©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Carboxyl-terminal Domain Truncation Alters Apolipoprotein A-I in Vivo Catabolism (*)

(Received for publication, September 29, 1994)

Hartmut H.-J. Schmidt (§) Alan T. Remaley John A. Stonik Rosemary Ronan Axel Wellmann Fairwell Thomas Loren A. Zech H. Bryan Brewer Jr. Jeffrey M. Hoeg (¶)

From the Molecular Disease Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Apolipoprotein A-I (apoA-I), the major protein of high density lipoproteins, facilitates reverse cholesterol transport from peripheral tissue to liver. To determine the structural motifs important for modulating the in vivo catabolism of human apoA-I (h-apoA-I), we generated carboxyl-terminal truncation mutants at residues 201 (apoA-I), 217 (apoA-I), and 226 (apoA-I) by site-directed mutagenesis. ApoA-I was expressed in Escherichia coli as a fusion protein with the maltose binding protein, which was removed by factor Xa cleavage. The in vivo kinetic analysis of the radioiodinated apoA-I in normolipemic rabbits revealed a markedly increased rate of catabolism for the truncated forms of apoA-I. The fractional catabolic rates (FCR) of 9.10 ± 1.28/day (±S.D.) for apoA-I, 6.34 ± 0.81/day for apoA-I, and 4.42 ± 0.51/day for apoA-I were much faster than the FCR of recombinant intact apoA-I (r-apoA-I, 0.93 ± 0.07/day) and h-apoA-I (0.91 ± 0.34/day). All the truncated forms of apoA-I were associated with very high density lipoproteins, whereas the intact recombinant apoA-I (r-apoA-I) and h-apoA-I associated with HDL(2) and HDL(3). Gel filtration chromatography revealed that in contrast to r-apoA-I, the mutant apoA-I associated with a phospholipid-rich rabbit apoA-I containing particle. Analysis by agarose gel electrophoresis demonstrated that the same mutant migrated in the pre-beta position, but not within the alpha position as did r-apoA-I. These results indicate that the carboxylterminal region (residue 227-243) of apoA-I is critical in modulating the association of apoA-I with lipoproteins and in vivo metabolism of apoA-I.


INTRODUCTION

The concentration of plasma high density lipoproteins (HDL) (^1)is inversely related to the risk of developing human cardiovascular disease(1, 2, 3) . ApoA-I is the major structural protein of HDL and an important determinant of the concentration of HDL in plasma(4) . ApoA-I binds and transports plasma lipid, serves as cofactor for the enzyme lecithincholesterol acyltransferase, and increases cholesterol efflux from peripheral tissues(5, 6, 7) . In addition, apoA-I has been reported to be an important ligand in the binding of HDL to cell membranes(8, 9) . All these properties are important in the ability of apoA-I to facilitate reverse cholesterol transport, the mechanism that has been proposed to account for the protective effect of HDL on cardiovascular disease.

ApoA-I is synthesized as a prepropeptide and is cotranslationally cleaved to proapoA-I, which then is secreted and endoproteolytically cleaved to form the mature 243 amino acid apoA-I protein(10) . The primary amino acid sequence of apoA-I (11, 12) has been suggested to contain highly conserved 11-amino acid repeats, which form amphipathic alpha-helices that interact with the lipid surface of lipoprotein particles(13, 14, 15, 16, 17, 18) . Structural analysis revealed that the carboxyl-terminal region of apoA-I plays an important role in lipid binding and in the interaction of HDL with cell membranes(9, 19, 20, 21) .

Analysis of patients with reduced plasma concentration of HDL has revealed that accelerated catabolism of apoA-I is the most common cause of low HDL levels(22, 23, 24, 25) . The mechanism, however, for the increased clearance of apoA-I in these various studies has not been delineated. Proteolysis is known to regulate the clearance of many other plasma proteins(26, 27, 28, 29, 30, 31) . The amino-terminal endoproteolytic cleavage of the propeptide, however, appears not to be important in modifying the plasma clearance of apoA-I(32) . Although the aminoterminal domain of the mature form of apoA-I can undergo limited proteolysis(33) , the carboxyl-terminal domain has been shown to be particularly sensitive to proteolysis(20, 34, 35, 36, 37, 38, 39, 40, 41) . These in vitro studies suggest that proteolysis of apoA-I may possibly occur in vivo. Preliminary data of a 26-kDa proteolyzed fragment of apoA-I with an intact amino-terminal domain showed rapid in vivo catabolism (42) . Kunitake et al.(34) reported on the occurrence of a 26-kDa and a 14-kDa proteolytic fragment isolated from pre-beta HDL, which was reduced by the presence of protease inhibitors. The amino-terminal sequence of both peptides were intact, suggesting a carboxyl-terminal in vivo proteolysis of apoA-I.

To address the role of structural motifs of apoA-I in its metabolic clearance, we have produced carboxyl-terminal truncation mutants of apoA-I and analyzed their metabolic clearance in vivo. These studies establish the importance of the carboxyl-terminal domain of apoA-I in its association with lipoprotein particles and catabolism.


