Structural studies of N- and C-terminally truncated human apolipoprotein A-I

Apolipoprotein A-I (apoA-I) plays an important structural and functional role in lipid transport and metabolism. This work is focused on the central region of apoA-I (residues 60-183) that is predicted to contain exclusively amphipathic alpha-helices. Six N- and/or C-terminally truncated mutants, delta(1-41), delta(1-59), delta(198-243), delta(209-243), delta(1-41,185-243), and delta(1-59,185-243), were analyzed in their lipid-free state in solution at pH 4.7-7.8 by far- and near-UV CD spectroscopy. At pH 7.8, all mutants show well-defined secondary structures consisting of 40-52% alpha-helix. Comparison of the alpha-helix content in the wild type and mutants suggests that deletion of either the N- or C-terminal region induces helical unfolding elsewhere in the structure, indicating that the terminal regions are important for the integrity of the solution conformation of apoA-I. Near-UV CD spectra indicate significant tertiary and/or quaternary structural changes resulting from deletion of the N-terminal 41 residues. Reduction in pH from 7.8 to 4.7 leads to an increase in the mutant helical content by 5-20% and to a large increase in thermal unfolding cooperativity. Van't Hoff analysis of the mutants at pH 4.7 indicates melting temperatures T(m) ranging from 51 to 59 degrees C and effective enthalpies deltaH(v)(T(m)) = 35 +/- 5 kcal/mol, similar to the values for plasma apoA-I at pH 7.8 (T(m) = 57 degrees C, deltaH(v) = 32 kcal/mol). Our results provide the first report of the pH effects on the secondary, tertiary, and/or quaternary structure of apoA-I variants and indicate the importance of the electrostatic interactions for the solution conformation of apoA-I.     

Surface behavior of apolipoprotein A-I and its deletion mutants at model lipoprotein interfaces

Apolipoprotein A-I (apoA-I) has a great conformational flexibility to exist in lipid-free, lipid-poor, and lipid-bound states during lipid metabolism. To address the lipid binding and the dynamic desorption behavior of apoA-I at lipoprotein surfaces, apoA-I, Δ(185-243)apoA-I, and Δ(1-59)(185-243)apoA-I were studied at triolein/water and phosphatidylcholine/triolein/water interfaces with special attention to surface pressure. All three proteins are surface active to both interfaces lowering the interfacial tension and thus increasing the surface pressure to modify the interfaces. Δ(185-243)apoA-I adsorbs much more slowly and lowers the interfacial tension less than full-length apoA-I, confirming that the C-terminal domain (residues 185-243) initiates the lipid binding. Δ(1-59)(185-243)apoA-I binds more rapidly and lowers the interfacial tension more than Δ(185-243)apoA-I, suggesting that destabilizing the N-terminal α-helical bundle (residues 1-185) restores lipid binding. The three proteins desorb from both interfaces at different surface pressures revealing that different domains of apoA-I possess different lipid affinity. Δ(1-59)(185-243)apoA-I desorbs at lower pressures compared with apoA-I and Δ(185-243)apoA-I indicating that it is missing a strong lipid association motif. We propose that during lipoprotein remodeling, surface pressure mediates the adsorption and partial or full desorption of apoA-I allowing it to exchange among different lipoproteins and adopt various conformations to facilitate its multiple functions.     

Structural organization of the N-terminal domain of apolipoprotein A-I: studies of tryptophan mutants

Site-directed mutagenesis and detailed fluorescence studies were used to study the structure and dynamics of recombinant human proapolipoprotein (proapo) A-I in the lipid free state and in reconstituted high-density lipoprotein (rHDL) particles. Five different mutants of proapoA-I, each containing a single tryptophan residue, were produced in bacteria corresponding to each of the naturally occurring Trp residues (position -3 in the pro-segment, 8, 50, 72, and 108) in the N-terminal half of the protein. Structural analyses indicated that the conservative Phe-Trp substitutions did not perturb the conformation of the mutants with respect to the wild-type protein. Steady-state fluorescence studies indicated that all of the Trp residues exist in nonpolar environments that are highly protected from solvent in both the lipid-free and lipid-bound forms. Time-resolved lifetime and anisotropy studies indicated that the shape of the monomeric form of proapoA-I is a prolate ellipsoid with an axial ratio of about 6:1. In addition, the region surrounding Trp 108 appears to be more mobile than the rest of the protein in the lipid-free state. However, in rHDL particles, no significant domain motion was detected for any of the Trp residues. The results presented in this work are consistent with a model for monomeric lipid-free proapoA-I in which the N-terminal half of the molecule is organized into a bundle of helices.     

