An initial event in atherosclerosis is the retention of lipoproteins within the intima of the vessel wall. The co-localization of apolipoprotein (apo) B and proteoglycans within lesions has suggested that retention is due to lipoprotein interaction with these highly electronegative glycoconjugates. Both apoB100-and apoB48-containing lipoproteins, i.e. low density lipoproteins (LDLs) and chylomicron remnants, are atherogenic. This suggests that retention is due to determinants in the initial 48% of apoB. To test this, the interaction of an apoB fragment (apoB17), and apoB48-and apoB100-containing lipoproteins with heparin, subendothelial matrix, and artery wall purified proteoglycans was studied. ApoB100-containing LDL from humans and human apoB transgenic mice and apoB48-containing LDLs from apoE knockout mice were used. Despite the lack of the carboxyl-terminal 52% of apoB, the apoB48-LDL bound to heparin-affinity gel as well as did apoB100-LDL. An NH 2 -terminal fragment containing 17% of full-length apoB was made using a recombinant adenovirus; apoB17 bound to heparin as well as did LDL. Monoclonal antibodies against the NH 2 -terminal region of apoB decreased apoB100 LDL binding to heparin, whereas antibodies against the LDL receptor-binding region did not alter LDL-heparin interaction. The role of the NH 2 -terminal region of apoB in LDL interaction with matrix molecules was also assessed. Media containing apoB17 decreased LDL binding to subendothelial matrix by 42%. Moreover, removal of the apoB17 by immunoprecipitation abrogated the inhibitory effect of these media. Antibodies to the NH 2 -terminal region decreased LDL binding to matrix and dermatan sulfate proteoglycans. Purified apoB17 effectively competed for binding of LDL to artery derived decorin and to subendothelial matrix. Thus, despite the presence of multiple basic amino acids near the LDL receptor-binding domain of LDL, the NH 2 -terminal region of apoB is sufficient for the interaction of lipoproteins with glycoconjugates produced by endothelial and smooth muscle cells. The presence of a proteoglycan-binding site in the NH 2 -terminal region of apoB may explain why apoB48-and apoB100-containing lipoproteins are equally atherogenic.

Viscoelastic behavior of proteins at interfaces is a critical determinant of their ability to stabilize emulsions. We therefore used air bubble surfactometry and drop volume tensiometry to examine the dynamic interfacial properties of two plasma apolipoproteins involved in chylomicron assembly: apolipoprotein A-IV and apolipoprotein B-17, a recombinant, truncated apolipoprotein B. At the air/ water interface apolipoproteins A-IV and B-17 displayed wide area-tension loops with positive phase angles indicative of viscoelastic behavior, and suggesting that they undergo rate-dependent changes in surface conformation in response to changes in interfacial area. At the triolein/ water interface apolipoprotein A-IV displayed maximal surface activity only at long interface ages, with an adsorption rate constant of 1.0 ؋ 10 ؊ 3 sec ؊ 1 , whereas apolipoprotein B-17 lowered interfacial tension even at the shortest interface ages, with an adsorption rate constant of 9.3 ؋ 10 ؊ 3 sec ؊ 1 . Apolipoprotein A-IV displayed an expanded conformation at the air/water interface and a biphasic compression isotherm, suggesting that its hydrophilic amphipathic helices move in and out of the interface in response to changes in surface pressure. We conclude that apolipoproteins A-IV and B-17 display a combination of interfacial activity and elasticity particularly suited to stabilizing the surface of expanding triglyceride-rich particles.

Previous studies demonstrated that structural perturbation of the ␣ 1 domain of apolipoprotein B (apoB) blocked the initiation of lipoprotein assembly. We explored the hypothesis that this domain may interact with the inner leaflet of the endoplasmic reticulum membrane in a manner that may nucleate microsomal triglyceride transfer proteindependent lipid sequestration. ApoB-17 (amino-terminal 17% of apoB), which contains most of the ␣ 1 domain, was expressed stably in rat hepatoma cells and recovered from medium in lipid-poor form. On incubation with phospholipid vesicles composed of 1-myristol-2-myristoyl-sn -glycero-3-phosphocholine or 1-palmitoyl-2-oleoyl-sn -gylycero-3-phosphocholine, apoB-17 underwent vesicle binding and was recovered in the d Ͻ 1.25 g/ml gradient fraction. To determine whether vesicle binding is disrupted by the same structural perturbations that block lipoprotein assembly in vivo, apoB-17 was subjected to partial and complete chemical reduction. Although normally a soluble peptide, mild reduction of apoB-17 caused its precipitation, suggesting that hydrophobic, solvent-inaccessible domains within the ␣ 1 domain of apoB are stabilized by intramolecular disulfide bonds. In contrast to apoB-17 chemically reduced in vitro, forms of apoB-17 bearing pairwise cysteine-to-serine substitutions were recovered in soluble form from transiently transfected COS-1 cell extracts. Although individual disruption of disulfide bond 2 or 4 in apoB-28 and apoB-50 was previously shown to block lipoprotein assembly in vivo, these alterations had no impact on the ability of apoB-17 to bind to phospholipid vesicles in vitro or on its capacity to form recombinant lipoprotein particles. These results suggest that while the vesicle/lipid-binding property of the ␣ 1 domain may reflect an essential role required for the initiation of lipoprotein formation, some other aspect of ␣ 1 domain function is perturbed by disruption of native disulfide bonds. -DeLozier, J. A., J. S. Parks, and G. S. Shelness. Vesicle-binding properties of wild-type and cysteine mutant forms of ␣ 1 domain of apolipoprotein B.

Previous studies have shown that cholesterol esterification activity by lecithin:cholesterol acyltransferase (LCAT) is progressively inhibited as up to three acidic acid residues are chemically modified. The purpose of this study was to determine whether three glutamic acid residues in LCAT (154, 155, and 165), that align exactly with three acidic acid residues (270, 271, and 281) in the amphipathic phospholipid binding region of apoE, were necessary for enzymatic activity. Site-directed mutagenesis was used to generate mutant constructs of LCAT in which glutamic acid residues 154, 155, and 165 were replaced with glutamine or lysine. Media harvested from transiently transfected COS cells was used as a source of LCAT for cholesterol esterification and phospholipase A 2 (PLA 2 ) assays. Cholesterol esterification for all mutant constructs (11-26 nmol CE/h/ g) was similar to or greater than that of wild type LCAT (16 nmol CE/h/ g), except for a triple mutant, in which glutamic acid residues 154, 155, and 165 were changed to lysines (5 nmol CE/h/ g). PLA 2 activity followed a similar trend. There was a significant decrease in the cholesterol esterification to PLA 2 activity ratio when residue 165 was mutated from its wild type negative charge (E) to an uncharged (Q) or positive (K) charged residue (10.2 vs. 6.0 vs. 4.3, respectively). We conclude that glutamic acid residues 154, 155, and 165 individually or collectively are not necessary for LCAT activity and that residue 165 may be in a region of LCAT that is involved with cholesterol binding or is sensitive to cholesterol binding at the active site of the enzyme.-