Oxidized lipoproteins inhibit surfactant phosphatidylcholine synthesis via calpain-mediated cleavage of CTP:phosphocholine cytidylyltransferase.
We investigated effects of pro-atherogenic oxidized lipoproteins on phosphatidylcholine (PtdCho) biosynthesis in murine lung epithelial cells (MLE-12). Cells surface-bound, internalized, and degraded oxidized low density lipoproteins (Ox-LDL). Ox-LDL significantly reduced [3H]choline incorporation into PtdCho in cells by selectively inhibiting the activity of the rate-regulatory enzyme, CTP:phosphocholine cytdylyltransferase (CCT). Ox-LDL coordinately increased the cellular turnover of CCTalpha protein as determined by [35S]methionine pulse-chase studies by inducing the calcium-activated proteinase, calpain. Forced expression of calpain or exposure of cells to the calcium ionophore, A23187, increased CCTalpha degradation, whereas overexpression of the endogenous calpain inhibitor, calpastatin, attenuated Ox-LDL-induced CCTalpha degradation. The effects of Ox-LDL on CCTalpha breakdown were attenuated in calpain-deficient cells. In vitro calpain digestion of CCTalpha isolated from cells transfected with truncated or internal deletion mutants indicated multiple cleavage sites within the CCTalpha primary structure, leading to the generation of a 26-kDa (p26) fragment. Calpain hydrolysis of purified CCTalpha generated p26, which upon NH2-terminal sequencing localized a calpain attack site within the CCTalpha amino terminus. Expression of a CCTalpha mutant where the amino-terminal cleavage site and a putative carboxyl-terminal hydrolysis region were modified resulted in an enzyme that was significantly less sensitive to proteolytic cleavage and restored the ability of cells to synthesize surfactant PtdCho after Ox-LDL treatment. Thus, these results provide a critical link between proatherogenic lipoproteins and their metabolic target, CCTalpha, resulting in impaired surfactant metabolism.
Zhou
Jiming
J
Department of Internal Medicine, The University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
Ryan
Alan J
AJ
Medh
Jheem
J
Mallampalli
Rama K
RK
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002154:Calpain / Q000502:physiology*; D019916:Choline-Phosphate Cytidylyltransferase / Q000737:chemistry / Q000378:metabolism*; D008077:Lipoproteins, LDL / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010713:Phosphatidylcholines / Q000096:biosynthesis*; D011663:Pulmonary Surfactants / Q000378:metabolism*; D051381:Rats
D008077:Lipoproteins, LDL; D010713:Phosphatidylcholines; D011663:Pulmonary Surfactants; C099252:oxidized low density lipoprotein; D019916:Choline-Phosphate Cytidylyltransferase; D002154:Calpain