Primary structure comparison of the proposed low density lipoprotein (LDL) receptor binding domain of human and pig apolipoprotein B: implications for LDL-receptor interactions

Apolipoprotein B (apoB) is the predominant protein in low density lipoprotein (LDL) and is responsible for LDL binding to the LDL receptor. Although the primary amino acid sequence of human apoB has been determined, little is known about the structural domains involved in mediating apoB binding to the LDL receptor. Amino acid sequence comparisons across species lines provide a means of defining structures that are essential for function. We have sequenced a l.l kb fragment of pig apoB genomic DNA, corresponding to a 363 amino acid segment proposed to mediate human apoB binding to the LDL receptor. In human apoB this domain contains two regions enriched in positively charged amino acids flanking two disulfide-linked cysteine residues. The pig amino acid sequence shared 72% identity with the human sequence. However, there were differences that have significant structural and functional implications. Human apoB arginine-3,359 corresponds to a critical arginine (position 142) residue in the apoE LDL receptor binding domain. In the pig, this arginine residue was not conserved. Also, the two disulfide-linked cysteine residues found near the proposed apoB binding domain were not conserved in the pig. Despite these differences, pig LDL had a higher affinity than human LDL for both the pig and human LDL receptor. Thus, these features are not required for high affinity binding of pig LDL to the LDL receptor, and may not be necessary for the binding of human LDL to the LDL receptor.     

A cross-species comparison of the apolipoprotein B domain that binds to the LDL receptor

Apolipoprotein (apo)-B-100 is the ligand that mediates the clearance of low density lipoprotein (LDL) from the circulation by the apoB,E (LDL) receptor pathway. Clearance is mediated by the interaction of a domain enriched in basic amino acid residues on apoB-100 with clusters of acidic residues on the apoB,E (LDL) receptor. A model has been proposed for the LDL receptor binding domain of apoB-100 based on the primary amino acid sequence (Knott, T. J., et al. 1986. Nature. 323: 734-738). Two clusters of basic residues (A: 3147-3157 and B: 3359-3367) are apposed on the surface of the LDL particle by a disulfide bridge between Cys 3167 and 3297. Support for this single domain model has been obtained from the mapping of epitopes for anti-apoB monoclonal antibodies that block the binding of apoB to the LDL receptor. Here we test this model by comparing the nucleotide (from 9623 to 10,442) and amino acid sequence (from 3139 to 3411) of apoB-100 in seven species (human, pig, rabbit, rat, Syrian hamster, mouse, and chicken). Overall, this region is highly conserved. Cluster B maintains a strong net positive charge and is homologous across species in both primary and secondary structure. However, the net positive charge of region A is not conserved across these species, but the region remains strongly hydrophilic. The secondary structure of the region between clusters A and B is preserved, but the disulfide bond is unique to the human sequence. This study suggests that the basic region B is primarily involved in the binding of apoB-100 to the apoB,E (LDL) receptor.     

Primary sequence mapping of human apolipoprotein B-100 epitopes. Comparisons of trypsin accessibility and immunoreactivity and implication for apoB conformation

Differential trypsin-accessibility and monoclonal antibodies (Mabs) to human apolipoprotein (apo) B-100 are both important tools for probing apoB structure and conformation on low-density lipoproteins (LDL). In this study, we have mapped greater than 80% of the C-terminal region (720 residues) of LDL apoB-100 using trypsin digestion. Our results extend our previous data [Yang et al. (1986) Nature (Lond.) 323, 738-742] confirming that the C-terminal region of about 420 residues of apoB-100 is largely inaccessible to trypsin, whereas the part just preceding this region has interspersed trypsin-accessible and inaccessible peptides. We have determined the amino acid sequence of specific apoB-100 peptides containing epitopes recognized by four separate Mabs: two epitopes have been mapped to within 20 residues, one has been mapped to 36 residues, and the last to 80 residues. We used polyclonal antisera to identify 16 overlapping clones of varying lengths of apoB-100 cDNAs extending from the C-terminus of apoB-100 cloned in the expression vector, lambda gt11. These clones were then tested against individual Mabs. By nucleotide sequence analysis of overlapping clones that show differential reactivities to different Mabs, we have mapped the individual epitopes of each Mab to within about 50-150 amino acid residues predicted from the DNA sequences. Confirmation and further fine mapping were accomplished by competition for LDL binding using partially purified fusion proteins and chemically synthesized oligopeptides. Two epitopes (Mabs 7 and 22) were mapped to the C-terminal 20 amino acids of apoB-100, one (Mab 16) to residues 4154-4189, and another (Mab 20) to residues 3926-4005. Mab 16 precipitates more than 80% of LDL particles. Mab 20 precipitates only denatured apoB but not native LDL apoB [Milne et al. (1987) Mol. Immunol. 24, 435]. Mabs 7 and 22 are unique in that they precipitate LDL apoB modified by storage much better than freshly isolated LDL-apoB. Although epitope expression and trypsin-accessibility represent two useful probes for the study of protein conformation, there was no obvious correlation between these two parameters when applied to LDL apoB for the antibodies we have examined.     