Cysteine mutants of human apolipoprotein A-I: a study of secondary structural and functional properties

Apolipoprotein A-I(Milano) (A-I(M)) (R173C), a natural mutant of human apolipoprotein A-I (apoA-I), and five other cysteine variants of apoA-I at residues 52 (S52C), 74 (N74C), 107 (K107C), 129 (G129C), and 195 (K195C) were generated. Cysteine residues were incorporated in each of the various helices at the same helical wheel position as for the substitution in A-I(M). The secondary structural properties of the monomeric mutants, their abilities to bind lipid and to promote cholesterol efflux from THP-1 macrophages, and the possibility of antiperoxidation were investigated. Results showed that the alpha helical contents of all of the cysteine mutants were similar to that of wild-type apoA-I (wtapoA-I). The cysteine variant of A-I(M) at residue 173 [A-I(M)(R173C)] exhibited weakened structural stability, whereas A-I(G129C) a more stable structure than wtapoA-I. A-I(G129C) and A-I(K195C) exhibited significantly impaired capabilities to bind lipid compared with wtapoA-I. A-I(K107C) possessed a higher capacity to promote cholesterol efflux from macrophages than wtapoA-I, and A-I(M)(R173C) and A-I(K195C) exhibited an impaired efflux capability. Neither A-I(M)(R173C) nor any other cysteine mutant could resist oxidation against lipoxygenase. In summary, in spite of the similar mutant position on the helix, these variants exhibited different structural features or biological activities, suggesting the potential influence of the local environment of mutations on the whole polypeptide chain.     

Protein heterogeneity of lipoprotein particles containing apolipoprotein A-I without apolipoprotein A-II and apolipoprotein A-I with apolipoprotein A-II isolated from human plasma

The protein heterogeneity of fractions isolated by immunoaffinity chromatography on anti-apolipoprotein A-I and anti-apolipoprotein A-II affinity columns was analyzed by high resolution two-dimensional gel electrophoresis. The two-dimensional gel electrophoresis profiles of the fractions were analyzed and automatically compared by the computer system MELANIE. Fractions containing apolipoproteins A-I + A-II and only A-I as the major protein components have been isolated from plasma and from high density lipoproteins prepared by ultracentrifugation. Similarities between the profiles of the fractions, as indicated by two-dimensional gel electrophoresis, suggested that those derived from plasma were equivalent to those from high density lipoproteins (HDL), which are particulate in nature. The established apolipoproteins (A-I, A-II, A-IV, C, D, and E) were visible and enriched in fractions from both plasma and HDL. However, plasma-derived fractions showed a much greater degree of protein heterogeneity due largely to enrichment in bands corresponding to six additional proteins. They were present in trace amounts in fractions isolated from HDL and certain of the proteins were visible in two-dimensional gel electrophoresis profiles of the plasma. These proteins are considered to be specifically associated with the immunoaffinity-isolated particles. They have been characterized in terms of Mr and pI. Computer-assisted measurements of protein spot-staining intensities suggest an asymmetric distribution of the proteins (as well as the established apolipoproteins), with four showing greater prominence in particles containing apolipoprotein A-I but no apolipoprotein A-II.     

Membrane insertion topology of the central apolipoprotein A-I region. Fluorescence studies using single tryptophan mutants

Apolipoprotein A-I (apoAI) contains several amphipathic α-helices. To carry out its function, it exchanges between lipid-free and different lipidated states as bound to membranes or to lipoprotein complexes of different morphology, size, and composition. When bound to membranes or to spherical lipoprotein surfaces, it is thought that most α-helices arrange with their long axis parallel to the membrane surface. However, we previously found that a central region spanning residues 87-112 is exclusively labeled by photoactivable reagents deeply located into the membrane (Co?rsico et al. (2001) J. Biol. Chem. 276, 16978-16985). A pair of amphipathic α-helical repeats with a particular charge distribution is predicted in this region. In order to study their insertion topology, three single tryptophan mutants, each one containing the tryptophan residue at a selected position in the hydrophobic face of the central Y-helices (W@93, W@104, and W@108), were used. From the accessibility to quenchers located at different membrane depths, distances from the bilayer center of 13.4, 10.5, and 15.7 ? were estimated for positions 93, 104, and 108, respectively. Reported data also indicate that distances between homologous positions (in particular for W@93 and W@104) are very short in dimers in aqueous solution, but they are larger in membrane-bound dimers. Data indicate that an intermolecular central Y-helix bundle would penetrate the membrane perpendicularly to the membrane surface. Intermolecular helix-helix interactions would occur through the hydrophilic helix faces in the membrane-bound bundle but through the hydrophobic faces in the case of dimers in solution.     

