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Lipid-free apolipoprotein (apo) A-I contributes to the reverse transport of cholesterol from the periphery to the liver by solubilizing plasma membrane phospholipid and cholesterol. The features of the apolipoprotein required for this process are not understood and are addressed in the current study. Membrane microsolubilization of human fibroblasts is not specific for apo A-I; unlipidated apos A-II, C, and E incubated with the fibroblast monolayers at a saturating concentration of 50 micrograms/ml are all able to release cholesterol and phospholipid similarly. To determine the properties of the apolipoprotein that drive the process, apo A-I peptides spanning the entire sequence of the protein were utilized; the peptides correspond to the 11- and 22-residue amphipathic alpha-helical segments, as well as adjacent combinations of the helices. Of the 20 helical peptides examined, only peptides representing the N-and C-terminal portions of the protein had the ability to solubilize phospholipid and cholesterol. Cholesterol efflux to the most effective peptides, 44-65 and 209-241, was approximately 50 and 70%, respectively, of that to intact apo A-I. Deletion mutants of apo E and apo A-I were constructed that have reduced lipid binding affinities as compared with the intact molecule. The proteins, apo A-I (Delta222-243), apo A-I (Delta190-243), apo E3 (Delta192-299) and apo E4 (Delta192-299) all exhibited a decreased ability to remove cellular cholesterol and phospholipid. These decreases correlated with the reduced ability of these proteins to penetrate into a phospholipid monomolecular film. Overall, the results indicate that insertion of amphipathic alpha-helices between the plasma membrane phospholipid molecules is a required step in the mechanism of apolipoprotein-mediated cellular lipid efflux. Therefore the lipid binding ability of the apolipoprotein is critical for efficient membrane microsolubilization.

To explore the potential of the common marmoset monkey (Callithrix jacchus) as a model for human plasma lipoprotein metabolism, several marmoset apolipoproteins were isolated and characterized in this study. Based on several properties, including molecular weight, amino acid composition, and sequence, the marmoset apolipoproteins are strikingly similar to human apolipoprotein (apo) A-I, A-II, C-III, and A-IV. The first 54 residues of marmoset apo A-I showed 87% sequence identity with the corresponding region of human apo A-I. Amino-terminal sequence analysis of a minor basic apo A-I isoform revealed that it contained an amino-terminal hexapeptide extension (Arg-His-Phe-Gln-Gln-) identical to that found in human proapo A-I. Like apo A-II in most nonhuman primates, marmoset apo-A-II differed from human apo A-II in that it did not contain cysteine and therefore existed as a monomer. The complete amino acid sequence of marmoset apo A-II was deduced. The protein contains 77 amino acids, as does human apo A-II, and showed an 82% identity with its human equivalent. In both species, apo C-III and E had similar amino-terminal sequences and amino acid compositions. Like human apo E, marmoset apo E contained minor sialylated isoforms. However, unlike human apo C-III, no sialyated isoforms of marmoset apo C-III were observed. In addition, the marmoset possessed an apolipoprotein whose molecular weight and amino acid composition were similar to those of human apo A-IV. The close structural similarities between corresponding marmoset and human apolipoproteins indicate that the marmoset monkey will be useful as a model for human lipoprotein metabolism.

A type III hyperlipoproteinemic subject having the apolipoprotein E (apo E) phenotype E3/2 was identified. From isoelectric focusing experiments in conjunction with cysteamine treatment (a method that measures cysteine content in apo E), the E2 isoform of this subject was determined to have only one cysteine residue, in contrast to all previously studied E2 apoproteins, which had two cysteines. This single cysteine was shown to be at residue 112, the same site at which it occurs in apo E3. From amino acid and sequence analyses, it was determined that this apo E2 differed from apo E3 by the occurrence of glutamine rather than lysine at residue 146. When phospholipid X protein recombinants of the subject's isolated E3 and E2 isoforms were tested for their ability to bind to the human fibroblast apo-B,E receptor, it was found that the E3 bound normally (compared with an apo E3 control) but that the E2 had defective binding (approximately 40% of normal). Although they contained E3 as well as E2, the beta-very low density lipoproteins (beta-VLDL) from this subject were very similar in character to the beta-VLDL from an E2/2 type III hyperlipoproteinemic subject; similar subfractions could be obtained from each subject and were shown to have a similar ability to stimulate cholesteryl ester accumulation in mouse peritoneal macrophages. The new apo E2 variant has also been detected in a second type III hyperlipoproteinemic subject.