EXPERIMENTAL PROCEDURES

Construction of the Expression Vector pMAL-c/A-I

PCR was utilized to introduce appropriate DNA sequences for constructing a fusion protein of apoA-I to the maltose binding protein. The following 5` and 3` primers were used in the study: 1) 5`-GCCGCCGCCGGATCCAACAACAACAACAACAACAACAACAACAACATCGAGGGTCGTGATGAACCCCCCCAGAGCCCCTGGGATCGAGTG-3`; 2) 5`-GGCGGCGGCAAGCTTTCACTGGGTGTTGAGCTTCTTAGTGTACTC-3`. The 5` primer contained a BamHI site (boldface), a poly-Asn domain, and a factor Xa cleavage site (underlined), which was immediately followed by the codons for the first 11 amino acids of mature apoA-I. The 3` primer has a HindIII site (boldface), a stop codon (underlined), and the codons for the last 9 carboxyl-terminal amino acids of apoA-I. The pLen-A-I plasmid(43) , which contains a full-length cDNA sequence of apoA-I, was used as a template for PCR. A PCR fragment of 0.8 kilobase pair was digested with BamHI and HindIII and ligated into the unique BamHI and HindIII sites of pMAL-c2 plasmid (New England Biolabs, Beverly, MA) to produce the plasmid designated pMAL-c/A-I (Fig. 1). In addition, pMAL-c/A-I vector was modified to create three carboxyl-terminal truncated apoA-I variants by introducing stop codons at residues 201 (Ser), 217 (Gly), and 226 (Lys) by using PCR. These apoA-I truncation mutants are designated apoA-I, apoA-I, and apoA-I. The 5` end primer for the PCR reactions was the same primer used for the full-length construct of apoA-I. The following are the 3` primers that were used for the truncation mutants: 1) 5`-GGCGGCGGCAAGCTTTCAGCTCAGATGCTCGGTGGCCTTGGCGTG-3` for apoA-I, 2) 5`GGCGGCGGCAAGCTTTCAGCCTTGGCGGAGGTCCTCGAGCGCGGG-3` for apoA-I, and 3) 5`-GGCGGCGGCAAGCTTTCACTTGAAGCTCTCCAGCACGGGCAGCAG-3` for apoA-I. All three primers contain a HindIII site (boldface), an in-frame stop codon (underlined), and the codons for the 9 carboxyl-terminal amino acids of each truncated apoA-I variant. The DNA sequence of all constructs was confirmed by the dideoxy chain termination method, using Sequenase T7 DNA polymerase (version 2, U. S. Biochemical Corp.).


Figure 1: Construction of the expression vector pMal-c/A-I. Recombinant apoA-I cDNA and truncation mutants were fused upstream to the maltose binding protein coding gene (malE). Expression of the fusion protein was driven by the isopropyl-1-thio-beta-D-galactopyranoside-inducible tac promoter (P).



Expression and Purification of Recombinant ApoA-I

DH5alpha Escherichia coli cells containing pMal-c/A-I were grown in LB-broth containing ampicillin (100 µg/ml) at 37 °C and were induced with 0.3 mM isopropyl-1-thio-beta-D-galactopyranoside. Bacteria were harvested by centrifugation at 4000 times g at 4 °C for 15 min, and the cell pellet was resuspended in 10 mM Tris (pH 7.4), 200 mM NaCl, 1 mM EDTA, 1 mM azide, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 0.2 mg/ml lysozyme, 2.5 µg/ml of leupeptin and aprotinin. After three cycles of freeze-thawing, the bacterial suspension was sonicated by short pulses of 10 s or less on ice (50 watts), and centrifuged at 9000 times g at 4 °C for 30 min. The supernatant was filtered (0.2 µm, Millipore) and affinity-purified with amylose resin (New England Biolabs). The fusion protein was dialyzed against 20 mM NH(4)HCO(3), lyophilized, and redissolved in 20 mM Tris (pH 7.4), 100 mM NaCl, 2 mM CaCl(2), and 1 mM azide (pH 8.0). Digestion with factor Xa (New England Biolabs) was performed using 10 mg/ml fusion protein and 0.05% factor Xa by weight at 4 °C for 72 h, and the digestions were terminated by adding 50 mM phenylmethylsulfonyl fluoride. The digested fusion protein was separated by electrophoresis on a 10-20% Tricine polyacrylamide gel containing SDS. ApoA-I was isolated from unstained gel slices, utilizing stained lanes of purified apoA-I as standard. Following electroelution (Bio-Rad) in 20 mM NH(4)HCO(3), apoA-I was lyophilized and redissolved in distilled H(2)0. Standard apoA-I was isolated from normolipemic human plasma by column chromatography, as described previously(11) . The purity of apoA-I samples were established by SDS-gel electrophoresis, immunoblotting, and isoelectric focussing, as described previously(11) .

In Vivo Metabolic Studies

ApoA-I was iodinated in 1 M glycine, 0.1 M Tris (pH 8.5) by the iodine monochloride method(44) , using either I or I as the radiotracer. Unbound iodine was removed by extensive dialysis against 0.85% (w/w) NaCl, 0.01% (w/w) EDTA, 0.1 M Tris (pH 7.4). All preparations were filter-sterilized (0.22-µm filters, Millipore) prior to injection and were used within 48 h of radiolabeling. New Zealand White female rabbits, 2.2-2.8 kg, were injected simultaneously with I and I radiolabeled apoA-I or apoA-I truncation mutants via the medial artery of the ear. Blood samples were collected from a catheter of the external jugular vein at 5 min and up to 5 days after injection and analyzed for radioactivity on a Packard Cobra counter (Packard Instrument Co., Downers Grove, IL). Residence times were obtained from the area under the plasma radioactivity decay curves, using a multiexponential computer curve-fitting technique(45) . Fractional catabolic rates (FCR) represent the reciprocal of residence times. Continuous density gradient ultracentrifugation was performed as described previously(46) . HDL density subfractions were defined the same as in humans (HDL(2), d = 1.063-1.125 g/ml; HDL(3), d = 1.125-1.210 g/ml; and VHDL, d = 1.21-1.25 g/ml, respectively).

Amino Acid Analysis

Approximately 10 µg of the purified proteins were electrophoresed on a SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membranes (Millipore). The membrane pieces were subjected to NH(2)-terminal sequence analysis on an Applied Biosystems model 610A sequencer. Amino acid analysis were performed after hydrolysis in 6 M HCl for 24 h on a Beckman model 6300 amino acid analyzer. Results from glycine determinations were excluded because of high background.

Gel Filtration Chromatography

One-hundred µl of rabbit plasma were analyzed by FPLC on two Superose-6 columns connected in series (Pharmacia, Uppsala, Sweden). Lipoproteins were eluted at 0.3 ml/min with phosphate-buffered saline, containing 1 mM EDTA and 0.02% (w/w) sodium azide(47) . After the first 10 ml were collected, the next 50 ml were collected in 0.5-ml fractions. Total cholesterol and triglyceride concentrations were determined enzymatically (Sigma). Free cholesterol and phospholipid concentrations were determined enzymatically (Wako Chemicals USA Inc.; Richmond, VA).


