The Carboxyl-terminal Hydrophobic Residues of Apolipoprotein A-I Affect Its Rate of Phospholipid Binding and Its Association with High Density Lipoprotein*

(Received for publication, March 3, 1997, and in revised form, April 25, 1997)

Maria Laccotripe Dagger , Savvas C. Makrides Dagger , Ana Jonas § and Vassilis I. Zannis Dagger

From the Dagger  Section of Molecular Genetics, Center for Advanced Biomedical Research, Departments of Medicine and Biochemistry, Boston University Medical Center, Boston, Massachusetts 02118-2394 and the § Department of Biochemistry, College of Medicine at Urbana-Champaign, University of Illinois, Urbana, Illinois 61801

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

We performed a series of mutations in the human apolipoprotein A-I (apoA-I) gene designed to alter specific amino acid residues and domains implicated in lecithin:cholesterol acyltransferase (LCAT) activation or lipid binding. We used the mutant apoA-I forms to establish nine stable cell lines, and developed strategies for the large scale production and purification of the mutated apoA-I proteins from conditioned media.

HDL and dimyristoyl phosphatidylcholine binding assays using the variant apoA-I forms have shown that replacement of specific carboxyl-terminal hydrophobic residues Leu222, Phe225, and Phe229 with lysines, as well as replacement of Leu211, Leu214, Leu218, and Leu219 with valines, diminished the ability of apoA-I to bind to HDL and to lyse dimyristoyl phosphatidylcholine liposomes. The findings indicate that Leu222, and Phe225, Phe229 located in the putative random coil region, and Leu211, Leu214, Leu218, and Leu219 located in the putative helix 8, are important for lipid binding. In contrast, substitutions of alanines for specific charged residues in putative helices 7, 8, or 9 as well as various point mutations in other regions of apoA-I, did not affect the ability of the variant apoA-I forms to bind to HDL or to lyse dimyristoyl phosphatidylcholine liposomes. Cross-linking experiments confirmed that the carboxyl-terminal domain of apoA-I participates in the self-association of the protein, as demonstrated by the inability of the carboxyl-terminal deletion mutants Delta 185-243 and Delta 209-243 to form higher order aggregates in solution. Lecithin:cholesterol acyltransferase analysis, using reconstituted HDL particles prepared by the sodium cholate dialysis method, has shown that mutants (Pro165 right-arrow Ala,Gln173 right-arrow Glu) (Leu311 right-arrow Val,Leu214 right-arrow Val,Leu318 right-arrow Val,Leu319 right-arrow Val), Leu222 right-arrow Lys,Phe255 right-arrow Lys,Phe290 right-arrow Lys) and Delta 209-243 reduced LCAT activation (38-68%). Mutant (Glu191 right-arrow Ala,His195 right-arrow Ala,Lys196 right-arrow Ala) enhanced LCAT activation (131%), and mutant (Ala162 right-arrow Leu,Leu189 right-arrow Trp) exhibited normal LCAT activation as compared with the wild type proapoA-I and plasma apoA-I forms. The apparent catalytic efficiency (Vmax(app)/Km(app)) of the apoA-I mutants ranged from 17.8 to 107.2% of the control and was the result of variations in both the Km and the Vmax in the different mutants. These findings indicate that putative helices 6 and 7, and the carboxyl-terminal helices 8 and 9 contribute to the optimum activation of lecithin:cholesterol acyltransferase. In addition to their use in the present study, the variant apoA-I forms generated will serve as valuable reagents for the identification of the domains and residues of apoA-I involved in binding the scavenger receptor BI, and facilitating cholesterol efflux from cells as well as aid in the structural analysis of apoA-I.


INTRODUCTION

Apolipoprotein A-I (apoA-I)1 is the major protein constituent of HDL and plays an important role in HDL stability, lipid transport, and metabolism (1). As a component of HDL, apoA-I is the principal physiological activator of lecithin:cholesterol acyltransferase (LCAT), particularly with physiological lecithins (2, 3). Reconstituted HDL (rHDL) particles, formed in vitro by mixing apoA-I with phospholipid-cholesterol vesicles, serve as substrates of LCAT and are converted into cholesteryl ester containing spheres upon incubation with LCAT (4). It was shown that rHDL particles have different sizes and that their LCAT activation ability correlates with the conformation of apoA-I on these particles (5-7). ApoA-I also promotes the efflux of cholesterol from peripheral cells, thus providing a substrate for the LCAT reaction (8). Finally, apoA-I plays a role in receptor-dependent or receptor-independent binding of HDL to cell surfaces (9-12). Such binding may contribute to either cholesterol efflux (8) or selective lipid uptake (13). As a result of these activities, apoA-I may play an important role in regulating the cholesterol content of peripheral tissues through the reverse cholesterol transport pathway (4, 8, 14).

Amino acid (15) and nucleotide (16, 17) sequence analyses of apoA-I have shown that both the protein and the gene contain repeated units. The protein contains 22- or 11-residue repeated units, which are organized into amphipathic alpha -helices (15-17). Models of secondary structure have been proposed that predict the presence of 8 or 9 helical regions in the apoA-I molecule (18, 19). The model proposed by Atkinson predicts the existence of 9 antiparallel helices and is consistent with the formation of an antiparallel alpha -helical bundle structure in solution (18).

Several studies have examined the ability of synthetic peptides and fragments of apoA-I to associate with phospholipid vesicles and to activate LCAT (20-22). Moderate reduction in LCAT activation in the range of 40-70% of normal was observed for a few naturally occurring mutants (23-28), whereas more substantial reduction was observed with deletion mutants in one or more of the apoA-I helices (29-31). These results, as well as studies using monoclonal antibodies (32, 33), suggest that several domains within the central region of apoA-I, between residues 95 and 185 are important for the activation of LCAT. These domains contain the putative helices 6 and 7 and the hinged domain, which includes the putative helices 4 and 5. The carboxyl-terminal region of apoA-I (residues 190-243) has been shown to be important for lipid and HDL binding as well as for binding to cell membranes (12, 29-31, 34, 35).

Although the data obtained with the carboxyl-terminal apoA-I mutants are informative, mapping of the apoA-I domains that are functionally important requires more precise point mutagenesis that disturbs minimally the three-dimensional structure of apoA-I. In the present study we describe the generation and characterization of novel apoA-I variants. Functional analysis established that specific hydrophobic residues in the putative loop region (223-231) and within putative helix 8 (187-223) are important for binding to HDL and for the initial association of apoA-I with multilamellar phospholipid vesicles. A relatively small reduction in LCAT activation was observed for several point mutants as a result of variations in both the apparent Km and Vmax.


EXPERIMENTAL PROCEDURES

Materials

The Klenow fragment of DNA polymerase I, T4 ligase, polynucleotide kinase, and restriction enzymes were purchased from New England Biolabs. Calf intestinal alkaline phosphatase was from Stratagene (La Jolla, CA). [35S]Methionine (>1000 Ci/mmol) and [14C]cholesterol (45-60 mCi/mmol) were from NEN Life Science Products. The Sequenase sequencing kit was from U. S. Biochemical Corp. Materials for the polymerase chain reaction were from Perkin-Elmer. Bactotryptone and Bacto-yeast extract were from VWR (Pittsburgh, PA). Dulbecco's modified Eagle's medium (DMEM) and methionine-free DMEM were from Life Technologies, Inc. Materials for the two-dimensional polyacrylamide gel electrophoresis were described previously (36). IgSorb was from the Enzyme Center (Boston, MA). BSA, POPC, DMPC, sodium cholate, aprotinin, and benzamidine were from Sigma. Materials for oligonucleotide synthesis were from Applied Biosystems.

Methods

Mutagenesis and Plasmid Construction

The oligonucleotides utilized as primers for the in vitro mutagenesis of the apoA-I gene were synthesized by the solid-phase phosphate triester method using an Automated Oligonucleotide Synthesizer (Applied Biosystems, model 380-B), according to the instructions provided by the manufacturer. The oligonucleotides were purified by electrophoresis on 20% polyacrylamide, 7 M urea gels.

The PstI-PstI fragment of the apoA-I gene was ligated to SalI linkers and inserted into the SalI polylinker site of the pUC19 vector. This new plasmid designated pUCA-IN (37) was mutagenized by polymerase chain reaction to introduce two new restriction sites, in intron 3 and at the 3' end of the apoA-I gene: a NotI site was introduced at nucleotides 1166-1173, and an XhoI site was introduced at nucleotides 2191-2196 of the pUCA-IN plasmid, creating plasmid pUCA-IN*.

The SalI-SalI fragment encompassing the apoA-I gene was excised from the pUCA-IN* plasmid and inserted into the unique XhoI site of the pBMT3X vector, thus placing the apoA-I gene under the control of the mouse metallothionine I promoter. This new vector, designated pBMT3X-AI, was digested with NotI and XhoI and was used to replace the normal with the corresponding mutated apoA-I segment, as described below.

