6-Aminohexanoic Acid as a Chemical Chaperone for Apolipoprotein(a)*

Jin WangDagger and Ann L. White§

From the § Department of Internal Medicine and Dagger  Center for Human Nutrition, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9052

    ABSTRACT
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INTRODUCTION
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Apolipoprotein (a) (apo(a)) is a component of the atherogenic lipoprotein, Lp(a). The efficiency with which apo(a) escapes the endoplasmic reticulum (ER) and is secreted by the liver is a major determinant of plasma Lp(a) levels. Apo(a) contains a series of domains homologous to plasminogen kringle (K) 4, each of which possesses a potential lysine-binding site. By using primary mouse hepatocytes expressing a 17K4 human apo(a) protein, we found that high concentrations (25-200 mM) of the lysine analog, 6-aminohexanoic acid (6AHA), increased apo(a) secretion 8-14-fold. This was accompanied by a decrease in apo(a) presecretory degradation. 6AHA inhibited accumulation of apo(a) in the ER induced by the proteasome inhibitor, lactacystin. Thus, 6AHA appeared to inhibit degradation by increasing apo(a) export from the ER. Significantly, 6AHA overcame the block in apo(a) secretion induced by the ER glucosidase inhibitor, castanospermine. 6AHA may therefore circumvent the requirement for calnexin and calreticulin interaction in apo(a) secretion. Sucrose gradients and a gel-based folding assay were unable to detect any influence of 6AHA on apo(a) folding. However, non-covalent or small, disulfide-dependent changes in apo(a) conformation would not be detected in these assays. Proline also increased the efficiency of apo(a) secretion. We propose that 6AHA and proline can act as chemical chaperones for apo(a).

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Apolipoprotein (a) (apo(a))1 is a component of lipoprotein (a) (Lp(a)). Lp(a) is an unusual lipoprotein found only in the plasma of humans, old world primates (1), and the hedghog (2). Lp(a) consists of low density lipoprotein (LDL) in which apoB100, the sole protein component of LDL, is attached to apo(a) by a disulfide bond (3). Apo(a) is synthesized by the liver (4-6) and associates with LDL in plasma to form Lp(a) (7-9). Plasma Lp(a) levels are determined by Lp(a) production rate (10, 11) and vary among individuals from <1 to >100 mg/dl (12). More than 90% of this variation is determined by inheritance at the apo(a) gene locus (13). High Lp(a) levels (>30 mg/dl) are associated with an increased incidence of various cardiovascular diseases (14). The mechanisms that determine the rate of apo(a) secretion by the liver are therefore of considerable interest.

Apo(a) is homologous to plasminogen and consists of multiple domains with homology to plasminogen kringle (K) 4, followed by domains homologous to the plasminogen K5 and protease domains (15). As many as 34 different apo(a) size isoforms (from <300 to >800 kDa) exist due to variation in the number of K4 domains encoded in the apo(a) gene (16). There is an inverse correlation between apo(a) size and plasma Lp(a) level, accounting for 19-70% of the variation in plasma Lp(a) levels depending on the ethnic group (17). Sequence polymorphism independent of K4 number also exists at the apo(a) gene locus, accounting for the remaining contribution of the apo(a) gene to plasma Lp(a) levels (18).

The K4 domains in apo(a) are of 10 types based on amino acid differences (19). Each contains a "lysine-binding pocket" that, depending on the K4 type, is predicted to bind with varying affinity to lysine, lysine analogs, and proline (20). These domains are important for mediating the initial interaction of apo(a) with apoB in the first step of Lp(a) assembly (21) and for the binding of Lp(a) to other proteins, such as plasminogen receptors and fibrin (22-24), which may contribute to the atherogenicity of Lp(a) (14).

Multiple factors are likely to contribute to the characteristic secretion rate of each apo(a) allelic variant (25-30). Hepatic apo(a) mRNA levels account for some of the variation in plasma Lp(a) concentration (25-27). Post-translational mechanisms also play an important role. Studies using primary baboon hepatocyte cultures demonstrated that apo(a) is synthesized as a lower molecular weight precursor with a prolonged residence time in the endoplasmic reticulum (ER) before maturation and secretion. Maturation of apo(a) involves addition of O-linked glycans and conversion of N-linked sugars to the complex form (7, 27). Mature apo(a) binds to the hepatocyte cell surface before release into the culture medium (8). Apo(a) is inefficiently secreted by the hepatocyte, and a portion of the precursor protein is retained inside the cell and is degraded by the proteasome (29, 30). Apo(a) allelic variants vary considerably in the extent of their intracellular retention and degradation. In particular, large apo(a) isoforms tend to be secreted less efficiently than small isoforms, accounting at least partially for the inverse correlation between apo(a) size and plasma Lp(a) levels (29). In addition, the absence of detectable plasma Lp(a) associated with some ("null") apo(a) alleles is explained by the production of apo(a) proteins which are unable to exit the ER and are completely retained inside the cells and degraded (29, 30).