An apolipoprotein (apo) A-I variant that has a relative charge of -1 compared to normal apo-A-I on isoelectric focusing gels has been identified in five unrelated families as a result of screening a large number of individuals. The cause of the electrophoretic abnormality has been examined by analyzing the variant apo-A-I structure. The evidence suggests that a single amino acid, lysine 107, has been deleted in the variant apo-A-I of all affected individuals studied from these families, with the remainder of the variant apo-A-I sequence being unaffected. The deletion of this single basic amino acid residue is sufficient to account for the charge difference between the variant and normal apo-A-I as seen on isoelectric focusing gels. This variant, previously referred to as A-I-Marburg or A-I-Münster-2, can now be designated by the structural abnormality apo-A-I(Lys107----0). The evidence from extensive pedigree analysis suggests the likelihood that the deletion mutant gene is allelic to the normal apo-A-I gene. At the same time, the kindred analyses have failed to yield a lipid abnormality that can be unequivocally related to the presence of this deletion mutant of apo-A-I. However, all subjects expressing apo-A-I(Lys107----0) also express normal apo-A-I, so that any abnormality caused by the variant apo-A-I might be adequately compensated for by the normal apo-A-I. To examine directly the functional consequence of the lysine deletion, the isolated variant was tested in vitro for its ability to activate lecithin:cholesterol acyltransferase, the principal cholesterol-esterifying enzyme in plasma. It was found that apo-A-I(Lys107----0) is deficient in its ability to activate lecithin:cholesterol acyltransferase, having only 40-60% of the cofactor activity of normal apo-A-I. The cofactor activity of the pro-apo-A-I component of the variant was also reduced to about 60% of either normal A-I or normal pro-apo-A-I. The functional defect is probably related to a disruption in the secondary and/or tertiary structure of the protein caused by the deletion of lysine 107 in the primary structure.

The three major isoforms of human apolipoprotein E (apo-E2, -E3, and -E4) are coded for by three alleles (epsilon 2, epsilon 3, and epsilon 4) which have a common genetic locus. Previously, we demonstrated that E2, E3, and E4 differ in primary structure from one another at two substitution sites, site A (residue 112) and site B (residue 158). At sites A/B, apo-E2, -E3, and -E4 contain cysteine/cysteine, cysteine/arginine, and arginine/arginine, respectively. We demonstrated that the substitution of cysteine for arginine at site B is at least partly responsible for the defective binding of apo-E2 to human fibroblast low density lipoprotein receptors, compared to the normal binding activity of apo-E3 or -E4. Subjects with the genetic disorder type III hyperlipoproteinemia are phenotypically homozygous for apo-E2, but the binding activity of apo-E to the fibroblast receptor differs considerably from one type III individual to another. We therefore undertook a partial comparative sequence analysis of apo-E2 from three type III subjects whose apo-E displayed this heterogeneity. The subject with the poorest binding apo-E2 was genotypically homozygous for an apo-E allele (epsilon 2); cysteine was found at sites A and B. The subject with the most active apo-E2 was genotypically homozygous for an apo-E allele (epsilon 2); cystine was found at site A and at a new site (site C, residue 145). The epsilon 2 allele specifies a protein that has arginine at site B (residue 158); the epsilon 2 allele specifies a protein that has arginine at site C (residue 145). Therefore, the two alleles differ from one another by cysteine/arginine interchanges at two positions, sites B and C. The third subject, whose apo-E2 displayed binding activity intermediate between the activities of the other two, was genotypically heterozygous, having one epsilon 2 allele and one epsilon 2 allele. The intermediate binding activity of apo-E2 from this subject resulted from having a mixture of severely defective apo-E (specified by epsilon 2) and slightly defective apo-E (specified by epsilon 2).

