Identification, characterization, and complete amino acid sequence of the conjugation-inducing glycoprotein (blepharmone) in the ciliate Blepharisma japonicum

Abstract

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-hydroxybenzoyl) 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 cDNA 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.

Developmentally and environmentally regulated expression of gamone 1: the trigger molecule for sexual reproduction inBlepharisma japonicum

“…Cells were cultured in WGP (Wheat Grass Powder, Pines) medium (Sugiura and Harumoto, 2001), concentrated by lowspeed centrifugation, washed with physiological balanced solution SMB -(synthetic medium for Blepharisma) and suspended in SMB -at a density of 2000-5000 cells/ml. T121 (mating type II) cells were also used to assay for gamone 1 activity, suspended in SMB -at a density of 1500-2000 cells/ml.…”

“…Because trace amounts of pigments often hamper the process of extraction of RNA, we prepared pigment-depleted cells by cold treatment as described (Sugiura and Harumoto, 2001). Cells were then suspended in TRIzol reagent (Lifetech) and total RNA was isolated by the acid guanidinium-phenol-chloroform method.…”

“…The mechanism of induction of conjugation in ciliates has been studied for nearly a century and the conjugation-inducing substances have been isolated from three species: Blepharisma japonicum, Euplotes raikovi and Euplotes octocarinatus (reviewed by Miyake, 1996;Sugiura and Harumoto, 2001). However, the molecular mechanisms relevant to the phenomenon of induction of conjugation have not been elucidated.…”

“…Both gamones are soluble and they have been isolated and characterized. Gamone 1 is the only glycoprotein that has ever been isolated in ciliate pheromones (Miyake and Beyer, 1974) consisting of 272 amino acids and six sugar residues (Sugiura and Harumoto, 2001). Gamone 2 is a small molecule derived from tryptophan, Sexual reproduction (conjugation) in protozoan ciliates is induced by specific cell-cell interactions between cells of complementary mating types.…”

“…Gamone 2 is the only non-peptide substance identified in ciliates and is considered to be a primitive form of pheromone (Miyake, 1996;Sugiura and Harumoto, 2001). The expression of gamone 1 by type I cells is the first essential event in the initiation of conjugation in B. japonicum.…”