We have recently characterized the secretion of human apo(a) from primary cultures of mouse hepatocytes transgenic for a 17K4 form of the human protein. We found that human apo(a) is also inefficiently secreted from primary hepatocytes, and a large portion of the protein is retained inside the cell and degraded (31).

The ER quality control machinery ensures that only correctly folded proteins exit this compartment and traverse the remainder of the secretory pathway (32). Apo(a) undergoes extensive post-translational modification in the ER; each kringle domain contains 3 disulfide bonds and an N-linked glycosylation site. The inefficient secretion of apo(a) may therefore result from an intrinsic difficulty in achieving a correct conformation. Indeed, newly synthesized apo(a) requires a prolonged period (30-60 min) to fold (33) and interacts with multiple ER chaperone proteins (30). In the current study, we demonstrate that the lysine analog, 6-aminohexanoic acid (6AHA), can increase the efficiency with which human apo(a) is secreted from transgenic mouse hepatocyte cultures. This increase in secretion is associated with a decrease in the portion of apo(a) targeted to the intracellular degradation pathway. We propose that 6AHA can act as a molecular chaperone for apo(a).

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Materials-- [35S]Cysteine and Expre35S35S label were from NEN Life Science Products. Protein A-agarose was from Repligen Corp. (Cambridge, MA). Rabbit anti-human Lp(a) was from Cortex Pharmaceuticals (San Leandro, CA), and sheep anti-human apoB was from Roche Molecular Biochemicals. Amino acids and their analogs were from Sigma. Lactacystin was from Calbiochem. All other reagents were of analytical grade.

Hepatocyte Isolation and Culture-- Lp(a) transgenic mice were obtained from Dr. Helen Hobbs. The mice were hemizygous for a human apo(a) cDNA encoding 17K4 domains, including all the unique K4 domains, under the control of the mouse transferrin promoter and were homozygous for a human apoB P1 phagemid clone spanning the entire human apoB gene (34). In addition, the mice were homozygous knock-outs for the LDL receptor.2 Throughout this paper, these mice are referred to as Lp(a) transgenic mice. Hepatocytes were isolated and cultured in a serum-free medium (SFM) formulation exactly as described previously (31). All experiments were performed with cells that had been in culture for 48-72 h.

Radiolabeling, Immunoprecipitation, and SDS-PAGE-- Steady-state labeling was performed as described previously (7). For pulse-chase studies, cells were preincubated for 1 h in methionine- and cysteine-free SFM, labeled for 15 min with the same medium supplemented with 125 µCi/ml each of [35S]cysteine and Expre35S35S label, and then chased for between 0 min and 6 h in complete SFM. Compounds were added during the preincubation, labeling, and chase periods as described for individual experiments. Labeled media were collected and clarified for 5 min at 2,000 × g. Cells were washed twice with PBS and then lysed in extraction buffer (EB, 1% Triton X-100, 0.3% CHAPS, 100 mM NaCl, 50 mM Tris, pH 9.0). Protease inhibitors (CompleteR, Roche Molecular Biochemicals) were added to all samples. Samples were immunoprecipitated and analyzed on 4-10% SDS-polyacrylamide gels, as described previously (7), with the exception that a rabbit anti-Lp(a) antibody was used. For quantitation of apo(a), autoradiographs were scanned with an Arcus II desktop scanner, and bands were quantified using Scanalytics ONE-Dscan software. Analysis of serially diluted samples demonstrated that this method was quantitative over at least a 16-fold range. Values obtained were normalized to total cell protein determined using the BCA assay (Pierce).

Analysis of Apo(a) Folding-- Apo(a) folding was examined as described previously (33). Briefly, cells were preincubated for 1 h and then labeled for 45 min, as described above. The media were then adjusted to 5 mM DTT, and the incubation was continued for a further 5 min. The cells were washed twice with PBS and chased for various periods in SFM. To harvest, the cells were placed on ice, washed 2 times with ice-cold PBS containing 20 mM N-ethylmaleimide (NEM), and then lysed in ice-cold EB + 20 mM NEM. Apo(a) was immunoprecipitated and analyzed by SDS-PAGE, with or without prior reduction with 2-mercaptoethanol, as described above.

    RESULTS
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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All experiments were performed using primary hepatocytes isolated from mice transgenic for human apoB and a 17K4 form of human apo(a) (Lp(a) transgenic mice; see "Experimental Procedures").