RESULTS

The predicted secondary structure of apoA-I based on the Chou and Fasman analysis(15, 16, 21, 48, 49, 50, 51) is illustrated in Fig. 2. The locations of the carboxyl-terminal truncations of apoA-I are indicated by arrows. ApoA-I is truncated within the carboxyl-terminal amphipathic alpha-helix. ApoA-I is truncated in the region between the eighth and ninth helix, whereas apoA-I is truncated within the eighth helix. These sites were chosen to span the region that has previously been shown to undergo carboxyl-terminal proteolysis(20, 34, 35) .


Figure 2: Schematic representation of the secondary structure of apoA-I. ApoA-I is composed of nine amphipathic alpha-helices (illustrated as rectangles) and two beta-sheets (zigzag lines). The locations of the three carboxyl-terminal deletion mutants at residues 226 (Lys), 217 (Gly), and 201 (Ser) are indicated by arrows.



Intact apoA-I and the truncation mutants were expressed in E. coli as a fusion protein with the maltose-binding protein and an unique factor Xa cleavage site between the two protein domains. This expression system produced high levels of the fusion protein (30-70 mg/liter), but digestion of the fusion protein with factor Xa in order to produce free apoA-I resulted in nonspecific degradation of apoA-I. Interestingly, apoA-I was much less susceptible to degradation compared to the other truncation mutants and r-apoA-I. The final yield of free apoA-I was 10 µg/l of purified apoA-I, and 0.1-0.5 µg/l for apoA-I, apoA-I, and r-apoA-I.

The purified recombinant intact apoA-I and the carboxyl-terminal truncation mutants were characterized by polyacrylamide gel electrophoresis, isoelectric focusing, and amino acid analysis. In addition, the PCR-amplified cDNA constructs were confirmed by DNA sequencing. Each of the apoA-I forms migrated as a single band (Fig. 3) with the expected molecular masses on polyacrylamide gel electrophoresis (r-apoA-I, 28 kDa; apoA-I 26 kDa; apoA-I, 25 kDa; and apoA-I, 23 kDa). The identity of each band was confirmed by immunoblot analysis, using a polyclonal rabbit anti-human apoA-I antibody (data not shown). Fig. 4shows the similar isoelectric focussing pattern of the two major isoforms of h-apoA-I and r-apoA-I, indicating that no significant charge modification of the recombinant apoA-I occurred during either the biosynthesis by the bacteria or during the protein purification. In Table 1, the predicted versus the observed amino acid composition of the purified apoA-I forms expressed as mole percent is shown. These data confirm the location of the apoA-I truncation and demonstrate the purity of the recombinant apoA-I forms. Amino-terminal sequence analysis of all the isolated apoA-I proteins revealed intact NH(2)-terminal domains, thus demonstrating the specificity of the factor Xa cleavage (data not shown).


Figure 3: SDS-gel electrophoresis of apoA-I. Human and r-apoA-I were isolated, radioiodinated, and electrophoresed on a 10-20% SDS acrylamide gel: lane 1, h-apoA-I; lane 2, r-apoA-I; lane 3, apoA-I; lane 4, apoA-I; and lane 5, apoA-I. The indicated molecular mass markers are phosphorylase b (97.4 kDa), bovine serum albumin (69 kDa), ovalbumin (46 kDa), carbonic anhydrase (30 kDa), and trypsin inhibitor (21.5 kDa).




Figure 4: Isoelectric focussing of apoA-I. Purified h-apoA-I (lane 1) and r-apoA-I (lane 2) were electrophoresed by isoelectric focussing (pH 5-7) and stained for protein. The major isoforms of apoA-I(0) and apoA-I are indicated.





The metabolic clearance of radiolabeled r-apoA-I and h-apoA-I was analyzed in rabbits (Fig. 5). The mean plasma decay curves of injected radiolabeled h-apoA-I (n = 6) and r-apoA-I (n = 6) were similar (Fig. 5A), demonstrating that r-apoA-I expressed in E. coli has similar in vivo kinetic behavior as h-apoA-I. The FCR using three-exponential equations with the SAAM31 program for r-apoA-I and h-apoA-I were 0.93 ± 0.07/day (mean ± S.D.) and 0.91 ± 0.34/day, respectively. Similar FCR values were previously reported for r-apoA-I and h-apoA-I in normolipemic male Japanese White rabbits(52) . Fig. 5, B-D, depicts the plasma decay curves of the three radiolabeled truncation mutants. All truncated apoA-I mutants had a markedly increased rate of catabolism when compared with normal apoA-I (Fig. 5A). Interestingly, the apoA-I mutant, which is missing only the carboxyl-terminal 17 amino acids, was rapidly catabolized, with an approximately five times higher FCR (FCR = 4.42 ± 0.51/day) than intact apoA-I. The FCR for apoA-I (FCR = 6.34 ± 0.81/day) and apoA-I (FCR = 9.10 ± 1.28/day) were even faster, suggesting that the more the carboxyl-terminal domain was deleted, the greater its catabolic rate. In addition, we evaluated the urine radioactivity during the first 24 h after the injection of the radiolabeled r-apoA-I and apoA-I. By monitoring radioactivity, we observed a urine/plasma ratio of 0.36 for r-apoA-I versus 5.82 for apoA-I during the 24-h collection period. By trichloroacetic acid precipitation and gel electrophoresis, we estimate that greater than 95% of the total counts were not protein bound. These results support the increase metabolic turnover of the truncated apoA-I forms.


Figure 5: In vivo metabolism of apoA-I. Proteins were labeled with either I or I and the in vivo metabolism of radiolabeled apoA-I forms was analyzed as described under ``Experimental Procedures.'' In A, the radioactivity decay curves for human (box) and recombinant (up triangle) apoA-I are illustrated using a two-log scale as the ordinate. Each time point represents the mean (±S.D.) for residual radioactivity from six independent experiments. S.D. of human apoA-I is indicated with upper error bars, and S.D. of recombinant apoA-I is indicated with lower error bars. B-D represent the radioactive decay curves for apoA-I (n = 3), apoA-I (n = 3), and apoA-I (n = 3), respectively, using a two-log scale as the ordinate.



