The amphipathic alpha-helical repeats of apolipoprotein A-I are responsible for binding of high density lipoproteins to HepG2 cells.

Nine monoclonal antibodies (mAbs) against apoA-I reacting with distinct but overlapping epitopes covering more than 90% of the sequence have been used to block the interaction of 125I-labeled high density lipoprotein (125I-HDL) with HepG2 cells in order to delineate the cell binding domain of apolipoprotein A-I (apoA-I). While 2 mAbs reacting with epitopes exclusively localized in the N-terminal region (residues 1 to 86) enhanced slightly association of 125I-HDL, all other mAbs, which react with epitopes localized in the regions of amphipathic alpha-helical repeats, inhibited that association by 9 to 15%. Although this inhibition is not significant compared to the effect of an irrelevant mAb, combination of these mAbs could significantly inhibit the association of 125I-HDL (32 to 43%) as could polyclonal antibodies (up to 95%). These results are compatible with the concept of HDL binding to these cells via the nonexclusive interaction of each of the amphipathic alpha-helical repeats of apoA-I. When the same approach was applied to block the association of 3H-cholesteryl ether (CE)-labeled HDL to HepG2 cells, each anti-apoA-I could inhibit by 15 to 25% the cellular association of cholesteryl ether while mAbs in combination or polyclonal antibodies could inhibit this association up to 45% or 60%, respectively. The cholesteryl ether radioactivity that remained associated with the cells (40%) in the presence of polyclonal antibodies could be effectively blocked by addition of an antibody against the receptor binding domain of apoE (1D7). Therefore, the differential cellular association of cholesteryl ether compared to apolipoprotein can be explained by the presence of apoE secreted by HepG2 and apoE or apoB/E receptors. Thus, we conclude that the optimum uptake of both cholesteryl ether and apoA-I of HDL by cells requires the accessibility of the entire apoA-I and the cooperative binding of the amphipathic alpha-helical repeats to HepG2 cell membranes. This type of interaction would explain the competitive binding observed for apoA-I, -A-II, and -A-IV by others.     

Epitope mapping of apolipoprotein A-I using endoproteinase cleavage and monoclonal antibodies in an enzyme-linked immunosorbent assay.

The epitopes for two monoclonal antibodies (MAbs) directed towards human apolipoprotein A-I (apoA-I), designated AI-1 and AI-3, have been more precisely defined. Previous work in our laboratory demonstrated that AI-1 and AI-3 recognize antigenic determinants located within cyanogen bromide (CNBr) fragments 1 (CF1) and 3 (CF3), respectively. Using peptides generated from endoproteinase cleavage of CF1 and CF3, we now report that both MAbs are specific for two previously unreported epitopes along the apoA-I molecule. The ability of whole endoproteinase digest mixtures to bind the MAbs, as determined by means of a competitive enzyme-linked immunosorbent assay (ELISA), indicated regions of CF1 and CF3 that were likely to form the epitopes. Purified peptides derived from the digests were then used to localize the epitopes recognized by MAbs AI-1 and AI-3 to within residues 28-47 and 140-147 of apoA-I, respectively. We have previously reported that the epitopes for both MAbs are exposed on HDL2, HDL3, and free apoA-I. Thus, the precise mapping of the binding sites recognized by AI-1 and AI-3 has enabled the identification of regions along apoA-I that are exposed on the surface of lipoprotein particles.     

Immunochemical characterization of six monoclonal antibodies to human apolipoprotein A-I: epitope mapping and expression.