Monoclonal antibodies to human low density lipoprotein identify distinct areas on apolipoprotein B-100 relevant to the low density lipoprotein-receptor interaction.

We have characterized the epitopes for ten murine monoclonal antibodies (Mabs) to human low density lipoprotein (LDL) and studied their ability to interfere with the LDL-receptor interaction. The epitopes for the antibodies were defined by using the following approaches: 1) interaction with apoB-48; 2) interaction with apoB-100 thrombolytic fragments; and 3) interaction with beta-galactosidase-apoB fusion proteins spanning different areas of the apoB-100 sequence. The results obtained are consistent with the following map of epitopes: Mab 6E, amino acids (aa) 1-1297, Mabs 5A and 6B, aa 1480-1693, Mabs 2A, 7A, 3B, and 4B, aa 2152-2377, Mabs 8A and 9A, aa 2657-3248 and 3H, aa 4082-4306. Four Mabs (2A, 5A, 7A, and 9A) whose epitopes are located in three different areas of apoB, dramatically reduced (up to 95%) the LDL-receptor interaction on cultured human fibroblasts; Fab fragments were as effective as the whole antibodies. Mab 3H, on the other hand, increased LDL binding up to threefold. These findings are consistent with the hypothesis that several areas of apoB-100 are involved independently or in concert in modulating the apoprotein B conformation required for interaction with the LDL receptor.     

Mutational analysis of the ligand binding domain of the low density lipoprotein receptor

The ligand binding domain of the low density lipoprotein (LDL) receptor contains seven imperfect repeats of a 40-amino acid cysteine-rich sequence. Each repeat contains clustered negative charges that have been postulated as ligand-binding sites. The adjacent region of the protein, the growth factor homology region, contains three cysteine-rich repeats (A-C) whose sequence differs from those in the ligand binding domain. To dissect the contribution of these different cysteine-rich repeats to ligand binding, we used oligonucleotide-directed mutagenesis to alter expressible cDNAs for the human LDL receptor which were then introduced into monkey COS cells by transfection. We measured the ability of the mutant receptors to bind LDL, which contains a single protein ligand for the receptor (apoB-100), and beta-migrating very low density lipoprotein (beta-VLDL), which contains apoB-100 plus multiple copies of another ligand (apoE). The results show that repeat 1 is not required for binding of either ligand. Repeats 2 plus 3 and repeats 6 plus 7 are required for maximal binding of LDL, but not beta-VLDL. Repeat 5 is required for binding of both ligands. Repeat A in the growth factor homology region is required for binding of LDL, but not beta-VLDL. Repeat B is not required for ligand binding. These results support a model for the LDL receptor in which various repeats play additive roles in ligand binding, each repeat making a separate contribution to the binding event.     

