In vitro characterisation of LYPLA1 and LYPLA2 as lyso-PS lipases.

Abstract

Lyso-phosphatidylserine (Lyso-PS) is a signalling lipid molecule which could trigger reaction cascades leading to various immunological and inflammatory diseases. Therefore, the necessity to control its levels in the body becomes essential, which could be done by converting lyso-PS back to glycerophosphoserine by lyso-PS-specific lipase-like activity. The main objective of this project was in vitro validation of generic lyso- phospholipid (lyso-PL) lipases, Lysophospholipase A1 (LYPLA1) and Lysophospholipase A2 (LYPLA2) as lyso-PS specific lipases. This was achieved by over-expressing LYPLA1 and LYPLA2 in mammalian cell line, Human embryonic kidney 293 cells (HEK293T cells), and checking activity using Activity-based protein profiling (ABPP). Inverse proportionality of concentration of protein-specific inhibitors N-[2-chloro-5-(trifluoromethyl)phenyl]-4-(2- furanylcarbonyl)-1-piperazineacetamid (ML348) and (5,5-Dioxido-4H-thieno[3, 2-c] [1]benzothiopyran-2-yl)[4-(4-methoxyphenyl)-1-piperazinyl]methanone (ML349) , respectively, to LYPLA1 and LYPLA2 activity in over-expressed model was also observed by incubating over-expressed lysates with gradually increasing concentrations of inhibitors. Lysates were incubated in different concentrations of protein-specific in vitro inhibitors, ML348 and ML349 , to check for IC50 values in over-expressed lysates. Ultimately, in vitro characterisation of LYPLA1 and LYPLA2 as a lyso-PS lipase was done using LC-MS based substrate assay. LYPLA1 and LYPLA2 was characterised as lyso-PS lipase in intro system. Lyso-PS lipase activity of LYPLA1 and LYPLA2 could be confirmed in an in vivo model like mouse model systems as well by isolating organs directly and performing substrate assay using inhibitors, to fully validate its function as a lyso-PS lipase in not only in vitro conditions but also in in vivo conditions.