Identification of phospholipase B from Dictyostelium discoideum reveals a new lipase family present in mammals, flies and nematodes, but not yeast

Abstract

The social amoeba Dictyostelium discoideum exhibits high activities of phospholipase and lysophospholipase [Ferber, Munder, Fischer and Gerisch (1970) Eur. J. Biochem. 14, 253-257]. We assayed Dictyostelium lysates to demonstrate the presence of a highly active phospholipase B (PLB) enzyme that removed both fatty-acid chains from phosphatidylcholine and produced the water-soluble glycerophosphorylcholine. We purified the PLB activity from Dictyostelium cytosol using standard agarose media (size exclusion and ion exchange), and combined this with an affinity purification step using myristoylated ARF1 (ADP-ribosylation factor 1), a protein which has a single fatty acid at its N-terminus. Two proteins co-purified (48 kDa and 65 kDa), and the 48 kDa protein was digested with trypsin, peptide fragments were separated by reverse-phase chromatography, and the resultant peptides were sequenced by Edman degradation. From the peptide sequences obtained, database searches revealed a gene which encodes a protein of 65 kDa with unknown function. The 48 kDa protein therefore appears to be a fragment of the fulllength 65 kDa product. Expression of the gene in Escherichia coli confirmed that it encodes a PLB. Characterization of its substrate specificity indicated that, in addition to phosphatidylcholine deacylation, the enzyme also hydrolysed phosphatidylinositol and phosphatidylethanolamine. The PLB identified in the present study is not related to existing PLBs found in bacteria, fungi or mammals. There are, however, genes similar to Dictyostelium PLB in mammals, flies, worms and Giardia, but not in yeast. We therefore have identified a novel family of intracellular PLBs.

The identification of a phospholipase B precursor in human neutrophils

“…However, the gene ( FLJ22662 ) is not related to any of these gene families and the coded protein lacks a typical GxSxG motif [20] found in lipases and phospholipases towards the C‐terminus. Our findings suggest that the PLB precursor is a member of a new gene family of PLB as described for Dictyostelium PLB [21]. Whether this protein is involved in arachidonic acid metabolism [22], atherosclerosis [23] and antibacterial defence [24,25], remains to be tested.…”

Partial venom gland transcriptome of a Drosophila parasitoid wasp, Leptopilina heterotoma, reveals novel and shared bioactive profiles with stinging Hymenoptera

“…PLB is a novel enzyme with both Phospholipase A1- and A2-like activities. It is widely encoded, except in yeast (Morgan, 2004). PLB is established as an important component of many venoms and was reported as early as 1964 for bee and various snake venoms (Doery, 1964).…”

Recent Progress on Phospholipases: Different Sources, Assay Methods, Industrial Potential and Pathogenicity

“…Many fungal species appear to produce PLB with both hydrolyzing and acyltransferase activity [4,59,116]. These activities have also been described in bacteria [2], Dictyostelium discoideum (protozoa) [84] and in mammalian cells [87,94,96]. The fungal PLBs range 600-700 amino acids in length and have a potential role in virulence and in fungal pathogenesis [4].…”

“…3a). Thus, it created confusion in nomenclature [84]; therefore, synonyms lysophospholipase and lysophospholipase-transacylase are used for PLB. The hydrolase activity allows the enzyme to cleave fatty acids from both phospholipids (PLB activity) and lysophospholipid (lysophospholipase activity; Fig.…”