Plasma lipoprotein metabolism is regulated and controlled by the specific apolipoprotein (apo-) constituents of the various lipoprotein classes. The major apolipoproteins include apoE, apoB, apoA-I, apoA-II, apoA-IV, apoC-I, apoC-II, and apoC-III. Specific apolipoproteins function in the regulation of lipoprotein metabolism through their involvement in the transport and redistribution of lipids among various cells and tissues, through their role as cofactors for enzymes of lipid metabolism, or through their maintenance of the structure of the lipoprotein particles. The primary structures of most of the apolipoproteins are now known, and various functional domains of these proteins are being mapped using selective chemical modification, synthetic peptides, and monoclonal antibodies. Furthermore, the establishment of structure-function relationships has been greatly advanced by the identification of genetically determined variants of specific apolipoproteins that are associated with a disorder of lipoprotein metabolism. Future studies will rely heavily on the use of recombinant DNA technology and site-specific mutagenesis to elucidate further the correlations between structure and function and the role of specific apolipoproteins in lipoprotein metabolism.

To investigate the potential of monoclonal antibodies as probes to determine the receptor-binding domain of apolipoprotein E (apo-E), five apo-E antibodies were tested to see if any of them inhibited 125I-apo-E3 . dimyristoylphosphatidylcholine binding to apo-B,E receptors on cultured fibroblasts. Only one of the five antibodies, referred to as 1D7, was found to inhibit binding, blocking greater than 90% of the receptor-binding activity of apo-E3 dimyristoylphosphatidyl-choline. The 1D7 Fab fragments were also effective inhibitors. The 1D7 bound to a Mr = 22,000 NH2-terminal thrombolytic fragment of apo-E (residues 1-191) and to a 93-residue cyanogen bromide fragment of apo-E (residues 126-218). The four noninhibitory antibodies bound only to the NH2-terminal thrombolytic fragment. These results suggested that the 1D7 epitope is contained between residues 126 and 191, and that the epitopes of the other antibodies are not contained in this region. The use of synthetic apo-E fragments, which cover various lengths of the sequence from residues 129-169, and human apo-E variants with substitutions at residues 145, 146, or 158, narrowed the location of the 1D7 epitope to residues 139-169 and, most likely, to the immediate vicinity of residues 140-150. It is of interest that 1D7 was found to bind to the same region of apo-E that has been implicated as the receptor-binding domain in receptor-binding studies using human apo-E variants and apo-E3 fragments.

An effective preparative isoelectric focusing method has been developed using the LKB Immobiline system in a vertical slab gel apparatus. Advantages of this procedure are ease of sample application, excellent resolution, and the direct visualization of focused bands. Narrow pH gradients have been used to separate apolipoprotein E3 isoforms (pH gradient 4.9-5.9) and to resolve the apolipoprotein C mixture (pH gradient 4.0-5.0). Recoveries ranged from 40 to 70%. The method should be valuable for protein and isoform purification.

A new apolipoprotein E (apo E) phenotype has been demonstrated in a Finnish hypertriglyceridemic subject (R.M.). At the time of this study, R.M.'s plasma triglyceride and cholesterol levels were 1,021 and 230 mg/dl, respectively. The subject's apo E isoelectric focusing pattern was characterized by two major bands, one in the E3 position and the other in the E1 position. Normally the E1 position is occupied by sialylated derivatives of apo E4, E3, or E2. The E1 band of subject R.M. is not a sialylated form, however, because it was not affected by neuraminidase digestion. The identity of the E1 variant as a genetically determined structure was established by amino acid and partial sequence analyses, confirming that the variant is an example of a previously uncharacterized apo E phenotype, E3/1. Both cysteamine modification and amino acid analysis demonstrated that this variant contains two cysteine residues per mole. Sequence analysis of two cyanogen bromide fragments and one tryptic fragment of the apo E3/1 showed that it differs from E2(Arg158----Cys) at residue 127, where an aspartic acid residue is substituted for glycine. This single amino acid interchange is sufficient to account for the one-charge difference observed on isoelectric focusing gels between E2(Arg158----Cys) and the E1 variant. The variant has been designated E1 (Gly127----Asp, Arg158----Cys). When compared with apo E3, the E1 variant demonstrated reduced ability to compete with 125I-LDL for binding to LDL (apo B,E) receptors on cultured fibroblasts (approximately 4% of the amount of binding of apo E3). This defective binding is similar to that of E2-(Arg158----Cys). Therefore, the binding defect of the variant is probably due to the presence of cysteine at residue 158, rather than aspartic acid at residue 127. In contrast, the apo E3 isoform from this subject demonstrated normal binding activity, indicating that it has a normal structure. In family studies, the vertical transmission of the apo E1 variant has been established. It is not yet clear, however, if the hypertriglyceridemia observed in the proband is associated with the presence of the E1(Gly127----Asp, Arg158----Cys) variant.

