Expression cloning of LDLB, a gene essential for normal Golgi function and assembly of the ldlCp complex.

The Chinese hamster ovary (CHO) cell mutants ldlC and ldlB, which exhibit almost identical phenotypes, define two genes required for multiple steps in the normal medial and trans Golgi-associated processing of glycoconjugates. The LDLC gene encodes ldlCp, an approximately 80-kDa protein, which in wild-type, but not ldlB, cells associates reversibly with the cytoplasmic surface of the Golgi apparatus. Here, we have used a retrovirus-based expression cloning system to clone a murine cDNA, LDLB, that corrects the pleiotropic mutant phenotypes of ldlB cells. The corresponding mRNA was not detected in ldlB mutants. LDLB encodes an approximately 110-kDa protein, ldlBp, which lacks homology to known proteins and contains no common structural motifs. Database searches identified short segments of homology to sequences from Drosophila melanogaster, Arabidopsis thaliana, and Caenorhabditis elegans, and the essentially full-length homologous human sequence (82% identity); however, as was the case for ldlCp, no homologue was identified in Saccharomyces cerevisiae. We have found that in wild-type cell cytosols, ldlCp is a component of an approximately 950-kDa "ldlCp complex," which is smaller, approximately 700 kDa, in ldlB cytosols. Normal assembly of this complex is ldlBp-dependent and may be required for Golgi association of ldlCp and for the normal activities of multiple luminal Golgi processes.

Structures of class A macrophage scavenger receptors. Electron microscopic study of flexible, multidomain, fibrous proteins and determination of the disulfide bond pattern of the scavenger receptor cysteine-rich domain.

Structures of secreted forms of the human type I and II class A macrophage scavenger receptors were studied using biochemical and biophysical methods. Proteolytic analysis was used to determine the intramolecular disulfide bonds in the type I-specific scavenger receptor cysteine-rich (SRCR) domain: Cys2-Cys7, Cys3-Cys8, and Cys5-Cys6. This pattern is likely to be shared by the highly homologous domains in the many other members of the SRCR domain superfamily. Electron microscopy using rotary shadowing and negative staining showed that the type I and II receptors are extended molecules whose contour lengths are approximately 440 A. They comprised two adjacent fibrous segments, an alpha-helical coiled-coil ( approximately 230 A, including a contribution from the N-terminal spacer domain) and a collagenous triple helix ( approximately 210 A). The type I molecules also contained a C-terminal globular structure ( approximately 58 x 76 A) composed of three SRCR domains. The fibrous domains were joined by an extremely flexible hinge. The angle between these domains varied from 0 to 180 degrees and depended on the conditions of sample preparation. Unexpectedly, at physiologic pH, the prevalent angle seen using rotary shadowing was 0 degrees , resulting in a structure that is significantly more compact than previously suggested. The apparent juxtaposition of the fibrous domains at neutral pH provides a framework for future structure-function studies of these unusual multiligand receptors.

Immunoelectron microscopy of low density lipoproteins yields a ribbon and bow model for the conformation of apolipoprotein B on the lipoprotein surface.

In the present study, the relative positions of 11 anti-apolipoprotein B monoclonal antibodies have been mapped onto the surface of human low density lipoproteins by electron microscopy. As the epitopes recognized by these antibodies have been previously located on the primary sequence of apoB, these data provide a map of the configuration of the protein on the surface of the LDL. The first 89% of apoB-100 may be modeled as a thick ribbon that wraps once around the LDL, completing the encirclement by about amino acid residue 4050. The thickness of the ribbon is sufficient to penetrate the monolayer, so that it makes contact with the core. There is a kink in the ribbon beginning almost halfway along its length at approximately apoB-48. The C-terminal 11% of apoB constitutes the "bow," an elongated structure of about 480 residues, beginning at 4050 and stretching back into one hemisphere and then crossing the ribbon into the other hemisphere between residues 3000 to 3500, thus bringing sequences in the C-terminal portion of apoB-100 near to the suggested binding site for the LDL receptor. The C-terminal sequences may act as a negative regulator of LDL receptor binding, in agreement with Parhofer et al, 1992. J. Clin. Invest. 89: 1931-1937, who reported the enhanced clearance from plasma of apoB-89-containing lipoproteins. It is proposed that in VLDL the bow could function to inhibit binding to the receptor; during lipolysis to form LDL, it is suggested that these C-terminal inhibitory sequences forming the bow would move sufficiently to allow interaction with the LDL-receptor.

Identification of apolipoprotein B100 polymorphisms that affect low-density lipoprotein metabolism: description of a new approach involving monoclonal antibodies and dynamic light scattering.