Optimized bacterial expression of human apolipoprotein A-I

Apolipoprotein A-I (apoA-I) serves critical functions in plasma lipoprotein metabolism as a structural component of high density lipoprotein, activator of lecithin:cholesterol acyltransferase, and acceptor of cellular cholesterol as part of the reverse cholesterol transport pathway. In an effort to facilitate structure:function studies of human apoA-I, we have optimized a plasmid vector for production of recombinant wild type (WT) and mutant apoA-I in bacteria. To facilitate mutagenesis studies, subcloning, and DNA manipulation, numerous silent mutations have been introduced into the apoA-I cDNA, generating 13 unique restriction endonuclease sites. The coding sequence for human apoA-I has been modified by the introduction of additional silent mutations that eliminate 18 separate codons that employ tRNAs that are of low or moderate abundance in Escherichia coli. Yields of recombinant apoA-I achieved using the optimized cDNA were 100+/-20 mg/L bacterial culture, more than fivefold greater than yields routinely obtained with the original cDNA. Site-directed mutagenesis of the apoA-I cDNA was performed to generate a Glu2Asp mutation in the N-terminal sequence of apoA-I. This modification, which creates an acid labile Asp-Pro peptide bond between amino acids 2 and 3, permits specific chemical cleavage of an N-terminal His-Tag fusion peptide used for rapid protein purification. The product protein's primary structure is identical to WT apoA-I in all other respects. Together, these changes in apoA-I cDNA and bacterial expression protocol significantly improve the yield of apoA-I protein without compromising the relative ease of purification.     

Self-association of human apolipoproteins A-I and A-II and interactions of apolipoprotein A-I with bile salts: quasi-elastic light scattering studies

We employed quasi-elastic light scattering (QLS) to systematically study the aqueous self-association of human apolipoproteins A-I and A-II (apo A-I and apo A-II) and the interactions of apo A-I with common taurine-conjugated bile salts. Self-association of apo A-I was promoted by increases in apolipoprotein concentration (0.09-2.2 mg/mL) and ionic strength (0.15-2.0 M NaCl), inhibited by increases in temperature (5-50 degrees C) and guanidine hydrochloride concentration (0-2.0 M), and unaffected by hydrostatic pressures up to 500 atm. The mean hydrodynamic radius (Rh) of apo A-I micelles ranged from 38 A to a maximum asymptotic value of 68 A. We examined several possible models of apo A-I self-association; the model that best fitted the Rh values assumed that apo A-I monomers first interacted at low concentrations to form dimers, which then further associated to form ring-shaped limiting octamers. Comparison of the temperature-dependent and ionic strength dependent free energy changes for the formation of octamers from apo A-I dimers suggested that hydrophobic forces strongly favored self-association and that electrostatic repulsive forces were only weakly counteractive. Apo A-II self-association was also promoted by increases in apolipoprotein concentration (0.2-1.8 mg/mL) and inhibited by increases in guanidine hydrochloride concentration (0-1.0 M) but was unaffected by variations in temperature (10-37 degrees C): the largest Rh values observed were consistent with limiting tetramers. As demonstrated by equilibrium dialysis, bile salts in concentrations below their critical micellar concentrations (cmc) bound to apo A-I micelles but had no effect upon apo A-I self-association, as inferred from constant Rh values. When bile salt concentrations exceeded their aqueous cmc values, a dissociation of apo A-I micelles resulted with the formation of mixed bile salt/apo A-I micelles. These studies support the concepts that apo A-I and apo A-II form small dimeric micelles at low concentrations that grow sharply to reach limiting sizes over a narrow concentration range. The influences of bile salt concentration and species upon these micelles have relevance to the plasma transport of bile salts in high-density lipoproteins and to the physical-chemical state of apo A-I and apo A-II molecules in native biles.     

