Isolation and characterization of the two major apoproteins in human lipoprotein [a].

Human Lp[a] was isolated in preparative amounts from two donors; the native lipoprotein and its constituent apoproteins, apo[a] and apoB, were characterized extensively. Based on differences in apparent molecular weight, four different isoforms of apo[a], a,-%, were observed between the two donors. The number and relative distribution of these isoforms varied between donors but were constant for each donor. Each apo[a] isoform was shown to be derived from a discrete apo[a]-B100 disulfide-linked complex present before reduction. Complete delipidation of Lp[a] was followed by solubilization, reduction, and carboxamidomethylation of the constituent apoproteins. These apoproteins were then separated by immunoaffinity chromatography using anti-apo(a1or anti-apoB-Sepharose; their purity and structural integrity were demonstrated by Western blot analysis. ApoB isolated by this procedure was essentially identical to apoB from autologous LDL with respect to molecular weight, secondary structure, amino acid composition, and sialic acid content. However, apo[a] differed from apoB in that it exhibited: I) a much less a-helical, less 8, but much more disordered structure; 2) a lower proportion of aspartate, isoleucine, leucine, phenylalanine, and lysine, but a higher proportion of proline, glycine, and threonine; and 3) a much higher content of sialic acid. I These results indicate that apo[a] is not a superglycosylated form of apoB but is distinctly different in its composition and structure.Gaubatz, J. W., M. V. Chari, M. L. Nava, J. R. Guyton, and J. D. Morrisett. Isolation and characterization of the two major apoproteins in human lipoprotein[a]. J. Lipid Res. 1987. 28: 69-79. Supplementary key words Lp[a] apo[a] apoB Western blot circular dichroism amino acid analysis isoforms SDS-PAGE immunoanity chromatography secondary structure properties of apo[a], the apoprotein moiety unique to Lp[a], had been rather difficult to study until it was shown that this protein was cross-linked to apoB in the same particle by one or more disulfide bonds (4-6). Reductive cleavage of these disulfides allows selective removal of apo[a] from the lipoprotein by ultracentrifugation or heparin-Sepharose chromatography, but not by gel filtration (7, 8). The resulting apo[a]-free lipoprotein closely resembles autologous LDL with respect to electrophoretic mobility, chemical composition, size, immunochemical reactivity, and LDL receptor binding (8). However, a rigorous comparison of the chemical and physical properties of apoB from these two populations has not been reported. The present study provides a detailed comparison of this apoprotein from these two sources. Apo[a] released from either Lp[a] or apoLp[a] exhibits molecular weight heterogeneity as determined by SDSPAGE (4). The chemical nature of this heterogeneity has not been firmly established, although preliminary data suggest it may be attributed to apoprotein molecules carrying different amounts of carbohydrate. The occurence of apo[a] and apoB in the same lipoprotein particle raises the important question of how these two apoproteins relate to each other chemically and physically. In this report, we present experimental evidence demonstrating that they are distinctly different apoproteins. Human Lp[ a] is a lipoprotein whose plasma levels have been highly correlated with coronary heart disease (1-3). Although this important correlation was demonstrated a number of years ago, only rather recently has the strutture of this lipoprotein received intensive study. This has been due, in part, to the lack of reliable methods for isolating preparative amounts of the lipoprotein in pure pared to most other naturally occurring lipoproteins. The ~ Abbreviations: apo[a], apolipoprotein[a]; apoB, apolipopmtein B; CD, circular dichroism; d, density; dss, decy1 sodium sulfate; m T , dithiothreitol; EDTA, ethylenediamine tetraacetate; HDL, high density lipopmtein; mu, m i n i n d m t o r unit; LDL, low density lipoprotein; LpIa], lipopmtein[a]; PAGE, polyacrylamide gel electmphoresis; PMSF, phenylmethanlsulfonyl fluoride; RCAM, s d u c e d and carboxamidomethylated; SDS, sodium dodecylsulfate. 'Address correspondence to: Joel D. Morrisett, Ph.D., Department of Medicine, The Methodist Hospital, MWA601, Houston, TX 77030. form, and to its low plasma 'Oncentration Journal of Lipid Research Volume 28, 1987 69