Since the disruption of the carboxyl-terminal apoA-I altered its plasma turnover, we examined the association of these various mutants with lipoprotein particles. The isolated apolipoproteins were radiolabeled, incubated in human plasma for 60 min at 37 °C, and analyzed by density gradient ultracentrifugation. The density distributions of h-apoA-I, r-apoA-I, apoA-I, apoA-I, and apoA-I are indicated in Fig. 6. Native apoA-I and r-apoA-I were primarily associated with HDL(2) (d = 1.063-1.125 g/ml) and HDL(3) (d = 1.125-1.210 g/ml) with their peak density at 1.13 g/ml. The truncation mutants were almost exclusively associated with VHDL (d = 1.21-1.25), with a peak density at 1.23 g/ml. Therefore, the loss of the carboxyl-terminal amphipathic helices of apoA-I, in addition to altering its plasma clearance, also altered its association with the HDL subfractions.


Figure 6: Density gradient ultracentrifugation of apoA-I in vitro. Radioiodinated proteins were incubated in human plasma for 60 min at 37 °C. Continuous density gradient ultracentrifugation was performed, and radioactivity of density fractions was determined for h-apoA-I (), r-apoA-I (+), apoA-I (times), apoA-I (), and apoA-I (). The density fractions corresponding to very low density lipoproteins (VLDL, d < 1.006 g/ml), low density lipoproteins (LDL, d = 1.006-1.063 g/ml), high density lipoproteins (HDL(2), d = 1.063-1.125; HDL(3), d = 1.125-1.210; VHDL, d = 1.21-1.25 g/ml), and lipoprotein-deficient serum (LPDS, d > 1.25) are indicated by arrows.



We further investigated the lipoprotein particle binding properties of the apoA-I mutants by examining the density distribution of radiolabeled h-apoA-I, r-apoA-I, and apoA-I following injection in vivo (Fig. 7). The density distributions of h-apoA-I (Fig. 7A) and r-apoA-I (Fig. 7B) were similar and were detected in both HDL(2) and HDL(3) density fractions. In contrast, apoA-I was found primarily within VHDL (Fig. 7C), as we observed in vitro (Fig. 6).


Figure 7: Density gradient ultracentrifugation of apoA-I in vivo. Radioiodinated h-apoA-I (top panel), r-apoA-I (middle panel), and apoA-I (lower panel) were injected into New Zealand White rabbits. Plasma obtained after 20 min (solid rectangle), 8 h (gray rectangle), and 24 h (open rectangle) were separated into the density fractions indicated by the arrows by continuous density gradient ultracentrifugation and analyzed for radioactivity.



These differences in distribution within particles defined ultracentrifugally are paralleled by differences in the particle distribution observed by electrophoretic separation (Fig. 8). Fasting human plasma has Fat 7B-stained lipoprotein particles of alpha and beta mobility (Fig. 8, lane 1). In contrast, a pre-beta mobility is present in rabbits (Fig. 8, lanes 2 and 3). The radiolabeled apoA-I was present in particles with a slower pre-beta migration (Fig. 8, lane 5) than the alpha mobility observed for r-apoA-I.


Figure 8: Agarose gel electrophoresis. Plasma was subjected to agarose gel electrophoresis. Human (lane 1) and rabbit plasma incubated with I-r-apoA-I (lanes 2 and 4) and apoA-I (lanes 3 and 5) were analyzed by staining with Fat Red 7B (lanes 1-3) and autoradiography (lanes 4 and 5). ApoA-I and r-apoA-I were analyzed by gel electrophoresis using a buffered agarose system (Ciba Corning Diagnostics Corp., Palo Alto, CA) and a Fat Red 7B stain.



We also analyzed the lipoprotein particle compositions of New Zealand White rabbit plasma using FPLC. Fig. 9A illustrates the analyses of total cholesterol, cholesteryl ester, phospholipids, and triglycerides for each fraction. The volumes 18-22 ml represent rabbit low density lipoprotein particle (LDL), whereas 26-32 ml represents HDL and 32-36 ml of phospholipid-rich HDL. The comparison of these HDL fractions isolated by gel filtration chromatography with the HDL(2), HDL(3), and VHDL isolated by sequential ultracentrifugation (Fig. 6), suggest that HDL(2) and HDL(3) elute with 26-32 ml, whereas VHDL elutes with 32-36 ml. We also characterized the lipoprotein-associated radioiodinated r-apoA-I and apoA-I after incubation in rabbit plasma by gel filtration chromatography. The highest radioactivity was in the fractions of the elution volume of 28-30 and 32-35 ml. The r-apoA-I primarily eluted with the 26-36-ml fractions, whereas the majority of apoA-I eluted with the 32-36-ml fractions. Plasma obtained 20 min and 4 h after injection of radioiodinated r-apoA-I and apoA-I into the rabbits were also analyzed by gel filtration chromatography as shown in Fig. 9, C and D. Twenty min after injection (Fig. 9C) r-apoA-I was associated with the HDL fraction, which elutes with the 26-32-ml fractions. ApoA-I is almost equally distributed within the HDL and the phospholipid-rich VHDL fraction. Four hours after injection, the total counts of apoA-I rapidly disappear, especially within the VHDL fraction (Fig. 9D). To verify that the radioactivity in each fraction is associated with apoA-I, we analyzed each fraction by polyacrylamide gel electrophoresis and immunoblot analysis. Within 26-36 ml of the eluted volume all fractions showed immunodetectable rabbit apoA-I with higher concentrations of apoA-I within the 26-32-ml fraction (data not shown). The same fractions were concentrated by trichloroacetic acid precipitation and analyzed by polyacrylamide gel electrophoresis. The autoradiogram showed identical distribution of r-apoA-I compared with rabbit apoA-I, which suggests that rabbit apoA-I and r-apoA-I associate with HDL in a similar pattern, which, in turn, is not surprising since there is a high sequence homology between rabbit and human apoA-I(54) .


