Ectophosphatase activity in the early-diverging fungus Blastocladiella emersonii: Biochemical characterization and possible role on cell differentiation

Blastocladiella emersonii is an interesting model for studding the evolution of cell differentiation in eukaryotic cell because of its taxonomic position towards the base of the fungal phylogenetic tree and because it undergoes radical morphological and biochemical changes throughout its life cycle. In this work, we biochemically characterized a high alkaline phosphotyrosine phosphatase activity present on the cell surface (ectophosphatase) of B. emersonii. The ectophosphatase activity was strongly inhibited at acidic pH values as well as by specific phosphatase inhibitors, such as sodium orthovanadate and bpv-PHEN. In addition, the enzyme activity was modulated by the extracellular concentration of inorganic phosphate (P_i) present in both reaction mixture and culture medium. Phosphotyrosine was hydrolysed at the same extent of its analog, p-NPP, while the hydrolysis of phosphothreonine was 2-fold lower, suggesting that a phosphotyrosine ectophosphatase activity is present on the cell surface of B. emersonii. The ectophosphatase activity was also strongly inhibited by EGTA, indicating the participation of Ca²⁺ ions on catalysis. The hydrolysis of p-NPP was differentially regulated throughout the B. emersonii life cycle, suggesting that the ectophosphatase activity could be involved in cell differentiation processes. In support of this, the addition of bpv-PHEN or vanadate at the beginning of germination inhibited the differentiation of zoospores to germ cells, compared to control or tartrate-treated cells. On the other hand, if the inhibitors are added 15 or 30 min after initiation of germination the inhibitory effect on zoospore germination decreases significantly, suggesting that the phosphotyrosine ectophosphatase activity is important at the first minutes of germination. The addition of vanadate, molybdate and bpv-PHEN during vegetative growth inhibited the enlargement of the cells compared to control or tartrate-treated cells. Finally, vanadate or bpv-PHEN added during sporulation strongly inhibited zoospore biogenesis, indicating an important role of such ectophosphatases in this differentiation process. Taken together, these data show the existence of a high alkaline ectophosphotyrosine phosphatase activity in B. emersonii that is probably tied to cell differentiation processes of the fungus.