Immunochemical analysis of the electronegative LDL subfraction shows that abnormal N-terminal apolipoprotein B conformation is involved in increased binding to proteoglycans

Electronegative LDL (LDL(-)) is a minor subfraction of modified LDL present in plasma. Among its atherogenic characteristics, low affinity to the LDL receptor and high binding to arterial proteoglycans (PGs) could be related to abnormalities in the conformation of its main protein, apolipoprotein B-100 (apoB-100). In the current study, we have performed an immunochemical analysis using monoclonal antibody (mAb) probes to analyze the conformation of apoB-100 in LDL(-). The study, performed with 28 anti-apoB-100 mAbs, showed that major differences of apoB-100 immunoreactivity between native LDL and LDL(-) concentrate in both terminal extremes. The mAbs Bsol 10, Bsol 14 (which recognize the amino-terminal region), Bsol 2, and Bsol 7 (carboxyl-terminal region) showed increased immunoreactivity in LDL(-), suggesting that both terminal extremes are more accessible in LDL(-) than in native LDL. The analysis of in vitro-modified LDLs, including LDL lipolyzed with sphingomyelinase (SMase-LDL) or phospholipase A(2) (PLA(2)-LDL) and oxidized LDL (oxLDL), suggested that increased amino-terminal immunoreactivity was related to altered conformation due to aggregation. This was confirmed when the aggregated subfractions of LDL(-) (agLDL(-)) and oxLDL (ag-oxLDL) were isolated and analyzed. Thus, Bsol 10 and Bsol 14 immunoreactivity was high in SMase-LDL, ag-oxLDL, and agLDL(-). The altered amino-terminal apoB-100 conformation was involved in the increased PG binding affinity of agLDL(-) because Bsol 10 and Bsol 14 blocked its high PG-binding. These observations suggest that an abnormal conformation of the amino-terminal region of apoB-100 is responsible for the increased PG binding affinity of agLDL(-).     

A role for apolipoprotein(a) in protection of the low-density lipoprotein component of lipoprotein(a) from copper-mediated oxidation

Low-density lipoprotein (LDL) oxidation is stimulated by copper. Addition of a recombinant form of apolipoprotein(a) (apo(a); the distinguishing protein component of lipoprotein(a)) containing 17 plasminogen kringle IV-like domains (17K r-apo(a)) protects LDL against oxidation by copper. Protection is specific to apo(a) and is not achieved by plasminogen or serum albumin. When Cu(2+) is added to 17K r-apo(a), its intrinsic fluorescence is quenched in a concentration-dependent and saturable manner. Quenching is unchanged whether performed aerobically or anaerobically and is reversible by ethylenediaminetetraacetate, suggesting that it is due to equilibrium binding of Cu(2+) and not to oxidative destruction of tryptophan residues. The fluorescence change exhibits a sigmoid dependence on copper concentration, and time courses of quenching are complex. At copper concentrations below 10 microM there is little quenching, whereas above 10 microM quenching proceeds immediately as a double-exponential decay. The affinity and kinetics of copper binding to 17K r-apo(a) are diminished in the presence of the lysine analogue epsilon -aminocaproic acid. We propose that copper binding to the kringle domains of 17K is mediated by a His-X-His sequence that is located about 5A from the closest tryptophan residue of the lysine binding pocket. Copper binding may account for the natural resistance to copper-mediated oxidation of lipoprotein(a) relative to LDL that has been previously reported and for the protection afforded by apo(a) from copper-mediated oxidation of LDL that we describe in the present study.     

Modifications of low-density lipoprotein induced by arterial proteoglycans and chondroitin-6-sulfate

Association of low-density lipoproteins (LDL) with arterial chondroitin sulfate proteoglycans (CSPG) appears to contribute to their deposition in the extracellular intimal compartment and to its internalization by macrophages. CSPG and LDL interact by ionic bridges with formation of soluble and insoluble complexes. We studied the alterations on LDL structure induced by its association with arterial CSPG and other glycosaminoglycans (GAG). In soluble complexes, at low and physiological ionic strength, arterial CSPG and sulfated GAG modify the kinetics of apoB-100 proteolysis by trypsin. However, less marked alterations in the peptide patterns were observed with proteinase V8 and almost none with thermolysin. This is indirect evidence that the presence of CSPG and GAG modified the exposure of polar regions of apoB-100 in LDL. Competitive binding experiments with agarose-bound heparin and soluble GAG also suggest that after formation of insoluble complexes with arterial CSPG and resolubilization the exposure of Lys, Arg-rich segments of apoB-100 is increased. Results from differential scanning calorimetry and differential thermal spectrophotometry showed that the CSPG and GAG-induced modifications reduced the thermal stability of the surface and core in LDL. If present in vivo, the structural alterations of polar segments of the LDL protein moiety may influence the outcome of its alteration with the arterial mesenchyma.     