Figure 9: Gel filtration chromatography. Composition analysis of New Zealand White rabbit plasma is illustrated in Fig. 9A, showing the concentrations of total cholesterol, cholesterol ester, phospholipids, and triglycerides in mg/dl. Radioiodinated apoA-I and r-apoA-I were analyzed prior to injection (Fig. 9B), 20 min (Fig. 9C), and 4 h (Fig. 9D) after injection into New Zealand White rabbits.




DISCUSSION

The concentration of plasma apoA-I, which is inversely correlated with the risk for CAD(1, 2, 3) , is determined by its rate of production and catabolism(55, 56) . The mechanism for the catabolism of apoA-I has not been completely delineated. We were interested in examining the effect of carboxyl-terminal proteolysis on the metabolic clearance of apoA-I, since this is a possible site for in vivo proteolysis(20, 34, 35, 36, 37, 38, 39, 40, 41, 42) .

In order to test the functional role of the carboxyl-terminal domain of apoA-I, we produced recombinant apoA-I containing various carboxyl-terminal deletions. The recombinant forms of apoA-I had the predicted size, charge, and amino acid composition as assessed by polyacrylamide gel electrophoresis (Fig. 3), isoelectric focusing (Fig. 4), and amino acid analysis (Table 1). The amino-terminal amino acid sequence of r-apoA-I was the same as h-apoA-I purified from plasma. The in vivo clearance rate of r-apoA-I was nearly identical to the clearance of h-apoA-I (r-apoA-I, FCR = 0.93 ± 0.07/day; h-apoA-I, FCR = 0.91 ± 0.34/day), thus validating the use of the recombinant truncation mutants in exploring the role of the carboxyl-terminal domain in the metabolic clearance of apoA-I. Similar results have been reported in a study of the in vivo conversion of recombinant proapoA-I to apoA-I in normolipemic male Japanese White rabbits (h-apoA-I, 0.98 ± 0.03/day; r-apoA-I, FCR = 1.02 ± 0.09/day) (52) . In addition to the intact r-apoA-I, we generated three truncation mutants (Fig. 2). ApoA-I had a stop codon in the middle of the last amphipathic helix of apoA-I. Residues from 226 to 243 have been proposed to play a role in apoA-I binding to cells(9, 57, 58) . For the apoA-I mutant, the truncation occurs in the hinge region between the eighth and ninth helix. In addition, we truncated apoA-I at residue 201, the site of an apoA-I phosphorylation (59) as well as a frameshift mutation which results in a truncated apoA-I and low levels of circulating apoA-I(60) . In contrast to the similar kinetics of clearance for r-apoA-I and h-apoA-I (Fig. 5A), all of the truncation mutants were catabolized significantly faster (Fig. 5, B-D). Moreover, the greater the carboxyl-terminal deletion, the greater the FCR (apoA-I, FCR = 4.42 ± 0.51/day; apoA-I, FCR = 6.34 ± 0.81/day; apoA-I, 9.10 ± 1.28/day). Urine analysis revealed that the rapid catabolism of apoA-I is associated with a dramatic increase of its free radiotracer within the urine.

It is known that radioiodination can interfere with the molecular properties of apolipoproteins as apoA-I(61, 62) ; however, we used a standardized radiolabeling method, that is reproducible and has been shown to yield kinetics of the radiolabeled protein that are almost identical to the kinetics obtained by endogenously labeled stable isotope technique(63) . In addition, to the amount of radioactivity, the concentration of the protein used for labeling, the ratio of the iodinated versus the non-iodinated protein and the handling of the iodinated proteins were the same for all of the labeled forms of apoA-I in this study. The proteins were injected within less than 24 h after radiolabeling and analyzed by polyacrylamide gel electrophoresis to exclude self-association (64) (Fig. 3). The different behavior of the truncation mutants versus h-apoA-I and r-apoA-I is, therefore, not due to the radiolabeling procedure.

Since the affinity of apolipoproteins for lipoprotein particles has been shown to inversely correlate with their metabolic clearance(65, 66) , we examined both the in vitro and in vivo lipoprotein association of the truncation mutants for lipid. After incubating the purified apoA-I forms in plasma, h-apoA-I and r-apoA-I associated with HDL(2) and HDL(3). In contrast, the truncated forms of apoA-I were relatively lipid-poor and associated with VHDL (Fig. 6). We defined the rabbit HDL subfractions by the same density fractions as in humans. We obtained similar results when the lipid-protein association of the apoA-I forms were examined in vivo (Fig. 7, A-C). Composition analysis of the rabbit VHDL revealed a phospholipid rich apoA-I containing particle (Fig. 9), which shows a similar composition as human VHDL(53) . The analysis of r-apoA-I and apoA-I before and after injection into rabbits by gel filtration chromatography revealed that apoA-I, in contrast to r-apoA-I, eluted in the fractions containing the phospholipid rich apoA-I containing particle. In addition, apoA-I associated with a lipoprotein particle that migrated in a pre-beta position on agarose gel electrophoresis (Fig. 8), in contrast to the alpha position observed for r-apoA-I (Fig. 8).