The fourth exon of the human apoA-I gene was amplified and mutagenized by polymerase chain reaction, using a set of specific mutagenesis primers, containing the mutation of interest, and a set of flanking universal primers, containing the restriction sites NotI and XhoI, using the pUCA-IN* vector as a template (38). The DNA fragment containing the mutation of interest, was digested with NotI and SalI and cloned into the NotI and XhoI sites of the pBMT3X-AI vector. The variant apoA-I sequences were verified by DNA sequencing.

Generation of Stable Cell Lines

The C127 cell line (ATCC CRC 1616) was maintained in DMEM supplemented with 10% fetal calf serum (Sigma), and grown at 37 °C, in 5% CO2. This cell line is a suitable host for transfection with the bovine papilloma virus-containing plasmids, described above.

To generate stable cell lines expressing the apoA-I variants, cells were transfected by the calcium chloride co-precipitation method (39). After selection for 10-15 days in media containing 10 µM CdCl2, surviving colonies were isolated with cloning cylinders. For protein labeling, 60-mm diameter cell cultures were rinsed twice with methionine-free DMEM containing 2 mM glutamine and 10 µM CdCl2, and preincubated in the same media for 2 h. After two additional rinses, the cells were labeled overnight with 0.25 mCi of [35S]methionine. Media were collected, immunoprecipitated with rabbit anti-human apoA-I antibodies, and analyzed by two-dimensional SDS-PAGE and autoradiography as described (40).

Large Scale Growth of Cell Cultures

Cell clones overproducing the variant apoA-I forms were grown for 5-7 days in two T-75 flasks, containing DMEM plus 10% fetal calf serum and 10 µM CdCl2. Confluent flasks were trypsinized, and the cells were placed into 850-cm2 roller bottles. The bottles were rotated at 1 rpm, to allow the cells to attach to the surface of the bottle. When the cells had grown to 80-85% confluence, 10 ml of packed Verax microcarriers were added and the speed of rotation was increased to 7 rpm. Prior to use the microspheres were autoclaved in phosphate-buffered saline solution, and subsequently incubated in media containing 1% fetal calf serum.

In the roller bottle system, the cells grow on 100-µm diameter collagen-coated porous lead microspheres. The cells attach to the collagen matrix and grow both on the surface and inside the beads. The porous nature of the microspheres greatly increases the surface area available to the cells allowing very high cell density. The constant rotation of the bottles, combined with the increase in the surface area, allows gas exchange to take place at an increased rate.

The cells were fed twice a week with 300 ml of DMEM medium containing 5% fetal calf serum and 10 µM CdCl2. For protein purification, the cells were rinsed twice with serum-free medium, and preincubated in the same medium for 2 h. After one additional rinse, the cells were incubated overnight in serum-free media. The conditioned medium was collected and stored in 1 mM EDTA, 0.01% NaN3, 10 µM aprotinin, and 10 µM benzamidine. Collection of media was repeated every 3-4 days.

Purification of Variant ApoA-I Forms

Medium (1 liter) collected from the roller bottles was concentrated down to 50 ml using an Amicon Ultrafiltration Cell and membranes with a 10,000 molecular weight cut-off. The concentrated medium was then dialyzed against 0.01 M Tris, pH 8, filtered, and passed through a DEAE HiTrapQ column (Pharmacia Biotech Inc.), which had been equilibrated with the same buffer. The protein was then eluted with a step gradient (5% right-arrow 20% right-arrow 50% right-arrow 100%) of M NH4CO3 in the Tris buffer. The fractions were analyzed by SDS-PAGE, and those containing the apoA-I protein were pooled and further concentrated down to 2 ml, using a Centricon concentrator (Amicon) with a molecular weight cut-off of 10,000. The concentrated sample was applied to a gel filtration column, HiPrep Sephacryl S-200 (Pharmacia), at a rate of 0.1 ml/min, and eluted with one column volume (320 ml) of 0.15 M NH4CO3, 0.02% NaN3 buffer at a rate of 0.1-0.5 ml/min. The purity of the apoA-I preparation was assessed by SDS-PAGE. Fractions greater than 95% pure were recovered. The concentration of the apolipoprotein was determined by the absorbance at 280 nm and an extinction coefficient of 1.15 mg-1 cm2.

Flotation Properties of the ApoA-I Variants

Cell cultures expressing the variant apoA-I proteins were labeled with [35S]methionine in methionine-free medium as described (41). A 2-ml aliquot of the culture medium was adjusted to a density of 1.21 g/ml with 0.65 g of potassium bromide, placed in a cellulose nitrate tube, mixed with 100 µg of human HDL, and overlaid sequentially with 1.75 ml of a potassium bromide solution of d = 1.15 g/ml, 3 ml of each of potassium bromide solutions of d = 1.063 g/ml and d = 1.019 g/ml, followed by normal saline.

The tubes were then centrifuged in a Beckman SW41 rotor at 35,000 rpm for 22 h. After centrifugation, 12 1-ml fractions were collected from the top of the tube using a Haake/Buchler fraction collector. The samples were then extensively dialyzed against a 1 mM solution of cold methionine, dried up in a Speed-Vac, resuspended in one-dimensional SDS-PAGE sample buffer, and analyzed by one-dimensional SDS-PAGE and autoradiography. To quantify the 35S-labeled apolipoproteins, the protein bands of the one-dimensional gels were excised and solubilized in 2.5 ml of 30% (w/v) H2O2, at 60 °C in scintillation vials. The solubilized acrylamide was then mixed with 15 ml of scintillation fluid and counted in an LKB scintillation counter.

Binding of ApoA-I to DMPC Liposomes

The binding of the wild type and the various mutant forms of apoA-I to DMPC multilamellar liposomes was studied by kinetic-turbidimetric methods (42). DMPC, dissolved in a glass-distilled chloroform:methanol (2:1) solution, was placed in a glass tube. The sample was dried under nitrogen, and the appropriate amount of a 5 mg/ml solution of apoA-I in buffer (10 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM NaN3, 0.01% EDTA) was added to it, to give a final DMPC:apoA-I ratio of 2.5:1 (w/w) at a final protein concentration of 0.2 mg/ml. The experiment was performed at 24 °C, and the absorbance at 325 nm was monitored at 5-min intervals using a Perkin-Elmer Lambda 3A spectrophotometer.

ApoA-I Cross-linking

The optimal concentration for cross-linking lipid-free apoA-I was 1.2-2.0 mg/ml. Prior to cross-linking the sample was dialyzed overnight in phosphate buffer (0.02 M sodium phosphate, 0.9% NaCl, 0.01% EDTA, 0.02% NaN3, pH 7.4) using dialysis tubing with a molecular weight cut-off of 3,500. An aliquot of 100 µl of each sample was placed into Eppendorf tubes and 50 µl of 10 mM bis(sulfosuccinimidyl)suberate (BS3) solution was added. The solution was vortexed, covered loosely, and incubated at 4 °C, for 3.5 h. At the end of the incubation time, 10 µl of 250 mM ethanolamine quenching solution was added to the reaction mixture. The solution was then concentrated to 1/4 of its original volume, using a Speed-Vac, and run on an 8-25% gradient Phast gel apparatus (Pharmacia) using SDS buffer strips, and electrophoresed for 76 V-h.

Preparation of rHDL

The rHDL complexes were prepared by the original sodium cholate dialysis method (43) using a molar ratio of 100:10:1:100 of POPC:cholesterol:apoA-I:sodium cholate. In a typical experiment, 0.14 mg of cholesterol (5,000-7,000 cpm of 14C-labeled cholesterol/nmol of cold cholesterol) and 2.71 mg of POPC were placed in glass tubes, vortexed gently, and dried under nitrogen. The dried lipid was dissolved in a 10 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM NaN3, and 0.01% EDTA buffer by repeated vortexing. The suspension was stored on ice for 1 h, sodium cholate was added, and the solution was kept on ice for 1 h more. Finally the apoA-I was added, and the incubation was continued for another 1 h. Sodium cholate was removed by dialysis at 4 °C against 5-6 liters of the 10 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM NaN3, and 0.01% EDTA buffer, using membranes with a molecular weight cut-off of 12,000-14,000. The rHDL particles were analyzed on a native 8-25% acrylamide gradient gel at 15 °C on a Pharmacia Phast-gel system. The rHDL particles were stored at 4 °C in nitrogen to prevent the oxidation of lipids.

Electron Microscopy

rHDL particles prepared with different variant apoA-I forms were negatively stained with potassium phosphotungstate using carbon-coated grids, and photographed with a Philips CM12 electron microscope (Philips Electron Optics, Eindhoven, The Netherlands).

LCAT Assay

The human LCAT enzyme was purified from normal plasma by ultracentrifugal flotation, followed by chromatography on Affi-Gel Blue, DEAE-Sepharose, and Phenyl-Sepharose columns as described (44). For LCAT analysis, the substrate rHDL particle was diluted in buffer (10 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM NaN3, and 0.01% EDTA), to give final apoA-I concentrations ranging from 10-7 to 10-6 M. Each reaction mixture contained 50 µl of a 40 mg/ml BSA, 20 µl of 100 mM beta -mercaptoethanol, and 50 µl of a 1-2 µg/ml LCAT solution. The reaction was carried out for 30 min at 37 °C.