Apolipoprotein complexity in Japanese eel Anguilla japonica: truncated apolipoprotein A-I and apolipoprotein A-I-like protein in plasma lipoproteins

A truncated apolipoprotein (apo) A-I with a molecular weight (M(r)) of 26 kDa was first isolated from the plasma high density lipoproteins of an atypical Japanese eel (Anguilla japonica). Interestingly, this eel contained a very small amount of intact apoA-I (M(r)28 kDa) in the plasma, although serine protease inhibitors were present throughout the plasma preparation. The N-terminal sequence of 20 amino acids in truncated apoA-I was completely identical with that of intact apoA-I. Another apolipoprotein with M(r)28 kDa, whose N-terminal amino acid sequence differed from apoA-I, was also found in high density lipoprotein and low density lipoprotein. The apolipoprotein profiles of Japanese eel plasma appear to be complicated.     

Studies on the polymorphism of human apolipoprotein A-I

Upon preparative isoelectric focussing of human apo-HDL, four major forms of apolipoprotein A-I have been isolated. As identified by the following nomenclature and pI, they comprise: apolipoprotein A-I1, pI 5.62; apolipoprotein A-I2, pI 5.53; apolipoprotein A-I3, pI 5.45; apolipoprotein A-I4 pI 5.36. These forms of apolipoprotein A-I were shown to have identical migration on polyacrylamide gel electrophoresis, molecular weights of 26 000 on sodium dodecyl sulfate gel electrophoresis and a common antigenicity with antisera against apolipoprotein A-I or A-I1. Each form had very similar amino acid compositions with the exception of form apolipoprotein A-I4 which contained one isoleucine residue per mol. All forms but apolipoprotein A-I4 were activators of lecithin:cholesterol acyltransferase, the latter was inhibitory to the reaction. From these results, it was concluded that apolipoprotein A-I1, A-I2 and A-I3 are equivalent forms of apolipoprotein A-I whereas apolipoprotein A-I4 is different or heterogeneous. Upon refocussing, the polymorphs were shown to be stable at their pI and not affected by changes in concentration and by the presence of urea or ampholytes. Exposure of a form of apolipoprotein A-I to alkaline pH partially regenerated the original heterogeneity; however, apolipoprotein A-I4 regenerated from apolipoprotein A-I1 did not contain isoleucine, which further demonstrates form apolipoprotein A-I4 heterogeneity.     

Expression and secretion of human apolipoprotein A-I in the heart

Nuclear envelope-peripheral heterochromatin fractions contain multiple histone kinase activities. In vitro assays and amino-terminal sequencing show that one of these activities co-isolates with heterochromatin protein 1 (HP1) and phosphorylates histone H3 at threonine 3. Antibodies recognizing this post-translational modification reveal that in vivo phosphorylation at threonine 3 commences at early prophase in the vicinity of the nuclear envelope, spreads to pericentromeric chromatin during prometaphase and is fully reversed by late anaphase. This spatio-temporal pattern is distinct from H3 phosphorylation at serine 10, which also occurs during cell division, suggesting segregation of differentially phosphorylated chromatin to different regions of mitotic chromosomes.     

Characterization of human apolipoprotein A-I expressed in Escherichia coli

Human apolipoprotein A-I (apoA-I), with an additional N-terminal extension (Met-Arg-Gly-Ser-(His)₆-Met) (His-apoA-I), has been produced in Escherichia coli with a final yield after purification of 10 mg protein/l of culture medium. We have characterized the conformation and structural properties of His-apoA-I in lipid-free form, and in reconstituted lipoproteins containing two apoA-I per particle (Lp2A-I) by both immunochemical and physicochemical techniques. The lipid-free forms of the two proteins present very similar secondary structure and stability, and have also very similar kinetics of association with dimyristoyl phosphatidylcholine. His-apoA-I and native apoA-I can be complexed with 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) to form similar, stable, either discoidal or spherical (sonicated) Lp2A-I particles. Lipid-bound native apoA-I and His-apoA-I showed very similar α-helical content (69% and 66%, respectively in discoidal Lp2A-I and 54% and 51%, respectively in spherical Lp2A-I). The conformation of His-apoA-I in lipid-free form and in discoidal or spherical Lp2A-I has also been shown to be similar to native apoA-I by immunochemical measurements using 13 monoclonal antibodies recognizing distinct apoA-I epitopes. In the free protein and in reconstituted Lp2A-I, the N-terminal has no effect on the affinity of any of the monoclonal antibodies and minimal effect on immunoreactivity values. Small differences in the exposure of some apoA-I epitopes are evident on discoidal particles, while no difference is apparent in the expression of any epitope of apoA-I on spherical Lp2A-I. The presence of the N-terminal extension also has no effect on the reaction of LCAT with the discoidal Lp2A-I or on the ability of complexes to promote cholesterol efflux from fibroblasts in culture. In conclusion, we show that His-apoA-I expressed in E. coli exhibits similar physicochemical properties to native apoA-I and is also identical to the native protein in its ability to interact with phospholipids and to promote cholesterol esterification and cellular cholesterol efflux.     