6-Aminohexanoic Acid (6AHA) Increases Apo(a) Secretion-- We have previously utilized the lysine analog, 6AHA, to examine the mechanism of association of newly synthesized apo(a) with the hepatocyte cell surface. 6AHA prevents apo(a) binding to the hepatocyte surface, presumably through its interaction with the apo(a) kringle domains (8, 31). In the course of these studies we sometimes observed that more apo(a) was secreted from hepatocytes incubated with 6AHA compared with control cultures. Fig. 1A illustrates this effect in Lp(a) transgenic mouse hepatocytes. In this experiment, hepatocytes were labeled overnight with [35S]cysteine and [35S]methionine in the presence or absence of 200 mM 6AHA. Human apo(a) and apoB were then immunoprecipitated from the cells and media and analyzed by SDS-PAGE (Fig. 1A).


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Fig. 1.   6AHA increases apo(a) secretion from primary hepatocytes. A, Lp(a) transgenic mouse hepatocytes were labeled for 16 h with 35S-labeled amino acids in control medium (-6AHA) or medium containing 200 mM 6AHA (+6AHA). Human apo(a) [(a)]and human apoB (B) were then immunoprecipitated from cells (c) and media (m) and were analyzed by 4-10% SDS-PAGE and fluorography, as described under "Experimental Procedures." B, hepatocytes were labeled as in A in the absence of 6AHA. The cells and media were harvested and divided into 2 equal aliquots. The samples were then immunoprecipitated with anti-apo(a) antibodies before (-) or after (+) adjustment to 200 mM 6AHA. The positions of the precursor (pr apo(a)) and mature (mt apo(a)) forms of apo(a), and of apoB100, apoB48, and the 200-kDa marker (myosin) are indicated. × denotes a protein nonspecifically recovered in immunoprecipitates of culture media.

The apo(a) precursor was observed in lysates of both control and 6AHA-treated hepatocytes. However, since 6AHA disrupts the association of apo(a) with the hepatocyte cell surface (8), mature apo(a) was only found in lysates from control cells (Fig. 1A). Mature apo(a) was present in the media of both cell types, but 4.5-fold more apo(a) was recovered from 6AHA-treated versus control media. This difference was only partially accounted for by the decrease in cell-surface associated mature apo(a) in 6AHA-treated cells, and total mature apo(a) (cells + media) was 4.3-fold higher in 6AHA treated versus control cultures (Fig. 1A). An additional protein (Fig. 1A, ×) was nonspecifically immunoprecipitated from the culture media. This protein binds to protein A and is consistently observed in the media of liver cell cultures (7).

The 6AHA-mediated increase in apo(a) secretion was specific since secretion of human apoB100 and apoB48 (Fig. 1A) and of endogenous albumin and transferrin (data not shown) was slightly decreased in 6AHA-treated cultures. The decrease in secretion of these proteins may reflect some sensitivity of the cells to the prolonged (overnight) incubation with 6AHA.

The increased recovery of apo(a) from the media of 6AHA-treated cells was not due to enhanced immunoprecipitation of apo(a) in the presence of the lysine analog, since addition of 200 mM 6AHA to labeled cell lysates or culture media did not increase the amount of apo(a) precipitated (Fig. 1B). In addition, immunoblotting of samples before and after incubation with antibody demonstrated essentially quantitative recovery of apo(a) in the immunoprecipitation (data not shown).

6AHA Increases Apo(a) Secretion Post-translationally-- To examine the mechanism of the 6AHA-induced increase in apo(a) secretion, pulse-chase experiments were performed. Hepatocytes were labeled for 15 min and then chased for up to 6 h in the presence or absence of 200 mM 6AHA. Apo(a) was immunoprecipitated from the cell lysates and culture media and analyzed by SDS-PAGE (Fig. 2).


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Fig. 2.   6AHA increases apo(a) secretion post-translationally. Lp(a) transgenic mouse hepatocytes were preincubated, labeled for 10 min, and then chased for up to 6 h in the absence of 6AHA (-6AHA) or with 200 mM 6AHA present in all incubations (+6AHA), exactly as described under "Experimental Procedures." Apo(a) in the cell lysates and culture media was then analyzed by immunoprecipitation and SDS-PAGE. The positions of the precursor (pr) and mature (mt) forms of apo(a) and a nonspecifically immunoprecipitated protein (×) are indicated.

