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Influence of Allelic Variation on Apolipoprotein(a) Folding in the Endoplasmic Reticulum

Influence of Allelic Variation on Apolipoprotein(a) Folding in the Endoplasmic Reticulum THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 8, Issue of February 21, pp. 5048–5055, 1997 © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Influence of Allelic Variation on Apolipoprotein(a) Folding in the Endoplasmic Reticulum* (Received for publication, May 17, 1996, and in revised form, October 29, 1996) Ann L. White‡, Bernadette Guerra, and Robert E. Lanford From the Department of Virology and Immunology, Southwest Foundation for Biomedical Research, San Antonio, Texas 78227 Plasma levels of lipoprotein(a) (Lp(a)) vary over 1000- approximately 12 to 51 (3). Each K4 repeat contains 3 internal fold between individuals and are determined by the disulfide bonds, 1 N-linked, and 6 potential O-linked glycosy- gene for its unique apolipoprotein, apo(a), which has lation sites (7). Thus, a large apo(a) isoform may contain in greater than 100 alleles. Using primary baboon hepato- excess of 150 disulfide bonds, 50 N-linked and 300 O-linked cyte cultures, we previously demonstrated that differ- carbohydrate side chains. ences in the ability of apo(a) allelic variants to escape Lp(a) is only found in the plasma of primates (12) and the the endoplasmic reticulum (ER) are a major determi- hedgehog (13, 14). In humans, plasma levels of Lp(a) vary from nant of Lp(a) production rate. To examine the reason for ,1to .100 mg/dl and are highly heritable (15). High plasma these differences, the folding of newly synthesized levels of Lp(a) are associated with an increased incidence of apo(a) was analyzed in pulse-chase experiments. Sam- cardiovascular diseases (for review, see Ref. 16). Greater than ples were harvested in the presence of N-ethylmaleim- 90% of the inter-individual variation in Lp(a) concentration is ide to preserve disulfide-bonded folding intermediates, attributable to the apo(a) gene locus (17). There is an inverse and apo(a) was analyzed by immunoprecipitation and correlation between apo(a) size and plasma Lp(a) level (5, 6, 10, SDS-polyacrylamide gel electrophoresis. Apo(a) re- 18); however, this relationship is not absolute (6, 10), and a quired a prolonged period (30–60 min) to reach its fully oxidized form. Multiple folding intermediates were re- particular isoform size may be associated with as much as a solved, including a disulfide-linked, apo(a)-containing 200-fold difference in plasma Lp(a) concentrations in different complex. Unexpectedly, all allelic variants examined individuals (19). Thus, sequence variations at the apo(a) locus showed similar patterns and kinetics of folding. Even independent of size also influence Lp(a) levels (20). “Null” “null” apo(a) proteins, which are unable to exit the ER, apo(a) alleles, which do not give rise to any detectable plasma appeared to fold normally. The ER glucosidase inhibi- protein (5, 9), and which are distributed throughout the apo(a) tor, castanospermine, prevented apo(a) secretion, but allele size range (18, 21), are an extreme example of apo(a) did not inhibit folding. This suggests that an event sequence variations that influence Lp(a) levels. A number of which is dependent on trimming of N-linked glucoses, polymorphisms have recently been identified at the apo(a) lo- and which occurs after the folding events detectable in cus (20, 22–31), and it is estimated that there are greater than our assay, is required for apo(a) secretion. Differences 100 apo(a) alleles (20). However, as yet, no identified polymor- in the ability to undergo this event may explain the phism other than K4 number has been demonstrated to di- variable efficiency with which apo(a) allelic variants rectly influence circulating Lp(a) levels. exit the ER. Lp(a) is synthesized by the liver (32–34), and the rate of Lp(a) production determines plasma Lp(a) concentration (35– 37). Due to the lack of a small animal model and the highly Apolipoprotein(a) (apo(a)) is a highly polymorphic, high mo- polymorphic nature of the apo(a) gene, the mechanisms gov- lecular weight glycoprotein that circulates in