RESULTS

Characterization of Secreted Wild Type and Variant ApoA-I Forms

To characterize the mutant apoA-I forms, cell clones expressing the mutant apoA-I genes (Table I) were labeled with [35S]methionine, immunoprecipitated, and analyzed by two-dimensional PAGE and autoradiography, using the plasma wild type apoA-I as an internal marker. The Coomassie-stained gel obtained from this analysis shows the position of the plasma apoA-I isoforms that were included in the sample, and the autoradiogram shows the position of the newly synthesized apoA-I. Superimposition of the gel on the autoradiogram establishes the charge and size differences between the plasma apoA-I and the newly synthesized variant apoA-I forms (37). Thus, using this analysis, mutants with charge or size differences from the wild type can be distinguished unequivocally.

Table I. List of the variant apoA-I forms and rationale for their selection

The assignment of the putative helices of apoA-I, based on the model proposed by Nolte and Atkinson, is shown in Fig. 3J.
Helix no. Repeat Mutations

6 6 Ala152 right-arrow Leu,Leu159 right-arrow Trpa
7 7 Pro165 right-arrow Ala,Gln172 right-arrow Glub
8 8 Glu191 right-arrow Ala,His193 right-arrow Ala,Lys195 right-arrow Alac
8 8 and 9 Glu212 right-arrow Ala,Asp213 right-arrow Ala,Arg215 right-arrow Alac
Leu211 right-arrow Val,Leu214 right-arrow Val,Leu218 right-arrow Val/Leu219 right-arrow Vald
9 10 Leu222 right-arrow Lys,Phe225 right-arrow Lys,Phe229 right-arrow Lysd
Glu234 right-arrow Ala,Glu235 right-arrow Ala,Lys238 right-arrow Ala,Lys239 right-arrow Alac
8 8 and 9 Gly185 right-arrow Stopd
9 10 Pro209 right-arrow Stopd

a Alter the putative bundle structure of apoA-I in solution.
b Alter the relative orientation of helices 6 and 7.
c Investigate the role of charged residues in inter or intrahelical ionic interactions.
d Investigate the role of specific domains and residues of apoA-I in lipid binding and LCAT activation.

Fig. 1 (A-I) shows the two-dimensional gel electrophoresis analysis for the following apoA-I variants: A, (Gly185 right-arrow Stop); B, (Pro209 right-arrow Stop); C, (Leu222 right-arrow Lys,Phe225 right-arrow Lys,Phe229 right-arrow Lys); D, (Leu211 right-arrow Val,Leu214 right-arrow Val,Leu218 right-arrow Val,Leu219 right-arrow Val); E, (Glu191 right-arrow Ala,His193 right-arrow Ala,Lys195 right-arrow Ala); F, (Pro165 right-arrow Ala,Gln172 right-arrow Glu); G, (Ala152 right-arrow Leu,Leu159 right-arrow Trp); H, (Glu212 right-arrow Ala,Asp213 right-arrow Ala,Arg215 right-arrow Ala); and I, (Glu234 right-arrow Ala,Glu235 right-arrow Ala,Lys238 right-arrow Ala,Lys239 right-arrow Ala). In panel A, the secreted radiolabeled variant form is smaller in size, and is two charge units more negative than the wild type proapoA-I (isoform 2). This is consistent with the net loss of two positive charges; the deleted sequence contains 8 negatively charged and 10 positively charged residues. The change in size is consistent with the deletion of 58 amino acids. In panel B, the secreted radiolabeled variant form is smaller in size, and is one charge unit more positive than the wild type proapoA-I. This is consistent with the net loss of one negative charge; the deleted sequence contains five negatively charged and four positively charged residues. The change in size is consistent with the deletion of 35 amino acids. In panel C, the secreted radiolabeled variant form has three additional positive charges as compared with the corresponding wild type proapoA-I. This is consistent with the acquisition of three positive charges due to the replacement of three neutral residues with three positively charged lysines. The additional acidic forms with the same Mr present on the gel are the result of deamidation or carbamylation of the major form, as described previously (45). The more basic, higher Mr isoform observed in this panel and in panels E, F, G, H, and I has been observed previously and represents the pre proapoA-I form (37). The acidic higher Mr isoforms of the newly secreted apoA-I present in this panel have been observed previously and may represent post-translationally modified forms of unknown nature (37, 40, 46). In panels D, G, and I, the secreted radiolabeled variant forms overlap completely with the wild type plasma proapoA-I. This is consistent with the lack of change in the total charge of these variant proteins due to the mutations. In panel H, the secreted radiolabeled variant form has one additional positive charge as compared with the wild type proapoA-I. This is consistent with the net loss of one negative charge due to the mutation. In panels E and F, the secreted radiolabeled variant forms have one additional negative charge as compared with the corresponding wild type proapoA-I form. This is consistent with the acquisition of one extra negative charge, due to the mutations.


Fig. 1. Analysis of variant apoA-I forms by two-dimensional polyacrylamide gel electrophoresis and autoradiography. Permanent C127 cell lines expressing the mutant proteins were labeled with [35S]methionine. The radiolabeled proteins were immunoprecipitated and analyzed by two-dimensional gel electrophoresis and autoradiography, as described under "Methods." The figure shows superimposition of the autoradiogram on the corresponding gels, stained for protein. Note that in panel A, the newly synthesized apoA-I is smaller in size, and is two charge units more negative than the wild type proapoA-I, consistent with the net loss of two positive charges. In panel B, the newly synthesized apoA-I is smaller in size, and is one charge unit more positive than the wild type proapoA-I, consistent with the net loss of one negative charge. In panel C, the newly synthesized apoA-I has three additional positive charges as compared with the corresponding wild type proapoA-I, confirming the replacement of three non-charged residues with three positively charged lysines. In panels D, G, and I, the newly synthesized apoA-I overlaps completely with the plasma proapoA-I, confirming the lack of change in the total charge, due to the mutations. In panel E, the newly synthesized apoA-I has one additional negative charge as compared with the wild type proapoA-I, confirming the net loss of one negative charge due to the mutation. In panel F, the newly synthesized apoA-I has one additional negative charge as compared with the corresponding wild type proapoA-I form, confirming the acquisition of one extra negative charge, due to the mutations.
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Isolation of the Wild Type and Variant ApoA-I Forms from Conditioned Medium

To obtain large quantities of apoA-I protein, the permanent cell lines expressing the different apoA-I forms were grown on a large scale using collagen-coated microspheres in roller bottles.

For protein purification, the cells were preincubated in 100 ml of serum-free media overnight. Approximately 1-1.5 liters of media thus collected was concentrated and passed through an anion exchange column (HiTrap-Q) on an FPLC, to remove the majority of the residual BSA. Fractions containing the majority of the apoA-I protein with the least amount of contaminants were pooled and concentrated to a small volume (1-2 ml) and chromatographed on a HiPrep Sephacryl S-200 column. Protein purity was 95-99% as determined by SDS-PAGE. Typical purification profiles of the different apoA-I mutants are shown in Fig. 2 (A-D).


Fig. 2. Representative purification profiles for the recombinant variant apoA-I proteins. Media from C127 cell lines expressing the mutant proteins were concentrated and purified by chromatography (FPLC) using a HiTrap-Q column to remove residual albumin, followed by a Sephacryl S-200 column, as described under "Methods." The figure shows the SDS-PAGE analysis of 5-µl aliquots of the indicated fractions obtained from the Sephacryl S-200 column. Panel A, variant apoA-I form (Delta 209-243); panel B, variant apoA-I form (Leu222 right-arrow Lys,Phe225 right-arrow Lys,Phe229 right-arrow Lys); panel C, variant apoA-I form (Pro165 right-arrow Ala,Gln172 right-arrow Glu); panel D, variant apoA-I form (Ala152 right-arrow Leu,Leu159 right-arrow Trp). The protein molecular size standards used are: maltose-binding protein-beta -galactosidase, 175 kDa; maltose-binding protein-paramyosin, 83 kDa; glutamic dehydrogenase, 62 kDa; aldolase, 47.5 kDa; triose-phosphate isomerase, 32.5 kDa; beta -lactoglobulin A, 25 kDa; lysozyme, 16.5 kDa.
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Binding of the Variant ApoA-I Forms to HDL

Previous studies have demonstrated that when soluble, lipid-free apoA-I is incubated with excess unlabeled plasma HDL, the exogenous apolipoprotein is incorporated into HDL by displacing another apoA-I molecule from the HDL surface (37). It has also been shown that radiolabeled apoA-I secreted by C127 cell lines can be incorporated into HDL particles, but in the absence of added HDL recombinant apoA-I expressed in C127 cells is distributed mostly in the lipoprotein-free fraction (d > 1.21 g/ml) (47).

