Identification, characterization, and complete amino acid sequence of the conjugation-inducing glycoprotein (blepharmone) in the ciliate Blepharisma japonicum.
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 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.
Sugiura
M
M
Department of Biological Science, Nara Women's University, Nara 630-8506, Japan.
Harumoto
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002241:Carbohydrates / Q000737:chemistry; D016798:Ciliophora / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D036341:Intercellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D012098:Reproduction / Q000235:genetics
D002241:Carbohydrates; D018076:DNA, Complementary; D016054:DNA, Protozoan; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; C006785:blepharmone protein, Blepharisma japonicum