At both 0 and 30 min of chase, the amount of apo(a) recovered from control and 6AHA-treated cultures was virtually identical (ratio of control:6AHA at 30 min = 1.1:1). Thus, 6AHA did not increase the rate of apo(a) synthesis. The amount of apo(a) precursor in the cells decreased with increasing chase times at a similar rate in control and 6AHA-treated cultures (Fig. 2; ratio of precursor in control:6AHA-treated cells at 6 h = 1.2:1). The decrease in apo(a) precursor over time was accompanied by the appearance of mature apo(a). For both sets of cultures mature apo(a) appeared in the cell lysates at 60 min of chase. Mature apo(a) also appeared in the media at 60 min in 6AHA-treated cultures but not until 120 min for control cells. The earlier appearance of apo(a) in the media of 6AHA-treated cells may be partially explained by the release of cell-surface associated apo(a) by the lysine analog. However, the total amount of mature apo(a) at 60 min was greater (by 2.9-fold) in 6AHA-treated versus control cultures. By 6 h of chase, approximately 8-fold more secreted apo(a) and 6.5-fold more total (cells + media) mature apo(a) was recovered from 6AHA-treated versus control cultures.

The increase in apo(a) secretion in the presence of 6AHA was accompanied by a decrease in apo(a) degradation. Over 6 independent experiments, 90 ± 3% (mean ± S.D.) and 27 ± 33% of apo(a) synthesized during a 15-min pulse was degraded in control and 6AHA-treated cells, respectively (p = 0.007). The extended kinetics of apo(a) secretion and degradation in both control and 6AHA-treated cultures are consistent with previous observations (7, 29-31).

6AHA Increases the Efficiency of Apo(a) Exit from the ER-- The increase in recovery of mature apo(a) from hepatocyte cultures incubated with 6AHA could be explained by two mechanisms. First, 6AHA may increase the efficiency with which apo(a) exits the ER and undergoes maturation and secretion. Second, since 6AHA disrupts the cell surface association of apo(a), it may prevent rapid degradation of a cell-surface associated pool of apo(a). To distinguish between these possibilities, we treated cells with 6AHA in the presence of either the proteasome inhibitor, lactacystin (Fig. 3), or the ER glucosidase inhibitor, castanospermine (CST) (Fig. 4).


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Fig. 3.   6AHA inhibits accumulation of the apo(a) precursor in lactacystin-treated cells. Lp(a) transgenic mouse hepatocytes were preincubated, labeled for 15 min, and chased for 30 min or 6 h, under control conditions or with either 10 µM lactacystin, 200 mM 6AHA, or both lactacystin and 200 mM 6AHA present in all incubations. Duplicate plates of cells were analyzed for each condition. Apo(a) was then immunoprecipitated and analyzed by SDS-PAGE. All procedures were performed as described under "Experimental Procedures." The positions of the precursor (pr) and mature (mt) forms of apo(a), of the 200 kDa marker, and of a nonspecifically immunoprecipitated protein (×) are indicated.


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Fig. 4.   Effect of 6AHA on the CST-induced inhibition of apo(a) secretion. A, schematic representation of the processing of N-linked glycans in the ER lumen. Steps inhibited by CST are indicated. Calnexin and calreticulin bind to monoglucosylated proteins. 6AHA increases apo(a) transport from the ER to the Golgi apparatus. GSD, glucosidase; MSD, mannosidase B. Hepatocytes were preincubated, labeled for 15 min, and chased for 30 min or 6 h, in the absence of CST (-/-), with CST present in all incubations (+/+), or with CST present in the chase medium only (-/+). 6AHA (200 mM) was absent (-6AHA) or present in all incubations (+6AHA). Apo(a) was immunoprecipitated from the cells and media and analyzed by SDS-PAGE, as described under "Experimental Procedures." The positions of the precursor (pr) and mature (mt) forms of apo(a) and of a protein (×) nonspecifically recovered in immunoprecipitates of culture media are indicated.

Lactacystin inhibits ER-associated pre-secretory degradation of apo(a) and allows accumulation of the apo(a) precursor inside the cell (Ref. 30 and Fig. 3). If 6AHA increases apo(a) secretion by enhancing its exit from the ER, it should inhibit the accumulation of the apo(a) precursor in lactacystin-treated cells. Hepatocytes were labeled for 15 min and then chased for 30 min (to measure apo(a) synthesis) or 6 h in the presence of 6AHA and/or lactacystin. Compared with untreated cells, 6AHA alone increased apo(a) secretion at 6 h by 11-fold (Fig. 3). No accumulation of the apo(a) precursor was observed at 6 h compared to control cells, consistent with the data in Fig. 2. When cells were treated with lactacystin alone, a modest 1.8-fold increase in apo(a) secretion was observed. However, the amount of apo(a) precursor remaining at 6 h was increased 12-fold relative to control cells (Fig. 3A). When both 6AHA and lactacystin were present, there was again a marked increase in apo(a) secretion (14-fold) but relatively little accumulation of the precursor (2.6-fold more than control cells at 6 h, but only 20% of that in cells treated with lactacystin alone). These results strongly suggest that 6AHA decreases apo(a) degradation by enhancing the efficiency with which apo(a) is transported out of the ER. The ability of 6AHA to increase recovery of mature apo(a) in these cultures therefore appears independent of its effect on the cell surface association of apo(a).