Identification of autoantibodies in human plasma recognizing an apoB-100 LDL receptor binding site peptide

The aim of this study was to test the hypothesis that autoantibodies recognize amino acid sequences in the LDL receptor binding region of apolipoprotein B-100 (apoB-100). Autoantibodies against an unmodified or malondialdehyde (MDA)-modified LDL receptor binding site peptide were determined by ELISA in baseline plasma samples of 78 cases with coronary events and 149 matched controls recruited from the prospective Malm? Diet Cancer Study. IgG and IgM recognizing this peptide were detected in all subjects but did not differ between cases and controls. Inverse associations were observed between IgG against the native binding site and plasma oxidized LDL (r = -0.21, P < 0.005), but there were no significant associations with total or LDL cholesterol levels. In univariate analyses, inverse associations were found between baseline carotid intima-media thickness and IgG against the MDA-modified binding site (r = -0.14, P < 0.05), but this association was lost when controlling for other major cardiovascular risk factors. Specificity studies demonstrated that the binding of autoantibodies to these sequences could be inhibited by oxidized but not by native LDL. Autoantibodies recognizing the LDL receptor binding site in apoB-100 are frequently expressed. Their association with plasma oxidized LDL suggests that they have been generated in response to breakdown products of LDL oxidation, but their influence on cholesterol metabolism and the development of atherosclerosis appears limited.     

A tandemly repeated sequence determines the binding domain for an erythrocyte receptor binding protein of P. falciparum

Erythrocyte invasion by the malarial merozoite is a receptor-mediated process, an obligatory step in the development of the parasite. The Plasmodium falciparum protein GBP-130, which binds to the erythrocyte receptor glycophorin, is shown here to encode the binding site in a domain composed of a tandemly repeated 50 amino acid sequence. The amino acid sequence of GBP-130, deduced from the cloned and sequenced gene, reveals that the protein contains 11 highly conserved 50 amino acid repeats and a charged N-terminal region of 225 amino acids. Binding studies on recombinant proteins expressing different numbers of repeats suggest that a correlation exists between glycophorin binding and repeat number. Thus, a repeat domain, a common feature of plasmodial antigens, has been shown to have a function independent of the immune system. This conclusion is further supported by the ability of antibodies directed against the repeat sequence to inhibit the in vitro invasion of erythrocytes by merozoites.     

Regional specificities of monoclonal anti-human apolipoprotein B antibodies

The usefulness of monoclonal antibodies as probes of protein structure is directly related to knowledge of the structures and locations of the epitopes with which they interact. In this report we provide a detailed map of 13 epitopes on apoB-100 defined by our anti-apoB monoclonal antibodies based on current information on the amino acid sequence of apoB-100. To localize antibody specificities to smaller regions along the linear sequence of the apoB-100 molecule we used a) thrombin- and kallikrein-generated fragments of apoB-100; b) beta-galactosidase- apoB fusion proteins; c) heparin; and d) antibody versus antibody competition experiments. Most of the monoclonal antibodies elicited by immunization with LDL were directed towards epitopes within the first 1279 amino terminal (T4/K2 fragments) or last 1292 carboxyl terminal amino acid residues (T2/K4 fragments) of apoB-100. One epitope localized to the mid-portion of apoB-100 was elicited by immunization with VLDL (D7.2). Saturating amounts of heparin bound to LDL did not inhibit the binding of any of the monoclonal antibodies to their respective epitopes on apoB-100, indicating that none of the antibody determinants is situated close to any of the reported heparin binding sites on LDL apoB. We examined the expression of apoB epitopes on VLDL subfractions and LDL isolated from a normolipidemic donor. The apparent affinities with which the antibodies interacted with their respective epitopes on the VLDL subfractions and LDL uniformly increased as follows: LDL greater than VLDL3 greater than VLDL2 greater than VLDL1, suggesting that each of the major regions of apoB-100 is progressively more exposed as normal VLDL particles become smaller in size and epitopes are most exposed in LDL. Previous experiments utilizing hypertriglyceridemic VLDL subfractions yielded similar results, but the rank order of VLDL subfractions and LDL was not the same for all antibodies tested. Thus, differences in apoB epitope expression on VLDL particles of differing sizes is a general phenomenon, but the expression of apoB epitopes in hypertriglyceridemic VLDL appears to be more heterogeneous than is the case for VLDL from normolipidemic donors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Locating a low density lipoprotein-targeting domain of human apolipoprotein B-100 by expressing a minigene construct in transgenic mice

Through its interaction with the low density lipoprotein (LDL) receptor, apolipoprotein (apo) B-100 is a major determinant of LDL metabolism and plasma cholesterol. Its receptor binding ability is conformation-dependent and requires its expression on the right lipoprotein particles. The structural signal that targets apoB-100 to LDL is unknown. We have microinjected a human apoB-100 minigene construct comprising less than 25% of the apoB-100 sequence driven by the natural apoB promoter to produce transgenic mice. The transgene product was expressed at a high level and was present exclusively in the LDL of these animals. Analysis of the responsible sequence (residues 2878-3925 of apoB-100) reveals unique structural features that may be important in its role as an LDL-targeting domain.     