Metallothionein and a peptide modeled after metallothionein, EmtinB, induce neuronal differentiation and survival through binding to receptors of the low-density lipoprotein receptor family

Accumulating evidence suggests that metallothionein (MT)-I and -II promote neuronal survival and regeneration in vivo . The present study investigated the molecular mechanisms underlying the differentiation and survival-promoting effects of MT and a peptide modeled after MT, EmtinB. Both MT and EmtinB directly stimulated neurite outgrowth and promoted survival in vitro using primary cultures of cerebellar granule neurons. In addition, expression and surface localization of megalin, a known MT receptor, and the related lipoprotein receptor-related protein-1 (LRP) are demonstrated in cerebellar granule neurons. By means of surface plasmon resonance MT and EmtinB were found to bind to both megalin and LRP. The bindings were abrogated in the presence of receptor-associated protein-1, an antagonist of the low-density lipoprotein receptor family, which also inhibited MT- and EmtinB-induced neurite outgrowth and survival. MT-mediated neurite outgrowth was furthermore inhibited by an anti-megalin serum. EmtinB-mediated inhibition of apoptosis occurred without a reduction of caspase-3 activity, but was associated with reduced expression of the pro-apoptotic B-cell leukemia/lymphoma-2 interacting member of cell death (Bim_S). Finally, evidence is provided that MT and EmtinB activate extracellular signal-regulated kinase, protein kinase B, and cAMP response element binding protein. Altogether, these results strongly suggest that MT and EmtinB induce their neuronal effects through direct binding to surface receptors belonging to the low-density lipoprotein receptor family, such as megalin and LRP, thereby activating signal transduction pathways resulting in neurite outgrowth and survival.     

Monoclonal antibodies to low density lipoprotein used for the study of low- and very-low-density lipoproteins, in "ELISA" and immunoprecipitation technics

Seven monoclonal antibodies to low-density lipoprotein were studied by the ELISA for their reactivity with LDL or VLDL. Cotitration experiments showed that five of them are addressed to different antigenic epitopes. Two of the monoclonal antibodies were temperature independent whereas the others had a decreased binding activity at 37 degrees C compared to that obtained at 25 degrees C or 4 degrees C, suggesting the presence of antibodies directed to sequence or conformation epitopes, respectively. All antibodies reacted with both LDL and VLDL; four of them had a higher affinity for LDL and two others for VLDL. Immunoprecipitation of LDL and/or VLDL was observed upon immunodiffusion with certain pairs of antibodies. This may allow the use of pairs of monoclonal antibodies to LDL for the quantitative determination of apolipoprotein B in serum LDL and VLDL.     

Molecular properties and preclinical pharmacology of JNJ-1250132, a steroidal progesterone receptor modulator that inhibits binding of the receptor to DNA in vitro

Progesterone receptor modulators have diverse potential therapeutic uses, including the treatment of endometriosis, uterine fibroids and breast cancer. Here we describe the molecular properties and preclinical pharmacology of a new steroidal progestin antagonist, JNJ-1250132. The compound is a high affinity ligand for the progesterone receptor, possessing cross-reactivity with other steroid receptors comparable to that of steroidal antagonists such as mifepristone. It inhibits progestin-inducible alkaline phosphatase gene expression in T47D human breast cancer cells, and also inhibits their in vitro proliferation. It inhibits gestation in rats and progesterone-dependent endometrial transformation in rabbits with efficacies comparable to mifepristone. Like mifepristone, it is a glucocorticoid antagonist in vivo. In cell-free DNA binding assays, the compound inhibits binding of the human progesterone receptor to a progesterone response element, and thus is similar to onapristone in this regard. In contrast, as judged by proteolytic analysis, JNJ-1250132 induces a receptor conformation more similar to that induced by mifepristone, which promotes receptor binding to DNA. Therefore, JNJ-1250132 has unique effects on the progesterone receptor that may translate into a novel clinical profile.     

Beneath the minerals, a layer of round lipid particles was identified to mediate collagen calcification in compact bone formation

Astronauts lose 1-2% of their bone minerals per month during space flights. A systematic search for a countermeasure relies on a good understanding of the mechanism of bone formation at the molecular level. How collagen fibers, the dominant matrix protein in bones, are mineralized remains mysterious. Atomic force microscopy was carried out, in combination with immunostaining and Western blotting, on bovine tibia to identify unrecognized building blocks involved in bone formation and for an elucidation of the process of collagen calcification in bone formation. Before demineralization, tiles of hydroxyapatite crystals were found stacked along bundles of collagen fibers. These tiles were homogeneous in size and shape with dimensions 0.69 x 0.77 x 0.2 micro m(3). Demineralization dissolved these tiles and revealed small spheres with an apparent diameter around 145 nm. These spheres appeared to be lipid particles since organic solvents dissolved them. The parallel collagen bundles had widths mostly <2 micro m. Composition analysis of compact bones indicated a high content of apolar lipids, including triglycerides and cholesterol esters. Apolar lipids are known to form lipid droplets or lipoproteins, and these spheres are unlikely to be matrix vesicles as reported for collagen calcification in epiphyseal cartilages. Results from this study suggest that the layer of round lipid particles on collagen fibers mediates the mineral deposition onto the fibers. The homogeneous size of these lipid particles and the presence of apolipoprotein in demineralized bone tissue suggest the possibility that these particles might be of lipoprotein origin. More studies are needed to verify the last claim and to exclude the possibility that they are secreted lipid droplets.