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Although oxidatively damaged lipoproteins are implicated in vascular injury, there is little information regarding the role of highdensity lipoprotein (HDL) oxidation in atherogenesis. One potential pathway involves hypochlorous acid (HOCl) produced by myeloperoxidase (MPO), a heme protein secreted by phagocytes. We previously showed that 3-chlorotyrosine is a specific product of HOCl. Therefore, to explore the role of oxidized HDL in the pathogenesis of vascular disease, we used MS to quantify 3-chlorotyrosine in HDL isolated from plasma and atherosclerotic tissue. HDL from human aortic atherosclerotic intima had an 8-fold higher level of 3-chlorotyrosine than plasma HDL. Tandem MS analysis identified MPO as a component of lesion HDL, suggesting that the two interact in the artery wall. Moreover, immunohistochemical studies found that specific epitopes derived from HOCl colocalized with apolipoprotein A-I, the major protein of HDL. These observations strongly support the hypothesis that MPO promotes HDL oxidation in the human artery wall. Levels of 3-chlorotyrosine were elevated in HDL isolated from the blood of humans with established coronary artery disease, suggesting that circulating levels of oxidized HDL represent a unique marker for clinically significant atherosclerosis. HDL or lipid-free apolipoprotein A-I exposed to HOCl was less able to remove cholesterol from cultured cells by a pathway requiring the cell membrane transporter ATP-binding cassette transporter A1. The detection of 3-chlorotyrosine in HDL isolated from vascular lesions raises the possibility that MPO, by virtue of its ability to form HOCl, may promote atherogenesis by counteracting the established antiatherogenic effects of HDL and the ATP-binding cassette transporter A1 pathway.

Abstract-When apolipoprotein A-I mimetic peptides synthesized from either D-or L-amino acids were given orally to LDL receptor-null mice, only the peptide synthesized from D-amino acids was stable in the circulation and enhanced the ability of HDL to protect LDL against oxidation. The peptide synthesized from L-amino acids was rapidly degraded and excreted in the urine. When a peptide synthesized from D-amino acids (D-4F) was administered orally to LDL receptor-null mice on a Western diet, lesions decreased by 79%. When added to the drinking water of apoE-null mice, D-4F decreased lesions by approximately 75% at the lowest dose tested (0.05 mg/mL). The marked reduction in lesions occurred independent of changes in total plasma or HDL-cholesterol. Key Words: atherosclerosis Ⅲ HDL Ⅲ apo A-I Ⅲ LDL oxidation I nfusion 1 or transgenic expression 2 of apo A-I, the major apolipoprotein of HDL, protects against atherosclerosis in animals. Proposed mechanisms by which apo A-I protects include reverse cholesterol transport 3 and removal of low levels of oxidized lipids, "seeding molecules" required to oxidize LDL. 4 -6 Apo A-I and class A amphipathic helical peptide analogs of apo A-I remove these "seeding molecules" and prevent LDL oxidation. 4,5 Intraperitoneal administration of an apo A-I mimetic peptide enhanced the ability of HDL to protect LDL against oxidation and protected mice from diet-induced atherosclerosis without changing plasma cholesterol levels. 7 The major limitation for the use of apo A-I or apo A-I mimetic peptides as pharmacological agents has been the need for parenteral administration. Mammalian enzymes such as proteases recognize peptides and proteins synthesized from L-amino acids but rarely recognize those synthesized from D-amino acids. We report here that orally administered apo A-I mimetic peptides synthesized from D-amino acids dramatically inhibit atherosclerosis in mice independent of changes in total plasma or HDL-cholesterol. Methods MiceFemale LDL receptor-null or apoE-null mice on a C57BL/6J background were from Jackson Laboratory, Bar Harbor, Maine. LDL receptor-null mice were maintained on chow diet (Ralston Purina) until they were 4-weeks old when they were switched to a Western diet (Teklad, Madison, WI, diet No. 88137) for 6 weeks. ApoE-null mice were maintained on chow diet throughout the study. LDL receptor-null mice received the test peptide or a vehicle control by gastric gavage twice daily for the periods indicated. At 4-weeks old, the test peptide was added to the drinking water of some of the apoE-null mice and the apoE-null mice were continued on the chow diet. The lyophilized peptide was easily dissolved in a measured quantity of drinking water resulting in a clear solution and was measured and replaced with fresh solution every other day.Mice were bled under anesthesia from the retroorbital venous plexus with Animal Research Committee approval. Atherosclerotic lesions were measured as described. 8 LipoproteinsLDL and HDL were isolated as described. 4 Blood was obtained fr...

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Binding of cells of Staphylococcus aureus to fibronectin, which may represent a mechanism of host tissue adherence, involves a fibronectin-receptor protein present on the bacterial surface. Cloning of a gene coding for a staphylococcal fibronectin-binding protein and construction of a fusion protein with fibronectin-binding properties was previously reported from our laboratory. We have now sequenced the gene and deduced a primary sequence of the fibronectin-binding protein. The protein resembles other cell-wall-associated proteins on Gram-positive bacteria in that it (i) appears to be anchored in the cell membrane via its C-terminal end, (ii) contains a proline-rich repeating unit outside the membrane anchor, and (iii) contains a long (36-amino acid) signal sequence at the N terminus. The fibronectin-binding activity has been localized to a domain composed of a 38-amino acid unit repeated completely three times and partially a fourth time; the identity between the three 38-amino acid sequences varies from 42 to 87%. Three synthetic peptides mimicking the structure of each 38-amino acid unit were constructed. All three peptides interacted with fibronectin, as indicated by their ability to inhibit binding of fibronectin to staphylococcal cells, whereas an unrelated 37-amino acid peptide showed no inhibitory activity.

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Despite three decades of extensive studies on human apolipoprotein A-I (apoA-I), the major protein component in high-density lipoproteins, the molecular basis for its antiatherogenic function is elusive, in part because of lack of a structure of the full-length protein. We describe here the crystal structure of lipid-free apoA-I at 2.4 Å. The structure shows that apoA-I is comprised of an N-terminal four-helix bundle and two C-terminal helices. The N-terminal domain plays a prominent role in maintaining its lipid-free conformation, indicating that mutants with truncations in this region form inadequate models for explaining functional properties of apoA-I. A model for transformation of the lipid-free conformation to the high-density lipoprotein-bound form follows from an analysis of solvent-accessible hydrophobic patches on the surface of the structure and their proximity to the hydrophobic core of the four-helix bundle. The crystal structure of human apoA-I displays a hitherto-unobserved array of positively and negatively charged areas on the surface. Positioning of the charged surface patches relative to hydrophobic regions near the C terminus of the protein offers insights into its interaction with cell-surface components of the reverse cholesterol transport pathway and antiatherogenic properties of this protein. This structure provides a much-needed structural template for exploration of molecular mechanisms by which human apoA-I ameliorates atherosclerosis and inflammatory diseases.