We have produced and characterized six murine monoclonal antibodies to human apolipoprotein A-I named A-I-9, A-I-12, A-I-15, A-I-16, A-I-19, and A-I-57. All monoclonal antibodies were specific for apolipoprotein A-I and bound between 55% and 100% of 125I-labeled high density lipoproteins (HDL) in a fluid phase radioimmunoassay. All antibodies possessed a higher affinity to apoA-I in HDL than to free, delipidated apoA-I. Two of them, particularly A-I-12 and A-I-15, which were directed to the same or very close epitopes on the molecule, recognized very poorly the delipidated protein. Binding of apoA-I to phospholipid restored the immunoreactivity of the monoclonal antibodies to the protein suggesting that lipids play an important role in determining the immunochemical structure of apoA-I. Using CNBr fragments and synthetic peptides, the epitopes for the antibodies were mapped as follows: A-I-19, CNBr fragment 1; A-I-12 and 15, CNBr fragment 2; A-I-9 and A-I-16, CNBr fragment 3; A-I-57, CNBr fragment 4. Antibody A-I-57 failed to recognized a mutant form of apoA-I, A-IMilano (Arg173----Cys) by immunoblotting and by competitive radioimmunoassay demonstrating that substitution of a single amino acid in human apoA-I may cause the loss of an antigenic determinant.     

Differential effects of lecithin and cholesterol on the immunoreactivity and conformation of apolipoprotein A-I in high density lipoproteins.

Recently identified epitopes in apoA-I define a distinct N-terminal region with a complex tertiary structure, characterized by multiple discontinuous epitopes. Other epitopes are constituted of short domains centered either on beta-turns or random coils or on the 22-mer amphipathic alpha-helices (Marcel, Y. L., Provost, P. R., Koa, H., Raffa?, E., Vu Dac, N., Fruchart, J.-C., and Rassart, E. (1991) J. Biol. Chem. 266, 3644-3653). The compared immunoreactivity of seven epitopes studies here in response first to delipidation of high density lipoprotein (HDL) apoA-I by detergents, and second to modifications of HDL lipid composition by phospholipase A2 or by enrichment in surface lipids demonstrates that apoA-I has a flexible conformation which is readily responsive to the nature and concentration of bound lipids and that the structure of lipid-free apoA-I is significantly different from that of HDL-bound apoA-I, possibly representing a condensed molecule with several masked domains. In HDL apoA-I, these epitopes define five distinct domains which are characterized by particular responses to lipid modifications. However, two domains, each starting at the N-terminal beta-turn of an amphipathic alpha-helical repeat (residues 99-121 and 186-209, respectively) have almost identical immunoreactivity whether after detergent treatment or after changes in cholesterol and phospholipid levels, a property which probably reflects the known periodicity of apoA-I structural 22-mers. The immunoreactivity of a discontinuous epitope, representative of the N-terminal domain, is inversely related to the concentration of phospholipids, a unique characteristic among the epitopes tested here which indicates that the complex N-terminal region interacts with phospholipids, either directly or indirectly. These studies demonstrate that the conformation of multiple domains of HDL apoA-I is dependent on lipid phase composition and differentially affected by cholesterol and phospholipids.     

Identification of domains in apoA-I susceptible to proteolysis by mast cell chymase. Implications for HDL function

When stimulated, rat serosal mast cells degranulate and secrete a cytoplasmic neutral protease, chymase. We studied the fragmentation of apolipoprotein (apo) A-I during proteolysis of HDL(3) by chymase, and examined how chymase-dependent proteolysis interfered with the binding of eight murine monoclonal antibodies (Mabs) against functional domains of apoA-I. Size exclusion chromatography of HDL(3) revealed that proteolysis for up to 24 h did not alter the integrity of the alpha-migrating HDL, whereas a minor peak containing particles of smaller size with prebeta mobility disappeared after as little as 15 min of incubation. At the same time, generation of a large (26 kDa) polypeptide containing the N-terminus of apoA-I was detected. This large fragment and other medium-sized fragments of apoA-I produced after prolonged treatment with chymase were found to be associated with the alphaHDL; meanwhile, small lipid-free peptides were rapidly produced. Incubation of HDL(3) with chymase inhibited binding of Mab A-I-9 (specific for prebeta(1)HDL) most rapidly (within 15 min) of the eight studied Mabs. This rapid loss of binding was paralleled by a similar reduction in the ability of HDL(3) to induce high-affinity efflux of cholesterol from macrophage foam cells, indicating that proteolysis had destroyed an epitope that is critical for this function. In sharp contrast, prolonged degradation of HDL(3) by chymase failed to reduce the ability of HDL(3) to activate LCAT, even though it led to modification of three epitopes in the central region of apoA-I that are involved in lecithin cholesterol acyltransferase (LCAT) activation. This differential sensitivity of the two key functions of HDL(3) to the proteolytic action of mast cell chymase is compatible with the notion that, in reverse cholesterol transport, intactness of apoA-I is essential for prebeta(1)HDL to promote the high-affinity efflux of cellular cholesterol, but not for the alpha-migrating HDL particles to activate LCAT.     