The receptor binding properties of lipoproteins derived from neonates and abetalipoproteinemic patients were examined. Compared to normal adults, the neonate plasma contained reduced cholesterol levels, with only 40% of the total cholesterol transported in the low-density lipoproteins (LDL). When compared at equal cholesterol concentrations, however, the total neonate lipoproteins (d less than 1.21) were as effective as adult d less than 1.21 lipoproteins in stimulating cholesteryl ester formation in cultured human fibroblasts. Analysis of the neonate lipoproteins explained their enhanced ability to deliver cholesterol to the cells via LDL (apoprotein B,E) receptors: the neonate d = 1.02-1.063 fraction contained, in addition to LDL, alpha 2-migrating, apoprotein E-rich high-density lipoproteins (HDL1), which were isolated by Geon-Pevikon electrophoresis. In binding studies performed with human fibroblasts at 4 degrees C, the neonate HDL1 were 14-fold more effective than either neonate or adult human LDL in displacing 125I-LDL from apo-B,E receptors. The neonate HDL (d = 1.063-1.21) contained a subfraction rich in apo-E and apo(E-A-II), which was isolated by heparin-Sepharose chromatography. This fraction was also active in displacing 125I-LDL from the receptors on cultured fibroblasts. Apoprotein E-containing HDL subclasses, similar to those described in the blood of neonates, were present in the d less than 1.063 and d = 1.063-1.21 lipoprotein fractions of patients with abetalipoproteinemia. These HDL with apo-E were enriched in cholesterol and were as effective as normal LDL in competing with 125I-LDL for apo-B,E receptor-mediated binding, internalization, and degradation. When incubated with cultured human fibroblasts, the HDL with apo-E from the abetalipoproteinemic subjects increased the cholesteryl ester mass three- to fourfold. These studies suggest that neonates and abetalipoproteinemic subjects may depend (at least in part) upon lipoproteins containing apo-E to deliver cholesterol to various tissues via the LDL (apo-B,E) receptor.

To identify the domain of apolipoprotein E (apo-E) involved in binding to low density lipoprotein (LDL) receptors on cultured human fibroblasts, apo-E was cleaved and the fragments were tested for receptor binding activity. Two large thrombolytic peptides (residues 1-191 and 216-299) of normal apo-E3 were combined with the phospholipid dimyristoylphosphatidylcholine (DMPC) and tested for their ability to compete with 125I-LDL for binding to the LDL (apo-B,E) receptors on human fibroblasts. The NH2-terminal two-thirds (residues 1-191) of apo-E3 was as active as intact apo-E3 . DMPC, while the smaller peptide (residues 216-299) was devoid of receptor-binding activity. When apo-E3 was digested with cyanogen bromide (CNBr) and the four largest CNBr fragments were combined with DMPC and tested, only one fragment competed with 125I-LDL for binding to cultured human fibroblasts (CNBr II, residues 126-218). This fragment possessed binding activity similar to that of human LDL. The 125I-labeled CNBr II . DMPC complex also demonstrated high affinity, calcium-dependent saturable binding to solubilized bovine adrenal membranes. The binding of CNBr II . DMPC was inhibited by 1,2-cyclohexanedione modification of arginyl residues or diketene modification of lysyl residues. In addition, the CNBr II had to be combined with DMPC before it demonstrated any receptor-binding activity. Pronase treatment of the membranes abolished the ability of this fragment to bind to the apo-B,E receptors. This same basic region in the center of the molecule has been implicated as the apo-B,E receptor-binding domain not only by this study but also by other studies showing that 1) natural mutants of apo-E that display defective binding have single amino acid substitutions at residues 145, 146, or 158; and 2) the apo-E epitope of the monoclonal antibody 1D7, which inhibits apo-E binding, is centered around residues 139-146.

