Oxidative events cause degradation of apoB-100 but not of apo[a] and facilitate enzymatic cleavage of both proteins

Lipoprotein [a] (Lp[a]) contains equimolar amounts of apoB-100 and apolipoprotein [a] (apo[a]). Both proteins are amenable to degradation in vivo by mechanisms yet to be clearly defined. In this study, we examined the in vitro susceptibility of LDL and Lp[a], obtained from the same donor, to oxidation by either Cu(2)+ or the combined Crotalus adamanteus phospholipase A2 and soybean lipoxygenase system, monitoring the course of the reaction by the generation of conjugated dienes and fatty acids. In some experiments, treatment with leukocyte elastase (LE) or matrix metalloproteinase 12 (MMP-12) was administered before and after the oxidative step. In the case of Lp[a] we found that with both oxidizing systems, conditions that caused the breakdown of apoB-100 did not degrade apo[a] although oxidation-mediated changes were detected in the latter by intrinsic tryptophan fluorescence spectroscopy. Similar results were obtained with a reassembled Lp[a] obtained by incubating free apo[a] with LDL. Both apo[a] and apoB-100 were cleaved by LE and MMP-12 but the enzymatic cleavage was more marked when the preoxidized proteins were used as a substrate. Taken together, our in vitro studies indicate that apo[a] but not apoB-100 resists oxidative fragmentation, whereas both proteins are cleaved by enzymes of the serine and metalloproteinase families. We speculate that the fragments of apo[a] observed in vivo may be preferentially generated by proteolytic rather than oxidative events, whereas apoB-100 can be degraded by both mechanisms.     

Recombinant kringle IV-10 modules of human apolipoprotein(a): structure, ligand binding modes, and biological relevance

The kringle modules of apolipoprotein(a) [apo(a)] of lipoprotein(a) [Lp(a)] are highly homologous with kringle 4 of plasminogen (75-94%) and like the latter are autonomous structural and functional units. Apo(a) contains 14-37 kringle 4 (KIV) repeats distributed into 10 classes (1-10). Lp(a) binds lysine-Sepharose via a lysine binding site (LBS) located in KIV-10 (88% homology with plasminogen K4). However, the W72R substitution that occurs in rhesus monkeys and occasionally in humans leads to impaired lysine binding capacity of KIV-10 and Lp(a). The foregoing has been investigated by determining the structures of KIV-10/M66 (M66 variant) in its unliganded and ligand [epsilon-aminocaproic acid (EACA)] bound modes and the structure of recombinant KIV-10/M66R72 (the W72R mutant). In addition, the EACA liganded structure of a sequence polymorph (M66T in about 42-50% of the human population) was reexamined (KIV-10/T66/EACA). The KIV-10/M66, KIV-10/M66/EACA, and KIV-10/T66/EACA molecular structures are highly isostructural, indicating that the LBS of the kringles is preformed anticipating ligand binding. A displacement of three water molecules from the EACA binding groove and a movement of R35 bringing the guanidinium group close to the carboxylate of EACA to assist R71 in stabilizing the anionic group of the ligand are the only changes accompanying ligand binding. Both EACA structures were in the embedded binding mode utilizing all three binding centers (anionic, hydrophobic, cationic) like plasminogen kringles 1 and 4. The KIV-10/T66/EACA structure determined in this work differs from one previously reported [Mikol, V., Lo Grasso, P. V. and, Boettcher, B. R. (1996) J. Mol. Biol. 256, 751-761], which crystallized in a different crystal system and displayed an unbound binding mode, where only the amino group of EACA interacted with the anionic center of the LBS. The remainder of the ligand extended into solvent perpendicular to the kringle surface, leaving the hydrophobic pocket and the cationic center of the LBS unoccupied. The structure of recombinant KIV-10/M66R72 shows that R72 extends along the ligand binding groove parallel to the expected position of EACA toward the anionic center (D55/D57) and makes a salt bridge with D57. Thus, the R72 side chain mimics ligand binding, and loss of binding ability is the result of steric blockage of the LBS by R72 physically occupying part of the site. The rhesus monkey lysine binding impairment is compared with that of chimpanzee where KIV-10 has been shown to have a D57N mutation instead.     

