Appearance of cross linked proteins in human atheroma and rat pre-fibrotic liver detected by a new monoclonal antibody

Abstract

A new monoclonal antibody against malondialdehyde (MDA)-treated low density lipoprotein (LDL) was raised using homogenate of human atheroma as immunogen. This antibody, DLH2, was obtained by selecting the clones which did not react to native LDL but did react to copper-induced oxidized LDL (OxLDL). DLH2 showed a greater reactivity to MDA-LDL than to OxLDL. When LDL was treated with various aldehyde containing reagents, treatment of LDL with glutaraldehyde or MDA greatly increased the reactivity to the antibody, while LDL treated with 2,4-hexadienal or 4-hydroxynonenal was not reactive. Among many proteins tested, high density lipoprotein, bovine serum albumin and hemoglobin showed significant reactivity to DLH2 after they were treated with MDA or glutaraldehyde. When low density and high density lipoproteins treated with MDA were subjected to immunoblot analysis, newly formed products larger than the original apolipoproteins were detected with the antibody, suggesting that this antibody recognizes aggregated proteins with divalent short chain cross linkers. The antigenic materials were shown by immunohistochemical analysis to be present in foamy macrophages in human atheromatous lesions. DLH2 antigen did not colocalize either with apolipoprotein B. Furthermore, we found a massive accumulation of the antigenic material in Kupffer cells in the liver of rats treated with alcohol and carbonyl iron, a model of hepatic fibrosis due to oxidative stress. These results suggest the presence of cross linked proteins in damaged tissues.

Oxidized low-density lipoprotein as a biomarker of in vivo oxidative stress: from atherosclerosis to periodontitis

“…(5–7) Among many antibodies against atherosclerotic lesions, a clone that strongly reacts with copper-induced OxLDL was raised. (8) The antibody, DLH3, recognizes oxidized phosphatidylcholines (OxPC), where 1-palmitoyl-2-(9-oxo)nonanoyl-PC (9CHO-PC, also called PONPC) is one of the potent antigenic molecules (Fig.…”

Curcumin/turmeric solubilized in sodium hydroxide inhibits HNE protein modification—An in vitro study

“…Immunization of rabbits with an autoantigen modified in vitro with HNE, results in rapid loss of tolerance to the immunogen compared to immunization with the unmodified autoantigen . There is increasing evidence for the presence of antibodies against HNE-modified proteins (Zheng et al, 1992;Maggi et al, 1994;Bergmark et al, 1995;Holvoet et al, 1995;Rhodes et al, 1995;Itabe et al, 1998;Chang et al, 1999;Allison and Fearon, 2000;Mottaran et al, 2002;Willis et al, 2002;Scofield et al, 2005). Therefore, in order to prevent or reduce the damage inflicted by HNE, naturally occurring antioxidants that can inhibit HNE-modification of proteins would be of great interest.…”

The Possible Origin of Oxidized Low-Density Lipoproteins in the Circulation

“…Biochemical identification of oxidized sterols, fatty acid derivatives, or short-chain aldehydes have also been reported [28][29][30][31]. In addition to the lipid components, dramatic changes in apoB protein such as aggregation or cleavage of peptide bonds occur during LDL oxidation [32][33][34].…”