Characterization of phospholipase C δ genes and their products in Paralichthys olivaceus

Abstract

Fishes possess more copies of genes than other vertebrates, possibly because of a genome duplication event during the evolution of the teleost fish (especially Actinopterygii: ray-finned fish) lineage. To further explore this idea, we cloned five genes encoding phosphoinositide-specific phospholipase C-δ (PLC-δ) from olive flounder (Paralichthys olivaceus), designated respectively PoPLC-δs (PoPLC-δ1A, PoPLC-δ1B, PoPLC-δ3A, PoPLC-δ3B and PoPLC-δ4), and we performed phylogenetic analysis and sequence comparison to compare our putative gene products (PoPLC-δs) with the sequences of known human PLC isoforms. Deduced amino acid sequences shared high sequence identity with human PLC-δ1, -δ3, and -δ4 isozymes and exhibited similar primary structures. Phylogenetic analysis of PoPLC-δs with PLC-δs of five teleost fishes (zebrafish, stickleback, medaka, tetraodon, and takifugu), three tetrapods (human, chicken, and frog), and two tunicates (sea squirt and pacific sea squirt), whose putative sequences of PLC-δ are available in Ensembl genome browser, indicated that the two paralogous genes corresponding to each PLC-δ isoform originated from fish-specific genome duplication prior to the divergence of teleost fish. Based on fish-specific genome duplication of PLC-δ genes and novel N-terminal extended alternative splice form of PLC-δ1 in vertebrate, we studied molecular, enzymatic characterization of duplicated and alternative splice form of PLC-δ1 gene from Paralichthys olivaceus. We identified a novel duplicated genes (PoPLC-δ1A and PoPLC-δ1B-Sf) and alternative splice variant (PoPLC-δ1B-Lf), which were shared from exon 3 (including PH domain) to exon 16, but differ at the exon 1 (Shrot form: Sf) and novel exon 2 (Long form:Lf) of the transcripts. We also compare the tissue type expression, identification and enzymatic characterization of three-types of olive flounder PLC-δ1 isoforms. Generally, expression of PoPLC-δ1 was widely detected in various tissues. However, PoPLC-δ1A was ubiquitously distributed in gill, kidney and spleen and PoPLC-δ1B-Lf was widely detected in various tissues, especially in the digestive system. PoPLC-δ1B-Sf was highly expression in stomach. The activity of recombinant PoPLC-δ1A, PoPLC-δ1B-Lf and PoPLC-δ1B-Sf proteins were expressed in E. coli, shown similar with mammalian PLC-δ1. Lipid binding activity was higher with PI binding than PIP2 binding. Although PLC-δ1 is generally found in the cytoplasm of quiescent cells, PoPLC-δ1A was localized in organelle and PoPLC-δ1B-Lf and PoPLC-δ1B-Sf were in plama membrane. Treatment of ionomycin and thapsigargin for shuttling, only PoPLC-δ1A was translocated to nuclei. We identified a novel exon of mouse PLC-δ1 gene located in the N-terminal and longer than N-terminal previously reported PLC-δ1 genes. The novel alternative splicing form(Long form: mPLC-δ1-Lf) and the previously reported form(Short form: mPLC-δ1-Sf) shared from exon 3(including PH domain) to exon 16. We also compare the expression analysis, identification and enzymatic characterization of two-types of mouse PLC-δ1 gene. Expression of mPLC-δ1-Lf was tissue specific as shown by its high expression in the stomach and large intestine in male and female distributed in tissues but mPLC-δ1-Sf was widely distributed. The recombinant mPLC-δ1-Lf and mPLC-δ1-Sf proteins were expressed in E. coli, and the activity of recombinant mPLC-δ1-Sf protein was shown higher than that of mPLC-δ1-Lf protein in given assay condition. Although the general catalytic and regulatory properties of mPLC-δ1-Lf are similar with those of mammalian PLC δ1(Short form) isozymes, but mPLC-δ1-Lf might have some distinctive in regulatory properties like tissue-specific expression and lipid binding specificity especially in the PS (phosphatidylserine) binding compare to mPLC-δ1-Sf.