Receptor binding activity of lipid recombinants of apolipoprotein B-100 thrombolytic fragments

Apolipoprotein (apo) B-100, the protein constituent of low density lipoproteins (LDL), is the determinant responsible for LDL binding to the apoB,E(LDL) receptor on cells. The current study was designed to identify the region(s) of apoB-100 that interact with the apoB,E(LDL) receptor. Apolipoprotein B-100 was fragmented by thrombin digestion, and the isolated fragments (T2, T3, T4) were recombined with cholesterol-induced canine high density lipoproteins (HDLc). Before the recombination, the receptor binding activity of apoE of the HDLc was abolished by reductive methylation and extensive trypsin treatment. This treatment permitted almost complete replacement of the small residual apoE fragments by the large apoB fragments. Recombinant apoB particles were isolated by ultracentrifugation and tested for binding to receptors on cultured human fibroblasts. The recombinant particles had chemical and physical properties similar to those of native HDLc. Recombinants of both the whole thrombolytic digest and of isolated fragments displayed specific binding to the apoB,E (LDL) receptor. Anti-apoB,E(LDL) receptor antibodies abolished 90% of the binding, and there was almost no specific binding to receptor-negative fibroblasts or to cells in which the receptors had been down-regulated. The binding of apoB-100 recombinants to the receptor also demonstrated calcium dependency; in addition, the surface binding of the recombinants was released by polyanionic compounds. All these recombinants had binding affinities comparable to one another but less than that of native LDL. Although T2, T3 and T4 recombinants can all bind specifically to the apoB,E(LDL) receptor, it remains to be established whether their activity represents physiologically relevant binding. Nevertheless, the present findings illustrate the potential of the recombinant method using HDLc lipids to reconstitute biological activity.     

Isolation and characterization of four heparin-binding cyanogen bromide peptides of human plasma apolipoprotein B

Apolipoprotein B-100 (apoB-100) is the major protein constituent of human plasma low-density lipoproteins (LDL). On the basis of its amino acid sequence [Chen, S.-H., Yang, C.-Y., Chen, P.-F., Setzer, D., Tanimura, M., Li, W.-H., Gotto, A. M., Jr., & Chan, L. (1986) J. Biol. Chem. 261, 12918-12921], apo B-100 is one of the largest monomeric proteins known with a calculated molecular weight of 512937. Heparin binds to the LDL surface by interacting with positively charged amino acid residues of apoB-100, forming soluble complexes in the absence of divalent metals and insoluble complexes in their presence. The purpose of this study was to isolate and characterize the heparin-binding domain(s) of apoB-100. Human plasma LDL were fragmented with cyanogen bromide (CNBr). After delipidation and reduction-carboxymethylation, the CNBr peptides were fractionated by sequential chromatography on DEAE-Sephacel, Mono S, and high reactive heparin (HRH) AffiGel-10; HRH was purified by chromatography of crude bovine lung heparin on LDL AffiGel-10. Heparin-binding peptides were further purified by reverse-phase high-performance liquid chromatography. Heparin-binding activity was monitored by a dot-blot assay with 125I-HRH. The amino-terminal sequences of four CNBr heparin-binding peptides (CNBr-I-IV) were determined. CNBr-I-IV correspond to residues 2016-2151, 3109-3240, 3308-3394, and 3570-3719, respectively, of the amino acid sequence of apoB-100. Each CNBr peptide contains a domain(s) of basic amino acid residues which we suggest accounts for their heparin-binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

The receptor binding domain of apolipoprotein E, linked to a model class A amphipathic helix, enhances internalization and degradation of LDL by fibroblasts