The ability of the variant apoA-I forms to bind to HDL was analyzed by KBr density gradient ultracentrifugation (41). The distribution of the radiolabeled recombinant variant and wild type apoA-I in different lipoprotein fractions, relative to the plasma apoA-I, is shown in Fig. 3 (A-I). The location of the mutations, in the model proposed by Nolte and Atkinson (18), described in this and subsequent figures is shown in panel J. The density distribution of the recombinant wild type apoA-I was similar to that of plasma apoA-I, suggesting an equilibrium between the recombinant and the plasma wild type forms. The majority of this recombinant protein floats in the HDL region, between densities 1.06 and 1.21 g/ml.


Fig. 3. Binding of wild type and variant apoA-I forms to human plasma HDL. Two ml of 35S-labeled culture media from permanent cell lines expressing the different forms of apoA-I were mixed with 100 µg of HDL and adjusted to a density of 1.21 g/ml with KBr and analyzed by discontinuous density gradient ultracentrifugation followed by 12% SDS-PAGE and autoradiography of the fractions obtained. The figure shows the distribution of the recombinant mutant apoA-I form as compared with the wild type proapoA-I. The distribution of plasma apoA-I (not shown) overlaps with that of the recombinant wild type apoA-I. Panel A, variant apoA-I form (Delta 185-243); panel B, variant apoA-I form (Delta 209-243); panel C, variant apoA-I form (Leu222 right-arrow Lys,Phe225 right-arrow Lys,Phe229 right-arrow Lys); panel D, variant apoA-I form (Leu211 right-arrow Val,Leu214 right-arrow Val,Leu218 right-arrow Val,Leu219 right-arrow Val); panel E, variant apoA-I form (Glu191 right-arrow Ala,His193 right-arrow Ala,Lys195 right-arrow Ala); panel F, variant apoA-I form (Pro165 right-arrow Ala,Gln172 right-arrow Glu); panel G, variant apoA-I form (Ala152 right-arrow Leu,Leu159 right-arrow Trp); panel H, (Glu212 right-arrow Ala,Asp213 right-arrow Ala,Arg215 right-arrow Ala); panel I, Glu234 right-arrow Ala,Glu235 right-arrow Ala,Lys238 right-arrow Ala,Lys239 right-arrow Ala). (Note the drastic changes in the density distribution of the variant proteins: (Delta 185-243), (Delta 209-243), (Leu222 right-arrow Lys,Phe225 right-arrow Lys,Phe229 right-arrow Lys), and (Leu211 right-arrow Val,Leu214 right-arrow Val,Leu218 right-arrow Val,Leu219 right-arrow Val.) Panel J, secondary structure model of apoA-I proposed by Nolte and Atkinson. Letters A-I show the position of the mutations described in the corresponding panels. Residues affected by mutations indicated C and L are critical for the ability of apoA-I to bind to HDL and to lyse multilamellar vesicles of DMPC.
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Deletion of the putative helices 8 and 9 (Delta 185-243), or part of helix 8 and helix 9 (Delta 209-243), affected dramatically the flotation properties of the mutant proteins. The majority of the protein was recovered in the non-lipoprotein fraction (d > 1.21 g/ml) (Fig. 3, A and B). The findings confirm previous findings (29) that the carboxyl-terminal region of apoA-I is necessary for its ability to bind to the lipoprotein surface.

Point mutations were used to identify the specific residues within the carboxyl terminus of apoA-I that are involved in HDL binding. Substitution of alanines for specific charged residues (Glu191 right-arrow Ala,His193 right-arrow Ala,Lys193 right-arrow Ala) or (Glu213 right-arrow Ala,Asp217 right-arrow Ala,Arg215 right-arrow Ala) in putative helix 8, or (Glu234 right-arrow Ala,Glu235 right-arrow Ala,Glu238 right-arrow Ala,Glu239 right-arrow Ala) in putative helix 9 did not affect the flotation properties of the mutant proteins, suggesting that these mutated residues are not crucial for the ability of the protein to associate normally with HDL (Fig. 3, E, H, and I).

However, substitution of lysines for three specific hydrophobic residues (Leu222, Phe225, and Phe229) located in the predicted random coil region between putative helices 8 and 9, altered dramatically the ability of the mutant protein to bind to HDL. As shown in Fig. 3C, the majority of the mutant protein is recovered in the lipoprotein-free fraction (d > 1.21 g/ml), indicating that these specific hydrophobic residues are critical for the ability of the protein to bind HDL.

Similarly, replacement of a series of leucine residues (Leu211, Leu214, Leu218, and Leu219) in putative helix 8 by valines, which have similar hydrophobicity but are less bulky, resulted in the same dramatic alteration in the flotation properties of the variant protein (Fig. 3D), indicating the importance of these residues for the binding of apoA-I to HDL.

Substitution of alanine for proline between putative helices 6 and 7 and the alteration of a neighboring glutamine to glutamate did not affect the binding of the variant apoA-I to HDL (Fig. 3F). These mutations were predicted to alter the orientation of these helices due to the elimination of the helix-breaking proline residue, and the change of the predicted A type half-repeat to a B type half-repeat (18).

Finally, a mutation in helix 6, which introduces amino acids at positions that are expected to disrupt the formation of the helical bundle in solution (Ala152 right-arrow Leu, Leu159 right-arrow Trp) (48, 49) does not affect the flotation properties of the mutant protein (Fig. 3G), suggesting that these substitutions do not affect the binding of apoA-I to HDL.

Lipid Binding Properties of the Variant ApoA-I Forms

DMPC binding experiments were performed to assess the effect of the mutations on the kinetics of interaction of apoA-I with DMPC multilamellar vesicles. The rate of the interaction was monitored by the change in absorbance at 325 nm. The experiments were performed at 24 °C, the transition temperature of the lipid, where the gel and liquid-crystalline phases co-exist, and where defects in the lipid matrix make it easier for apoA-I to interact.

As illustrated in Fig. 4 (A-G), both plasma and recombinant wild type proapoA-I bind and solubilize DMPC rapidly, as indicated by the dramatic decrease in turbidity of the DMPC dispersions. In contrast, the apoA-I mutants in which the putative helices 8 and 9 (Delta 185-243), or part of helix 8 and helix 9 (Delta 209-243) were deleted, interacted extremely slowly with the phospholipid (Fig. 4, A and B). After incubation at 24 °C for 8 h, the reaction mixture was adjusted to a density of 1.21 g/ml and was fractionated by ultracentrifugation. Approximately 85% of the wild type apoA-I was recovered in the top fraction, bound to the DMPC, whereas only approximately 20% of the deletion mutant (Delta 209-243) protein was found in the top fraction (data not shown).


Fig. 4. A-G, solubilization of multilamellar vesicles of DMPC by wild type and variant apoA-I forms, monitored by the turbidity change as a function of time, at 24 °C. Multilamellar vesicles of DMPC were combined with wild type or variant apoA-I forms at a ratio of DMPC:apoA-I of 2.5/1 (w/w). The change in turbidity was monitored by the change in absorbance at 325 nm, at 5-min intervals, and was plotted as a function of time. Panel A, (Delta 185-243); panel B, (Delta 209-243); panel C, (Leu222 right-arrow Lys,Phe225 right-arrow Lys,Phe229 right-arrow Lys); panel D, (Leu211 right-arrow Val,Leu214 right-arrow Val,Leu218 right-arrow Val,Leu219 right-arrow Val); panel E, Glu191 right-arrow Ala,His193 right-arrow Ala,Lys195 right-arrow Ala); panel F, (Pro165 right-arrow Ala,Gln172 right-arrow Glu), panel G, (Ala152 right-arrow Leu,Leu159 right-arrow Trp).
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The same slow kinetics of interaction were observed for the mutant in putative helix 8, where four valines were substituted for leucines (Leu211 right-arrow Val,Leu214 right-arrow Val,Leu218 right-arrow Val,Leu219 right-arrow Val) (Fig. 4D). It is worth noting that even when the ratio of protein to lipid was doubled (2.5:2, DMPC:protein) the reaction was still extremely slow (data not shown).

Similarly slow kinetics of interaction with DMPC were obtained for the mutant in which hydrophobic residues in the predicted random coil region between helices 8 and 9 were changed to charged residues (Leu222 right-arrow Lys,Phe225 right-arrow Lys,Phe229 right-arrow Lys) (Fig. 4C). This variant associates initially with the phospholipid at a rate that is slightly higher than the rate of association of the mutants of Fig. 4 (A, B, and D). However, the rate of association is still slow compared with that observed for the plasma and the recombinant wild type apoA-I.

In contrast to the slow rate of solubilization of multilamellar vesicles of DMPC observed in apoA-I mutants with substitutions of hydrophobic carboxyl-terminal amino acids, other point mutants in the central and carboxyl-terminal region of the molecule do not affect the ability of the protein to solubilize efficiently the multilamellar vesicles of DMPC.

Fig. 4 (E-G) shows the kinetics of interaction of the mutant apoA-I forms (Glu191 right-arrow Ala,His193 right-arrow Ala,Lys195 right-arrow Ala), (Pro165 right-arrow Ala,Gln172 right-arrow Glu), and (Ala152 right-arrow Leu,Leu159 right-arrow Trp) with DMPC. All of these mutants interact spontaneously and solubilize DMPC with a rate comparable to those of the recombinant wild type proapoA-I.