A recently discovered familial lipoprotein disorder is characterized by reduced plasma levels of high density lipoproteins (HDL) and elevated triglyceride levels. The clinical aspects of this disorder are presented in an accompanying article (Franceschini et al. 1980. J. Clin. Invest. 66: 892-900). The apoprotein content of the HDL isolated from these patients differed markedly from that of normal HDL in that three apoprotein bands not previously described in man were present as major protein components. As determined by sodium dodecyl sulfate (SDS) gel electrophoresis, the relative molecular weights (Mr) of these new apoprotein bands were 55,000, 35,000, and 28,000. Although the Mr 28,000 apoprotein coelectrophoresed with authentic A-I on SDS polyacrylamide gels and showed immunochemical identity with the A-I apoprotein when tested with monospecific apo-A-I antiserum, it contained two amino acid residues, cysteine and isoleucine, which were not present in the amino acid sequence of normal human apo-A-I. This variant form of the A-I apoprotein was designated the A-IMilano apoprotein and denoted A-Icys. By virtue of the presence of cysteine (2 mol/mol A-Icys), the A-Icys apoprotein was capable of forming intermolecular disulfide bonds, and dimer formation of A-Icys produced the Mr 55,000 apoprotein. The Mr 35,000 apoprotein was composed of two different subunits, A-Icys and A-II. By analogy to the apo(E--A-II) complex, which also occurs in human HDL, this mixed disulfide complex was designated as the apo(A-Icys--A-II) complex. The A-IMilano (A-Icys) is the first example of a variation in the primary sequence of a protein of plasma lipoproteins.

Cholesterol-enriched, apolipoprotein E-containing high density lipoproteins (apo E HDLc), which were isolated from the plasma of cholesterol-fed dogs by using agarose column chromatography or ultracentrifugation, possessed essentially identical biochemical and metabolic characteristics. Radioiodinated (125I) apo E HDLc isolated by either method gave identical rates of clearance from the plasma, i.e., greater than 50% of the injected dose was cleared from the plasma within 5 to 10 minutes, principally by the liver. Detailed studies localizing apo E HDLc uptake to specific cell types within the liver were performed in both normal and cholesterol-fed rats. The validity of using the canine apo E HDLc in the rat was supported by observations of marked similarities in plasma clearance, i.e., a rapid acute phase of disappearance, and a near-quantitative hepatic uptake of lipoproteins in both species. Canine apo E HDLc (fluorescently labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine), which were injected into normal rats, appeared to be taken up primarily by parenchymal cells, as determined by fluorescence microscopy. Light microscopic autoradiography also revealed that the uptake of 125I apo E HDLc was principally carried out by parenchymal cells in rat liver. Likewise, the uptake of apo E HDLc by the liver of cholesterol-fed rats was extensively localized to parenchymal cells. An in situ, single-pass perfusion of a lobule of the liver of a normal dog with iodinated and fluorescently labeled apo E HDLc confirmed that the uptake of the lipoproteins was principally carried out by parenchymal cells. The plasma clearance of apo E HDLc by hepatic parenchymal cells, even in cholesterol-fed animals in which the apo B,E (LDL) receptors were markedly down-regulated (undetectable), suggests that the apo E receptor, presumably the remnant receptor, is localized in the parenchymal cells.

Selective chemical modification of lysine residues of lipoproteins by acetoacetylation dramatically altered the metabolism of the lipoproteins without significantly altering other physical or chemical properties. Modification of 30-60% of the total lysine residues of iodinated rat or human low-density lipoproteins ((125)I-LDL) resulted in a rapid removal of these acetoacetylated lipoproteins from the plasma of rats. Within minutes after intravenous injection into intact rats, greater than 80% of the total injected dose disappeared from the plasma. The rapidly cleared acetoacetylated LDL appeared in the liver, and within 6-30 min as much as 50-80% of the total injected dose of modified LDL could be accounted for in the liver. Furthermore, it was possible to demonstrate in the isolated perfused rat liver that the Kupffer cells were responsible for the lipoprotein uptake. Human high-density lipoproteins (HDL(3)) were also rapidly removed from the plasma after acetoacetylation. In striking contrast, acetoacetylation (30-60%) of two E apoprotein-containing lipoproteins (rat HDL(1) and dog HDL(c)) retarded their removal from the plasma. The accelerated removal of modified LDL and HDL(3), in contrast to the retarded removal of modified HDL(1) and HDL(c), suggests that the recognition and removal process is specific for a property acquired by only certain lipoproteins after acetoacetylation. Moreover, these results suggest that lysine residues of the E apoprotein may play a functional role in the recognition process for the normal clearance of HDL(1) and HDL(c), a process that is interfered with after acetoacetylation.