Identification of homozygosity for a human apolipoprotein A-I variant

An apolipoprotein (apo) A-I variant, previously described in two Norwegian families (Schamaun et al. 1983. Hum. Genet. 64: 380-383), represents a mutation in apoA-I in which a single amino acid substitution of lysine for glutamic acid has taken place at residue 136. An offspring resulting from intermarriage between the two families is genotypically homozygous for this variant. He is the first individual discovered to be homozygous for any of the apoA-I variants. Analysis of lipid data collected from these families indicates one or more lipid abnormalities. The low density lipoproteins (LDL) of subjects having this apoA-I variant demonstrate a compositional abnormality. The plasma cholesterol concentration in the homozygous subject is low because of the extremely reduced levels of LDL and apoB, a property shared by some of his first-degree relatives. However, because of the presence of apoE2 in this family, it is not possible to definitively link these lipid abnormalities to the presence of the A-I variant.     

Denaturation of apolipoprotein A-I and the monomer form of apolipoprotein A-I(Milano).

In this study the thermal and denaturant induced unfolding of apolipoprotein A-I (apo A-I) and the monomer form of apolipoprotein A-I(Milano) (apo A-I(M)) was followed. Dimer apo A-I(M) was reduced with dithiothreitol, which was present in the protein solutions in all experiments. Thermal denaturation is followed by differential scanning calorimetry (DSC) and far-UV and near-UV CD. Both apo A-I and monomer apo A-IM have a broad asymmetric DSC peak that could be deconvoluted into three non two-state transitions, apo A-I being more stable than the monomer apo A-IM. Estimation of melting of tertiary structure by near-UV CD is lower than that for secondary structure determined from far-UV. This together with the non two-state unfolding of the proteins observed with DSC is indicative of unfolding via a molten globular-like state. Apo A-I and monomer apo A-I(M) are equally susceptible to guanidinum chloride, half-unfolded at 1.2 M denaturant. The presence of 0.5 and 1.0 M denaturant, lower and equalize the denaturation temperatures of the proteins, respectively.     

Quantitative determination of apolipoprotein A-I in high-density lipoproteins and 'free' apolipoprotein A-I by two-dimensional agarose gel lipoprotein-'rocket' immunoelectrophoresis of human serum

A method has been developed for quantitative analysis of 'free' apolipoprotein A-I and apolipoprotein A-I associated with high-density lipoprotein (HDL) in serum. The method utilizes the difference between the rate of electrophoretic migration of apolipoprotein A-I associated with HDL (alpha) and 'free' apolipoprotein A-I (pre-beta) in agarose gel. Apolipoprotein A-I is subsequently quantitated by electrophoresis in a second dimensional gel containing anti-apolipoprotein A-I antibodies. Using this method all apolipoprotein A-I of normal fasting serum was found associated with HDL (n = 16). By contrast, 'free' apolipoprotein A-I accounted for up to 12% of the total in the serum of patients with isolated hypertriglyceridemia (n = 8) or mixed hyperlipoproteinemia (n = 8). Between 30 and 35% of 'free' apolipoprotein A-I was found in one patient afflicted with the apolipoprotein C-II deficiency syndrome. Also, 'free' apolipoprotein A-I could be detected in normal postabsorptive serum. 30 and 90 min following heparin-enhanced lipolysis 'free' apolipoprotein A-I accounted for 23 and 20%, respectively, of the total apolipoprotein A-I of serum. Apolipoprotein A-I associated with HDL remained unaltered. It appears, therefore, that 'free' apolipoprotein A-I is liberated from triglyceride-rich lipoproteins during lipolysis.     

Accumulation of cholestatic lipoproteins in ANIT-treated human apolipoprotein A-I transgenic rats is diminished through dose-dependent apolipoprotein A-I activation of LCAT