Monoclonal antibodies as probes of high-density lipoprotein structure: identification and localization of a lipid-dependent epitope

Eight stable murine monoclonal antibodies (mabs) were raised against human high-density lipoproteins (HDL). Three different antibody reactivities were demonstrated by immunoblotting. A group of five antibodies were specific for apolipoprotein A-I (apoA-I) and bound to similar or overlapping epitopes. The second type of reactivity, shown by mab-32, was specific for apoA-II. In the third group, two antibodies showed high reactivity with apoA-II and slight cross-reactivity with apoA-I. The properties of two antibodies, mab M-30 specific for apoA-I and mab M-32 specific for apoAII, were characterized in detail as probes of HDL structure. The association of 125I-labeled HDL or synthetic complexes of apoA-I and phosphatidylcholine with mab M-30 was lipid dependent. Mab M-32 binding to apoA-II was independent of lipid. The lipid-dependent epitope bound by mab M-30 has been localized to an 18 amino acid synthetic apoA-I peptide. Moreover, studies with HDL2, HDL3, and immunoadsorbed HDL subfractions indicate that binding of mab M-30 to HDL is influenced by some component within the microenvironment individual HDL particles. These lines of evidence suggest that the molar ratio of apoA-I to apoA-II is the critical determinant. Binding of mab M-32 to HDL increased the reactivity of HDL to mab M-30 in a dose-dependent manner, indicating an unusual form of cooperativity between two mabs that recognize different proteins in HDL. These monoclonal antibodies will be valuable in studies of the metabolic significance of protein-protein and lipid-protein interactions in HDL.     

Comparison of the cytolytic effects in vitro on Trypanosoma brucei brucei of plasma, high density lipoproteins, and apolipoprotein A-I from hosts both susceptible (cattle and sheep) and resistant (human and baboon) to infection.

The African trypanosome, Trypanosoma brucei brucei causes a fatal wasting disease in livestock but does not ordinarily infect humans, apparently because this unicellular parasite is lysed by high density lipoproteins (HDL) in human serum. To assess whether there is a specific active constituent in trypanolytic HDL, we have systematically compared the cytotoxic action on T.b.brucei in vitro of native and delipidated HDL, and of individual apolipoproteins, from nonpermissive hosts (human and baboon) with their counterparts from susceptible hosts (cattle and sheep). When suspensions of trypanosomes were incubated for 2 h at 37 degrees C with human or baboon plasma most cells were lysed, but not with bovine or sheep plasma. Similarly, HDL isolated from human and baboon plasma were trypanolytic (typically about 95% and 60% lysis, respectively, at 1 mg protein/ml), whereas bovine and sheep HDL were benign (less than 8% lysis). Subfractionation of human HDL by serial isopycnic ultracentrifugation and by heparin-Sepharose affinity chromatography established that the denser and smaller particles had greater trypanolytic activity both in vitro and in vivo. When human HDL was delipidated, the trypanocidal activity was associated with the water-soluble protein (apolipoprotein) fraction and not with the lipid constituents. Bovine apolipoproteins were also weakly trypanolytic in free solution (20-40% lysis), but not when complexed with cholesterol-phospholipid liposomes (less than 10% lysis). The major apolipoprotein of human HDL, apolipoprotein (apo) A-I had full trypanolytic activity (89-95% lysis at 1 mg protein/ml) when purified, whether in solution or incorporated into liposomes, but other apolipoproteins isolated from human HDL, including apoA-II, apoC, and apoE, were nontrypanolytic. Purified baboon apoA-I was also trypanolytic, though less potent than human apoA-I, but apoA-I from permissive hosts (cattle and sheep) was inactive when presented in liposomes. Incubation of bovine or sheep HDL with purified human apoA-I, and subsequent separation of the HDL by ultracentrifugation, produced chimeric HDL containing significant amounts of the human apolipoprotein; these particles showed appreciable trypanolytic activity. By contrast, human HDL particles in which about 70% of the apoA-I had been displaced with apoA-II had markedly reduced lytic properties compared to the native HDL (30% versus 80% lysis at 0.6 mg total protein/ml). We tentatively conclude that the trypanolytic activity of native human or baboon plasma resides in the apoA-I content of the HDL particles and that, conversely, bovine and sheep plasma are inactive because the apoA-I polypeptide present in their HDL lacks trypanocidal activity.     