The major sites of apoA-I degradation are the kidney and liver(67) . It has been proposed that once apoA-I dissociates from HDL, it is quickly removed from the circulation by filtration through the kidney(68, 69) . Renal tubular cells have been shown to uptake apoA-I from urine(68) . We demonstrate that intact apoA-I does not appear within the urine, which supports the tissue uptake of apoA-I and liberation of the iodide radiotracers. Horowitz (70) demonstrated an increased plasma and renal clearance of an exchangeable pool of apoA-I in individuals with low plasma HDL concentrations. The authors suggested that these subjects have an increased cholesteryl ester transfer protein-mediated exchange of HDL cholesteryl ester for very low density lipoprotein triglyceride, resulting in a triglyceride-enriched HDL. Human triglyceride-rich HDL(2) has been shown to form pre-beta HDL after incubation with hepatic lipase(71) . Similarly, HDL can transform into discoidal HDL particles by cholesteryl ester transfer protein and hepatic lipase activity(72) . This, in turn, may lead to a more loosely bound apoA-I that is susceptible to increased renal clearance, due to the altered surface properties of lipase modified HDL. The predicted structure of apoA-I, as illustrated in Fig. 2, consists of amphipathic alpha-helices that span the surface of the highly curved discoidal HDL(14, 16) . Sparks et al.(73) examined the structural stability of discoidal and spherical HDL particles and concluded that discoidal HDL is unstable and, therefore, has the tendency to interact with cell membranes and other lipoproteins, thus facilitating cholesterol efflux. The transition of discoidal HDL particles into less dense HDL particles may be accompanied by dissociation of lipid poor apoA-I, which is then rapidly catabolized. The concept of increased clearance of loosely bound apoA-I is also supported by studies of model peptides that differ in their affinity for HDL(64) . Synthetic peptides that have decreased lipid association have increased catabolism in rats. In addition, Westerlund (65) showed that carboxyl-terminal truncation mutants of apoE, an apolipoprotein with similar structural features of apoA-I, have different lipid association properties, which result in more non-lipoprotein bound apoE and an increased metabolic clearance. Similarly, our data suggest that the apoA-I truncation mutants are relatively lipid poor and bound to a phospholipid rich apoA-I containing particle that migrates in pre-beta position. The increased metabolic clearance we observed for apoA-I truncation mutants is likely to be related to their decreased ability to associate with HDL(2) and HDL(3). Either the smaller lipoprotein particles itself or the increased dissociation of apoA-I from the smaller HDL lipoprotein particles contributes to the rapid clearance of the truncated forms of apoA-I.

ApoA-I is synthesized as a prepropeptide and normally undergoes several proteolytic events before mature apoA-I is produced(32) . The prepeptide signal sequence is cleaved cotranslationally in the endoplasmic reticulum(74) . The amino-terminal propeptide is then cleaved by an endoprotease rapidly after secretion(74, 75) . The rate of conversion of proapoA-I to apoA-I is much faster than the clearance of mature apoA-I(32) . The in vivo proteolytic cleavage of apoA-I at other sites has not been fully demonstrated, but it has been shown that apoA-I, particularly apoA-I on pre-beta HDL, is cleaved by an EDTA sensitive protease shortly after phlebotomy(34) . This cleavage has been shown to occur at the carboxyl-terminal domain of the protein, because the resulting 26-kDa fragment has a complete amino-terminal domain(34) . The carboxyl-terminal domain is also susceptible to cleavage in vitro by several different proteases(36, 37, 38, 39, 40, 41) . This suggests that apoA-I has a site in the carboxyl-terminal domain, which is near the site of the apoA-I truncation mutants, that is particularly susceptible to proteolysis. Our results suggest that cleavage by a protease in the region between the middle of the eighth amphipathic helix and the ninth amphipathic helix would produce an apoA-I fragment with altered functional properties: decreased lipid association and increased metabolic clearance. This suggests a possible mechanism for the regulation of the catabolism of apoA-I by proteolysis, which is known to occur for other plasma proteins (26, 27, 28, 29, 30, 31) .

In summary, our results are consistent with the hypothesis that carboxyl-terminal proteolysis of apoA-I leads to rapid catabolism. The smallest deletion of 17 residues, which disrupts the last amphipathic alpha-helical domain markedly altered the lipoprotein association properties and increased catabolism of apoA-I. The more the carboxyl-terminal domain was truncated, the faster apoA-I was catabolized. The truncated forms of apoA-I appeared on smaller and denser phospholipid rich HDL particles, which suggests a possible mechanism for increased clearance of truncated apoA-I.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a Postdoctoral Fellowship from the Deutsche Forschungsgemeinschaft.

To whom correspondence should be addressed: Section of Cell Biology, NHLBI, Bldg. 10, Rm. 7N115, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-494-1500; Fax: 301-402-0190.

(^1)
The abbreviations used are: HDL, high density lipoproteins; apoA-I, apolipoprotein A-I; PCR, polymerase chain reaction; VHDL, very high density lipoproteins; h-apoA-I, human apoA-I; r-apoA-I, recombinant apoA-I; FCR, fractional catabolic rate; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; FPLC, fast protein liquid chromatography.


ACKNOWLEDGEMENTS

We acknowledge Drs. Douglas Powell, Robert F. Hoyr, Jr., and the other members of the Section on Laboratory Animal Medicine and Surgery, NHLBI for help in critical catheter placement as well as in the care of these animals; Susan Meyn and Glenda Talley for excellent technical support, and Nysa Sanders for help in preparing the manuscript.