plasma as a com- erning Lp(a) production rate have remained poorly character- ponent of lipoprotein(a) (Lp(a)). Lp(a) is composed of low den- ized. Some of the variation in Lp(a) production rate is deter- sity lipoprotein in which apoB is attached to apo(a) by disulfide mined by differences in hepatic apo(a) mRNA concentration (9, linkage (1, 2). As many as 34 different isoforms of apo(a) have 38). “Transcript negative null” alleles that do not produce any been identified in human plasma, which vary in size from ,300 detectable hepatic apo(a) mRNA transcript (9, 39) represent an to .800 kDa (3–6). Apo(a) has a highly complex, repetitive extreme example of this type of regulation. Post-transcriptional structure and is homologous with plasminogen (7). Apo(a) con- mechanisms are also important (38). A sequence polymorphism tains an inactive copy of the plasminogen protease domain and in the 59-untranslated region of human apo(a) was recently a single plasminogen kringle 5 (K5) domain, preceded by mul- shown to influence the translation efficiency of apo(a) mRNA in tiple domains with homology to plasminogen K4 (7). The size vitro (31). variation of apo(a) is due to differences in the number of K4 Baboons show very similar characteristics to humans in domains encoded in the apo(a) gene (8–11), which varies from terms of plasma Lp(a) levels and apo(a) isoform sizes (40, 41). We have established primary cultures of baboon hepatocytes as * This research was supported by National Institutes of Health Grant a model system for the analysis of Lp(a) biogenesis (34). Since HL50426. The costs of publication of this article were defrayed in part hepatocytes can be isolated from animals with selected Lp(a) by the payment of page charges. This article must therefore be hereby phenotypes, this provides us with a unique opportunity to marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. examine the influence of apo(a) allelic variation on Lp(a) pro- ‡ To whom correspondence should be addressed. duction rate. Using this system, we have demonstrated that, in The abbreviations used are: apo, apolipoprotein; CST, castanosper- comparison to other secretory proteins, newly synthesized mine; PBS, phosphate-buffered saline; DTT, dithiothreitol; ER, endo- apo(a) has a prolonged residence time in the endoplasmic re- plasmic reticulum; Lp, lipoprotein; NEM, N-ethylmaleimide; PAGE, polyacrylamide gel electrophoresis; TM, tunicamycin. ticulum (ER) before it is processed to its mature form and 5048 This paper is available on line at http://www-jbc.stanford.edu/jbc/ This is an Open Access article under the CC BY license. Apo(a) Folding 5049 FIG.1. Folding of newly synthesized apo(a). Hepatocytes expressing an I isoform of apo(a) were labeled for 10 min with 125 mCi/ml each of 35 35 35 [ S]cysteine and Expre S S label and then chased for 0, 10, 30, or 60 min in the presence of 0.5 mM cycloheximide. Cells were then harvested in the presence of 20 mM NEM, and apo(a) was immunoprecipitated. Samples were analyzed by 3–10% SDS-PAGE, with (reduced) or without (non-reduced) prior reduction with 2-mercaptoethanol, as described under “Experimental Procedures.” The positions of the precursor (pr) and mature (mt) forms of apo(a) are indicated, as well as a high molecular weight apo(a)-containing complex (comp.) and nonspecifically immunopre- cipitated proteins (ns) in the nonreduced samples. approximately the size of an I isoform. transported to the cell surface (21, 39, 42, 43). Differences in Steady-state Labeling—For steady-state labeling experiments, hepa- the efficiency with which apo(a) allelic variants undergo post- tocytes were incubated for 20 h in SFM containing 0.1 3 the normal translational processing accounts, at least partially, for the concentration of methionine and cysteine plus 125 mCi/ml each of inverse correlation between apo(a) size and plasma Lp(a) level 35 35 35 [ S]cysteine and Expre S S label. Culture media were clarified at (21). In addition, “transcript positive null” apo(a) phenotypes, 2000 3 g for 10 min and adjusted to 1% Nonidet P-40. Cells were in which there is no detectable plasma Lp(a), but which are washed with PBS and lysed in 2 ml of extraction buffer (50 mM Tris, 150 mM