Rare mutations in apolipoprotein B (apoB) can cause defective binding of low-density lipoproteins (LDLs) to the LDL receptor, leading to elevated plasma cholesterol levels and premature atherosclerosis. This communication describes a novel approach to study the effects of apoB mutations on LDL metabolism. Monoclonal antibody MB19 identifies a common polymorphism in apoB, an Ile/Thr substitution at residue 71, by binding with a 60-fold higher affinity to apoB(Ile71)-containing LDL. Because each LDL contains a single apoB, a maximum of two LDLs may be bound by the bivalent monoclonal antibody. Thus, at the appropriate concentration, an equivalent amount of MB19 will promote substantial dimer formation of LDL containing the strongly binding apoB(Ile71), but little dimer formation of LDL containing the weakly binding apoB(Thr71). For LDL isolated from heterozygous individuals, the amount of dimer formed, determined by dynamic light scattering, yields an estimate of the allelic ratio of the two forms of LDL. For such individuals, not only the effect of the polymorphism recognized by MB19 but also the effects of other polymorphisms on the LDL allelic ratio can be determined. Examination of six normolipemic MB19 heterozygotes gave percent allelic ratios between 48:52 and 51:49 tight:weak-binding LDL, not significantly different from a 50:50 ratio. These individuals were also heterozygous for six common apoB polymorphisms, allowing calculation of the odds that each of these polymorphisms caused significant alterations in lipid levels. In contrast, the rare mutation at residue 3500 causing defective binding to the LDL receptor and familial defective apoB100 (FDB) resulted in substantial changes (26:74 and 13:87) in LDL allelic ratio in both of two FDB individuals examined.

Familial ligand-defective apolipoprotein B. Identification of a new mutation that decreases LDL receptor binding affinity.

Detection of new ligand-defective mutations of apolipoprotein B (apoB) will enable identification of sequences involved in binding to the LDL receptor. Genomic DNA from patients attending a lipid clinic was screened by single-strand conformation polymorphism analysis for novel mutations in the putative LDL receptor-binding domain of apoB-100. A 46-yr-old woman of Celtic and Native American ancestry with primary hypercholesterolemia (total cholesterol [TC] 343 mg/dl; LDL cholesterol [LDL-C] 241 mg/dl) and pronounced peripheral vascular disease was found to be heterozygous for a novel Arg3531-->Cys mutation, caused by a C-->T transition at nucleotide 10800. One unrelated 59-yr-old man of Italian ancestry was found with the same mutation after screening 1,560 individuals. He had coronary heart disease, a TC of 310 mg/dl, and an LDL-C of 212 mg/dl. A total of eight individuals were found with the defect in the families of the two patients. They had an age- and sex-adjusted TC of 240 +/- 14 mg/dl and LDL-C of 169 +/- 10 mg/dl. This compares with eight unaffected family members with age- and sex-adjusted TC of 185 +/- 12 mg/dl and LDL-C of 124 +/- 12 mg/dl. In a dual-label fibroblast binding assay, LDL from the eight subjects with the mutation had an affinity for the LDL receptor that was 63% that of control LDL. LDL from eight unaffected family members had an affinity of 91%. By way of comparison, LDL from six patients heterozygous for the Arg3500-->Gln mutation had an affinity of 36%. The percentage mass ratio of the defective Cys3531 LDL to normal LDL was 59:41, as determined using the mAb MB19 and dynamic laser light scattering. Thus, the defective LDL had accumulated in the plasma of these patients. Using this mass ratio, it was calculated that the defective Cys3531 LDL particles bound with 27% of normal affinity. Deduced haplotypes using 10 apoB gene markers showed the Arg3531-->Cys alleles to be different in the two kindreds and indicates that the mutations arose independently. The Arg3531-->Cys mutation is the second reported cause of familial ligand-defective apoB.

Apolipoprotein B and low-density lipoprotein structure: implications for biosynthesis of triglyceride-rich lipoproteins.

ApoB100 is a very large glycoprotein essential for triglyceride transport in vertebrates. It plays functional roles in lipoprotein biosynthesis in liver and intestine, and is the ligand recognized by the LDL receptor during receptor-mediated endocytosis. ApoB100 is encoded by a single gene on chromosome 2, and the message undergoes a unique processing event to form apoB48 message in the human intestine, and, in some species, in liver as well. The primary sequence is relatively unique and appears unrelated to the sequences of other serum apolipoproteins, except for some possible homology with the receptor recognition sequence of apolipoprotein E. From its sequence, structure prediction shows the presence of both sheet and helix scattered along its length, but no transmembrane domains apart from the signal sequence. The multiple carbohydrate attachment sites have been identified, as well as the locations of most of its disulfides. ApoB is the single protein found on LDL. These lipoproteins are emulsion particles, containing a core of nonpolar cholesteryl ester and triglyceride oil, surrounded by an emulsifying agent, a monolayer of phospholipid, cholesterol, and a single molecule of apoB100. An emulsion particle model is developed to predict accurately the physical and compositional properties of an LDL of any given size. A variety of techniques have been employed to map apoB100 on the surface of the LDL, and all yield a model in which apoB surrounds the LDL like a belt. Moreover, it is concluded that apoB100 folds into a long, flexible structure with a cross-section of about 20 x 54 A2 and a length of about 585 A. This structure is embedded in the surface coat of the LDL and makes contact with the core. During lipoprotein biosynthesis in tissue culture, truncated fragments of apoB100 are secreted on lipoproteins. Here, it was found that the lipoprotein core circumference was directly proportional to the apoB fragment size. A cotranslational model has been porposed for the lipoprotein assembly, which includes these structural features, and it is concluded that in permanent hepatocyte cell lines, apoB size determines lipoprotein core circumference.

Lipoprotein assembly. Apolipoprotein B size determines lipoprotein core circumference.

Apolipoprotein B (apoB) is an essential structural protein for the two triglyceride-rich lipoproteins synthesized by humans: chylomicrons and very low density lipoproteins. Although much is known about the role of apoB in clearance of lipoproteins from the circulation, relatively little is known about its role in the assembly of nascent lipoproteins. Therefore, we have investigated the relationship between the length of various N-terminal apoB fragments and the characteristics of the lipoproteins with which these fragments were associated. After the addition of puromycin, HepG2 cells secreted a discrete series of C-terminally truncated apoB fragments on lipoprotein particles including apoB25, apoB29, apoB31, apoB33, apoB36, apoB38, apoB42, apoB45, apoB49, apoB51, apoB55, apoB70, and apoB80. Also, using plasmids encoding apoB26, apoB33, apoB37, apoB42, and apoB48, C-terminally truncated apoB fragments were expressed and secreted after transient transfection of HepG2 cells. Lipoproteins bearing the metabolically labeled apoB fragments were isolated from the cell culture media and characterized in terms of size, density, flotation coefficient, and composition. Lipoprotein radii, calculated from their flotation coefficients and buoyant densities, were used to derive the circumference of the non-polar core of each lipoprotein species. When plotted as a function of apoB size, core circumference defined a straight line of near-zero intercept. The slope of this line was approximately 1 A of core circumference/1 kDa of apoB molecular mass. A model for the mechanism of lipoprotein assembly in HepG2 cells, consistent with the concept that apoB size determines lipoprotein core circumference, is proposed.

Mapping apolipoprotein B on the low density lipoprotein surface by immunoelectron microscopy.

Apolipoprotein B (apoB) was mapped using electron microscopy to visualize pairs of monoclonal antibodies binding to the low density lipoprotein (LDL) surface. The sites at which these monoclonals bind the apoB polypeptide sequence had already been established. The angular distances between all possible pairs of binding sites except one allowed the relative placement of six epitopes on the LDL sphere. We conclude that apoB extends over at least a hemisphere of the LDL surface since four epitopes are located in the Northern Hemisphere at sites arbitrarily designated as the North Pole, the Aleutian Islands, Bogotá, and in the Atlantic Ocean, while two are found in the Southern Hemisphere at Buenos Aires and at Madagascar. ApoB appears to possess a restricted flexibility, since these relative epitope locations show a substantial standard deviation in latitude and longitude. Mapping of additional epitopes may provide an answer to the question of whether apoB circumnavigates the LDL sphere.

Plasma very low density lipoproteins contain a single molecule of apolipoprotein B.

Rat and human very low density lipoproteins (VLDL) were fractionated by zonal ultracentrifugation, yielding sharply defined fractions with narrow sedimentation limits. Sedimentation coefficients for the individual fractions were determined at two densities with the analytical ultracentrifuge, and the results were analyzed to yield buoyant densities and molecular weights for the particles in each fraction. For the rat lipoproteins, the weight concentrations of triglycerides, cholesterol, phospholipid, and protein were determined for each fraction, and their molar concentrations of apolipoprotein B were measured with a radioimmunoassay. For the human lipoproteins the corresponding values were taken from Patsch et al. (Patsch, W., J. R. Patsch, G. M. Kostner, S. Sailer, and H. Braunsteiner. 1978. Isolation of subfractions of human very low density lipoproteins by zonal ultracentrifugation. J. Biol. Chem. 253:4911-4915). From these data, a ratio of the number of apoB peptides to the number of lipoprotein particles was calculated for each fraction. This ratio was close to 1 for all VLDL fractions, ranging in particle diameter from about 40 to 80 mm and 30 to 50 mm, respectively, for rat and human VLDL. The majority rat VLDL contain B-48 rather than B-100 as their (single) apoB peptide. Based on these data, we proposed that only a single copy of B-48 is required for VLDL assembly in rat liver, unless nascent hepatic VLDL contain additional apoB peptides which are uniformly lost from the plasma VLDL particles when they are analyzed.