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Y L Marcel

Publications and source records attributed to Y L Marcel.

At least 91 records · Page 5Linked to original sources
PubMed1988

Apolipoprotein distribution in human lipoproteins separated by polyacrylamide gradient gel electrophoresis.

The heterogeneity of serum lipoproteins (excluding very low density (VLDL) and intermediate density (IDL) lipoproteins) and that of lipoproteins secreted by HepG2 cells has been studied by immunoblot analysis of the apolipoprotein composition of the particles separated by polyacrylamide gradient gel electrophoresis (GGE) under nondenaturing conditions. The reactions of antibodies to apoA-I, apoA-II, apoE, apoB, apoD, and apoA-IV have revealed discrete bands of particles which differ widely in size and apolipoprotein composition. GGE of native serum lipoproteins demonstrated that apoA-II is present in lipoproteins of limited size heterogeneity (apparent molecular mass 345,000 to 305,000) and that apoB is present in low density lipoproteins (LDL) and absent from all smaller or denser lipoproteins. In contrast, serum apoA-I, E, D, and A-IV are present in very heterogeneous particles. Serum apoA-I is present mainly in particles of 305 to 130 kDa where it is associated with apoA-II, and in decreasing order of immunoreactivity in particles of 130-90 kDa, 56 kDa, 815-345 kDa, and finally within the size range of LDL, all regions where there is little detectable apoA-II. Serum apoE is present in three defined fractions, one within the size range of LDL, one containing heterogeneous particles between 640 and 345 kDa, and one defined fraction at 96 kDa. Serum apoD is also present in three defined fractions, one comigrating with LDL, one containing heterogeneous particles between 390 and 150 kDa, and one band on the migration front. Most of serum apoA-IV is contained in a band comigrating with albumin. GGE of centrifugally prepared LDL shows the presence of apoB, apoE, and apoD, but not that of apoA-I. However, the particles containing apoA-I, which, in serum, migrated within the LDL size range and as bands of 815 to 345 kDa, were recovered upon centrifugation in the d greater than 1.21 g/ml fraction. GGE of high density lipoproteins (HDL) indicated that most of apoA-I, A-II, and A-IV were present in lipoproteins of the same apparent molecular mass (390-152 kDa). ApoD tended to be associated with large HDL, and this was also significant for HDL apoE, which is present in lipoproteins ranging from 640 to 275 kDa. GGE of very high density lipoproteins (VHDL) presented some striking features, one of which was the occurrence of apolipoproteins in very discrete bands of different molecular mass. ApoA-II was bimodally distributed at 250-175 kDa and 175-136 kDa, the latter fraction also containing apoA-I.(ABSTRACT TRUNCATED AT 400 WORDS)

Apolipoproteins↗
PubMed1987

Absence of intestinal synthesis of apolipoprotein B-48 in two cases of abetalipoproteinemia.

Previous studies have reported that the absence of chylomicron, very-low-density lipoprotein, and low-density lipoprotein in abetalipoproteinemia is a consequence of apoprotein B (apo B) deficiency. Although the absence of apo B from the intestine has been shown by immunofluorescence, the antiserum used was raised against low-density lipoprotein apo B. Therefore, the precise nature of the underlying defect remains unknown, given that the postulated gene mutation could prevent the synthesis of the molecular form of apo B specific for chylomicrons, apo B-48, or produce an unstable aberrant form of apo B particle. This report concerns 2 girls aged 5.5 and 4.75 with well-documented clinical and biological manifestations of the disease in whom there was no immunologically detectable plasma apo B-48 and apo B-100. Their cultured jejunal explants incubated with [14C]palmitate showed slight decrease in the esterification of triglycerides, phospholipids, and cholesteryl esters. However, only traces of triglycerides and small amounts of cholesteryl esters were found in the culture medium in contrast to phospholipids, which were readily exported. Protein synthesis as assessed by [3H]leucine incorporation by explants was normal and only modestly diminished in the fat chylomicronlike fraction floated from the sonicated explants. However, there was no radioactivity at the electrophoretic position of apo B-100 and apo B-48. Immunologic confirmation of the absence of these two apoproteins was obtained by Western blots. These data confirm the hypothesis that in certain cases of abetalipoproteinemia the intestinal defect results from the lack of synthesis of apo B-48.