Human apolipoprotein E (apo E) consists of two distinct domains, the lipid-associating domain (residues 192-299) and the globular domain (residues 1-191) which contains the LDL receptor (LDLR) binding site (residues 129-169). To test the hypothesis that an arginine-rich apo E receptor binding domain (residues 141-150) is sufficient to enhance low-density lipoprotein (LDL) uptake and clearance when covalently linked to a class A amphipathic helix, a peptide in which the receptor binding domain of human apo E, LRKLRKRLLR (hApoE[141-150]), is linked to 18A, a well-characterized high-affinity lipid-associating peptide (DWLKAFYDKVAEKLKEAF), we synthesized the peptide hApoE[141-150]-18A (hE18A) and its end-protected analogue, Ac-hE18A-NH(2). The importance of positively charged residues and the role of the hydrophobic residues in the receptor binding domain were also studied using four analogues. Ac-LRRLRRRLLR-18A-NH(2) [Ac-hE(R)18A-NH(2)] and Ac-LRKMRKRLMR-18A-NH(2) (Ac-mE18A-NH(2)) contained an extended hydrophobic face, including the receptor binding region. Control peptides, Ac-LRLLRKLKRR-18A-NH(2) [Ac-hE(Sc)18A-NH(2)], had the amino acid residues of the apo E receptor binding domain scrambled to disrupt the extended hydrophobic face, and Ac-RRRRRRRRRR-18A-NH(2) (Ac-R(10)18A-NH(2)) had only positively charged Arg residues as the receptor binding domain. The effect of the dual-domain peptides on the uptake and degradation of human LDL by fibroblasts was determined in murine embryonic fibroblasts (MEF1). LDL internalization was enhanced 3-, 5-, and 7-fold by Ac-mE18A-NH(2), Ac-hE18A-NH(2), and Ac-hE(R)18A-NH(2), respectively, whereas the control peptides had no significant biological activity. All three active peptides increased the level of degradation of LDL by 100%. The LDL binding and internalization to MEF1 cells in the presence of these peptides was not saturable over the LDL concentration range that was studied (1-10 microgram/mL). Furthermore, a similar enhancement of LDL internalization was observed independent of the presence of the LDL receptor-related protein (LRP), LDLR, or both. Pretreatment of cells with heparinase and heparitinase abolished more than 80% of the enhanced peptide-mediated LDL uptake and degradation by cells. We conclude that the dual-domain peptides enhanced LDL uptake and degradation by fibroblasts via a heparan sulfate proteoglycan (HSPG)-mediated pathway.     

Different combinations of cysteine-rich repeats mediate binding of low density lipoprotein receptor to two different proteins

Seven imperfect repeats of a 40-amino acid cysteine-rich sequence constitute the ligand binding domain of the low density lipoprotein (LDL) receptor. To assess the contribution of each repeat, three site-directed mutations were made individually in each repeat: 1) deletion of the repeat, 2) substitution of a conserved isoleucine with aspartic acid, and 3) substitution of a conserved aspartic acid with tyrosine. cDNAs containing these mutations were transfected into simian COS cells and assayed for their ability to bind LDL, which contains a 500-kDa protein ligand (apoB-100), and beta-migrating very low density lipoprotein (beta-VLDL), which contains multiple copies of a 33-kDa ligand (apoE). The results showed that binding of the two ligands required different combinations of repeats. LDL binding required repeats 3-7; deletion of any one of these repeats markedly reduced LDL binding. In contrast, beta-migrating very low density lipoprotein binding was insensitive to the loss of any single repeat with the important exception of repeat 5, whose loss reduced binding by 60%. The same effects were obtained when each of the repeats was altered by either of the two substitution mutations. The current findings suggest that a multiplicity of cysteine-rich repeats may allow a single protein to bind several different protein ligands by employing different combinations of repeats.     

