Alteration of plasma HDL cholesteryl ester composition with transgenic expression of a point mutation (E149A) of human LCAT.

We have previously identified a single amino acid mutation (hE149A) in human LCAT that increases its in vitro reactivity with phosphatidylcholine species containing sn-2 arachidonate (Wang et al. 1997. J. Biol. Chem. 272: 280-286). The purpose of the present study was to determine whether in vivo overexpression of hE149A compared with human wild-type LCAT (hLCAT-wt) would be sufficient to enrich the steady state composition of plasma HDL cholesteryl esters (CE) with long chain (>18 carbon) polyunsaturated fatty acyl species. Transgenic lines with 20-fold overexpression of hLCAT were created and studied between 12 and 16 weeks of age while consuming a chow diet. Transgenic overexpression of hE149A compared with hLCAT-wt significantly enriched HDL with CE species containing 20:4 (45%) and 22:6 n-3 (108%), at the expense of those containing 18:2, without a significant change in the plasma HDL concentration, particle size, or phospholipid fatty acyl composition. Removing the contribution of endogenous mouse LCAT by crossing the transgenic mice into the mouse LCAT knockout background resulted in even greater changes in HDL CE composition, with a 2.4-, 5-, and 5-fold increase in 20:4, 20:5 n-3, and 22:6 n-3 cholesteryl esters in the hE149A mice compared with hLCAT-wt Tg mice, respectively. Our results demonstrate that in vivo expression of hE149A significantly enriches HDL cholesteryl esters in 20- and 22-carbon fatty acyl species without affecting HDL concentration or size. Furthermore, the data suggest that endogenous mouse LCAT in hLCAT transgenic mice contributes to the plasma HDL CE pool out of proportion to its mass, presumably because the hLCAT transgene is poorly activated by mouse apolipoprotein A-I.

Evaluation of common electrophoretic methods in determining the molecular weight of apolipoprotein(a) polymorphs.

Five different gel systems were evaluated for their utility in determining the molecular weights of apolipoprotein(a) (apo(a)) polymorphs by SDS polyacrylamide or agarose gel electrophoresis. Three linear polyacrylamide gradient gels (2-16% from Isolab (Akron, OH), 4-15% from Pharmacia (Piscataway, NJ), and 2.5-6% homemade), a 4% polyacrylamide, and a 1.5% agarose gel were examined. Crosslinked phosphorylase B oligomers served as molecular weight standards. Molecular weights of four different apo(a) polymorphs were determined in each gel system and compared to values measured previously by sedimentation equilibrium. The results indicate that molecular weights obtained by gradient polyacrylamide gel electrophoresis were within 10% and often not statistically different from values acquired by sedimentation equilibrium. The use of homogenous 4% polyacrylamide and 1.5% agarose gels led to molecular weights that were overestimated by 20 and 60-70%, respectively. ApoB100, which is a commonly used molecular weight marker, was found to have anomalously fast mobility in each of the four polyacrylamide gel systems. Because its use would lead to overestimated apo(a) molecular weights, it was not useful as a molecular weight standard. Our results indicate that SDS-gradient polyacrylamide gel electrophoresis with cross-linked phosphorylase B as standard is a suitable gel system for evaluating apo(a) molecular weights.

Effect of glycation on the properties of lipoprotein(a).

Lipoprotein(a) [Lp(a)] was glycated by incubation in vitro with glucose (0 to 200 mmol/L), and its properties were compared with native Lp(a) and native and glycated LDL. Glucose was incorporated into Lp(a) in proportions that mirrored the distribution of lysines between apolipoprotein (apo) B-100 and apo(a). Because the kringle IV domains of apo(a) are lysine poor, only 10% of glucose bound to apo(a), whereas 90% was attached to the apoB-100 of Lp(a). Approximately 3% of the lysines of both Lp(a) and LDL were modified, which is a level comparable with that observed in LDL isolated from diabetic individuals. Glucose uptake by Lp(a) and LDL was almost identical and was linear as a function of concentration and time. Glycation increased the negative charge of Lp(a) and LDL as monitored by electrophoresis and ion-exchange chromatography and also reduced the affinity of Lp(a) and LDL for heparin-Sepharose. Glycation did not affect the lysine-binding property of Lp(a) or generate measurable malondialdehyde oxidation adducts. The catabolism of glycated Lp(a) by human monocyte-derived macrophages (HMDMs), like that of native Lp(a), was largely LDL receptor independent. Both glycated Lp(a) and LDL were degraded at a comparatively faster rate and stimulated greater cholesteryl ester formation than their unmodified counterparts. However, the degradation rate of glycated Lp(a) was approximately four- to fivefold slower and its stimulation of cholesteryl ester formation was ninefold lower than that of either form of LDL. These results show that Lp(a) can be glycated nonenzymatically in vitro, that the incorporation of glucose is dependent on the distribution of lysines between apo(a) and apoB-100, and that glycation does not affect the lysine-binding properties of Lp(a). Furthermore, glycation produced modest increases in the degradation rate of Lp(a) and associated cholesteryl ester synthesis by HMDMs. Based on these data, glycation does not appear to significantly enhanced the atherogenic potential of unmodified Lp(a).

Subunit composition of lipoprotein(a) protein.

We determined the molecular weight of four different apo(a) polymorphs by sedimentation equilibrium in 6 M guanidine hydrochloride in order to estimate the molar ratio of apo(a) to apoB in Lp(a). They had molecular weights of 289,000, 310,000, 341,000, and 488,000 and 15, 16, 18, and 27 kringle 4 domains, respectively. Their carbohydrate content was similar (23.2 wt %), as was their partial specific volume (0.682 mL/g). Knowing the mass of apo(a), we estimated the molar ratio of apo(a) to apoB from (1) the molecular weight of the protein moiety of the four respective parent Lp(a) particles as calculated from their mass and percentage composition and the mass of apoB, (2) the mass of apo(a) lost from Lp(a) upon its reduction and carboxymethylation, by determining the difference in mass between Lp(a) and Lp(a-), and (3) from the mass (measured by sedimentation equilibrium in 6 M guanidine hydrochloride) of the lipid-free apoB-apo(a) complex (1.06 x 10(6) daltons) of the Lp(a) particle with the smallest apo(a) polymorph by subtracting the mass of apoB. Our results obtained with each of the three different physicochemical methods indicated that the protein moiety of each of the four Lp(a) particles that was investigated consisted of a complex of two molecules of apo(a) and one molecule of apoB.

Fractured carbon fiber-based biosensor for glucose.

A microsensor for glucose was constructed using DuPont E120 fibers, which exhibit extensive fracturing upon severe anodic pretreatment. The fracturing is accompanied by an increase in the electrochemical surface area of several orders of magnitude, which is exploited here in the design of a microsensor. Following fracture, the fibers were platinized to enhance their ability to detect enzymatically produced hydrogen peroxide, and glucose oxidase was immobilized at the surface of the fiber in polypyrrole. When operated amperometrically in flow injection analysis, the treated electrodes showed a linear response to injected glucose concentration up to 10 mM, with an observed Km' near 20 mM. The sensors were found to be stable for up to 2 months when stored dry at 4 degrees C.