Abetalipoproteinemia↗
PubMed1987

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.

Animals↗
PubMed1987

Structural relationship of human apolipoprotein B48 to apolipoprotein B100.

Although the complete amino acid sequence of human apolipoprotein (apo) B100 is known (4536 amino acids), the structure of apo B48 has not been defined. The objective of our study was to define the structure of apo B48 and its relationship to apo B100. Antibodies were produced against 22 synthetic peptides corresponding to sequences in human apo B100. The levels of immunoreactivity of the antipeptides to apo B100 and apo B48 were used to define the structural relationship between these two species of apo B. Six antibodies from sequences in the amino-terminal half of apo B100, including antipeptide 2110-2129, bound to both apo B100 and apo B48. 15 other apo B-specific antipeptides from sequences carboxyl-terminal to residue 2152 bound to apo B100, but not to apo B48. Immunoblots of cyanogen bromide digests of apo B100 and apo B48 with antipeptides 2068-2091 and 2110-2129 detected a 16-KD fragment (residues 2016-2151) in the apo B100 digest and a fragment of identical size in the apo B48 digest. Because apo B48 appears to contain the apo B100 cyanogen bromide fragment 2016-2151 and because an antiserum specific for the peptide 2152-2168 does not bind to apo B48, we conclude that apo B48 represents the amino-terminal 47% of apo B100 and that the carboxyl terminus of apo B48 is in the vicinity of residue 2151 of apo B100.

Amino Acids↗
PubMed1987

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.

Antibodies, Monoclonal↗
PubMed1986

Apolipoprotein D and cross-reacting human plasma apolipoproteins identified using monoclonal antibodies.

We have produced five hybridomas which secreted monoclonal antibodies that reacted with human plasma apolipoprotein D. On analysis by polyacrylamide gel electrophoresis (PAGE) high density lipoproteins and lecithin:cholesterol acyltransferase (EC 2.3.1.43)-enriched fractions of plasma contained many protein bands that reacted with the antibodies. Purified apolipoprotein D had the lowest Mr (29,000), the lowest pI (4.8-5.2), and the greatest migration on alkaline urea-PAGE of all the immunoreactive bands. These characteristics agreed with those described for apolipoprotein D in the literature. The other immunoreactive proteins had apparent Mr from about 39,000 to 98,000, they migrated more slowly than apolipoprotein D on alkaline urea-PAGE, and there were 10 polymorphs on isoelectric focusing. These cross-reacting proteins were present in the high density lipoproteins of each of four individuals sampled on several occasions and in pooled plasma. All of the monoclonal antibodies reacted both with apo-D and the higher Mr cross-reacting proteins. Each of our five monoclonal antibodies bound to one of two distinct antigenic sites on apo-D, determined by antibody competition immunoassays. Neither of these two sites was composed of carbohydrate, but expression of both sites seemed to be influenced by thiol-reducing agents: site 5G10 gained but 4E11 either lost immunoreactivity or was unchanged by reduction according to the conditions. We conclude that apolipoprotein D is only one of several plasma proteins, which contain two homologous polypeptide antigenic sites, recognized by monoclonal antibodies and also by a specific goat antiserum. Apolipoprotein D had the least Mr of these proteins.

Animals↗
PubMed1986

Human apolipoprotein E. Determination of the heparin binding sites of apolipoprotein E3.

The interaction of human apolipoprotein (apo-) E3 with heparin was examined using heparin-Sepharose as a model system. The approach taken to determine the region of apo-E that is responsible for binding to heparin was to identify apo-E monoclonal antibodies that inhibited heparin binding, to determine the epitopes of the inhibiting antibodies, and finally to examine the heparin binding of fragments containing the inhibiting antibody epitopes. Three antibodies, designated 1D7, 6C5, and 3H1, were found to inhibit binding, suggesting that multiple heparin binding sites were present on apo-E. The epitopes of the inhibiting antibodies were determined by immunoblot analysis of synthetic or proteolytic fragments of apo-E. Measurement of the heparin binding activity of fragments containing epitopes of the inhibiting antibodies demonstrated that apo-E3 contains two heparin binding sites. The first site is located in the vicinity of residues 142-147 and coincides with the 1D7 epitope. The second binding site is contained in the carboxyl-terminal region of apo-E and is inhibited by 3H1, the epitope of which is located between residues 243 and 272. The epitope of the third inhibiting antibody, 6C5, is located at the amino terminus of apo-E; however, this antibody inhibits the second heparin binding site located in the carboxyl-terminal region. A head-to-tail association of apo-E, in which the 6C5 epitope and the second heparin binding site would be in close proximity, is proposed to account for this observation. In the lipid-free state both heparin binding sites on apo-E are expressed; however, when apo-E is complexed to phospholipid or on the surface of a lipoprotein particle, only the first binding site (residues 142-147) is expressed.