Functional characterization of the 180-kD ribosome receptor in vivo

A cDNA encoding the 180-kD canine ribosome receptor (RRp) was cloned and sequenced. The deduced primary structure indicates three distinct domains: an NH2-terminal stretch of 28 uncharged amino acids representing the membrane anchor, a basic region (pI = 10.74) comprising the remainder of the NH2-terminal half and an acidic COOH- terminal half (pI = 4.99). The most striking feature of the amino acid sequence is a 10-amino acid consensus motif, NQGKKAEGAP, repeated 54 times in tandem without interruption in the NH2-terminal positively charged region. We postulate that this repeated sequence represents a ribosome binding domain which mediates the interaction between the ribosome and the ER membrane. To substantiate this hypothesis, recombinant full-length ribosome receptor and two truncated versions of this protein, one lacking the potential ribosome binding domain, and one lacking the COOH terminus, were expressed in Saccharomyces cerevisiae. Morphological and biochemical analyses showed all proteins were targeted to, and oriented correctly in the ER membrane. In vitro ribosome binding assays demonstrated that yeast microsomes containing the full-length canine receptor or one lacking the COOH-terminal domain were able to bind two to four times as many human ribosomes as control membranes lacking a recombinant protein or microsomes containing a receptor lacking the NH2-terminal basic domain. Electron micrographs of these cells revealed that the expression of all receptor constructs led to a proliferation of perinuclear ER membranes known as "karmellae." Strikingly, in those strains which expressed cDNAs encoding a receptor containing the putative ribosome binding domain, the induced ER membranes (examined in situ) were richly studded with ribosomes. In contrast, karmellae resulting from the expression of receptor cDNA lacking the putative ribosome binding domain were uniformly smooth and free of ribosomes. Cell fractionation and biochemical analyses corroborated the morphological characterization. Taken together these data provide further evidence that RRp functions as a ribosome receptor in vitro, provide new evidence indicating its functionality in vivo, and in both cases indicate that the NH2-terminal basic domain is essential for ribosome binding.     

The receptor binding domain of apolipoprotein E is responsible for its antioxidant activity

Apolipoprotein E (apoE) is a 34-kDa lipid-associated protein present in plasma and in the central nervous system. Previous studies have demonstrated that apoE has multiple functions, including the ability to transport lipids, regulate cell homeostasis, and inhibit lipid oxidation. The lipid binding domain of apoE has been localized to the carboxyl-terminal domain, whereas a cluster of basic amino acid residues within the N-terminal domain is responsible for its receptor binding activity. This study was undertaken to identify the domain in apoE responsible for its antioxidant activity. Results showed that apoE inhibits Cu(2+)-induced LDL oxidation by delaying conjugated diene formation in a concentration-dependent manner. Reductive methylation of lysine residues or cyclohexanedione modification of arginine residues in apoE abolished its ability to inhibit LDL oxidation. Additional studies showed that a 22-kDa peptide containing the N-terminal domain of apoE3 was more effective than a similar peptide with the apoE4 sequence in inhibiting Cu(2+)-induced LDL oxidation. In contrast, the 10-kDa peptide that contains the C-terminal domain of apoE was ineffective. Inhibition of Cu(2+)-induced LDL oxidation can also be accomplished with a peptide containing either a single sequence or a tandem repeat sequence of the receptor binding domain (residues 141-155) of apoE. Taken together, these results localized the antioxidant domain of apoE to its receptor binding domain and the basic amino acids in this domain are important for its antioxidant activity.     

Mutation of lysine residues in apolipoprotein B-100 causes defective lipoprotein[a] formation

Lipoprotein[a] (Lp[a]) is assembled by a two-step process involving an initial lysine-dependent binding between apolipoprotein B-100 (apoB-100) and apolipoprotein[a] (apo[a]) that facilitates the formation of a disulphide bond between apoB-100Cys4,326 and apo[a]Cys4,057. Previous studies of transgenic mice expressing apoB-95 (4,330 amino acids) and apoB-97 (4,397 amino acids) have shown that apoB-100 amino acids 4,330-4,397 are important for the initial binding to apo[a]. Furthermore, a lysine-rich peptide spanning apoB-100 amino acids 4,372-4,392 has recently been shown to bind apo[a] and inhibit Lp[a] assembly in vitro. This suggests that a putative apo[a] binding site exists in the apoB-4,372-4,392 region. The aim of our study was to establish whether the apoB-4,372-4,392 sequence was important for Lp[a] assembly in the context of the full-length apoB-100. Transgenic mice were created that expressed a mutant human apoB-100, apoB-100K4-->S4, in which all four lysine residues in the 4,372-4,392 sequence were mutated to serines. The apoB-100K4-->S4 mutant showed a reduced capacity to form Lp[a] in vitro compared with wild-type human apoB-100. Double transgenic mice expressing both apoB-100K4-->S4 and apo[a] contained significant amounts of free apo[a] in the plasma, indicating a less-efficient assembly of Lp[a] in vivo. Taken together, these results clearly show that the apoB-4,372-4,392 sequence plays a role in Lp[a] assembly.     