The apolipoprotein E2 ( apoE2 ) variant that possesses a cysteine substituted for an arginine at residue 158 in the amino acid sequence E2( Arg158 ----Cys) can be distinguished by sodium dodecyl sulfate-polyacrylamide gel electrophoresis from other forms of apoE, including E3 (the parent form), E4( Cys112 ----Arg), E2( Arg145 ----Cys), and E2( Lys146 ----Gln). The E2( Arg158 ----Cys) migrates as a distinctly separable band with a higher apparent molecular weight than the other forms. Chemical modification of apoE2 ( Arg158 ----Cys) with sulfhydryl reagents (2-bromoethyl)-trimethylammonium bromide or cysteamine, which convert cysteine to arginyl or lysyl analogues, respectively, abolishes the difference in apparent molecular weight and results in the co-electrophoresis of E2( Arg158 ----Cys) with other apoE forms. The mobilities of the other apoE variants are not affected by these modifications. These results suggest that the substitution site at residue 158 is a key location, important in modifying the behavior of apoE and in modulating its apparent molecular weight on sodium dodecyl sulfate-polyacrylamide gels. Furthermore, the technique used in this study may be very helpful in distinguishing specific mutant forms of apoE2 .

The beta-very low density lipoproteins (beta-VLDL) that accumulate in type III hyperlipoproteinemic subjects can be divided into two fractions (fraction I and fraction II), which differ in size, lipid composition, and the type of apolipoprotein B (apo-B) present in the particles. The apo-B48-containing particles (fraction I) are of intestinal origin, while apo-B100-containing particles (fraction II) are derived from the liver. Both fractions contain a defective form of apo-E referred to as apo-E2. Intravenous infusion of heparin into two subjects with type III hyperlipoproteinemia resulted in the complete removal of fraction II particles from density less than 1.006 g/ml, while fraction I particles remained at this density. In vitro studies confirmed that fraction I particles did not change density when subjected to hydrolysis with lipoprotein lipase, while fraction II particles shifted to the intermediate density lipoprotein range (approximately equal to 1.02 g/ml). When the beta-VLDL were hydrolyzed by lipoprotein lipase in the presence of density greater than 1.21 g/ml lipoprotein-deficient plasma, the addition of normal apo-E (apo-E3), but not apo-E2, resulted in a shift of fraction II particles to the low density lipoprotein (LDL) range (approximately equal to 1.05 g/ml). Fraction I particles did not undergo a shift to this higher density, supporting previous observations that apo-B48-containing particles are not converted to LDL. The demonstration that apo-B100-containing particles in type III hyperlipoproteinemic subjects could be converted to particles with the density of LDL suggests that apo-E plays a role in the normal conversion of VLDL to LDL. The mutant form of apo-E (apo-E2) found in the beta-VLDL from type III hyperlipoproteinemic subjects appears to impede this conversion, whereas the addition of normal apo-E (apo-E3) allows the processing to occur.