Animals↗
PubMed1986

Epitopes of apolipoprotein B-100 and B-48 in both liver and intestine. Expression and evidence for local synthesis in recessive abetalipoproteinemia.

The presence of apolipoprotein (apo) B in liver and intestine from a patient with abetalipoproteinemia was evaluated by immunohistochemistry with a polyclonal and six monoclonal antibodies to different apo B-48 and B-100 epitopes. In normal liver, apo B was present inside and outside hepatocytes. The patients liver exhibited staining in the cytoplasm with the polyclonal and three monoclonal antibodies. By immunoelectron-microscopy, apo B was found to be present in the smooth endoplasmatic reticulum and the Golgi complex. Normal intestinal epithelium was labeled with polyclonal and all monoclonal antibodies, including those specific for apo B-100. The patients epithelium stained with polyclonal and six monoclonals, and apo B was present in the Golgi complex. Thus, normal intestinal mucosa expressed apo B-48 and B-100 epitopes, which indicates apo B-100 synthesis in the gut. The synthesis of the apo B molecule in the patient seems to be retained in both liver and gut, which suggests a posttranslational defect.

Abetalipoproteinemia↗
PubMed1986

[Application of monoclonal antibodies to the assay of apolipoproteins].

Our present understanding of the dyslipoproteinaemias comes mainly from recent advances in our knowledge of the apolipoproteins and their metabolic roles. The concentrations of apo Al and apo B in plasma are accepted to be the best measures of lipoproteins as risk factors for cardiovascular disease. This review describes the advantages and limitations of monoclonal antibodies in the immunoassay of apo Al and apo B. Two different molecular forms of apo B are present in plasma: B-100 secreted by the liver, and the smaller B-48 from the intestine. Some, but not all, of the antigenic sites of B-100 are present on B-48, which allows us to measure all apo B or apo B-100 alone. The expression of some antigenic sites is influenced by the type of lipids present with apo B. These sites are variably expressed across the spectrum of apo-B containing lipoproteins. Some antigenic sites on apo Al vary in their expression but, while lipids have affected the reactivity of apo Al with some polyclonal antisera, our studies with monoclonal antibodies have shown that all antigenic sites are present independently of the presence of lipids. Some antigenic sites are fully expressed on all HDL but the expression of a number of sites increases during storage of samples, and at a rate which depends on the storage conditions, and probably on chemical changes in apo Al. Thus, we can choose to assay either all or only a selection of apo Al and apo B in a plasma sample, according to the antibody and conditions of assay which are chosen.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1985

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.

Animals↗
PubMed1985

Tangier disease apolipoprotein A-I compared with normal plasma A-I using monoclonal antibodies.

The molecular defect in Tangier disease is unknown. We have compared the electrophoretic and immunoreactive properties of Tangier disease and normal apolipoprotein A-I using four monoclonal antibodies. We verified that the molecular weight, pI and CNBr-cleaved fragments of Tangier disease and normal apolipoprotein A-I were not different, excluding the possibility that dimers, aggregates or fragments of apolipoprotein A-I could be responsible for its rapid catabolism in this disease.

Antibodies, Monoclonal↗
PubMed1985

Modulation of apolipoprotein B antigenic determinants in human low density lipoprotein subclasses.