The results obtained with the DMPC binding assay are in agreement with the results from the HDL binding assay and indicate that those mutants (both the deletion and the point mutations) that lost their ability to associate normally with the HDL, also displayed slow kinetics of association with the multilamellar vesicles of DMPC. The findings show that the carboxyl terminus of apoA-I is important for lipid binding, and indicate for the first time that specific hydrophobic residues in the predicted random coil region between putative helices 8 and 9, as well as the leucine residues present in putative helix 8, are critical for the ability of the protein to bind to both the HDL surface as well as to bind and solubilize multilamellar vesicles of DMPC.

Self-association Properties of the Variant ApoA-I Forms

To study the self-association properties of the mutants in comparison with the plasma apoA-I protein, we performed cross-linking experiments using the cross-linking reagent BS3.

Plasma apoA-I formed dimers, trimers, and tetramers in addition to monomers, when it was present in solution at a concentration of 1.5 mg/ml (Fig. 5). In contrast, both of the carboxyl-terminal deletion mutants (Delta 185-243) and (Delta 209-243) formed predominantly monomers, and a few dimers. The formation of the dimer was concentration-dependent, but even at concentrations as high as 2 mg/ml, the dimer represented only a minor component.


Fig. 5. Cross-linking of wild type and variant apoA-I proteins, as analyzed by SDS-PAGE. Lipid-free wild type or variant apoA-I forms were cross-linked with BS3 and analyzed on an 8-25% Phast gel (Pharmacia). Lanes 1 and 6 contain plasma apoA-I, lane 2 contains the variant (Leu222 right-arrow Lys,Phe225 right-arrow Lys,Phe229 right-arrow Lys), lanes 3, 4, and 7 contain the variant (Delta 209-243), lane 5 contains the variant (Delta 185-243), lane 8 contains the variant (Ala152 right-arrow Leu,Leu159 right-arrow Trp), lane 9 contains the variant (Pro165 right-arrow Ala,Gln172 right-arrow Glu), and lane 10 contains the variant (Asp191 right-arrow Ala,His193 right-arrow Ala,Lys195 right-arrow Ala). Note that the deletion of the carboxyl-terminal residues 185-243 or 209-243 prevents oligomerization of the mutant apoA-I protein. However, the point mutation (Leu222 right-arrow Lys,Phe225 right-arrow Lys,Phe229 right-arrow Lys), which affected lipid binding, did not affect oligomerization.
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The mutants (Ala152 right-arrow Leu,Leu159 right-arrow Trp) in putative helix 6, (Pro165 right-arrow Ala,Gln172 right-arrow Glu) in putative helix 7 and the turn preceding it, as well as the mutant (Asp191 right-arrow Ala,His193 right-arrow Ala,Lys195 right-arrow Ala) in putative helix 8, all formed higher order oligomers similar to the ones observed for the plasma apoA-I. These results show that alterations in these regions do not affect the self-association of apoA-I.

The variant (Leu222 right-arrow Lys,Phe225 right-arrow Lys,Phe229 right-arrow Lys) in the random coil region between putative helices 8 and 9, which did not bind to HDL and which interacted slowly with multilamellar vesicles of DMPC, self-associated to form dimers, trimers, and tetramers. This suggests that either these residues are not involved in the self-association of apoA-I or that self-association requires more than one region of the apoA-I molecule.

The observation that deletion of a small part of the carboxyl-terminal region of apoA-I prevents the protein from self-associating at high concentrations in solution is of great importance. In the future it may be possible, using these mutants or derivatives, to determine the structure of apoA-I by x-ray crystallography or NMR spectroscopy.

Generation of rHDL Substrates for the LCAT Reaction

The mutant proteins were reconstituted in particles containing POPC, and cholesterol (cold and labeled) at a ratio of 100:10:1 of POPC:cholesterol:apoA-I using the sodium cholate dialysis method (43), and were used as substrates for the LCAT reaction. The sodium cholate dialysis method allowed the formation of HDL particles, even with the mutants which interacted very slowly with phospholipid. These particles were sized by native gradient gel electrophoresis (Fig. 6). This analysis identified two populations of particles at a ratio of approximately 3:1, with diameters of 96 Å and 109 Å. LCAT assays were performed with the mixed particle population.


Fig. 6. Characterization of rHDL particles by native gradient gel electrophoresis. The rHDL particles were prepared from a molar ratio of 100:10:1:100 POPC:cholesterol:apoA-I:sodium cholate. Lane 1, plasma apoA-I rHDL; lane 2, variant apoA-I form (Pro165 right-arrow Ala,Gln172 right-arrow Glu) rHDL; lane 3, markers (thyroglobulin, 170 Å; ferritin, 122 Å; catalase, 104 Å; LDH, 81 Å; albumin, 71 Å); lane 4, recombinant wild type proapoA-I rHDL; lane 5, markers (thyroglobulin, 170 Å; ferritin, 122 Å; catalase, 104 Å; LDH, 81 Å; albumin, 71 Å); lane 6, plasma apoA-I rHDL; lane 7, variant apoA-I form (Leu222 right-arrow Lys,Phe225 right-arrow Lys,Phe229 right-arrow Lys) rHDL; lane 8, variant apoA-I form (Delta 209-243) rHDL; lane 9, recombinant wild type apoA-I rHDL.
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The rHDL particles were also negatively stained with potassium phosphotungstate, overlaid on carbon-coated grids, and photographed with a Philips CM12 electron microscope.

Fig. 7 (A-H) shows formation of the rHDL particles with all of the variant apoA-I protein samples tested for LCAT activation. Under the negative staining conditions used, these particles form the typical "rouleaux" indicating that they are discoidal in shape and that they have the thickness of a phospholipid bilayer. The number of rouleaux observed depends on the concentration of the sample on the carbon grid. In the samples that are less concentrated, a large number of round particles that lie flat on the grid are also observed. These particles do not seem to pack hexagonally on the grid during aggregation (a characteristic of spherical structures), providing further evidence that these particles are, in fact, discoidal in shape.


Fig. 7. Electron microscopy of the rHDL particles used as substrates for the LCAT reaction. The apoA-I forms used for the formation of rHDL particles are as follows. Panel A, variant apoA-I form (Delta 209-243); panel B, variant apoA-I form (Leu222 right-arrow Lys,Phe225 right-arrow Lys,Phe229 right-arrow Lys); panel C, variant apoA-I form (Leu211 right-arrow Val,Leu214 right-arrow Val,Leu218 right-arrow Val,Leu219 right-arrow Val); panel D, variant apoA-I form (Glu191 right-arrow Ala,His193 right-arrow Ala,Lys195 right-arrow Ala); panel E, variant apoA-I form (Pro165 right-arrow Ala,Gln172 right-arrow Glu); panel F, variant apoA-I form (Ala152 right-arrow Leu,Leu159 right-arrow Trp); panel G, plasma apoA-I discoidal particles; panel H, recombinant wild type proapoA-I. Note that all the discoidal particles formed are relatively uniform in size, and similar to the ones observed for plasma apoA-I.
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Activation of LCAT by the Variant ApoA-I Forms

Even though other apolipoproteins like apoC-I, apoA-IV, and apoE can activate the LCAT reaction in vitro using rHDL particles as substrates, none is as effective as apoA-I, when physiological lecithins are used as substrates (3). To identify the domains and residues of apoA-I that are responsible for LCAT activation, the mutant proteins were reconstituted in rHDL particles. The LCAT activity was assayed as the rate of production of labeled cholesterol esters from the rHDL particles. The labeled cholesterol esters were separated from the free cholesterol by thin layer chromatography. All LCAT assays were standardized by adding fixed amounts of apoA-I reconstituted in the HDL particles, and LCAT enzyme.

The ability of the wild type proapoA-I secreted from C127 cells to activate LCAT is comparable with that of plasma apoA-I (Fig. 8). The mutant in helix 6, which contains amino acid substitutions designed to destabilize the bundle structure of apoA-I in solution (Ala152 right-arrow Leu,Leu159 right-arrow Trp), was also able to activate LCAT to levels comparable with those of plasma apoA-I. However, the mutant apoA-I form (Pro165 right-arrow Ala,Gln172 right-arrow Glu), activates LCAT to approximately 55% as compared with the wild type apoA-I. The substitutions of alanine for proline and glutamate for glutamine are predicted to change the orientation of these helices. These results indicate that the putative helices 6 and 7 contribute to the efficient activation of LCAT.


Fig. 8. LCAT activation by the wild type and the indicated mutant apoA-I proteins. The LCAT activity was assayed as the rate of production of labeled cholesterol esters from the rHDL vesicles, as described under "Methods." The labeled cholesterol esters were separated from the free cholesterol by thin layer chromatography. All LCAT assays were standardized by adding fixed amounts of apoA-I (reconstituted in the HDL particles), and LCAT enzyme. Error bars represent standard deviation for n = 3 or n = 4 experiments.
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The mutant form (Glu191 right-arrow Ala,His193 right-arrow Ala,Lys195 right-arrow Ala) activates LCAT to levels slightly higher than the ones observed for the wild type apoA-I.