Apolipoprotein (apo-) E3, when combined with the phospholipid dimyristoylphosphatidylcholine (DMPC), binds avidly to apo-B,E (low density lipoprotein) receptors on human fibroblasts. Apolipoprotein E2 isolated from type III hyperlipoproteinemic subjects, which differs from apo-E3 by the presence of cysteine instead of arginine at residue 158, possesses only about 1% of the receptor binding activity of apo-E3. Modification of apo-E2 with cysteamine, which converts the cysteine at position 158 to a positively charged lysine analogue, activates receptor binding approximately 13-fold. In the present experiments, thrombin was used to cleave apo-E2 into two fragments (Mr = 22,000 and Mr = 10,000). The larger fragment, which has been shown to possess the receptor binding domain, displayed binding activity up to 12-fold greater than intact apo-E2 or equivalent to apo-E2 treated with cysteamine. When the Mr = 22,000 fragment was modified with cysteamine and combined with DMPC, receptor binding was further enhanced, attaining the level of activity of normal apo-E3 X DMPC, a 100-fold increase over apo-E2 X DMPC binding. When the cysteamine modification was reversed by incubation with beta-mercaptoethanol, the Mr = 22,000 fragment retained most of its binding activity. However, when the same sample was tested 24 h later, the level of binding activity dropped significantly. The receptor binding of apo-E2-containing beta-very low density lipoproteins could also be activated by cysteamine treatment, with the same retention of enhanced binding activity occurring after the reversal of the modification. These results indicate that apo-E2 can attain full binding activity by the removal of the carboxyl-terminal one-third of the molecule and the addition of a positive charge at residue 158 of the molecule. The retention of enhanced binding after the reversal of the cysteamine modification indicates that the enhanced binding is probably due to conformational changes induced in the binding domain (and maintained by the phospholipid) and not merely to the presence of the positive charge at residue 158.

Variant forms of apolipoprotein A-I (apo-A-I) have been shown to exist in the human population. One mutant form, referred to as apo-A-I-Münster-3, is one charge unit more basic than normal apo-A-I on isoelectric focusing gels. This variant has the same immunologic characteristics and molecular weight as normal apo-A-I. The apo-A-I-Münster-3 from subjects in three unrelated families (in two of which the trait has been shown to be transmitted as an autosomal co-dominant) has been analyzed by partial amino acid sequencing to define the cause of the electrophoretic abnormality. In the apo-A-I of family A, the abnormality was shown to occur in the smallest cyanogen bromide fragment, CB-2 (residues 87-112), and amino acid sequencing revealed asparagine instead of the usual aspartic acid at residue 103. Subjects with this mutant form have shown no signs of dyslipoproteinemia. The NH2-terminal cyanogen bromide fragment (CB-1, residues 1-86) from the apo-A-I of family B was shown to differ electrophoretically from normal CB-1, and amino acid sequencing revealed that a substitution of arginine for proline at residue 4 was responsible for this variant form. Analysis of the plasma lipids of one affected family B member demonstrated that the percentage of the total cholesterol that was esterified was somewhat lower than that normally observed. In a third family, family C, a variant having the same electrophoretic abnormality as the other two was determined to have an amino acid substitution at yet a different position. In this variant, histidine was found at residue 3 in the apo-A-I sequence, rather than the usual proline. In all three cases, the substitution could account for the electrophoretic abnormality. It is proposed that these three apo-A-I-Münster-3 variants be designated apo-A-I(Asp103----Asn), apo-A-I(Pro4----Arg), and apo-A-I(Pro3----His), respectively, to indicate the substitution that accounts for the abnormality in isoelectric focusing gels.

Certain proteins (called apolipoproteins B and E) on the surface of lipoprotein particles are responsible for mediating the binding of cholesterol-rich particles to specific lipoprotein receptors on the surface of cells and represent a major pathway controlling blood cholesterol levels. Three important disorders of lipoprotein metabolism, which provide insights into the molecular mechanisms responsible for the elevation of specific atherogenic lipoproteins, are the following: (1) Type III hyperlipoproteinemia results from specific mutations in apolipoprotein E that prevent the normal binding of chylomicron remnants and very-low-density lipoprotein remnants to lipoprotein receptors. Patients with this disorder who have elevated levels of these remnant lipoproteins develop atherosclerosis. (2) Familial defective apolipoprotein B-100 results from a single amino acid substitution in apolipoprotein B that prevents low-density lipoprotein from binding normally to the low-density lipoprotein receptor and elevates plasma cholesterol levels. (3) Familial hypercholesterolemia, which results in elevated levels of plasma low-density lipoprotein and premature atherosclerosis, is caused by a variety of mutations in the low-density lipoprotein receptor that interfere with the normal binding of lipoproteins to this receptor. These observations not only provide insights into the mechanisms responsible for normal lipoprotein metabolism, but also highlight the potential role of specific lipoproteins in atherogenesis. [ABSTRACT FROM AUTHOR]