Fusion proteins encoded by orf 129L of ectromelia and orf A30L of smallpox viruses cross-react with neutralizing monoclonal antibodies

Open reading frame (orf) 129L of ectromelia (EV) and orf A30L of smallpox viruses (SPV) encoding fusion proteins were cloned and expressed in E. coli cells. The recombinant polypeptides (prA30L H pr129L) were purified from cell lysates by Ni-NTA chromatography. Recombinant polypeptides were able to form trimers in buffered saline and they destroyed under treatment with SDS and 2-mercaptoethanol. Reactivity of prA30L, pr129L and orthopoxvirus proteins was analyzed by ELISA and Western blotting with panel of 22 monoclonal antibodies (MAbs) against orthopoxviruses (19 against EV, 2 MAbs against vaccinia virus and 1 Mabs against cowpox virus). This data allowed us to conclude that there are 12 EV-specific epitopes of pr129L and EV fusion proteins, ten orthopox-specific epitopes of EV, VV, CPV fusion proteins, from them 9 orthopox-specific epitopes of prA30L and SPV fusion proteins. Five Mabs, which cross-reacted with orthopox-specific epitopes, were able to neutralize the VV on Vero cells and from them two MAbs has neutralizing activity against smallpox virus. Our findings demonstrate that 129L fusion protein have EV-specific epitopes, that EV 129L and SPV A30L fusion proteins have a several orthopox-specific epitopes to induce a neutralizing antibodies against human pathogenic orthopoxviruses.     

Novel potent apoA-I peptide mimetics that stimulate cholesterol efflux and pre-β particle formation in vitro

Reverse cholesterol transport (RCT) is believed to be the primary mechanism by which HDL and its major protein apoA-I protect against atherosclerosis. Starting from the inactive 22-amino acid peptide representing the consensus sequence of the class A amphipathic helical repeats of apoA-I, we designed novel peptides able to mobilize cholesterol from macrophages in vitro, and to stimulate the formation of ‘nascent HDL’ particles, with potency comparable to the entire apoA-I protein.     

Neutralization of Chlamydia psittaci with Monoclonal Antibodies

Neutralization of Chlamydia (C.) psittaci avian strain P-1041 was examined in vitro using monoclonal antibodies (MAbs). Of the 10 MAbs used, 6 were found to exhibit neutralizing capability. These include 3 against major outer membrane protein (MOMP), 1 against lipopolysaccharide (LPS) and 2 against other protein molecules [90 kilodalton (kDa) and 90/50 kDa]. Most neutralizing MAbs were dependent on complement for efficient neutralization, while a strain-specific MAb (2B5) against the 90 kDa protein displayed a different requirement for complement and neutralized the infectivity of the P-1041 at high concentrations without complement. By competitive inhibition enzyme-linked immunosorbent assay (competitive inhibition ELISA), all 3 neutralizing anti-MOMP MAbs were demonstrated to recognize different epitopes found in very close proximity to each other on the outer membrane.     

Expression of human apolipoprotein A-I epitopes in high density lipoproteins and in serum