To investigate the effect of low density lipoprotein (LDL) heterogeneity on the conformation of LDL apolipoprotein B (apo-B), the immunoreactivities of 6 monoclonal antibodies against LDL apo-B were measured in 3 LDL subfractions isolated by equilibrium density gradient ultracentrifugation. To ensure a broad range of LDL particles, the LDL subfractions were prepared from normal subjects and patients with hyperapobetalipoproteinemia. With 3 of the antibodies, 1D1, 5E11, and 3A10, LDL fractions 1 (the most buoyant), 2 (the intermediate), and 3 (the densest) were equally immunoreactive and competed similarly with reference whole LDL. In contrast, with 3 other antibodies, 2D8, 3F5, and 4G3, fraction 1 was significantly more reactive than fraction 3; that is for each in turn, 290, 250, and 150% more of the densest LDL protein was required to achieve the same displacement as with fraction 1. Further, the immunoreactivities of the 3 LDL fractions with antibodies 2D8, 3F5, and 4G3 were negatively correlated with their LDL cholesterol to LDL protein ratio with r values of 0.727, 0.898, and 0.870, respectively, suggesting that as LDL particle size decreases, the conformation of the LDL apo-B changes progressively. It is of interest that the antigenic determinants recognized by 3F5 and 4G3 are close to the LDL receptor recognition site on LDL apo-B. Therefore, it is possible that the reduced immunoreactivity of these determinants in dense LDL may be the in vitro correlate of the reduced fractional catabolics rate of dense LDL compared to buoyant LDL previously observed in vivo.

Antibodies, Monoclonal↗
PubMed1985

Molecular cloning and expression of partial cDNAs and deduced amino acid sequence of a carboxyl-terminal fragment of human apolipoprotein B-100.

Apolipoprotein (apo) B-100 cDNAs were identified in a human liver cDNA library cloned in the expression vector lambda gt11. The beta-galactosidase-apoB-100 fusion protein was detected by two independently produced low density lipoprotein polyclonal antisera and by three apoB-100 monoclonal antibodies that crossreact with apoB-74. It was not recognized by two apoB-100 monoclonal antibodies that crossreact with apoB-26. The longest clone, lambda B8, was completely sequenced. It contains a 2.8-kilobase DNA fragment containing the codons for the carboxyl-terminal 836 amino acid residues of apo-B-100, as well as the 3' untranslated region of apoB-100 mRNA. We have thus mapped apoB-74 to the carboxyl-terminal portion of apoB-100. The deduced amino acid sequence of the cloned DNA matches the sequences of 14 apoB-100 peptides determined in our laboratory. Minor differences in amino acid sequence were noted in three of the peptides, suggesting polymorphism of apoB-100 at the protein and DNA levels. Secondary structure predictions reveal an unusual pattern for apolipoproteins, consisting of beta-structure (24%), alpha-helical content (33%), and random structure (30%). Ten amphipathic helical regions of 10-24 residues were identified. This carboxyl-terminal fragment of apoB-100 is considerably more hydrophobic than other apolipoproteins with known structure. Its lipid binding regions might include stretches of highly hydrophobic beta-sheets as well as amphipathic helices. Our findings on apoB structure might be important for understanding the role of apoB-100-containing lipoproteins in atherosclerosis.

Amino Acid Sequence↗
PubMed1985

The use of monoclonal antibodies to probe human apolipoprotein B structure and function.

Apolipoprotein (apo) B plays an important role in plasma lipid transport and in the maintenance of cholesterol homeostasis. Attempts to determine the structure of apo B have been hampered by technical obstacles resulting from its chemical and physical properties. Recently monoclonal antibodies (Mabs) against human apo B have been used as probes to study apo B structure and heterogeneity. Certain Mabs are capable of blocking binding of low density lipoprotein (LDL) apo B to the cell surface LDL receptor, which presumably reflects the proximity of their antigenic determinants to the receptor recognition domain. The distribution of antigenic determinants recognized by Mabs has been studied on the hepatic (apo B-100) and intestinal (apo B-48) forms of apo B and on fragments generated by limited proteolysis of apo B. Some Mabs are specific for apo B-100, whereas others cross-react with apo B-48. Apo B-100 specific Mabs coupled to Sepharose have been used to isolate separately apo-B-containing lipoproteins of intestinal and hepatic origin and their respective lipid and apolipoprotein compositions have been determined. Using the separated fractions it has been shown that apo B-100, but not apo B-48, can react with the LDL receptor. Most Mabs failed to react with apo B which had been delipidated and resolubilized, but in some cases immunoreactivity could be recovered if the solubilized apo B were reincorporated into lipid vesicles. These experiments showed that different determinants had different lipid requirements for their expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