As indicated above, the variant apoA-I forms (Leu211 right-arrow Val,Leu214 right-arrow Val,Leu218 right-arrow Val,Leu219 right-arrow Val), (Leu222 right-arrow Lys,Phe225 right-arrow Lys,Phe229 right-arrow Lys), and (Delta 209-243), were able to form reconstituted HDL particles of similar sizes to those formed by the wild type apoA-I (Figs. 6 and 7). The ability of these mutants to activate LCAT was reduced to 68, 38, and 58%, respectively (Fig. 8). This analysis indicates that the carboxyl-terminal region of apoA-I contributes to the efficient activation of LCAT.

The apparent kinetic parameters for LCAT activation were also calculated by measuring the initial velocity of the LCAT reaction as a function of the apoA-I concentration in the rHDL substrate particles. Four different concentrations of apoA-I were used ranging from 4.57 × 10-6 to 5.71 × 10-7 M apoA-I. The kinetic parameters are summarized in Table II.

Table II. Kinetic parameters for LCAT activation for plasma, wild type, and variant apoA-I

The apparent kinetic parameters were determined by nonlinear least squares fit of initial velocity and apoA-I concentrations to the expected hyperbola using the KaleidaGraph program (59).
rHDL substrate Apparent Km Apparent Vmax Apparent Vmax/apparent Km

M nmol CE/h nmol CE/h · M
Plasma A-I 9.79  ± 0.47 × 10-7 8.4  ± 0.18 8.58  × 106
Ala152 right-arrow Leu,Leu159 right-arrow Trp 15.7  ± 1.98 × 10-7 10.8  ± 0.72 6.88  × 106
Pro165 right-arrow Ala,Gln172 right-arrow Glu 9.71  ± 2 × 10-7 5.19  ± 0.48 5.34  × 106
Glu191 right-arrow Ala,His193 right-arrow Ala,Lys195 right-arrow Ala 21.5  ± 4.2 × 10-7 19.78  ± 3.71 9.2  × 106
Leu211 right-arrow Val,Leu214 right-arrow Val,Leu218 right-arrow Val,Leu219 right-arrow Val 11.7  ± 4 × 10-7 5.74  ± 1.07 4.9  × 106
Leu222 right-arrow Lys,Phe225 right-arrow Lys,Phe229 right-arrow Lys 43  ± 1.57 × 10-7 6.58  ± 0.38 1.53  × 106
 Delta 209-243 8.65  ± 0.4 × 10-7 4.04  ± 0.97 4.67  × 106

The apparent Km values reflect the binding affinity of the enzyme for the rHDL substrate, and the apparent Vmax values reflect the activation of the enzyme and the catalytic rate constants (50). The apparent catalytic efficiency (apparent Vmax/Km) of the apoA-I mutants ranged from 17.8 to 107.2% of the control and was the result of variations in both the Km and the Vmax (Table II).


DISCUSSION

Background

Epidemiological and genetic data have shown convincingly that low levels of HDL or apoA-I are associated with an increased risk of developing coronary heart disease (51). In a systematic effort to map the domains of apoA-I important for its functions, we have mutagenized the human apoA-I gene and we have created permanent cell lines expressing the variant apoA-I proteins. Large scale cultures of the permanent cell lines in roller bottles containing collagen-coated microspheres allowed us to obtain sufficient quantities of the apoA-I substrate and analyze its functions.

Lipid and Lipoprotein Binding Properties of the Variant ApoA-I Forms

Consistent with previous observations, deletion of residues 185-243 (Delta 185-243) or 209-243 (Delta 209-243) of apoA-I severely altered the ability of the mutant proteins to bind to HDL (29). To identify specific residues within the carboxyl terminus of apoA-I involved in lipid and/or lipoprotein binding, we introduced a series of point mutations in this region. Analysis of the ability of these mutants to bind to HDL has shown that substitution of a series of charged amino acids between residues 195 and 238 did not affect the ability of the mutant proteins to bind to HDL, indicating that inter- or intrahelical ionic interactions may not be essential for the binding of apoA-I to HDL. This is consistent with the presence, in the general population, of several substitutions of charged for neutral amino acids that do not affect HDL levels (52). This finding indicates that apoA-I may have the ability to tolerate substitutions of charged amino acids without adverse physiological consequences.

In contrast, substitution of the positively charged lysine for specific hydrophobic residues (Leu222, Phe225, and Phe229) located in the predicted random coil region (18), altered dramatically the binding of the mutant protein to HDL. It is reasonable to assume that this highly hydrophobic domain of apoA-I has the ability to insert into the phospholipid component of HDL and initially anchor the protein on to the lipoprotein surface. Following the initial attachment, the rest of the molecule might subsequently "wrap" around the HDL particle with the non-polar phases of the alpha  helices oriented inside toward the phospholipid side chains, and the polar phases oriented outside facing the aqueous environment. The involvement of this highly hydrophobic region of apoA-I in lipid binding was also suggested by Fourier power spectra analysis of the hydropathy profile along the apoA-I sequence (18). This analysis showed that the sequence between residues 222-230 has a hydropathy profile between what is expected for an alpha  helix and a beta  sheet structure (18). This region that does not participate in the formation of a defined secondary structure, may serve to attach apoA-I to HDL. Similar conclusions were reached by CD and denaturation studies of deletion mutants of apoA-I, which showed that the COOH-terminal region of apoA-I is largely unstructured and assumes an amphipathic alpha -helical structure upon binding to lipid (53).

X-ray crystallography has demonstrated that leucine residues are located in topologically distinct positions and contribute to the stabilization of the helical bundle structure of apoE in solution (54). It has also been shown that helices which participate in a four-helix bundle structure display a pattern of hydrophobic and hydrophilic residues, which can be described as a seven-residue repeat of the type (a, b, c, d, e, f, g, h)n (48, 49). Leucine residues are found mostly at positions a and d and may serve to stabilize the tertiary structure of the bundle by hydrophobic interactions. Computer analysis of the apoA-I sequence indicated that such heptad repeats are encountered in the apoA-I sequence between residues 115 and 180 (helices 4-7) and residues 188 and 243 (helices 8 and 9).2

Replacement of Leu211, Leu214, Leu218, and Leu219 by valines in helix 8 altered dramatically the ability of the mutant protein to bind to HDL. The presence of valines in these positions, which have similar hydrophobicity to but are less bulky than leucines, might prevent leucine zipper type hydrophobic interactions between juxtapositioned leucine residues (55). This may cause conformational changes in the random coil region that diminish the ability of this region to attach to the surface of HDL. The structural alterations associated with these mutants are the subject of ongoing research.

Preliminary analysis of several other point mutations, along the predicted helices 1-6 of apoA-I, has shown that these mutations did not have any effect on the ability of the protein to bind to HDL, thus reinforcing the notion that the carboxyl-terminal region of apoA-I plays a unique role in the binding of apoA-I to HDL.3

The results obtained from the kinetic analysis of DMPC binding are consistent with the results obtained from the HDL binding assay. Mutants that failed to bind to HDL also lysed multilamellar vesicles of DMPC very slowly, pointing to the possibility that the mechanism of the interaction of apoA-I with the HDL surface and the multilamellar phospholipid vesicles may be similar.

The carboxyl-terminal domain (putative helices 8 and 9 and the random coil region), however, is most likely only involved in the initial penetration of the protein into the phospholipid bilayer, since the mutant proteins that did not bind to HDL and interacted very slowly with the DMPC bilayers were able to form rHDL particles when the sodium cholate reconstitution method was used. These results are in agreement with previously published studies, where a proteolytic fragment of apoA-I (1-192) interacted slowly with DMPC, but did form rHDL particles when the sodium cholate method was used (34).

Activation of LCAT by Variant ApoA-I Forms

rHDL particles formed using the sodium cholate dialysis method have been shown before to be excellent substrates for the LCAT reaction (56). In the present study, these particles were visualized by electron microscopy and sized by native gradient gel electrophoresis. This latter analysis identified two populations of particles with diameters of 96 Å and 109 Å. LCAT assays were performed with the mixed particle population. The ratio of the 96-Å to the 109-Å particle present in the mixture is 3:1 and is the same for the plasma, the recombinant wild type, and the mutant pro-apoA-I proteins. Therefore these two different size particles contribute equally to the total LCAT activity in the various samples. Since the ability of the 96-Å particles to activate LCAT is 10-fold higher than that of the 109-Å particle, the contribution of the 109-Å particle is only 1/30th of the overall activation. Thus our results, expressed as percent activation relative to the wild type apoA-I, reflect the relative change in LCAT activation of the 96-Å substrate.