The expression and immunoreactivity of apolipoprotein (apo) A-I epitopes in high density lipoproteins (HDL) and serum has been investigated using two series of monoclonal antibodies (Mabs) which have been described elsewhere. Series 1 Mabs, identified as 3D4, 6B8, and 5G6, were obtained by immunization and screening with apoA-I, and series 2 Mabs, identified as 2F1, 4H1, 3G10, 4F7, and 5F6, were obtained by immunization and screening with HDL. These Mabs were characterized with respect to their binding to HDL particles in solution. In series 2 Mabs, 2F1, 3G10, and 4F7, which react with apoA-I CNBr-fragments 1 and 2, could precipitate 100% of 125I-labeled HDL, while 4H1 and 5F6, which react with CNBr fragments 1 and 3, precipitated 90 and 60% of 125I-labeled HDL, respectively. Therefore, three distinct epitopes mapped to CNBr fragments 1 and 2 have been identified which are expressed on all HDL particles, indicating that several antigenic do mains exist on apoA-I which have the same conformation on all apoA-I-containing lipoproteins. The Mabs reacting at these sites have significantly higher affinity constants for 125I-labeled HDL than those that failed to precipitate 100% of HDL. This suggests that the high affinity Mabs react with apoA-I epitopes that are both expressed on all lipoproteins and located in thermo-dynamically stable regions of the molecules. All Mabs from series 1 precipitated 35% or less of 125I-labeled HDL prepared from freshly collected serum, but the proportion of HDL particles expressing the epitopes for these Mabs doubled or more upon serum storage at 4 degrees C. The time course of the alteration of apoA-I antigen in vitro was measured in three normolipemic donors. Upon storage of serum at 4 degrees C, the immunoreactivity of series 2 Mabs (4H1, 3G10) remained unchanged. However, the immunoreactivity of series 1 Mab 3D4 increased linearly at 38%/day for 4 weeks and by 12 weeks had plateaued at about 280-fold compared to day 1. The immunoreactivity of other series 1 Mabs also increased significantly with time in vitro. This process was partially inhibited in the presence of EDTA and by addition of antioxidants, however, the exact molecular nature of this in vitro alteration of apoA-I antigen was not identified.     

PLTP secreted by HepG2 cells resembles the high-activity PLTP form in human plasma

Plasma phospholipid transfer protein (PLTP) is an important regulator of plasma HDL levels and HDL particle distribution. PLTP is present in plasma in two forms, one with high and the other with low phospholipid transfer activity. We have used the human hepatoma cell line, HepG2, as a model to study PLTP secreted from hepatic cells. PLTP activity was secreted by the cells into serum-free culture medium as a function of time. However, modification of a previously established ELISA assay to include a denaturing sample pretreatment with the anionic detergent sodium dodecyl sulphate was required for the detection of the secreted PLTP protein. The HepG2 PLTP could be enriched by Heparin-Sepharose affinity chromatography and eluted in size-exclusion chromatography at a position corresponding to the size of 160 kDa. PLTP coeluted with apolipoprotein E (apoE) but not with apoB-100 or apoA-I. A portion of PLTP was retained by an anti-apoE immunoaffinity column together with apoE, suggesting an interaction between these two proteins. Furthermore, antibodies against apoE but not those against apoB-100 or apoA-I were capable of inhibiting PLTP activity. These results show that the HepG2-derived PLTP resembles in several aspects the high-activity form of PLTP found in human plasma.     

Immunochemical characterization of mucins. Polypeptide (M1) and polysaccharide (A and Leb) antigens.

Seven monoclonal antibodies (MAbs) reacting with high-molecular-mass components (greater than 20,000 kDa) isolated from an ovarian mucinous cyst of an A Le(a-b+) patient are described. By the use of immunoradiometric methods, these MAbs characterized seven different epitopes associated with components having a density of 1.45 g/ml by CsCl-density-gradient ultracentrifugation, like mucins. Two MAbs reacted with A and Lewis blood-group antigens respectively (polysaccharide epitopes). The five other MAbs characterized five M1 epitopes (called a, b, c, d and e), mainly associated with components of more than 20,000 kDa and 2000 kDa. They were completely destroyed by papain and 2-mercaptoethanol treatment (polypeptide epitopes). Moreover, timed trypsin digestion of native mucin resulted in a progressive loss of M1 activity and degraded these mucins into smaller M1-positive fragments. The a and c epitopes were partially degraded from relatively high-molecular-mass fragments (2000 kDa to 500 kDa) into a 100 kDa fragment. The b and d epitopes were completely degraded into smaller fragments ranging from 100 kDa to 40 kDa. The e epitope was completely destroyed by trypsin. These different pathways of M1 antigen degradation suggest the occurrence of different epitopes located in separate regions of the mucin molecules.     