6AHA Overcomes the Castanospermine-induced Block in Apo(a) Secretion-- N-Linked carbohydrate is added to secretory proteins co-translationally in the ER lumen as GlcNAc2Mn9Glc3 precursors (where GlcNAc is N-acetylglucosamine, Man is mannose, and Glc is glucose). While the protein is still being translated, the outer and two inner glucoses are trimmed by ER glucosidases I and II, respectively (Ref. 35 and Fig. 4A). Incorrectly folded proteins are recognized by the enzyme UDP-Glucose:glycoprotein glucosyltransferase which re-attaches a single glucose to the N-linked sugar chain (36). Monoglucosylated glycans are then recognized by the lectin-like ER chaperones, calnexin (CNX) and calreticulin (CRT) which bind to the proteins and help them fold (Ref. 37 and Fig. 4A). The re-attached glucose is then removed by glucosidase II. Depending on the folded state of the protein, it will then either continue down the secretory pathway or will enter a re-glucosylation/deglucosylation cycle (involving cyclic interactions with CNX and CRT) until it is fully folded and can be secreted (Fig. 4A).

CST inhibits glucosidases I and II (38). If CST is added prior to the label in a pulse-chase experiment, it prevents the co-translational removal of the 3 glucose residues from radiolabeled apo(a) and prevents the interaction of apo(a) with CNX and CRT (Ref. 30 and data not shown). Apo(a) secretion is almost completely prevented under these conditions, but its intracellular degradation is not affected (30, 33). Treatment with lactacystin causes accumulation of the apo(a) precursor in these CST-treated cells but does not increase its secretion (Ref. 30 and data not shown), confirming that addition of CST prior to labeling inhibits secretion of the radiolabeled apo(a) by preventing its transport out of ER. If CST is added after apo(a) has been radiolabeled (i.e. after co-translational removal of the initial 3 glucose residues), it can trap apo(a) in its monoglucosylated form and enhance the interaction of apo(a) with CNX and CRT (30, 39). Under these conditions, both secretion and degradation of apo(a) are blocked (Ref. 30 and Fig. 4B).

The effect of 6AHA on the secretion of the differentially glucosylated forms of apo(a) was examined in hepatocytes treated with CST either before (pre-translation) or after (post-translation) a 15-min pulse to trap radiolabeled apo(a) in its tri- and mono-glucosylated forms, respectively (Fig. 4B). An increase in the molecular weight of the apo(a) precursor was observed in cells treated with CST pre-translation, confirming the retention of the 3 glucose residues on its N-linked glycans. As observed previously (33), maturation and secretion of this form of apo(a) was almost entirely prevented and an increased portion was degraded (total apo(a) remaining at 6 h 56% of that for control cells; Fig. 4B). Treatment with 6AHA overcame the CST-induced ER retention and degradation of apo(a), and increased secretion of triglucosylated apo(a) 14-fold, to levels greater (by 52%) than for untreated cells (Fig. 4B). Thus, 6AHA can surmount the block in apo(a) secretion induced by CST, providing definitive evidence that 6AHA increases apo(a) secretion by enhancing the efficiency with which it exits the ER. As far as we are aware, this is the first documentation that the inhibitory effect of CST on glycoprotein secretion can be overcome.

When CST was added to hepatocytes post-translation, a smaller increase in apo(a) molecular weight was observed, due to retention of a single glucose residue on each N-linked glycan (Fig. 4B). In the absence of 6AHA, both secretion and degradation of monoglucosylated apo(a) were blocked (Fig. 4B). In the presence of 6AHA, a small amount (15% of that observed for control cells) of apo(a) was able to escape the ER and undergo maturation and secretion. However, the vast majority was again retained in an undegraded form inside the cell. Thus, 6AHA is unable to overcome the block in apo(a) secretion induced by conditions that enhance the interaction of apo(a) with CNX and CRT (30).

Influence of 6AHA on Apo(a) Folding-- Since only correctly folded proteins are permitted to exit the ER, we hypothesized that 6AHA may enhance apo(a) folding efficiency. Apo(a) folding can be analyzed using an assay that detects variously disulfide bonded apo(a) folding intermediates through differences in their electrophoretic mobility on non-reducing SDS-PAGE (33). The sensitivity of the assay is enhanced when radiolabeled apo(a) is allowed to accumulate in the ER and is then chemically reduced and unfolded and allowed to re-fold (33). Apo(a) folding after reduction with dithiothreitol (DTT) was examined in cultures incubated in the presence or absence of 200 mM 6AHA (Fig. 5).


