Oxidized Phosphatidylcholines That Modify Proteins

doi:10.1074/jbc.271.52.33208

Oxidized Phosphatidylcholines That Modify Proteins

ANALYSIS BY MONOCLONAL ANTIBODY AGAINST OXIDIZED LOW DENSITY LIPOPROTEIN*

From the Department of Microbiology and Molecular Pathology, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-01, Japan, the
^¶ School of Pharmaceutical Sciences, Kitasato University, Shirokane, Minato-ku, Tokyo 108, Japan, and the
^§ Department of Biomembranes, The Tokyo Metropolitan Institute of Medical Science, Komagome, Bunkyo-ku, Tokyo 113, Japan

^‡To whom correspondence should be addressed.
Dept. of Microbiology and Molecular Pathology, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-01, Japan
. Tel: +81-426-85-3739; Fax: +81-426-85-3508.

Abstract

Oxidatively modified low density lipoprotein (OxLDL) is known to be involved in atherogenesis. We have previously developed a murine monoclonal antibody, FOH1a/DLH3, which recognized oxidatively modified lipoproteins as well as foam cells in human atherosclerotic lesions (Itabe, H., Takeshima, E., Iwasaki, H., Kimura, J., Yoshida, Y., Imanaka, T., and Takano, T. (1994) J. Biol. Chem. 269, 15274-15279). The antigen of this monoclonal antibody was formed by peroxidation of phosphatidylcholine (PC), and the antigenic oxidized PC (OxPC) derivatives are thought to form complexes with polypeptides including apolipoproteins. OxLDL was measured by a sensitive sandwich enzyme-linked immunosorbent assay using the monoclonal antibody and anti-human apolipoprotein B antibody, in which antigenic OxPC competed with OxLDL. When antigenic activities of PC analogs were tested by the competition assay, 1-palmitoyl-2-(9-oxononanoyl) PC (9-CHO PC) and the hydroperoxide of egg PC potently inhibited the detection of OxLDL. 1-Palmitoyl-2-linoleoyl PC was oxidized with ferrous ion and ascorbic acid, and the antigenic products were purified from the OxPC extracts on high pressure liquid chromatography columns and subsequently analyzed by laser desorption mass spectrometry. Molecular weight determination and retention times of high pressure liquid chromatography suggest that one of these products was 9-CHO PC. Other products are thought to be 8-carbon aldehyde, dihydroxy, and ketohydroxy derivatives of PC. When a C-terminal 16-mer synthetic peptide of the 70-kDa peroxisomal membrane protein was simply incubated with 9-CHO PC, it was found to be reactive in a sandwich enzyme-linked immunosorbent assay using FOH1a/DLH3 and an anti-peptide antiserum. These results suggest that the anti-OxLDL monoclonal antibody FOH1a/DLH3 reacts with several oxidized products of PC including aldehyde derivatives of PC, which covalently modify polypeptides.

Footnotes

↵* This work was supported in part by a research fund from the Kanagawa Academy of Science and Grants-in-aid for Scientific Research 06454602 and 07772183 from the Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

OxLDL

oxidized LDL

LDL

low density lipoprotein

apoB

apolipoprotein B

BSA

bovine serum albumin

MDA

malondialdehyde

PC

phosphatidylcholine

OxPC

oxidized phosphatidylcholine

5-CHO PC

1-palmitoyl-2-(5-oxovaleroyl) phosphatidylcholine

8-CHO PC

1-palmitoyl-2-(8-oxooctanoyl) phosphatidylcholine

9-CHO PC

1-palmitoyl-2-(9-oxononanoyl) phosphatidylcholine

5-COOH PC

1-palmitoyl-2-glutaroyl phosphatidylcholine

PCOOH

phosphatidylcholine hydroperoxide(s); PMP70-C; 70-kDa peroxisomal membrane protein C-terminal synthetic peptide

TLC

thin layer chromatography

ELISA

enzyme-linked immunosorbent assay

HPLC

high performance liquid chromatography

RP

reverse phase

PBS

phosphate-buffered saline (pH 7.4)

PAF

platelet activating factor.
↵² H. Itabe, S. Jimi, S. Kamimura, K. Suzuki, N. Uesugi, T. Imanaka, H. Shijo, and T. Takano, submitted for publication.
↵³ J. Kimura, I. Michishita, K. Shimamura, H. Uchiyama, H. Itabe, and T. Takano, unpublished observation.
- Received May 23, 1996.
- Revision received August 20, 1996.