Binding and cross-linking studies show that scavenger receptor BI interacts with multiple sites in apolipoprotein A-I and identify the class A amphipathic alpha-helix as a recognition motif

Scavenger receptor, class B, type I (SR-BI) mediates the selective uptake of high density lipoprotein (HDL) cholesteryl ester without the uptake and degradation of the particle. In transfected cells SR-BI recognizes HDL, low density lipoprotein (LDL) and modified LDL, protein-free lipid vesicles containing anionic phospholipids, and recombinant lipoproteins containing apolipoprotein (apo) A-I, apoA-II, apoE, or apoCIII. The molecular basis for the recognition of such diverse ligands by SR-BI is unknown. We have used direct binding analysis and chemical cross-linking to examine the interaction of murine (m) SR-BI with apoA-I, the major protein of HDL. The results show that apoA-I in apoA-I/palmitoyl-oleoylphosphatidylcholine discs, HDL(3), or in a lipid-free state binds to mSR-BI with high affinity (K(d) congruent with 5-8 microgram/ml). ApoA-I in each of these forms was efficiently cross-linked to cell surface mSR-BI, indicating that direct protein-protein contacts are the predominant feature that drives the interaction between HDL and mSR-BI. When complexed with dimyristoylphosphatidylcholine, the N-terminal and C-terminal CNBr fragments of apoA-I each bound to SR-BI in a saturable, high affinity manner, and each cross-linked efficiently to mSR-BI. Thus, mSR-BI recognizes multiple sites in apoA-I. A model class A amphipathic alpha-helix, 37pA, also showed high affinity binding and cross-linking to mSR-BI. These studies identify the amphipathic alpha-helix as a recognition motif for SR-BI and lead to the hypothesis that mSR-BI interacts with HDL via the amphipathic alpha-helical repeat units of apoA-I. This hypothesis explains the interaction of SR-BI with a wide variety of apolipoproteins via a specific secondary structure, the class A amphipathic alpha-helix, that is a common structural motif in the apolipoproteins of HDL, as well as LDL.     

Quantitation of plasma apolipoprotein A-I using two monoclonal antibodies in an enzyme-linked immunosorbent assay

A rapid sandwich enzyme-linked immunosorbent assay (ELISA) for the quantitation of human apolipoprotein (apo) A-I was developed. The assay uses a pair of noncompeting purified monoclonal antibodies to detect apoA-I in plasma. The antibodies used in this assay were selected because they bind greater than 90% of radioiodinated high density lipoprotein (HDL), they identify "fresh" nondeamidated epitopes on apoA-I, and they have comparable binding affinities for isolated HDL and HDL in plasma. The assay was standardized with a plasma secondary standard composed of lyophilized human serum. The assay was used to measure the apoA-I levels in normal subjects, patients with coronary artery disease, and patients with familial hypercholesterolemia. The results indicate that certain monoclonal antibodies can be used to reliably measure plasma levels of apoA-I in diverse groups of subjects.     

The small molecular mass ubiquinone-binding protein (QPc-9.5 kDa) in mitochondrial ubiquinol-cytochrome c reductase: isolation, ubiquinone-binding domain, and immunoinhibition