Enzyme-linked immunoassay for Lp[a]

Based on our findings that rabbit antisera raised against human Lp[a] or apo[a] have the potential to cross-react with plasminogen, and in some cases have nearly equal affinities for plasminogen and Lp[a], we have developed an assay for plasma Lp[a] based on a "sandwich" ELISA that is insensitive to the presence of plasminogen. This was accomplished through the use of anti-apo[a] as a capture antibody and quantitation of the bound Lp[a], i.e., the apoB-100-apo[a] complex, with an anti-apoB antibody. Although apo[a] is heterogeneous in size, all Lp[a] particles tested, either in pure form or contained in whole plasma, gave parallel dose-response curves and were immunologically equivalent. However, when purified Lp[a] particles with different apo[a] isoforms were studied, those having larger isoforms were, on a weight basis, less reactive than those having a smaller size. Nearly equivalent reactivity was observed when protein concentration was expressed on a molar basis. The distribution of Lp[a] in a population of 84 subjects was skewed with one-third of the individuals having less than 1 mg/dl Lp[a] protein. All subjects tested had measurable concentrations of Lp[a] with a lower limit of detection of 0.030 mg/dl Lp[a] protein. The mean level was 3.2 mg/dl with a range of 0.045 to 13.3 mg/dl. These studies demonstrate the successful development of an ELISA for Lp[a] protein that is insensitive to the presence of plasminogen; that heterogeneity of Lp[a] and apo[a] are an important source of variation in the assay; and the need for an appropriate Lp[a] standard in order to minimize this variation.     

Extracellular processing of proapolipoprotein A-II in Hep G2 cell cultures is mediated by a 54-kDa protease immunologically related to cathepsin B

Apolipoprotein A-II is the second most abundant polypeptide found in human plasma high density lipoprotein particles. The primary translation product of human apo-A-II mRNA is a prepropolypeptide. We have previously reported (Gordon, J. I., Sims, H. F., Edelstein, C., Scanu, A. M., and Strauss, A. W. (1984) J. Biol. Chem. 259, 15556-15563) that the prosegment of apo-A-II was removed following export from a human hepatoma cell line (Hep G2). This represented a novel processing compartment for prosegments terminating with paired basic residues and differed from the processing of proalbumin which occurred with high efficiency prior to export from these cells. We have now characterized the enzyme responsible for this extracellular cleavage. The proapo-A-II converting activity is blocked by the thiol protease inhibitors antipain, E-64, leupeptin, and Ala-Lys-Arg chloromethyl ketone. Incubation of 125I-iodotyrosylated Ala-Lys-Arg chloromethyl ketone with serum-free media harvested from cell cultures over a 12-h period revealed a time-dependent accumulation of a 54-kDa protease. Although small quantities of the 54-kDa protease were detected in cell lysates, the major intracellular sequences labeled by the affinity probe had masses of 31.5 and 6 kDa. The 54-kDa extracellular, as well as 31.5- and 6-kDa intracellular, species were all immunoprecipitated by monospecific anti-human liver cathepsin B IgG. Addition of this antibody to media inhibited extracellular conversion of proapo-A-II to the mature protein. Based on these observations, we conclude that a "pro" cathepsin B-like protease exported by Hep G2 cells is responsible for proapo-A-II prosegment removal. It appears that cathepsin B-like proteases exhibit a complex pattern of segregation within the secretory pathway and that larger molecular weight forms of cathepsin B-like proteases are capable of accurately processing propolypeptides.     

Isolation of apolipoprotein(a) from lipoprotein(a)

An easy method was developed for the rapid and selective isolation of apo(a) from human plasma Lp(a). This procedure was applied to a "low density" Lp(a) subspecies (usually found in the density interval 1.050 to 1.070 g/ml) from a single individual whose apo(a) was of a size smaller than apoB-100. After reduction with 0.01 M dithiothreitol, apo(a) was separated from the Lp(a) particle by rate zonal centrifugation on a 7.5-30% NaBr density gradient. Two completely water-soluble products were recovered: apo(a), which contained less than 1% each of phospholipid and cholesterol, remained at the bottom of the gradient, and a lipid-rich floating LDL-like particle which contained apoB but not apo(a) and which we referred to as Lp(a-). The separation of these two components was also achieved by subjecting reduced Lp(a) to electrophoresis on 2.5-16% polyacrylamide gradient gels. However, dissociation of reduced Lp(a) could not be achieved by gel filtration in either low or high salt solutions. These observations indicate that apo(a) is associated to Lp(a) by non-covalent interactions in addition to its disulfide linkage to apoB. The latter is sensitive to chemical reduction whereas the former are broken through the action of a gravitational or electrical field.     

Amplification of human APO(a) kringel 4–37 from blood lymphocyte DNA

We have been able to amplify the lysine binding pocket region of human apo(a) kringle type 5 starting from the DNA isolated from peripheral blood lymphocytes. This development now permits the identification of Lp(a) mutants that by lacking their ability of bind to lysine/fibrin would have a lesser thrombogenic potential.