Conformation of apolipoprotein B-100 in the low density lipoproteins of tangier disease. Identification of localized conformational response to triglyceride content.

The low density lipoproteins (LDL) from patients with Tangier disease are enriched in triglycerides, 27% of LDL mass versus 7% for normal LDL. To study whether this unique LDL core lipid composition affects the surface disposition of apolipoprotein (apo) B-100, we analyzed the LDL by protease digestion and in competitive radioimmunoassays. Limited proteolytic digestion of Tangier LDL by Staphylococcus aureus V8 protease generated a prominent fragment of 120 kDa (cleavage site at residue 1076), which was not visible in similarly digested normal LDL. In competitive radioimmunoassay, Tangier LDL bound weakly to the apoB-specific monoclonal antibody MB20, compared with control LDL. We localized the MB20 epitope between residues 1031 and 1084 of apoB-100, probably very near residue 1076. DNA sequencing of exon 21 of apoB genomic clones (coding for residues 1014-1084) from a Tangier patient revealed no difference from the normal DNA sequence, thus eliminating a protein polymorphism as a basis for the altered protease sensitivity and antibody binding. When the triglyceride contents of Tangier LDL were reduced to 10% of mass by incubation with normal high density lipoproteins, production of the 120-kDa fragment by proteolysis decreased and MB20 binding increased in affinity, implying a change toward normal conformation of apoB-100. Thus, using two independent techniques, proteolytic digestion and binding of monoclonal antibodies, we have demonstrated an alternative conformation of apoB-100 in the vicinity of residue 1076, which reflects the content of triglycerides in the LDL particle.     

Partial apolipoprotein E-beta-galactosidase fusion protein expressed in Escherichia coli retains binding activity to the LDL(B/E) receptor

A partial rat apo E-beta-galactosidase fusion protein was produced in Escherichia coli Y1089 infected with recombinant lambda GT11 obtained by immunoscreening of a rat liver cDNA library with an anti-rat LDL antiserum. Partial cDNA overlapped the apo E mRNA sequence coding for apo E binding domain towards the LDL(B/E) receptor up to codon for Arg-139. Fusion protein specifically bound to human fibroblasts. The high-affinity component exhibited a Kd of 5 x 10(-8) M and 4.1 x 10(5) sites per cell. Fusion protein binding to fibroblasts was mediated by their apo E moiety and not by beta-galactosidase since: (1) specific binding of fusion protein was competed out by human LDL; (2) beta-galactosidase did not compete with fusion protein binding; and (3) human fibroblasts from a patient with familial hypercholesterolemia, deficient in LDL(B/E) receptor, bound fusion protein 10-times lower than control fibroblasts. It was demonstrated that partial fusion protein retained the functional activity of the native apo E. However, compared to full-length native or engineered apo E, fusion protein was able to bind fibroblasts without being complexed with phospholipids. Fusion proteins might be a useful tool for studying the functional efficiency of the LDL(B/E) receptor and for mapping residues and domains involved in the binding process.     

Structure and developmental expression of the nerve growth factor receptor in the chicken central nervous system

Chicken nerve growth factor (NGF) receptor cDNAs have been isolated and sequenced in an effort to identify functionally important receptor domains and as an initial step in determining the functions of the NGF receptor in early embryogenesis. Comparisons of the primary amino acid sequences of the avian and mammalian NGF receptors have identified several discrete domains that differ in their degree of conservation. The highly conserved regions include an extracellular domain, likely to be involved in ligand binding, in which the positions of 24 cysteine residues and virtually all negatively charged residues are conserved; a transmembrane region, including flanking stretches of extracellular and cytoplasmic amino acids, which has properties suggesting it interacts with other proteins; and a cytoplasmic PEST sequence, which may regulate receptor turnover. Transient expression of NGF receptor mRNA has been seen in many regions of the developing CNS. Experiments suggest that both NGF and its receptor help regulate development of the retina.