Previous studies have pointed out the importance of putative helices 6 and 7 (residues 145-183) of apoA-I in the activation of LCAT (21, 29, 30). Specifically, deletion of residues 148-186 or residues 143-164 and 165-185 reduced the capacity of the mutant proteins to activate LCAT to background levels (29, 30). The possibility exists, however, that these results were due to dramatic alterations in the protein structure caused by the large deletions.

The point mutant (Pro165 right-arrow Ala,Gln172 right-arrow Glu) eliminated a helix-breaking proline residue, and also changed a glutamine to glutamate. According to the model proposed by Nolte and Atkinson (18), type A half-repeats have neutral residues at the eighth position, whereas type B have negatively charged residues. Thus, the glutamine to glutamate substitution converts the predicted type A repeat to a type B repeat. It has been suggested that beta  turns occur mostly between A and B, rather than AA or BB repeats (18). Thus, the 45% reduction in the LCAT activation ability of this mutant suggested that the two amino acid substitutions may have distorted the orientation of the putative helices 6 and 7, which were shown previously to be important for LCAT activation (29, 30, 33). Consistent with this finding, a naturally occurring apoA-I variant (Pro165 right-arrow Arg) has a 45-55% LCAT activation ability as compared with wild type apoA-I (57). It is possible that one or both of the helices interact electrostatically with the polar face of the predicted amphipathic alpha -helical segment found close to the active site of the LCAT enzyme (residues 151 and 174 of LCAT), as it was proposed by Fielding (14). Ionic interactions between LCAT and apoA-I have also been suggested by the quantitative dissociation of LCAT from HDL in concentrated salt solutions (58). Alterations in the ionic interactions between apoA-I and the LCAT enzyme may lead to a less efficient activation.

The 42% decrease in the LCAT activation ability of the deletion (Delta 209-243) mutant could be attributed mainly to the change in the apparent Vmax. This is consistent with previous studies (29, 30, 31), which showed that deletion of residues 209-219, 220-241, 212-233, and 213-243 resulted in LCAT activation of 11.2, 16, 28, and 13%, respectively, as compared with the wild type apoA-I. The variation in the extent of inhibition is probably due to either the difference in the phospholipid used for the formation of the reconstituted particles (egg phosphatidylcholine in the previous studies, versus POPC in this study), or the difference in the size of the particles formed. In addition, the deletion (Delta 213-243) mutant used in the previous study contained 12 residues of unrelated carboxyl-terminal sequence (29). Substitution of valines for leucines 211, 214, 218, and 219 or substitution of lysines for leucine 222 and phenylalanines 225 and 229, resulted in 32 and 68% reduction, respectively, in the capacity of the mutant proteins to activate LCAT. This impairment was the result of a decrease in the apparent Vmax for the former mutant and mainly due to an increase in the apparent Km for the latter mutant. The combined data from this and the previous studies (29-31) suggest that domains and residues within the carboxyl-terminal region of apoA-I contribute to the optimal activation of LCAT. It is possible that the presence of the carboxyl-terminal domain allows apoA-I to acquire a proper conformation, which facilitates the postulated specific interaction of the middle region (residues 145-183) of apoA-I with the LCAT enzyme.

A mutant in the putative helix 6 containing amino acids in positions that are expected to destabilize the bundle structure in solution (49) binds normally to HDL and DMPC and also activates LCAT normally. These findings suggest that potential structural alterations of the bundle structure in solution have no consequences for lipid binding and LCAT activation, where the lipid bound protein assumes a new conformation.

Substitution of alanines for charged residues in putative helix 8 enhanced slightly the capacity of the variant protein to activate LCAT as compared with the wild type proapoA-I. The significance of this increased activation is not clear. It is possible that the removal of charged residues in putative helix 8 may allow a stronger electrostatic interaction to occur between the putative alpha -helical segment of LCAT (residues 151 and 174) and putative helices 6 and 7 of apoA-I. Alternatively, the absence of these charged residues may allow the cholesterol substrate to position itself more favorably relative to the active site serine residue of the LCAT enzyme. The catalytic efficiency of the last two mutants was comparable with that of the wild type pro apoA-I (Table II) due to concomitant increases in both the Vmax and the Km.

The lack of dramatic changes in the apparent Vmax for any of the mutations tested strongly suggests that none of the apoA-I residues altered participate in the catalytic mechanism of LCAT.

Domains of ApoA-I Involved in Self-association

In addition to its role in the initial anchoring of the protein into the phospholipid bilayer, the carboxyl-terminal domain of apoA-I also appears to participate in the self-association of the protein. The deletion mutants (Delta 185-243) and (Delta 209-243) existed only as monomers and dimers in solution rather than as a mixture of oligomers. Similar conclusions were reached with the amino-terminal proteolytic fragment (1-192) of apoA-I (34). It has been suggested that self-association of apoA-I may promote stabilization of the potential amphipathic alpha -helical segments of the carboxyl-terminal region which is less organized in the monomeric form (53). Overcoming the oligomerization problem and achieving high concentrations of the monomeric apoA-I fragment in solution can facilitate efforts to derive the three-dimensional structure of apoA-I by x-ray crystallography or NMR spectroscopy.

Overall, the present study shows that specific hydrophobic residues in the predicted random coil region between helices 8 and 9, and in the putative helix 8 (Fig. 3), are critical for the ability of apoA-I to bind to HDL and to lyse multilamellar vesicles of DMPC. It is possible that the formation of a "leucine zipper-like" structure between the putative helices 8 and 9 may stabilize this random coil region during its interaction with lipids and lipoproteins and allow its association with the phospholipid surface. Although the carboxyl-terminal region of apoA-I is required for both the lipid binding and self-association, the residues which participate in these two functions appear to be different.

Several amino acid substitutions in the carboxyl-terminal domain of apoA-I cause a moderate reduction in the catalytic efficiency of LCAT, suggesting that residues in this region contribute to optimum activation of LCAT, without a direct participation in the catalytic mechanism.

In addition to their use in the present study, the variant apoA-I forms generated will serve as valuable reagents for future studies to identify the domains and residues of apoA-I involved in binding to scavenger class B1 receptor, in promoting cholesterol efflux from cells, as well as for the structural analysis of apoA-I.


FOOTNOTES

*   This work was supported by Grants HL48739 and HL16059 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   To whom correspondence should be addressed: Section of Molecular Genetics, Center for Advanced Biomedical Research, Depts. of Medicine and Biochemistry, Boston University Medical Center, 700 Albany St., W-509, Boston, MA 02118-2394. Tel.: 617-638-5085; Fax: 617-638-5141.
1   The abbreviations used are: apoA-I, apolipoprotein A-I; HDL, high density lipoprotein; LCAT, lecithin:cholesterol acyltransferase; DMPC, dimyristoyl phosphatidylcholine; POPC, palmitoyl oleyl phosphatidylcholine; rHDL, reconstituted high density lipoprotein; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium; BS3, bis(sulfosuccinimidyl)suberate.
2   M. Kokkinidis and V. I. Zannis, unpublished data.
3   M. Laccotripe and V. I. Zannis, unpublished observations.

ACKNOWLEDGEMENTS

We thank Donald Gantz for electron microscopic images; Cheryl England, Michael Gigliotti, and Gayle Forbes for excellent technical assistance; and Anne Plunkett for typing the manuscript.


