Expression, Purification, and Characterization of Human Diacylglycerol Kinase ?.

ABSTRACT: Diacylglycerol kinase ? (DGK?) phosphorylates diacylglycerol (DG) to generate phosphatidic acid. The dysfunction of DGK? has been linked to several diseases, such as cardiac hypertrophy, ischemia, and seizures. Moreover, much attention has been paid to DGK?, together with DGK?, as a potential target for cancer immunotherapy. However, DGK? has never been purified and, thus, neither its enzymatic properties nor its structure has yet been reported, hindering our understanding of the catalytic mechanism of DGK? and the development of a reasonable structure-based drug design. In the present study, we generated a full-length DGK? using a baculovirus-insect cell expression system for enzymological and structural studies. Full-length DGK? remained soluble and was purified to near homogeneity as a monomer with yields suitable for protein crystallization (0.63 mg/1 L culture). Enzymatic characterization showed that the purified DGK? is in a fully functional state. The K m values for adenosine triphosphate (ATP) and DG were 0.05 mM and 1.5 mol %, respectively, and the EC50 for the activator phosphatidylserine was 8.6 mol %, indicating that its affinity for ATP is moderately higher than those of DGK? and DGK?, and its affinities for DG and phosphatidylserine are comparable to those of DGK?/DGK?. We further confirmed that the purified enzyme could be concentrated without any significant aggregation. Circular dichroism revealed that DGK? is comprised of 25% ?-helices and 18% ?-strands. This is the first successful purification and characterization of the enzymatic and conformational properties of DGK?. The purification of DGK? allows detailed analyses of this important enzyme and will advance our understanding of DGK?-related diseases and therapies.

SUBMITTER: Saito T 

PROVIDER: S-EPMC6935893 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature