Philip K. Weech

Distribution and concentration of cholesteryl ester transfer protein in plasma of normolipemic subjects.

A MAb (TP-2) directed against human cholesteryl ester transfer protein (CETP) has been applied to the development of a competitive solid-phase RIA. Experiments with immobilized CETP have shown that upon incubation with plasma or HDL in the presence of Tween (0.05%) apo A-I (but not apo A-II) binds to CETP while TP-2 binding to CETP is concomitantly decreased. With high detergent concentration (0.5% Triton), the interference is eliminated and a specific RIA in which all plasma CETP fractions have the same affinity can be obtained. Plasma levels of CETP, apo A-I, lipids, and lipoproteins were measured in 50 normolipemic, healthy subjects of both sexes. CETP levels varied nearly fourfold with a mean value of 1.7 micrograms/ml. CETP was positively correlated only with apo A-I (r = 0.38) and HDL-triglyceride (r = 0.39). In 29 other normolipemic subjects, where several apolipoproteins were also measured, significant correlations of CETP with apo A-I (0.41), apo E (0.43), and HDL-cholesterol (0.41) were observed, but there was no significant relationship between CETP and either apo A-II, B, or D. In other experiments CETP was shown to be present mostly in HDL3 and VHDL, to display exclusively an alpha 2-electrophoretic migration, and to occur within discrete particles ranging in size from 129 to 154 kD. In conclusion, the association of CETP with apo A-I-containing lipoproteins probably explains the correlation between CETP and apo A-I levels. The relationship between CETP and apo E suggests either a common metabolism or a specific cooperative role in cholesterol ester transport for these proteins.

STRUCTURE ACTIVITY RELATIONSHIPS OF TETRAHYDROCANNABINOL ANALOGUES ON HUMAN CANNABINOID RECEPTORS

A series of Δ8-tetrahydrocannabinol (THC) and biphenylic derivatives were prepared and their binding affinity for both human cannabinoid receptors hCB1 and hCB2 evaluated.

Apolipoprotein D transcription occurs specifically in nonproliferating quiescent and senescent fibroblast cultures

We studied apolipoprotein D (apoD) mRNA in primary cultures of human diploid fibroblasts (HDF). In early‐passage HDF no apoD mRNA was detected in replicating cells in sparse culture, but the gene was expressed in quiescent cells in confluent and in serum‐starved cultures. In contrast, late‐passage HDF expressed apoD mRNA in sparse culture, but the level increased after attainment of confluence. Thus fibroblasts, the common cell‐type expressing apoD mRNA in vivo, express this characteristic following growth‐arrest. The same pattern of activation was found in another fibroblast cell line deficient in apoB/E (LDL) receptors, excluding a role for cellular cholesterol delivery by the LDL‐receptor pathway controlling apoD expression

Isolation and characterization of apolipoprotein B-48 and B-100 very low density lipoproteins from type III hyperlipoproteinemic subjects.

Two major species of human apolipoprotein (apo) B have been identified, apo B-48 and apo B-100, which are the predominant forms in chylomicrons and very low density lipoproteins (VLDL), respectively. Due to defective hepatic clearance, apo B-48 containing lipoproteins accumulate in the plasma of subjects with type III hyperlipoproteinemia. In the present study, we have used immunoaffinity chromatography to separate type III VLDL into a nonretained (apo B-48 VLDL) and a retained (apo B-100 VLDL) fraction. To achieve complete separation, as determined by electrophoresis and radioimmunoassay, it was necessary to employ two different insolubilized anti-apo B-100 monoclonal antibodies because of immunochemical heterogeneity within the apo B-100 VLDL fraction. The ability to separate apo B-100 VLDL from apo B-48 VLDL shows that the two apo B species are found on different particles. The apo B-48 VLDL had an electrophoretic mobility similar to chylomicrons, whereas the apo B-100 VLDL migrated similarly to total type III VLDL. Both fractions showed a concentration of particles with diameters approximately 100 nm, with apo B-48 VLDL being somewhat more heterogeneous in particle size. The two fractions were qualitatively similar in apolipoprotein composition but apo B-48 VLDL was enriched in apo E, relative to apo B-100 VLDL. Apo B-48 VLDL was enriched in cholesterol esters and deficient in triglycerides and phospholipids when compared with apo B-100 VLDL. The existence of immunochemical heterogeneity in the apo B-100 VLDL may reflect different functional subpopulations of particles within this fraction.

Expression of apolipoprotein B epitopes in lipoproteins. Relationship to conformation and function.

The immunochemical properties of apolipoprotein (apo) B have been studied in very low density lipoprotein (VLDL)1 (Sf 100 to 400), VLDL2 (Sf 60 to 100), VLDL3 (Sf 20 to 60), different intermediate density lipoprotein (IDL), and low density lipoprotein (LDL) subfractions isolated from patients with type IV hypertriglyceridemia. In these lipoproteins, we characterized the association of apo B with other apolipoproteins and the expression and immunoreactivity of several apo B epitopes close to the apo B receptor binding sites (3F5, 4G3, 3A8, and 5E11) and of other epitopes located on the apo B100-B48 common region (1D1 and 2D8). Immunoprecipitation showed that the proportion of lipoprotein particles expressing each apo B epitope increased from VLDL1 to LDL2; this was more apparent with 3A8 and 5E11 than with 3F5. The VLDL that were negative for apo E epitopes (60% or more of the total) were enriched in apo C. The lipoprotein particles containing apo E and/or apo C-III decreased progressively from VLDL1 (30% and 85%, respectively) to LDL2 (10% and 25%, respectively). Similar observations were made for apo C-I and apo D, demonstrating that apolipoprotein heterogeneity is greatest in the lightest lipoproteins. By competitive radioimmunoassay, the epitope for 4G3 was equally immunoreactive in each lipoprotein subclass, and the affinity constant (Ka) of 4G3 for different lipoproteins showed little variation. In contrast, both immunoreactivity and Ka of 3A8 and 5E11 increased progressively and significantly with the increasing density of the lipoprotein subclasses. This phenomenon is correlated with the increasing binding affinity of apo B in these lipoprotein subclasses to the LDL receptor of fibroblasts. We conclude that, as the apo B-containing lipoproteins become smaller, the conformation of specific regions of apo B is modified: in the receptor binding domain, the conformation of epitope 4G3, which is mapped between residues 2980 and 3080, remains constant, while that of 3A8 and 5E11 (residues 3441 to 3568) changes progressively. We propose the theory that the change in conformation in the domain spanning residues 3441 and 3568 allows the maximum expression of epitopes 3A8 and 5E11 and of the receptor binding site.

Apolipoprotein D—An atypical apolipoprotein

The structure of ApoD and its sites of synthesis have been discovered. These characteristics differ from those of the other apolipoproteins. The role of ApoD in the plasma lipoprotein system remains to be discovered, but the recent, rapid increase in our knowledge of this protein suggests that it plays an important role in the homeostasis or housekeeping of probably all organs. One of its functions is likely to be the transport of a hydrophobic ligand (a lipid) in a one-to-one molar ratio with itself. This transport is likely to occur unidirectionally between neighboring cells in an organ, and between perivascular cells and the blood circulation. The chemical structure of the natural ligand, or ligands, of ApoD in normal cells in vivo or in culture is not known, but ApoD has been shown to bind some steroids and bilirubin. Remarkable upregulation of synthesis of ApoD has been observed during regeneration of injured peripheral nerves. Perhaps the physiologic role of ApoD will prove to be more interesting and of equal importance in biology to the roles of the other apolipoproteins in cardiovascular disease.

Apolipoprotein A-I from normal human plasma: definition of three distinct antigenic determinants

We have prepared, selected and cloned four mouse hybridomas that secreted monoclonal antibodies against human plasma apolipoprotein A-I. These antibodies are all of the IgG-I subclass, and were named anti-A-I 6B8, 5G6, 3D4 and 5A6. We characterized the specificity of the antibodies, finding that all four of them reacted similarly, and with only the major proteins having the molecular weight and isoelectric focusing characteristics of apolipoprotein A-I. The antibodies reacted with all known charge-polymorphs of apolipoprotein A-I and pro apolipoprotein A-I. Thus, the polymorphs of apolipoprotein A-I are alike in that they all contain the antigenic sites of these four antibodies. In a solid-phase, antibody competition radioimmunoassay we found inhibition or enhancement of antibody binding to apolipoprotein A-I, according to the pair of antibodies tested. Antibodies 6B8, 5G6 and 3D4 were different from one another and reacted with different antigenic determinants, but 5A6 was similar to 3D4 and reacted at the same site. We compared the reactions of the four antibodies with CNBr-cleaved fragments of apolipoprotein A-I separated by polyacrylamide gel electrophoresis. We found three different patterns of reaction with the apolipoprotein A-I fragments; 6B8, 5G6 and 3D4 were different, but 5A6 resembled 3D4. Thus, the four antibodies reacted with at least three different antigenic sites in apolipoprotein A-I, which were present in different CNBr fragments of apolipoprotein A-I, but not on fragment 4 which forms the carboxy-terminal segment.

Immunochemical characterization of apolipoprotein A-I from normal human plasma. In vitro modification of apo A-I antigens.

Two series of monoclonal antibodies (MAB) directed against apolipoprotein A-I (apo A-I) reacted differentially with freshly prepared sera or plasma. MAB from Series 1 were obtained after immunization and screening with purified apo A-I, while those from Series 2 were obtained by immunization with high density lipoprotein (HDL) and screening with both HDL and apo A-I. Series 2 MAB 5F6, 3G10, and 4F7 reacted significantly better with fresh material than material stored at 4°C for longer than 1 month. Conversely, Series 1 MAB 3D4, and 688, and Series 2 MAB 2F1 reacted more strongly with the stored preparations. Series 2 MAB 4H1 reacted equally with stored or fresh material. The inability of 3D4 and 6B8 to react with fresh sera or plasma was not due to an inhibitor in the fresh material. Preparation of HDL from serum or plasma and delipidation of this material had no effect on the above phenomena, which appears related directly to apo A-I and not to the interaction of apo A-I with lipids. The variation of immunoreactivity with storage at 4°C was also shown to be unrelated to proteolysis of apo A-I. Alkaline treatment of freshly prepared sera, HDL, or apo HDL with sodium hydroxide simulated the effect of storage, allowing this material to react strongly with MAB 3D4, 688. As expected, there was a decrease in the reactivity of MAB 5F6 with alkali-treated fresh material compared to untreated fresh material. However, further investigation showed that alkaline treatment does not completely mimic the effect of storage. MAB 2F1, which reacts poorly with fresh material, reacted very poorly with alkali-treated fresh material, and MAB 3G10 and 4F7, which react well with fresh material, reacted even better with alkali-treated fresh material. While isoelectric focusing of apo HDL prepared from stored serum showed a redistribution of apo A-I toward more acidic isoforms compared to fresh serum, alkaline treatment of apo HDL resulted in partial cleavage of apo A-I and in the generation of isoforms more acidic than known apo A-I isomorphs. Therefore, alkaline treatment of serum is only a partial model for antigenic modification of apo A-I upon storage. These results demonstrate that in vitro conditions (storage or alkaline treatment) selectively modify certain antigenic sites of apo A-I, but not others. This phenomenon, which is probably related to deamidation, may affect apo A-I immunoassays with either monoclonal or polyclonal antibodies.

Monoclonal antibodies distinguish between lipid-dependent and reversible conformational states of human apolipoprotein B☆

Monoclonal antibodies (MAbs) have been prepared against apolipoprotein (apo) B which had been delipidated and resolubilized (apo Bsol). The MAbs were classified into nine groups according to their behavior in competitive binding assays and, following SDS electrophoresis and immunoblots, their corresponding epitopes were assigned to apo B48, apo B74, apo B26 and to fragments of LDL apo B (apo BLDL) generated by limited tryptic proteolysis. In addition to their reactivity with soluble apo B all antibodies also reacted with LDL which had been adsorbed to polystyrene. Competitive binding of MAbs to insolubilized antigen also indicated conformational similarities between apo Bsol and apoBLDL. These similarities were, however, less apparent when the respective antigens were in solution. In competitive radioimmunoassays the majority of anti-apo Bsol MAbs reacted better with apo Bsol than with LDL although the opposite was true for one group of MAbs. In immunoprecipitation studies the MAbs which preferentially recognize apo BLDL could precipitate 95% of 125I-LDL whereas the immunoprecipitation with those MAbs which preferentially recognize soluble apo B varied between 0 and 95%. Between 65 and 82% of 125I-apo Bsol was immunoprecipitated under the same conditions. Thus, epitopes defined by MAbs prepared against apo Bsol may be expressed on apo BLDL when it is adsorbed to plastic but not necessarily when it is in solution. For those epitopes preferentially expressed on apo BLDL, reincorporation of soluble apo B into phospholipid-cholesteryl ester microemulsions or phospholipid-cholesterol liposomes increased their immunoreactivity, whereas, the reincorporation of apo B into lipid vesicles resulted in a decreased reactivity with those MAbs which recognized better apo Bsol than apo BLDL. Thus, while the respective conformations of apo Bsol and apo BLDL are only partially similar, they can be reversibly interchanged by delipidation and relipidation.

Characterization of Antigenic Determinants of Human Apolipoprotein B Distribution on Tryptic Fragments of Low Density Lipoprotein

In the hope of obtaining useful probes to study the structure of human apolipoprotein B (apo B), we characterized monoclonal antibodies against low density lipoprotein (LDL). We examined the distribution of their corresponding antigenic determinants on tryptic fragments of LDL separated by monodimensional (SDS) or twodimensional electrophoresis. Each antibody reacted with several different fragments even when the proteolysis was apparently complete. A peptide of 125,000 daltons was the smallest fragment recognized by all the antibodies. The antibody, 2D8, which cross-reacts with apo B-48 and 3A8 which blocks the LDL pathway both reacted with the same 43,000 dalton fragment. Two other antibodies, 3F5 and 4G3, previously shown to be close together in LDL, also appeared close together in the primary structure of apo B. A determinant present on apo B-26 (1D1) was dissociated from all others examined on fragments of less than 125,000. Similarities in the patterns of reactivities with LDL-tryptic fragments between certain monoclonal antibodies and the lectins Concanavalin A and Limax flavus agglutinin indicated the proximity of the corresponding antigenic determinants to carbohydrate moieties. Competition studies suggested that the two major carbohydrate chains of LDL do not participate in the determinants themselves.