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Lipidomic analysis of the molecular specificity of a cholinephosphotransferase in situ

Lipidomic analysis of the molecular specificity of a cholinephosphotransferase in situ
Lipidomic analysis of the molecular specificity of a cholinephosphotransferase in situ
Dynamic lipidomics using ESI–MS (tandem electrospray ionization mass spectrometry) of 9-deuterated choline (choline-d9) incorporation into mammalian cell PtdCho (phosphatidylcholine) permits assessment of the molecular specificity of synthesis. Bulk cell PtdCho synthesis occurs in spatially distinct locations, using separate CPTs (1,2 diacylglycerol CDP:choline cholinephosphotransferases). We assessed whether in vitro molecular selectivity of DAG (diacylglycerol) incorporation between CPTs is manifest in situ, by monitoring choline-d9 incorporation into PtdCho and lyso-PtdCho molecular species over 3 h in control cells and in CHO-K1 cells overexpressing hCEPT1. Compared with controls, the basal molecular species composition of hCEPT1 overexpressors was significantly enriched in arachidonate. This was not due to net accretion of cellular PtdCho arguing against effects of inadequate unsaturated PtdCho degradation or remodelling. Rather, time-course analyses of PtdCho and lyso-PtdCho pools showed that both arachidonate-containing DAG incorporation and turnover of PtdCho is increased in hCEPT1 overexpressors. Increased choline-d9 incorporation into arachidonyl lyso-PtdCho shows that both phospholipase A1- and A2-mediated turnover is involved. Spatially distinct molecular specificity of DAG incorporation into cellular PtdCho at the level of hCEPT1 exists in situ.
cho-k1 cells, cholinephosphotransferase, lipidomics, molecular specificity, ms, phosphatidylcholine
0300-5127
1060-1062
Hunt, A.N.
95a3e223-da96-40e7-b47d-27dce014e305
Fenn, H.C.
8fd898e4-3a8b-421b-85b4-3c4f7e9fa52a
Clark, G.T.
7a3aed07-f59f-4a97-92ad-1658c89c8a57
Wright, M.M.
51d98643-de79-45b8-8c94-e66231a525b4
Postle, A.D.
0fa17988-b4a0-4cdc-819a-9ae15c5dad66
McMaster, C.R.
f85e5869-acc9-4c58-afe4-1f10fe58539f
Hunt, A.N.
95a3e223-da96-40e7-b47d-27dce014e305
Fenn, H.C.
8fd898e4-3a8b-421b-85b4-3c4f7e9fa52a
Clark, G.T.
7a3aed07-f59f-4a97-92ad-1658c89c8a57
Wright, M.M.
51d98643-de79-45b8-8c94-e66231a525b4
Postle, A.D.
0fa17988-b4a0-4cdc-819a-9ae15c5dad66
McMaster, C.R.
f85e5869-acc9-4c58-afe4-1f10fe58539f

Hunt, A.N., Fenn, H.C., Clark, G.T., Wright, M.M., Postle, A.D. and McMaster, C.R. (2004) Lipidomic analysis of the molecular specificity of a cholinephosphotransferase in situ. Biochemical Society Transactions, 32 (Part 6), 1060-1062.

Record type: Article

Abstract

Dynamic lipidomics using ESI–MS (tandem electrospray ionization mass spectrometry) of 9-deuterated choline (choline-d9) incorporation into mammalian cell PtdCho (phosphatidylcholine) permits assessment of the molecular specificity of synthesis. Bulk cell PtdCho synthesis occurs in spatially distinct locations, using separate CPTs (1,2 diacylglycerol CDP:choline cholinephosphotransferases). We assessed whether in vitro molecular selectivity of DAG (diacylglycerol) incorporation between CPTs is manifest in situ, by monitoring choline-d9 incorporation into PtdCho and lyso-PtdCho molecular species over 3 h in control cells and in CHO-K1 cells overexpressing hCEPT1. Compared with controls, the basal molecular species composition of hCEPT1 overexpressors was significantly enriched in arachidonate. This was not due to net accretion of cellular PtdCho arguing against effects of inadequate unsaturated PtdCho degradation or remodelling. Rather, time-course analyses of PtdCho and lyso-PtdCho pools showed that both arachidonate-containing DAG incorporation and turnover of PtdCho is increased in hCEPT1 overexpressors. Increased choline-d9 incorporation into arachidonyl lyso-PtdCho shows that both phospholipase A1- and A2-mediated turnover is involved. Spatially distinct molecular specificity of DAG incorporation into cellular PtdCho at the level of hCEPT1 exists in situ.

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More information

Published date: 2004
Keywords: cho-k1 cells, cholinephosphotransferase, lipidomics, molecular specificity, ms, phosphatidylcholine

Identifiers

Local EPrints ID: 27169
URI: http://eprints.soton.ac.uk/id/eprint/27169
ISSN: 0300-5127
PURE UUID: 29b1ea77-1faf-47d3-8b70-f055aed7d043
ORCID for A.N. Hunt: ORCID iD orcid.org/0000-0001-5938-2152
ORCID for A.D. Postle: ORCID iD orcid.org/0000-0001-7361-0756

Catalogue record

Date deposited: 25 Apr 2006
Last modified: 09 Jan 2022 02:47

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Contributors

Author: A.N. Hunt ORCID iD
Author: H.C. Fenn
Author: G.T. Clark
Author: M.M. Wright
Author: A.D. Postle ORCID iD
Author: C.R. McMaster

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