REFERENCES

  1. Glomset, J. A. (1968) J. Lipid Res. 9, 155-167 [Abstract/Free Full Text]
  2. Miller, N. E., Thelle, D. S., Forde, O. H., and Mjos, O. D. (1977) Lancet 1, 965-968 [Medline] [Order article via Infotrieve]
  3. Castelli, W. P., Garrison, R. J., Wilson, P. W., Abbott, R. D., Kalousdian, S., and Kannel, W. B. (1986) J. Am. Med. Assoc. 256, 2835-2838 [Abstract]
  4. Assmann, G., and Brewer, H. B., Jr. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 989-993 [Abstract]
  5. Fielding, C. J., Shore, V. G., and Fielding, P. E. (1972) Biochem. Biophys. Res. Commun. 46, 1493-1498 [Medline] [Order article via Infotrieve]
  6. Oram, J. F., McKnight, G. L., and Hart, C. B. (1990) Atherosclerosis Rev. 20, 103-107
  7. Hara, H., and Yokoyama, S. (1991) J. Biol. Chem. 266, 3080-3086 [Abstract/Free Full Text]
  8. Slotte, J. P., Oram, J. F., and Bierman, E. L. (1987) J. Biol. Chem. 262, 12904-12907 [Abstract/Free Full Text]
  9. Morrison, J. R., McPherson, G. A., and Fidge, N. H. (1992) J. Biol. Chem. 267, 13205-13209 [Abstract/Free Full Text]
  10. Zannis, V. I., Karathanasis, S. K., Keutmann, H. T., Goldberger, G., and Breslow, J. L. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2574-2578 [Abstract]
  11. Brewer, H. B., Jr., Fairwell, T., LaRue, A., Ronan, R., Houser, A., and Bronzert, T. J. (1978) Biochem. Biophys. Res. Commun. 80, 623-630 [Medline] [Order article via Infotrieve]
  12. Cheung, P., and Chan, L. (1983) Nucleic Acids Res. 11, 3703-3715 [Abstract]
  13. 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]
  14. Segrest, J. P., De Loof, H., Dohlman, J. G., Brouillette, C. G., and Anantharamaiah, G. M. (1990) Proteins Struct. Funct. Genet. 8, 103-117 [Medline] [Order article via Infotrieve]
  15. Segrest, J. P., Jones, M. K., De Loof, H., Brouillette, C. G., Venkatachalapathi, Y. V., and Anantharamaiah, G. M. (1992) J. Lipid Res. 33, 141-166 [Abstract]
  16. Boguski, M. S., Freeman, M., Elshourbagy, N. A., Taylor, J. M., and Gordon, J. I. (1986) J. Lipid Res. 27, 1011-1034 [Abstract]
  17. Nolte, R. T., and Atkinson, D. (1992) Biophys. J. 63, 1221-1239 [Abstract]
  18. Mishra, V. K., Palgunachari, M. N., Segrest, J. P., and Anantharamaiah, G. M. (1994) J. Biol. Chem. 269, 7185-7191 [Abstract/Free Full Text]
  19. Morrison, J., Fidge, N. H., and Tozuka, M. (1991) J. Biol. Chem. 266, 18780-18785 [Abstract/Free Full Text]
  20. Dalton, M. B., and Swaney, J. B. (1993) J. Biol. Chem. 268, 19274-19283 [Abstract/Free Full Text]
  21. 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]
  22. Rader, D. J., Gregg, R. E., Meng, M. S., Schaefer, J. R., Zech, L. A., Benson, M. D., and Brewer, H. B., Jr. (1992) J. Lipid Res. 33, 755-763 [Abstract]
  23. 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]
  24. Brinton, E. A., Eisenberg, S., and Breslow, J. L. (1991) J. Clin. Invest. 87, 536-544 [Medline] [Order article via Infotrieve]
  25. Le, N. A., and Ginsberg, H. N. (1988) Metabolism 37, 614-617 [Medline] [Order article via Infotrieve]
  26. Ponting, C. P., Marshall, J. M., and Cederholm-Williams, S. A. (1992) Blood Coagul. & Fibrinolysis 3, 605-614
  27. Morris, B. J. (1992) J. Hypertens. 10, 209-214 [Medline] [Order article via Infotrieve]
  28. Rieger, K. J., Saez-Servent, N., Papet, M. P., Wdzieczak-Bakala, J., Morgat, J. L., Thierry, J., Voelter, W., and Lenfant, M. (1993) Biochem. J. 296, 373-378 [Medline] [Order article via Infotrieve]
  29. Ward, P. E., Chow, A., and Drapeau, G. (1991) Biochem. Pharmacol. 42, 721-727 [Medline] [Order article via Infotrieve]
  30. Liu, J. N. (1993) Blood 81, 980-987 [Abstract]
  31. Skidgel, R. A., and Tan, F. (1992) Agents Actions Suppl. 38, 359-367 [Medline] [Order article via Infotrieve]
  32. Bojanovski, D., Gregg, R. E., Zech, L. A., Meng, M. S., Bishop, C., Ronan, R., and Brewer, H. B., Jr. (1987) J. Clin. Invest. 80, 1742-1747 [Medline] [Order article via Infotrieve]
  33. Lins, L., Piron, S., Conrath, K., Vanloo, B., Brasseur, R., Rosseneu, M., Baert, J., and Ruysschaert, J. M. (1993) Biochim. Biophys. Acta 1151, 137-142 [Medline] [Order article via Infotrieve]
  34. Kunitake, S. T., Chen, G. C., Kung, S. F., Schilling, J. W., Hardman, D. A., and Kane, J. P. (1990) Arteriosclerosis 10, 25-30 [Abstract]
  35. Scanu, A. M. (1987) Atherosclerosis Rev. 16, 9-18
  36. Lijnen, H. R., and Collen, D. (1981) Thromb. Res. 24, 151-156 [Medline] [Order article via Infotrieve]
  37. Bausserman, L. L., and Herbert, P. N. (1984) Biochemistry 23, 2241-2245 [Medline] [Order article via Infotrieve]
  38. Shore, V. G., Sae, A. S., and Shore, B. (1978) Biochim. Biophys. Acta 529, 319-330 [Medline] [Order article via Infotrieve]
  39. Byrne, R. E., and Scanu, A. M. (1983) Biochemistry 22, 2894-2903 [Medline] [Order article via Infotrieve]
  40. Jeng, I., Steelman, R., Reilly, P., Jeng, Y., and Schonfeld, G. (1980) Biochem. Biophys. Res. Commun. 92, 876-882 [Medline] [Order article via Infotrieve]
  41. Swaney, J. B. (1993) J. Lipid Res. 24, 245-52 [Abstract/Free Full Text]