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Fig. 5.   Influence of 6AHA on apo(a) folding. Hepatocytes were labeled for 45 min, treated with 5 mM DTT for 5 min, then chased between 0 and 120 min in the absence of 6AHA (-6AHA) or with 6AHA present in all incubations (+6AHA). Cells were harvested in the presence of 20 mM N-ethylmaleimide. Apo(a) was then immunoprecipitated and analyzed by SDS-PAGE with (reduced) or without (non-reduced) prior reduction with 2-mercaptoethanol. An additional sample chased for 0 min was immunoprecipitated with preimmune serum (ns) as a control. The positions of the precursor (pr), mature (mt), and aggregated (aggregates) forms of apo(a) are indicated.

Cells were labeled for 45 min, treated with 5 mM DTT for 5 min, and then chased for various periods up to 2 h. After harvesting the cells in the presence of N-ethylmaleimide to preserve variously disulfide bonded folding intermediates, apo(a) was immunoprecipitated and analyzed reduced and non-reduced by SDS-PAGE (Fig. 5). As previously observed (31, 33), for control cells, a single form of apo(a), representing the reduced, unfolded protein, was observed immediately after DTT treatment on non-reduced gels. By 10 min of chase, the mobility of the majority of apo(a) on the gel had increased, reflecting the formation of intramolecular disulfide bonds (Fig. 5). However, a portion of apo(a) entered into high molecular weight aggregates represented by a smear on the gel. These aggregates were disulfide-linked since they disappeared on reduction (Fig. 5, compare reduced and non-reduced). The mobility of the monomeric form of apo(a) continued to increase up to the 60-min chase time, at which point the mature form of apo(a) also appeared in the cell lysates (Fig. 5). The apo(a) aggregates persisted throughout the 120-min chase period. The pattern of apo(a) folding was almost identical when examined in 6AHA-treated cultures. No change in apo(a) folding kinetics or extent of aggegation was detected in the presence of 6AHA (Fig. 5)

Kringle motifs often mediate protein-protein interactions. We therefore considered the possibility that 6AHA enhances the efficiency of apo(a) secretion by decreasing interactions between the apo(a) kringles and other proteins in the ER lumen. Such interactions could lead to the incorporation of apo(a) into non-covalently-linked aggregates that would not be detected in the gel-based folding assay. To investigate this issue, we analyzed the distribution of the apo(a) precursor on sucrose gradients under conditions known to maintain apo(a)-protein interactions (30). We observed no effect of 6AHA on the distribution of apo(a) on these gradients (data not shown).

6AHA Increases Apo(a) Secretion after Folding Is Complete-- Since 6AHA did not have a detectable effect on apo(a) folding, we examined its effect on apo(a) secretion when added 2 h after apo(a) synthesis (Fig. 6). Apo(a) folding should be complete by this time (Fig. 5), but apo(a) should not yet have been targeted to the degradation pathway (30). Hepatocytes were labeled for 15 min and then chased for 30 min or 6 h with 6AHA present in all incubations or added only after 2 h of chase (Fig. 6). When added after 2 h, 6AHA increased apo(a) secretion almost as much as when added to all incubations (10- and 12-fold increase over control, respectively; Fig. 6). The small amount of mature apo(a) in lysates of cells chased for 2 h before addition of 6AHA (Fig. 6) presumably represents apo(a) that bound to the cell surface during the initial 2-h chase, since 6AHA more easily prevents than disrupts apo(a) association with the cell surface (8).


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Fig. 6.   6AHA can increase apo(a) secretion after apo(a) folding is complete. Hepatocytes were preincubated, labeled for 15 min, and then chased for 30 min or 6 h as described under "Experimental Procedures," under control conditions (control), with 200 mM 6AHA present in all incubations (6AHA) or with 200 mM 6AHA added only after 2 h of chase (6AHA @ 2 h). Apo(a) was then immunoprecipitated from the cells and media and analyzed by SDS-PAGE. The positions of the precursor (pr) and mature (mt) forms of apo(a) and of a protein (×) nonspecifically immunoprecipitated from culture media are indicated.

Proline Also Increases Apo(a) Secretion-- In addition to 6AHA, lysine and proline can bind to apo(a) and prevent its interaction with other proteins (21-24) and with the hepatocyte cell surface (8). To determine whether these amino acids could increase apo(a) secretion, hepatocytes were labeled and then chased in the presence of 200 mM 6AHA, lysine, or proline (Fig. 7). 6AHA and proline increased apo(a) secretion 6- and 5-fold over control cells, respectively. The lysine analog, tranexamic acid, also increased apo(a) secretion (data not shown). In contrast, lysine prevented both secretion and degradation of apo(a) (Fig. 7). Arginine also inhibited apo(a) exit from the ER (data not shown). The inhibition of apo(a) secretion was not specific, as apoB secretion was almost completely abolished by 200 mM lysine (Fig. 7) and was probably due to cell toxicity. Titration of lysine to concentrations that did not inhibit apo(a) degradation (25 mM) had no influence on apo(a) secretion (data not shown). In contrast, 25 mM 6AHA increased apo(a) secretion 4-fold (data not shown). The difference in ability of 6AHA and lysine to enhance apo(a) secretion at this concentration may reflect their different affinities for the apo(a) kringle domains (24).