The small molecular mass ubiquinone-binding protein (QPc-9.5 kDa) was purified to homogeneity from 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyl[3H]octyl)-1,4-benzoquinol+ ++- labeled bovine heart mitochondrial ubiquinol-cytochrome c reductase. The N-terminal amino acid sequence of the isolated protein is Gly-Arg-Gln-Phe-Gly-His-Leu-Thr-Arg-Val-Arg-His-, which is identical with that of a Mr = 9500 protein in the reductase [Borchart et al. (1986) FEBS Lett. 200, 81-86]. A ubiquinone-binding peptide was prepared from [3H]azidoubiquinol-labeled QPc-9.5 kDa protein by trypsin digestion followed by HPLC separation. The partial N-terminal amino acid sequence of this peptide, Val-Ala-Pro-Pro-Phe-Val-Ala-Phe-Tyr-Leu-, corresponds to amino acid residues 48-57 in the reported Mr = 9500 protein. According to the proposed structural model for the Mr = 9500 protein, the azido-Q-labeled peptide is located in the membrane on the matrix side. These results confirm our previous assessment that the Mr = 13,400 subunit is not the small molecular weight Q-binding protein. Purified antibodies against QPc-9.5 kDa have a high titer with isolated QPc-9.5 kDa protein and complexes that contain it. Although antibodies against QPc-9.5 kDa do not inhibit intact succinate- and ubiquinol-cytochrome c reductases, a decrease of 85% and 20% in restoration of succinate- and ubiquinol-cytochrome c reductases, respectively, is observed when delipidated succinate- or ubiquinol-cytochrome reductases are incubated with antibodies prior to reconstitution with ubiquinone and phospholipid, indicating that epitopes at the catalytic site of QPc-9.5 kDa are buried in the phospholipid environment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monoclonal antibodies against chordin. Use in structural and immunohistochemical studies

Eight MAbs have been developed against chordin and designated as At2-At9. It is shown that all antibodies are directed against identical, spatially overlapping or closely positioned epitopes of chordin. The chordin molecule has repetitive sites wherein epitopes for the eight MAbs are located. This site lies within a proteinase-resistant fragment of chordin, presumably a glycopeptide, of molecular mass between 2 and 10 kDa. Fluorescence staining of cryostat sections from stellate sturgeon with the use of At5 (indirect Coons' method) has revealed a positive reaction with notochord cells and sheath and with the spinal cord. No significant reaction with cartilage, muscle and kidney was detected.     

Megalin acts in concert with cubilin to mediate endocytosis of high density lipoproteins

Cubilin has recently been shown to function as an endocytic receptor for high density lipoproteins (HDL). The lack of apparent transmembrane and cytoplasmic domains in cubilin raises questions as to the means by which it can mediate endocytosis. Since cubilin has been reported to bind the endocytic receptor megalin, we explored the possibility that megalin acts in conjunction with cubilin to mediate HDL endocytosis. While megalin did not bind to HDL, delipidated HDL, or apoA-I, it was found to copurify with cubilin isolated by HDL-Sepharose affinity chromatography. Cubilin and megalin exhibited coincident patterns of mRNA expression in mouse tissues including the kidney, ileum, thymus, placenta, and yolk sac endoderm. The expression of both receptors in yolk sac endoderm-like cells was inducible by retinoic acid treatment but not by conditions of sterol depletion. Suppression of megalin activity or expression by treatment with either megalin antibodies or megalin antisense oligodeoxynucleotides resulted in inhibition of cubilin-mediated endocytosis of HDL. Furthermore, megalin antisense oligodeoxynucleotide treatment resulted in reduced cell surface expression of cubilin. These data demonstrate that megalin acts together with cubilin to mediate HDL endocytosis and further suggest that megalin may play a role in the intracellular trafficking of cubilin.     

Localization of two epitopes of apolipoprotein A-I that are exposed on human high density lipoproteins using monoclonal antibodies and synthetic peptides

To understand the structure of apolipoprotein A-I, we have used an immunochemical approach and identified specific regions of apoA-I that may be exposed on the apoprotein as it exists on high density lipoprotein (HDL). Twelve mouse monoclonal antibodies specific for human apoA-I were generated from six fusions. Thirteen synthetic peptides of between 5 and 16 amino acid residues in length, which span the amino-terminal two-thirds of apoA-I, were tested for their ability to react with each of the 12 antibodies. In a competitive solid-phase radioimmunoassay, a synthetic peptide, which represented residues 1-15 of mature apoA-I, inhibited the binding of antibody AI-16 to immobilized HDL. Similarly, a synthetic peptide, which represented residues 90-105 of apoA-I, inhibited the binding of antibody AI-18 to immobilized HDL. Using systematic changes in the size and sequence of the oligopeptides, the limits and essential amino acid residues of these epitopes were defined. Comparisons of the slopes of the competition curves obtained with immunoreactive peptides, isolated apoA-I, and HDL verified that these two regions of apoA-I are exposed on the surface of apoA-I as it exists on native HDL.