  42. Gregg, R. E., Zech, L. A., Bojanovski, D., Meng, M. A., Ronan, R., Bishop, C., and Brewer, H. B., Jr. (1985) Circulation 72, Suppl. III, 364 (abstr.) [Abstract]
  43. Mallory, J. B., Kushner, P. J., Protter, A. A., Cofer, C. L., Appleby, V. L., Lau, K., Schilling, J. W., and Vigne, J.-L (1987) J. Biol. Chem. 262, 4241-4247 [Abstract/Free Full Text]
  44. McFarlane, A. S. (1958) Nature 182, 53
  45. Berman, M., and Weiss, M. (1978) DHEW (NIH) 78, 180
  46. Remaley, A. T., Wong, A. W., Schumacher, U. K., Meng, M. S., Brewer, H. B., Jr., and Hoeg, J. M. (1993) J. Biol. Chem. 268, 6785-6790 [Abstract/Free Full Text]
  47. Jia, S., Cole, T. G., Kitchens, R. T., Pfleger, B., and Schonfeld, G. (1990) Metabolism 39, 155-160 [Medline] [Order article via Infotrieve]
  48. Chou, P. Y., and Fasman, G. D. (1978) Annu. Rev. Biochem. 47, 251-276 [CrossRef][Medline] [Order article via Infotrieve]
  49. Marcel, Y. L., Provost, P. R., Koa, H., Raffai, E., Vu Dac, N., Fruchart, J.-C., and Rassart, E. (1991) J. Biol. Chem. 266, 3644-3653 [Abstract/Free Full Text]
  50. Brasseur, R., De Meutter, J., Vanloo, B., Goormaghtigh, E., Ruysschaert, J. M., and Rosseneu, M. (1990) Biochim. Biophys. Acta 1043, 245-252 [Medline] [Order article via Infotrieve]
  51. Calabresi, L., Meng, Q.-H., Castro, G. R., and Marcel, Y. L. (1993) Biochemistry 32, 6477-6484 [Medline] [Order article via Infotrieve]
  52. Saku, K., Liu, R., Ohkubo, K., Bai, H., Hirata, K., Yamamoto, K., Morimoto, Y., Yamada, K., and Arakawa, K. (1993) Biochim. Biophys. Acta 1167, 257-263 [Medline] [Order article via Infotrieve]
  53. Chapman, M. J. (1986) Methods Enzymol. 128, 70-143 [Medline] [Order article via Infotrieve]
  54. Pan, T., Hao, Q., Yamin, T., Dai, P., Chen, B., Chen, S., Kroon, P. A., and Chao, Y. (1987) Eur. J. Biochem. 170, 99-104 [Abstract]
  55. Schaefer, E. J., Heaton, W. H., Wetzel, M. G., and Brewer, H. B., Jr. (1982) Arteriosclerosis 2, 16-26 [Abstract]
  56. Roma, P., Gregg, R. E., Meng, M. S., Ronan, R., Zech, L. A., Franceschini, G., Sirtori, C. R., and Brewer, H. B., Jr. (1993) J. Clin. Invest. 91, 1445-1452 [Medline] [Order article via Infotrieve]
  57. Vadiveloo, P. K., Allan, C. M., Murray, B. J., and Fidge, N. H. (1993) Biochemistry 32, 9480-9485 [Medline] [Order article via Infotrieve]
  58. Allan, C. M., Fidge, N. H., and Kanellos, J. (1992) J. Biol. Chem. 267, 13257-13261 [Abstract/Free Full Text]
  59. Beg, Z. H., Stonik, J. M, Hoeg, J. M., Demosky, S. J., Jr., Fairwell, T., and Brewer, H. B., Jr. (1989) J. Biol. Chem. 264, 6913-6921 [Abstract/Free Full Text]
  60. Funke, H., von Eckardstein, A., Pritchard, P. H., Karas, M., Albers, J. J., Assmann, G., Reckwerth, A., and Welp, S. (1991) J. Clin. Invest. 87, 371-376 [Medline] [Order article via Infotrieve]
  61. Osborne, J. C., Schaefer, E. J., Powell, G. M., Lee, N. S., and Zech, L. A. (1984) J. Biol. Chem. 259, 347-353 [Abstract/Free Full Text]
  62. Khouw, A. S., Parthasarathy, S., and Witztum, J. L. (1993) J. Lipid Res. 34, 1483-1496 [Abstract]
  63. Formisano, S., Brewer, H. B., Jr., and Osborne, J. C., Jr. (1978) J. Biol. Chem. 253, 354-358 [Abstract]
  64. Ikewaki, K., Rader, D. J., Schaefer, J. R., Fairwell, T., Zech, L. A., and Brewer, H. B., Jr. (1993) J. Lipid Res. 34, 2207-2215 [Abstract]
  65. Garber, D. W., Venkatachalapathi, Y. V., Gupta, K. B., Ibdah, J., Phillips, M. C., Hazelrig, J. B., Segrest, J. P., and Anantharamaiah, G. M. (1992) Arteriosclerosis Thromb. 12, 886-894 [Abstract]
  66. Westerlund, J. A., and Weisgraber, K. H. (1993) J. Biol. Chem. 268, 15745-15750 [Abstract/Free Full Text]
  67. Glass, C. K., Pittman, R. C., Keller, G. A., and Steinberg, D. (1983) J. Biol. Chem. 258, 7161-7167 [Abstract/Free Full Text]
  68. Glass, C., Pittman, R. C., Civen, M., and Steinberg, D. (1985) J. Biol. Chem. 260, 744-750 [Abstract/Free Full Text]
  69. Saku, K., Reddy, G. S., Hynd, B. A., and Kashyap, M. L. (1984) Metabolism 33, 432-8 [Medline] [Order article via Infotrieve]
  70. Horowitz, B. S., Goldberg, I. J., Merab, J., Vanni, T. M., Ramakrishnan, R., and Ginsberg, H. N. (1993) J. Clin. Invest. 91, 1743-1752 [Medline] [Order article via Infotrieve]
  71. Barrans, A., Collet, X., Barbaras, R., Jaspard, B., Manent, J., Vieu, C., Chap, H., and Perret, B. (1994) J. Biol. Chem. 269, 11572-11577 [Abstract/Free Full Text]
  72. Clay, M. A., Newnham, H. H., Forte, T. M., and Barter, P. I. (1992) Biochim. Biophys. Acta 1124, 52-58 [Medline] [Order article via Infotrieve]
  73. Sparks, D. L., Lund-Katz, S., and Phillips, M. C. (1992) J. Biol. Chem. 267, 25839-25847 [Abstract/Free Full Text]
  74. Gordon, J. I., Sims, H. F., Lentz, S. R., Edelstein, C., Scanu, A. M., and Strauss, A. W. (1983) J. Biol. Chem. 258, 4037-4044 [Abstract/Free Full Text]
  75. Edelstein, C., Gordon, J. I., Toscas, K., Sims, H. F., Strauss, A. W., and Scanu, A. M. (1983) J. Biol. Chem. 258, 11430-11433 [Abstract/Free Full Text]

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