly novel protein. We also estimated the arrangement of sugars by comparing the affinity to various lectin columns.