Products of lipid peroxidation induce missorting of the principal lysosomal protease in retinal pigment epithelium.

Phagocytosis of photoreceptor outer segments (OS) by retinal pigment epithelium (RPE) is essential for OS renewal and survival of photoreceptors. Internalized, oxidatively modified macromolecules perturb the lysosomal function of the RPE and can lead to impaired processing of photoreceptor outer segments. In this study, we sought to investigate the impact of intracellular accumulation of oxidatively damaged lipid-protein complexes on maturation and distribution of cathepsin D, the major lysosomal protease in the RPE. Primary cultures of human RPE cells were treated with copper-oxidized low density lipoprotein (LDL) and then challenged with serum-coated latex beads to stimulate phagocytosis. Three observations were noted to occur in this experimental system. First, immature forms of cathepsin D (52 and 46 kDa) were exclusively associated with latex-containing phagosomes. Second, maturation of cathepsin D was severely impaired in RPE cells loaded with oxidized LDL (oxLDL) prior to the phagocytic challenge. Third, pre-treatment with oxLDL caused sustained secretion of pro-cathepsin D and the latent form of gelatinase A into the extracellular space in a dose-dependent manner. These data stimulate the hypothesis that intracellular accumulation of poorly degradable, oxidized lipid-protein cross-links, may alter the turnover of cathepsin D, causing its mistargeting into the extracellular space together with the enhanced secretion of a gelatinase.

Phospholipids in oxidized low density lipoproteins perturb the ability of macrophages to degrade internalized macromolecules and reduce intracellular cathepsin B activity.

Previous studies showed that pre-treatment of mouse peritoneal macrophages (MPM) with oxidized low density lipoprotein (oxLDL) repressed subsequent degradation of oxLDL following uptake. Parallel studies on the activity of the lysosomal protease, cathepsin B in MPM and in vitro indicate that oxLDL also induces a reduction in this activity. We now report that pre-treatment of MPM with the lipid portion of oxLDL induced a reduction both in the degradation of internalized small macromolecules such as maleylated (mal) BSA (30%) or larger ones such as aggregated LDL (100%), and in cellular cathepsin B activity (42%). Binding and uptake of malBSA were not affected. Pre-treatment of MPM for 2 h with oxidized phosphatidylcholine (oxPC) isolated from oxLDL or generated from Cu2+-treated 1-palmitoyl-2-linoleoyl phosphatidylcholine (oxPLPC), also inhibited 125I-malBSA degradation and reduced cathepsin B activity in MPM and in vitro. Further separation of oxPLPC and oxPC from oxLDL by thin layer chromatography led to the isolation of a polar lipid fraction possessing most of the biological activity in oxPC. Partial characterization of this fraction from oxPLPC using liquid chromatography/electrospray ionization/mass spectrometry indicated that this polar fraction containing fragmentation products of linoleate, was still comprised of multiple bioactive molecular ions. Collectively, these results suggest that specific oxPC fractions in oxLDL are partially responsible for the alterations in MPM metabolism under study induced by oxLDL.

Phospholipid hydroxyalkenals: biological and chemical properties of specific oxidized lipids present in atherosclerotic lesions.

OBJECTIVE: Phosphatidylcholine hydroxyalkenals (PC-HAs) are a class of oxidized PCs derived from lipid peroxidation of arachidonate or linoleate at the sn-2 position to form terminal gamma-hydroxy, alpha-, and beta-unsaturated aldehydes. The aim of this study was to characterize some of their biological properties, ascertain the mechanism of their action, and assess whether they have in vivo relevance. METHODS AND RESULTS: Combinations of cell biological approaches with radiolabels, mass spectroscopy, and immunochemical as well as immunohistochemical techniques were used to show that PC-HAs reduce the proteolytic degradation by mouse peritoneal macrophages (MPMs) of internalized macromolecules, such as maleylated bovine serum albumin, and that the activity of the lysosomal protease, cathepsin B, in MPMs form Michael adducts with MPM proteins and with N-acetylated cysteine in vitro form pyrrole adducts with MPM proteins and reduce the maturation of Rab5a, thereby impairing phagosome-lysosome fusion (maturation) in phagocytes; they are present unbound and as pyrrole adducts in human atherosclerotic lesions. CONCLUSIONS: PC-HAs are present in vivo and possess multiple functions characteristic of oxidized LDL and 4-hydroxynonenal.

Phospholipids in oxidized LDL not adducted to apoB are recognized by the CD36 scavenger receptor.

Previous studies have shown that oxidation of low-density lipoprotein (oxLDL) results in its recognition by scavenger receptors on macrophages. Whereas blockage of lysyl residues on apoB-100 of oxLDL by lipid peroxidation products appears to be critical for recognition by the scavenger receptor class A (SR-A), modification of the lipid moiety has been suggested to be responsible for recognition by the scavenger class B receptor, CD36. We studied the recognition by scavenger receptors of oxidized LDL in which lysyl residues are blocked prior to oxidation through methylation [ox(m)LDL]. This permits us to minimize any contribution of modified apoB-100 to the recognition of oxLDL, but does not disrupt the native configuration of lipids in the particle. We found that ox(m)LDL was recognized by receptors on mouse peritoneal macrophages (MPM) almost as well as oxLDL. Ox(m)LDL was recognized by CD36-transfected cells but not by SR-A-transfected cells. Oxidized phospholipids (oxPC) transferred from oxLDL or directly from oxPC to LDL, conveyed recognition by CD36-transfected cells, confirming that CD36 recognized unbound oxidized phospholipids in ox(m)LDL. Collectively, these results suggest that oxPC not adducted to apoB within the intact oxLDL particle are recognized by the macrophage scavenger receptor CD36, that these lipids are not recognized by SR-A, and that they can transfer from oxidized to unoxidized LDL and induce CD36 recognition.

Hydroxynonenal inactivates cathepsin B by forming Michael adducts with active site residues.

Oxidation of plasma low-density lipoprotein (oxLDL) generates the lipid peroxidation product 4-hydroxy-2 nonenal (HNE) and also reduces proteolytic degradation of oxLDL and other proteins internalized by mouse peritoneal macrophages in culture. This leads to accumulation of undegraded material in lysosomes and formation of ceroid, a component of foam cells in atherosclerotic lesions. To explore the possibility that HNE contributes directly to the inactivation of proteases, structure-function studies of the lysosomal protease cathepsin B have been pursued. We found that treatment of mouse macrophages with HNE reduces degradation of internalized maleyl bovine serine albumin and cathepsin B activity. Purified bovine cathepsin B treated briefly with 15 microM HNE lost approximately 76% of its protease activity and also developed immunoreactivity with antibodies to HNE adducts in Western blot analysis. After stabilization of the potential Michael adducts by sodium borohydride reduction, modified amino acids were localized within the bovine cathepsin B protein structure by mass spectrometric analysis of tryptic peptides. Michael adducts were identified by tandem mass spectrometry at cathepsin B active site residues Cys 29 (mature A chain) and His 150 (mature B chain). Thus, covalent interaction between HNE and critical active site residues inactivates cathepsin B. These results support the hypothesis that the accumulation of undegraded macromolecules in lysosomes after oxidative damage are caused in part by direct protease inactivation by adduct formation with lipid peroxidation products such as HNE.