Preferential association of apolipoprotein E Leiden with very low density lipoproteins of human plasma

Apolipoprotein (apo) E Leiden is a rare variant of human apoE characterized by defective receptor binding and associated with dominant transmission of type III hyperlipoproteinemia. In heterozygotes, apoE Leiden is present in higher concentrations in both total plasma and very low density lipoproteins (VLDL) than the other apoE allele product. In the present study we analyzed cell expression and plasma lipoprotein association of apoE Leiden to determine whether the unequal concentration of the two apoE allele products could be explained by differences in secretion rate from the hepatocyte or by preferential association with VLDL. We transfected the rat hepatoma cell line McA-RH7777 with apoE Leiden or normal human apoE3, and studied their secretion and media distribution. In pulse-chase experiments, the secretion of apoE Leiden was comparable to that of both human apoE3 and rat endogenous apoE, approaching 100% in 90 min. In similar transfection experiments, secreted apoE Leiden was significantly less glycosylated than normal apoE3 (21.7% vs. 36.6%, P < 0.005, n = 4), a finding also noted for apoE Leiden in human plasma. In in vitro incubation experiments, apoE Leiden showed a markedly higher preference for VLDL of normolipidemic human plasma when compared to both apoE3 (2.6-fold, P < 0.001) and apoE4 (1.6-fold, P < 0.001). These results suggest that the accumulation of apoE Leiden in VLDL derives from a high affinity of the mutant protein for the VLDL. This enrichment in defective apoE probably exacerbates impairment of VLDL removal from the circulation, thus contributing to the dominant transmission of type III hyperlipoproteinemia

Apolipoprotein A-IMilano. Detection of normal A-I in affected subjects and evidence for a cysteine for arginine substitution in the variant A-I

The A-I(Milano) contains a variant form of apolipoprotein A-I, which unlike normal A-I, has cysteine. We have characterized the A-I(Milano) isoforms in two affected subjects, D.M. and D.G. In these subjects, the two most prominent A-I(Milano) isoforms were displaced from the corresponding normal A-I isoforms by a single charge unit toward the anode, as determined by isoelectric focusing. The amino acid compositions of the purified isoforms, which were separated by either preparative isoelectric focusing or thiopropyl-Sepharose 6B chromatography, indicated that the A-I(Milano) from these subjects contained both a normal A-I (A-I(N)) and a cysteine-containing variant A-I (A-I(Cys)). Furthermore, amino acid analyses suggested that the A-I(Cys) differed from normal A-I by a single cysteine for arginine substitution, which was sufficient to account for the charge difference between the two proteins. Partial sequence analysis revealed that an arginine in normal A-I was replaced by cysteine in the variant A-I at residue 173. Consistent with the amino acid analyses, the cysteine-containing isoforms shifted one charge unit toward the cathode after modification of the cysteine residue with cysteamine. Quantitation of the relative amounts of the A-I(N) and A-I(Cys) in D.M. and D.G. by thiopropyl-Sepharose 6B chromatography revealed that the relative levels were different in each subject. The percentage of the total A-I represented by the A-I(N) in D.M. and D.G. was 16.1 and 25.7%, respectively. The demonstration of variable amounts of normal A-I in A-I(Milano) subjects raises some interesting questions regarding the genetics, regulation, and metabolism of apolipoprotein A-I

Structural characterization of a low density lipoprotein receptor-active apolipoprotein E peptide, ApoE3-(126-183).

Apolipoprotein E (apoE) plays a critical role in lipoprotein particle clearance from blood plasma through its interaction with the low density lipoprotein (LDL) receptor and other related receptors. Here, we studied a 58-residue peptide encompassing the receptor binding region of apoE. ApoE3-(126-183) was generated by cyanogen bromide cleavage of recombinant apoE3-(1-183), purified by reversed-phase high pressure liquid chromatography, and characterized by mass spectrometry. Far UV CD spectroscopy of the peptide showed that it is unstructured in aqueous solution. The addition of trifluoroethanol or dodecylphosphocholine induces the peptide to adopt an alpha-helical conformation. ApoE3-(126-183) efficiently transforms dimyristoylphosphatidylglycerol (DMPG) vesicles into peptide-lipid complexes. Analysis of apoE3-(126-183). DMPG complexes by electron microscopy revealed disc-shaped particles with an average diameter of 13 +/- 3 nm. Flotation equilibrium analysis yielded a particle molecular mass of 252 kDa. Far UV CD analysis of apoE3-(126-183).DMPG discs provided evidence that the peptide adopts a helical conformation. Competition binding experiments with (125)I-labeled low density lipoprotein (LDL) were conducted to assess the ability of apoE3-(126-183).DMPG complexes to bind to the LDL receptor. Both N-terminal apoE and the peptide, when complexed with DMPG, competed with (125)I-LDL for binding sites on the surface of cultured human skin fibroblasts. Under the conditions employed, apoE3-(126-183).DMPG complexes were similar to apoE3-(1-183).DMPG discs in their ability to bind to the receptor, demonstrating that the peptide represents a good model to study the interaction between apoE and the LDL receptor. Preliminary NMR results indicated that a high resolution structure of the apoE3-(126-183) peptide is obtainable.Journal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S.info:eu-repo/semantics/publishe