Administration of alpha-naphthylisothiocyanate (ANIT) to rats induces changes to plasma lipids consistent with cholestasis. We have previously shown (J. Lipid Res. 37 (1996) 1086) that animals treated with ANIT accumulate large amounts of free cholesterol (FC) and phospholipid (PL)-rich cholestatic lipoproteins in the LDL density range by 48 h. This lipid was cleared by 120 h through apparent movement into HDL with concomitant cholesteryl ester (CE) production. It was hypothesised that the clearance was mediated through the movement of the PL and FC into apolipoprotein A-I (apo A-I) containing lipoproteins followed by LCAT esterification to form CE. To test this hypothesis, rats overexpressing various amounts of human apo A-I (TgR[HuAI] rats) were treated with ANIT (100 mg/kg) and the effect of plasma apo A-I concentration on plasma lipids and lipoprotein distribution was examined. In untreated TgR[HuAI] rats, human apo A-I levels were strongly correlated to plasma PL (r(2)=0. 94), FC (r(2)=0.93) and CE (r(2)=0.90), whereas in ANIT-treated TgR[HuAI] rats, human apo A-I levels were most strongly correlated to CE levels (r(2)=0.80) and an increased CE/FC ratio (r(2)=0.62) and the movement of cholestatic lipid in the LDL to HDL. Since LCAT activity was not affected by ANIT treatment, these results demonstrate that the ability of LCAT to esterify the plasma FC present in cholestatic liver disease is limited by in vivo apo A-I activation of the cholestatic lipid and not by the catalytic capacity of LCAT.     

Secretion of apolipoprotein A-I in lipoprotein particles following transfection of the human apolipoprotein A-I gene into 3T3 cells

Apolipoprotein A-I (apoA-I) is the major protein constituent of plasma high density lipoproteins (HDL). To examine apoA-I processing and secretion, the human apoA-I gene (2.2-kilobase PstI-PstI fragment) linked to the mouse metallothionein promoter was transfected by electroporation into NIH 3T3 fibroblasts along with the plasmid pSV2 neo, which confers neomycin resistance. Transfected cells were selected for neomycin resistance and screened for the ability to produce apoA-I by enzyme-linked immunosorbent assay. In the absence of lipids in the medium, selected 3T3 cells secreted apoA-I, mainly in the proprotein form, at density greater than 1.25 g/ml. Following incubation of cells with lipids, and subsequent washing with lipid-free medium, apoA-I was recovered in the HDL region (1.063-1.21 g/ml) as well as in the 1.21 g/ml infranatant. Examination of the HDL fraction by electron microscopy revealed round particles, 10-21 nm in diameter. These data indicate that human apoA-I secreted by transfected 3T3 fibroblasts can assemble into lipoprotein particles under the appropriate conditions.     

Structural analysis of human apolipoprotein A-I variants. Amino acid substitutions are nonrandomly distributed throughout the apolipoprotein A-I primary structure

In the course of an electrophoretic mutation screening program of 32,000 dried blood samples from newborns, 17 genetic variants of apolipoprotein A-I (apoA-I) were found and structurally analyzed. The following defects were identified by the combined use of high performance liquid chromatography, time-of-flight secondary ion mass spectrometry, and sequence analysis: Pro3----Arg (1 x), Pro4----Arg (1 x), Asp89----Glu (1 x), Lys107----0 (4 x), Lys107----Met (2 x), Glu139----Gly (2 x), Glu147----Val (1 x), Pro165----Arg (4 x), and Glu198----Lys (1 x). The distribution of point mutations in the apoA-I gene leading to these 9 and 11 other variants of apoA-I reported previously was statistically analyzed. Substitutions are overrepresented in the 10 amino-terminal amino acids (p less than 0.001, chi 2-test) and in residues 103-177 (p less than 0.025, chi 2-test) or residues 103-198 (p less than 0.05, chi 2-test), respectively. We further noted the following. (i) Prolines were substituted by arginine or histidine residues at a frequency much higher than expected on the basis of random nucleotide substitutions (5 out of 18 "electrically non-neutral" amino acid substitutions, p less than 0.001, chi 2-test). These substitutions are the result of transversions of cytosines contained within stretches of at least 5 consecutive cytosines in the apoA-I gene. The observed hypervariability of the apoA-I amino terminus, therefore, might be caused by a hot spot for mutation formed by the 7 subsequent cytosines in codons 3, 4, and 5. (ii) CpG dinucleotides were overrepresentatively affected by C----T transitions (5 out of 18 electrically nonneutral amino acid substitution, p less than 0.001, chi 2-test). The hypervariability of the apoA-I alpha-helical domain might therefore be caused by CpG dinucleotides predominantly occurring in codons 120-208 of apoA-I (82 out of 125). (iii) Comparison of mutation sites in the human apoA-I gene with sites of nonsynonymous substitutions revealed that amino acid substitutions found in human apoA-I were predominantly localized in areas that were little conserved during mammalian evolution. These regions may therefore represent areas of less structural constraint for the function of apoA-I.