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Fig. 7.   Influence of other amino acids on apo(a) secretion. Hepatocytes were preincubated, labeled for 15 min, and then chased for 30 min or 6 h under control conditions or with 200 mM 6AHA, proline (pro.), or lysine (lys.) present in all incubations. Apo(a) and human apoB were then immunoprecipitated from the cells (c) and media (m) and analyzed by SDS-PAGE, exactly as described under "Experimental Procedures." The positions of the precursor (pr) and mature (mt) forms of apo(a), of apoB100 and apoB48, and of a protein (×) non-specifically recovered in immunoprecipitates of culture media are indicated.


    DISCUSSION
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ABSTRACT
INTRODUCTION
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RESULTS
DISCUSSION
REFERENCES

Differences in the efficiency with which apo(a) allelic variants undergo post-translational processing and secretion from the liver contribute significantly to inter-individual differences in plasma Lp(a) levels (29). A variable portion of apo(a) is not secreted and is retained inside the hepatocyte in an ER-associated compartment and is degraded by the proteasome (30). In the current study, we demonstrate that the efficiency of apo(a) secretion can be markedly increased by the lysine analog 6AHA. 6AHA increases transport of newly synthesized apo(a) out of the ER and decreases the portion of apo(a) targeted to the proteasome for destruction. We propose that 6AHA can act as a chaperone for apo(a).

Incubation of primary hepatocytes from Lp(a) transgenic mice with 200 mM 6AHA resulted in a specific increase (up to 12-fold) in apo(a) secretion. Pulse-chase studies demonstrated that the effect was post-translational and was accompanied by a decrease in apo(a) intracellular degradation. Co-incubation of cells with 6AHA and either the proteasome inhibitor, lactacystin, or the ER glucosidase inhibitor, CST, conclusively demonstrated that the effect of 6AHA was mediated at the level of the ER. Thus, 6AHA decreased apo(a) presecretory degradation by enhancing the efficiency with which the apo(a) precursor underwent maturation and secretion.

Other small molecules have also been demonstrated to increase export of certain proteins from the ER. For example, glycerol, trimethylamine N-oxide, and dimethyl sulfoxide can correct certain temperature-sensitive protein folding defects when added to cells in culture (40-42). The Delta F508 mutant form of the cystic fibrosis transmembrane conductance regulator (CFTR) is normally unable to exit the ER and is targeted to the proteasome for destruction (43). Incubation of cells expressing this mutant with any of the above three compounds allows a portion of the CFTR protein to traffic to the cell surface as a functional chloride channel (41). Another example is the influence of calcium on the intracellular transport of the LDL receptor-related protein (LRP). In the absence of calcium, LRP aggregates in the ER. Calcium is believed to enhance LRP folding by stabilizing its calcium binding domains (44). LRP misfolding can be reversed on calcium restoration. In addition to calcium, LRP secretion from cells that express ligands for this receptor requires co-expression of the receptor-associated protein, RAP (45). RAP acts as a specific chaperone for LRP by preventing association of the newly synthesized receptor with its ligands in the ER and subsequent aggregation of LRP (45).

We considered the possibility that 6AHA may increase apo(a) secretion by a mechanism similar to those described above. Apo(a) is a large glycoprotein that undergoes extensive post-translational modification in the ER and requires a prolonged period (approximately 60 min) to fold (33). Binding of 6AHA to apo(a) kringle domains can induce a conformational change in apo(a) (46) and prevent its interaction with other proteins, such as apoB and plasminogen receptors (21-24). We hypothesized that 6AHA may increase apo(a) folding efficiency by stabilizing the folded apo(a) kringle domains or by preventing non-productive interactions of apo(a) with other proteins in the ER lumen. The involvement of the apo(a) kringle domains in the 6AHA-mediated effect was supported by the ability of proline to increase apo(a) secretion. Proline is predicted to bind to the repeated K4 type 2 domain in apo(a) (47) and can prevent the interaction of apo(a) with the cell surface (8) and of Lp(a) with other apoB-containing lipoproteins (48). In addition, at 25 mM, 6AHA but not lysine increased apo(a) secretion, which is consistent with the apparent relative affinity of apo(a) for these compounds (24).

