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J. Biol. Chem., Vol. 278, Issue 39, 37032-37040, September 26, 2003

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Oxidized Lipoproteins Inhibit Surfactant Phosphatidylcholine Synthesis via Calpain-mediated Cleavage of CTP:Phosphocholine Cytidylyltransferase^*

Jiming Zhou , Alan J. Ryan  , Jheem Medh  and Rama K. Mallampalli   ¶ ||

From the Departments of Internal Medicine and ^¶Biochemistry and Department of Veterans Affairs Medical Center, The University of Iowa College of Medicine, Iowa City, Iowa 52242

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 CCT 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 CCT degradation, whereas overexpression of the endogenous calpain inhibitor, calpastatin, attenuated Ox-LDL-induced CCT degradation. The effects of Ox-LDL on CCT breakdown were attenuated in calpain-deficient cells. In vitro calpain digestion of CCT isolated from cells transfected with truncated or internal deletion mutants indicated multiple cleavage sites within the CCT primary structure, leading to the generation of a 26-kDa (p26) fragment. Calpain hydrolysis of purified CCT generated p26, which upon NH₂-terminal sequencing localized a calpain attack site within the CCT amino terminus. Expression of a CCT 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, CCT, resulting in impaired surfactant metabolism.

Received for publication, April 24, 2003 , and in revised form, July 7, 2003.

^* This study was supported by a Merit Review Award from the Office of Research and Development, Department of Veterans Affairs, and National Institutes of Health R01 Grants HL55584, HL68135, and HL71040 (to R. K. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed: Division of Pulmonary Diseases, Critical Care, and Occupational Medicine, Dept. of Internal Medicine, University of Iowa College of Medicine, Iowa City, IA 52242. Tel.: 319-356-1265; Fax: 319-353-6406; E-mail: rama-mallampalli{at}uiowa.edu.

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