REFERENCES

  1. Zannis, V. I., Kardassis, D., and Zanni, E. E. (1993) Adv. Hum. Genet. 21, 145-319 [Medline] [Order article via Infotrieve]
  2. Fielding, C. J., Shore, V. G., and Fielding, P. E. (1972) Biochem. Biophys. Res. Commun. 46, 1493-1498 [Medline] [Order article via Infotrieve]
  3. Soutar, A. K., Garner, C. W., Baker, H. N., Sparrow, J. T., Jackson, R. L., Gotto, A. M., and Smith, L. C. (1975) Biochemistry 14, 3057-3064 [Medline] [Order article via Infotrieve]
  4. Fielding, C. J. (1990) in Advances in Cholesterol Research (Esfahani, M., and Swaney, J. B., eds), pp. 271-314, Telford Press , NJ
  5. Jonas, A., Kezdy, K. E., and Wald, J. H. (1989) J. Biol. Chem. 264, 4818-4824 [Abstract/Free Full Text]
  6. Wald, J. H., Krul, E. S., and Jonas, A. (1990) J. Biol. Chem. 265, 20037-20043 [Abstract/Free Full Text]
  7. Jonas, A., Wald, J. H., Toohill, K. L. H., Krul, E. S., and Kezdy, K. E. (1990) J. Biol. Chem. 265, 22123-22129 [Abstract/Free Full Text]
  8. Bakria, A., Puchois, P., Ghalim, P., Torpier, N., Barbaras, G., Ailhaud, R., and Fruchard, J. C. (1991) Atherosclerosis 87, 135-146 [Medline] [Order article via Infotrieve]
  9. McKnight, G. L., Reasoner, J., Gilbert, T., Sundquist, K. O., Hokland, B., McKernan, P. A., Champagne, J., Johnson, C. J., Bailey, M. C., Holly, R., O'Hara, P. J., and Oram, J. F. (1992) J. Biol. Chem. 267, 12131-12141 [Abstract/Free Full Text]
  10. Graham, D. L., and Oram, J. F. (1987) J. Biol. Chem. 262, 7439-7442 [Abstract/Free Full Text]
  11. Morrison, J. R., McPherson, G. A., and Fidge, N. H. (1992) J. Biol. Chem. 267, 13205-13209 [Abstract/Free Full Text]
  12. Allan, C. M., Fidge, N. H., Morrison, J. R., and Kanellos, J. (1993) Biochem. J. 290, 449-455 [Medline] [Order article via Infotrieve]
  13. Glass, C., Pittman, R. C., Weinstein, D. B., and Steinberg, D. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 5435-5439 [Abstract]
  14. Fielding, C. J., and Fielding, P. E. (1995) J. Lipid Res. 36, 211-228 [Abstract]
  15. Barker, W. C., and Dayhoff, M. O. (1977) Comp. Biochem. Physiol. 57, 309-315
  16. Karathanassis, S. K., Zannis, V. I., and Breslow, J. L. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 6147-6151 [Abstract]
  17. Li, W. H., Tanimura, M., Luo, C. C., Datta, S., and Chan, L. (1988) J. Lipid Res. 29, 245-271 [Medline] [Order article via Infotrieve]
  18. Nolte, R. T., and Atkinson, D. (1992) Biophys. J. 63, 1221-1239 [Abstract]
  19. Boguski, M. S., Freeman, M., Elshourbagy, N. A., Taylor, J. M., and Gordon, J. I. (1986) J. Lipid Res. 27, 1011-1034 [Abstract]
  20. Sparrow, J. T., and Gotto, A. M., Jr. (1980) Ann. N. Y. Acad. Sci. 348, 187-211 [Medline] [Order article via Infotrieve]
  21. Fukushima, D., Yokoyama, S., Kroon, D. J., Kezdy, F. J., and Kaiser, E. T. (1980) J. Biol. Chem. 255, 10651-10657 [Abstract/Free Full Text]
  22. Jonas, A., von Eckardstein, A., Kezdy, K. E., Steinmetz, A., and Assmann, G. (1991) J. Lipid Res. 32, 97-106 [Abstract]
  23. Jonas, A., von Eckardstein, A., Churgay, L., Mantulin, W. W., and Assmann, G. (1993) Biochim. Biophys. Acta 1166, 202-210 [Medline] [Order article via Infotrieve]
  24. Utermann, G., Haas, J., Steinmetz, A., Paetzold, R., Rall, S. C., Weisgraber, K. H., and Mahley, R. W. (1984) Eur. J. Biochem. 144, 325-331 [Abstract]
  25. Franceschini, G., Baio, M., Calabresi, L., Sirtori, C. R., and Cheung, M. C. (1990) Biochim. Biophys. Acta 1043, 1-6 [Medline] [Order article via Infotrieve]
  26. Steinmetz, A., Utermann, G., Haas, J., Menzel, H. J., Rall, S. C., Weisgraber, K. H., and Mahley, R. W. (1984) Agents Actions Suppl. 16, 217-224 [Medline] [Order article via Infotrieve]
  27. Deeb, S. S., Cheung, M. C., Peng, R., Wolf, A. C., Stern, R., Albers, J. J., and Knopp, R. H. (1991) J. Biol. Chem. 266, 13654-13660 [Abstract/Free Full Text]
  28. Schmitz, G., Bruning, T., Williamson, E., and Nowicka, G. (1990) Eur. Heart J. 11, 197-211 [Medline] [Order article via Infotrieve]
  29. Minnich, A., Collet, X., Roghani, A., Cladaras, C., Hamilton, R. L., Fielding, C. J., and Zannis, V. I. (1992) J. Biol. Chem. 267, 16553-16560 [Abstract/Free Full Text]
  30. Sorci-Thomas, M., Kearns, M. W., and Lee, J. P. (1993) J. Biol. Chem. 268, 21403-21409 [Abstract/Free Full Text]
  31. Holvoet, P., Zhao, Z., Vanloo, B., Vos, R., Deridder, E., Dhoest, A., Taveirne, J., Brouwers, E., Demarsin, E., Engelborghs, Y., Rossenau, M., Collen, D., and Brasseur, R. (1995) Biochemistry 34, 13334-13342 [Medline] [Order article via Infotrieve]
  32. Banka, C. L., Bonnet, D. J., Black, A. S., Smith, R. S., and Curtiss, L. K. (1991) J. Biol. Chem. 266, 23886-23892 [Abstract/Free Full Text]
  33. Meng, Q.-H., Calabresi, L., Fruchart, J.-C., and Marcel, Y. L. (1993) Biol. Chem. 268, 16966-16973 [Abstract/Free Full Text]
  34. Ji, Y., and Jonas, A. (1995) J. Biol. Chem. 270, 11290-11297 [Abstract/Free Full Text]
  35. Morrison, J., Fidge, N. H., and Tozuka, M. (1991) J. Biol. Chem. 266, 18780-18785 [Abstract/Free Full Text]
  36. Zannis, V. I. (1986) Methods Enzymol. 128, 823-851 [Medline] [Order article via Infotrieve]
  37. Roghani, A., and Zannis, V. I. (1988) Biochemistry 27, 7428-7435 [Medline] [Order article via Infotrieve]
  38. Mullis, K. B., and Faloona, F. A. (1987) Methods Enzymol. 155, 335-350 [Medline] [Order article via Infotrieve]
  39. Graham, F. L., and van der Eb, A. J. (1973) Virology 52, 456-467 [Medline] [Order article via Infotrieve]
  40. Zannis, V. I., Breslow, J. L., and Katz, A. J. (1980) J. Biol. Chem. 255, 8612-8617 [Free Full Text]
  41. Redgrave, T. G., Roberts, D. C., and West, C. E. (1975) Anal. Biochem. 64, 42-49
  42. Pownall, H. J., Massey, J. B., Kusserow, S. K., and Gotto, A. M. (1978) Biochemistry 17, 1183-1188 [Medline] [Order article via Infotrieve]
  43. Matz, C. E., and Jonas, A. (1982) J. Biol. Chem. 257, 4535-4540 [Abstract/Free Full Text]
  44. Albers, J. J., Chen, C. H., and Lacko, A. G. (1986) Methods Enzymol. 129, 763-783 [Medline] [Order article via Infotrieve]
  45. Ghiselli, G., Rohde, M. F., Tanenbaum, S., Krishnan, S., and Gotto, A. M., Jr. (1985) J. Biol. Chem. 260, 15662-15668 [Abstract/Free Full Text]
  46. Zannis, V. I., Kurnit, D. M., and Breslow, J. L. (1982) J. Biol. Chem. 257, 536-544 [Free Full Text]
  47. Hussain, M. M., Roghani, A., Cladaras, C., Zanni, E. E., and Zannis, V. I. (1991) Electrophoresis 12, 273-283 [Medline] [Order article via Infotrieve]
  48. Banner, D. W., Kokkinidis, M., and Tsernoglou, D. (1987) J. Mol. Biol. 196, 657-675 [Medline] [Order article via Infotrieve]
  49. Paliakasis, C. D., and Kokkinidis, M. (1992) Protein Eng. 5, 739-748 [Abstract]
  50. Bolin, D. J., and Jonas, A. (1994) J. Biol. Chem. 269, 7429-7434 [Abstract/Free Full Text]
  51. Castelli, W. P., Doyle, J. T., Gordon, T., Hames, C. G., Hjortland, M. C., Hulley, S. B., Kagan, A., and Zukel, W. J. (1977) Circulation 55, 767-772 [Abstract]
  52. von Eckardstein, A., Funke, H., Walter, M., Altland, K., Benninghoven, A., and Assmann, G. (1990) J. Biol. Chem. 265, 8610-8617 [Abstract/Free Full Text]
  53. Davidson, W. S., Hazlett, T., Mantulin, W. W., and Jonas, A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13605-13610 [Abstract/Free Full Text]
  54. Wilson, C., Wardell, M. R., Weisgraber, K. H., Mahley, R. W., and Agard, D. A. (1991) Science 252, 1817-1822 [Medline] [Order article via Infotrieve]
  55. Landschultz, W. H., Johnson, P. F., and McKnight, S. L. (1988) Science 240, 1759-1764 [Medline] [Order article via Infotrieve]
  56. Jonas, A., Sweeny, S. A., and Herbert, P. N. (1984) J. Biol. Chem. 259, 6369-6375 [Abstract/Free Full Text]
  57. von Eckardstein, A., Funke, H., Henke, A., Altland, K., Benninghoven, A., and Assmann, G. (1989) J. Clin. Invest. 84, 1722-1730 [Medline] [Order article via Infotrieve]
  58. Jonas, A. (1987) in Plasma Lipoproteins (Gotto, A. M., ed), pp. 299-333, Elsevier Press, Amsterdam
  59. Cleland, W. W. (1979) Methods Enzymol. 63, 103-138 [Medline] [Order article via Infotrieve]

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