However, using a gel-based assay, we were unable to detect any influence of 6AHA on disulfide-dependent apo(a) folding, and sucrose gradients were unable to reveal any influence of 6AHA on non-covalent interactions between apo(a) and other proteins in the ER lumen. In addition, 6AHA was able to increase apo(a) secretion when added 2 h after apo(a) synthesis, a time point at which apo(a) folding is apparently complete. These results suggest that the effect of 6AHA may be independent of apo(a) folding. It is possible that 6AHA modulates the interaction of apo(a) with a specific "receptor" that normally retains misfolded apo(a) in the ER. In this case, 6AHA may allow the secretion of misfolded apo(a). It will be interesting to determine whether apo(a) secreted in the presence of 6AHA is functional (for example whether it can still bind apoB). Another possibility is that 6AHA may promote interaction of apo(a) with a protein required for transport of apo(a) out of the ER. The ER-Golgi intermediate compartment protein, ERGIC53, is required for transport of the coagulation factors V and VIII from the ER to the Golgi (49). It is possible that interaction with this or a similar protein may be required for apo(a) intracellular transport.

Alternatively, it is possible that 6AHA does affect apo(a) folding but that we are unable to detect the effect. The gel-based folding assay and sucrose gradients we used to analyze apo(a) folding will not detect non-covalent changes in apo(a) conformation, such as those induced by binding of 6AHA to the apo(a) kringles (46). Disulfide bond-dependent changes in conformation that affect only one or a few of the many kringle domains in apo(a) may also go undetected. "Null" apo(a) proteins that are not secreted, but are completely retained and degraded in the ER, show patterns of folding indistinguishable from those of secreted apo(a) proteins (33). Recent studies by Cox et al. (50), however, demonstrated a deletion of 100 amino acids in the protease domain of one of these null proteins, which would clearly prevent proper folding of this domain. In addition, treatment of hepatocytes with CST does not produce a detectable effect on apo(a) folding in these assays, despite inhibiting apo(a) secretion and interaction with the lectin-like ER chaperones (30, 33). That 6AHA can increase apo(a) secretion after apo(a) folding is apparently complete is not inconsistent with an effect of 6AHA on apo(a) folding. Similar to the effect of calcium on LRP, it is possible that 6AHA can rescue misfolded apo(a) in the ER. Clearly, further studies are required to determine the precise mechanism of the 6AHA-induced increase in apo(a) secretion.

A particularly interesting and novel observation made in the current study was the ability of 6AHA to overcome a block in apo(a) secretion induced by the ER glucosidase inhibitor, castanospermine (CST). Addition of CST to cells either prior to or after addition of label can trap radiolabeled apo(a) in a tri- or monoglucosylated form, respectively (Fig. 4A). In this way, CST can be used to prevent or enhance, respectively, interaction of apo(a) with the lectin-like ER chaperones, calnexin and calreticulin (Ref. 30 and data not shown). Under both conditions, apo(a) secretion is prevented, presumably due to misfolding when chaperone interactions are blocked, and retention of apo(a) in the ER by the chaperones when their interaction is enhanced. 6AHA was unable to overcome the block in apo(a) secretion induced by conditions that enhance interaction of apo(a) with calnexin and calreticulin, suggesting that 6AHA cannot disrupt the apo(a)-chaperone interactions. However, 6AHA did overcome the block in apo(a) secretion induced when CST was added to prevent apo(a)-chaperone interactions. It is possible that under these conditions 6AHA enabled apo(a) to overcome the ER quality control mechanisms that normally retain misfolded apo(a) in the ER. Alternatively, if 6AHA enhances apo(a) folding, it may eliminate the dependence of apo(a) folding on interaction with calnexin and calreticulin.

In conclusion, we have demonstrated that 6AHA and related compounds can increase the efficiency of apo(a) secretion from primary hepatocyte cultures. We propose that 6AHA acts as a chaperone for apo(a), increasing its transport out of the ER and decreasing the portion of apo(a) targeted to intracellular degradation. Future studies will be designed to determine the precise mechanism(s) of action of 6AHA. These studies will provide important insight into the factors that determine plasma levels of the atherogenic lipoprotein, Lp(a).

    FOOTNOTES

* This work was supported by National Institutes of Health Grant HL59541.

To whom correspondence should be addressed: Center for Human Nutrition, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9052. E-mail: awhite{at}crcdec.swmed.edu.

2 H. Hobbs, personal communication.

    ABBREVIATIONS

The abbreviations used are: apo(a), apolipoprotein (a); 6AHA, 6-aminohexanoic acid; CHAPS, 3-[(3-Cholamidopropyl)dimethylammonio]-1-propansulfonate; CNX, calnexin; CRT, calreticulin; CST, castanospermine; ER, endoplasmic reticulum; K, kringle; LDL, low density lipoprotein; Lp(a), lipoprotein (a); LRP, LDL receptor related protein; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; SFM, serum-free medium.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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