Apolipoprotein (apo) B-100, the major protein component in low density lipoprotein (LDL), is the ligand that binds to the LDL receptor. It is important in the metabolism of LDL and elevated plasma levels of LDL-apo B are strongly associated with increased risk of coronary artery disease. Although apo B-100 is of great clinical and biological importance its primary structure has defied chemical elucidation, mainly because of its enormous size, insolubility, and tendency to aggregate. Less than 5% of the apo B-100 sequence has been reported, despite the efforts of many laboratories over the past twenty years. Here we report the complete amino acid sequence of human apo B-100 as deducted by sequence analysis of complementary DNA clones; 2,366 of the 4,536 residues were also confirmed by direct sequencing of apo B-100 tryptic peptides. The distribution of trypsin-accessible and -inaccessible peptides of the protein on LDL is non-random and they can be grouped into 5 hypothetical domains. Of 20 potential N-glycosylation sites identified in the sequence, 13 were found by direct peptide sequencing to be glycosylated, and 4 unglycosylated. Examination of the primary structure of apo B-100 reveals that it contains a large number of long (greater than 70 residues) internal repeats and an even larger number of shorter ones, suggesting that the apo B-100 sequence was derived largely from internal duplications. Finally, using synthetic peptides of a specific region of apo B-100, we have identified a potential LDL receptor-binding domain (residues 3,345-3,381) which can bind to the LDL receptor and suppress 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase activities in cultured human fibroblasts.

The human plasma apoproteins apoA-I and apoC-I enhanced the activity of partially purified lecithin: cholesterol acyltransferase five to tenfold with chemically defined phosphatidylcholine:cholesterol single bilayer vesicles as substrates. By contrast, apoproteins apoA-II, apoC-II, and apoC-III did not give any enhancement of enzyme activity. The activation by apoA-I and apoC-I differed, depending upon the nature of the hydrocarbon chains of phosphatidylcholine acyl donor. ApoA-I was most effective with a phosphatidylcholine containing an unsaturated fatty acyl chain. ApoC-I activated LCAT to the same extent with both saturated and unsaturated phosphatidylcholine substrates. Two of the four peptides obtained by cyanogen bromide cleavage of apoA-I retained some ability to activate LCAT. The efficacy of each of these peptides was approximately 25% that of the whole protein. Cyanogen bromide fragments of apoC-I were inactive. The apoproteins from HDL, HDL2, and HDL3, at low protein concentrations, were equally effective as activators of LCATand less effective than apoA-I. Higher concentrations of apoHDL, apoHDL2, and apoHDL3 inhibited LCAT activity. ApoC and apoA-II were both found to inhibit the activation of LCAT by apoA-I. The inhibition of LCAT by higher concentrations of apoHDL was not correlated with the aopA-II and apoC content.

Two synthetic peptides containing amino acid sequences analogous to the envelope glycoprotein of human T‐lymphotropic virus (HTLV) type III (HTLV‐III) and lymphadenopathy associated virus (LAV) were produced and used to immunize rabbits. The subsequent rabbit antisera neutralized HTLV‐III infectivity in vitro. The two synthetic peptides corresponded to regions associated with the gp120 or gp41 subunits respectively, of human immunodeficiency virus (HIV). This data indicates that at least two neutralizing epitopes are present on the envelope glycoprotein of HIV and these epitopes are associated with two distinct virus envelope glycoproteins. Antisera generated against these peptides neutralized infectivity of two different isolates of HTLV‐III. The data is discussed in terms of possible strategy for developing an effective vaccine against the etiologic agents of acquired immune deficiency syndrome (AIDS).

Apolipoprotein C-II (apoC-II), a protein constituent of human very low density lipoproteins, is the activator for lipoprotein lipase (LPL; triacylglycerol acyl-hydrolase, EC 3.1.1.3). The amino acid sequence of the 78 residues of apoC-II has recently been established in this laboratory. To determine the minimal sequence requirements for activation, we have prepared both native and synthetic fragments of apoC-I and tested them for their ability to activate LPL. Cyanogen bromide fragments of apoC-II coresponding to residues 1-9 and 10-59 had little ability to activate LPL. However, the COOH-terminal cyanogen bromide fragment corresponding to residues 60-78 increased hydrolysis 4fold compared to an average of 9-fold activation for the same concentration of apoC-II. The synthetic peptide containing residues 60-78 prepared by solid-phase techniques enhanced the lipolysis 3-fold. Addition of five residues produced a synthetic fragment 55-78 that enhanced the release of fatty acid 12-fold compared to 13-fold for intact apoC-I. By contrast, the synthetic peptide containing residues 66-78 did not activate. Removal ofthe three COOH-terminal residues, Gly-Glu-Glu, from fragment 60-78 decreased the ability to activate LPL by >95%. These studies suggest that the maximal activation of LPL by apoC-I1 requires a minimal sequence contained within residues 55-78.Chylomicrons and very low density lipoproteins (VLDL) are the vehicles for the transport of plasma triglycerides (1, 2). Transfer of fatty acid from the triglyceride-rich lipoproteins to the tissue requires hydrolysis of the triglyceride by lipoprotein lipase at the capillary walls. Apoliprotein C-II from human VLDL (apoC-II) plays an important role in triglyceride metabolism by serving as an activator of lipoprotein lipase (LPL; triacylglycerol acyl-hydrolase, EC 3.1.1.3) (3, 4). The amino acid sequence of apoC-I1 (Fig. 1) has recently been determined (5). The protein consists of 78 amino acid residues and is lacking cysteine, cystine, and histidine.We now present results of preliminary studies to define the sequence requirement in apoC-II necessary for the activation of LPL. In addition to testing the three cyanogen bromide (CNBr) fragments corresponding to residues 1-9, 10-59, and 60-78, we have also synthesized and tested the fragments representing residues 66-78, 60-78, and 55-78. On the basis of the results of these studies, we suggest that the sequence determinant in apoC-I1 that is required for maximal activation of LPL is contained between residues 55 and 78. MATERIALS AND METHODSIsolation of apoC-II and CNBr Fragments. apoC-II was isolated as described (6, 7) from VLDL obtained from fasting subjects with type IV or type V hyperlipoproteinemia (8). The isolated apoprotein was homogeneous by polyacrylamide gel electrophoresis in urea and sodium dodecyl sulfate and by amino acid analysis. The three CNBr fragments of apoC-II were prepared by chromatography on Bio-Gel P-30 (5).The peptide corresponding to residues 60-75 was prepared from a tryptic digest of ...