NaCl, 1% Nonidet P-40, pH 9.0). associated with substantial levels of apo(a) mRNA in the liver Effect of Glycosylation Inhibitors—To analyze the influence of glyco- (9, 39), are explained by the production of defective apo(a) sylation inhibitors on apo(a) maturation and secretion, hepatocytes proteins that are unable to exit the ER and are retained and were preincubated for1hin methionine and cysteine-free SFM in the degraded inside the cell (21). Thus, the efficiency with which presence or absence of tunicamycin (5 mg/ml), castanospermine, or apo(a) leaves the ER is a major determinant of plasma Lp(a) deoxymannojirimycin (1 mM each). The cells were then labeled for 30 levels. min in the same media containing 125 mCi/ml each of [ S]cysteine and 35 35 Expre S S label and then chased in complete SFM plus drugs for 5 h. The ER lumen provides a specialized environment to pro- Culture media and cells were harvested as described above. mote the folding and assembly of newly synthesized transmem- Analysis of Apo(a) Folding—Apo(a) folding was initially analyzed brane and secretory proteins. Misfolded and unassembled pro- essentially as described by Braakman et al. (46). Hepatocytes were teins are retained and degraded in the ER (44). Considering the preincubated for1hin methionine and cysteine-free SFM and then complex structure of the apo(a) glycoprotein, we hypothesized labeled for 10 min in the same medium containing 125 mCi/ml each of 35 35 35 that the long ER residence time of apo(a) may be due to the [ S]cysteine and Expre S S label. The cells were then either har- vested immediately or were washed once with PBS and chased for extended time required to reach its correct conformation. Dif- various periods in complete SFM containing 0.5 mM cycloheximide. To ferences in the proficiency with which apo(a) allelic variants harvest, the cells were placed on ice, washed twice with ice-cold phos- fold may account for their varying abilities to escape the ER. To phate-buffered saline (PBS) containing 20 mM N-ethylmaleimide test this hypothesis, we analyzed the folding of a number of (NEM), and then lysed in 1 ml of ice-cold extraction buffer containing 20 apo(a) allelic variants in the ER. mM NEM. In later experiments, this protocol was modified to optimize detection of apo(a) folding intermediates. The experiments were per- EXPERIMENTAL PROCEDURES formed as described above, except that the cells were labeled for 1 h. 35 35 35 Materials—[ S]Cysteine and Expre S S label were from DuPont Five minutes before the end of labeling, 5 mM dithiothreitol (DTT) was NEN. Protein A-agarose was from Repligen Corp. (Cambridge, MA). added to the cultures. The hepatocytes were then washed twice with Sheep anti-human apoB was from Boehringer Mannheim, and goat PBS, chased, and harvested, as above. anti-human Lp(a) was from Biodesign (Kennebunkport, ME). Methio- Immunoprecipitation and SDS-PAGE—Samples were immunopre- nine and cysteine-free Williams medium E was purchased from Life cipitated exactly as described previously (42). Prior to electrophoresis, Technologies, Inc. Tunicamycin was from Sigma, and castanospermine immunoprecipitates were heated to 100 °C for 5 min in gel sample and deoxymannojirimycin were from Genzyme Corp. (Cambridge, MA). buffer with (reduced samples) or without (nonreduced samples) 10% All other chemicals were of analytical grade. 2-mercaptoethanol. Proteins were resolved by 3–10% SDS-PAGE and Hepatocyte Isolation and Culture—Hepatocytes were isolated as de- fluorography (42). scribed previously (45). Lobectomy was performed according to institu- Immunoblotting—Aliquots of cell lysates were resolved by 3–10% tional guidelines under general anesthesia; ketamine hydrochloride SDS-PAGE, either with or without prior reduction with 2-mercaptoeth- was used as an immobilizing agent, and anesthesia was maintained anol, and immunoblotted for apo(a), exactly as described previously with sodium pentobarbital. Analgesics were provided 48 h post-opera- (42). tively. Cells were cultured in a serum-free medium (SFM) formulation (formula III) as described previously (43), except for the omission of RESULTS thyrotropin releasing factor. All experiments were performed using Folding of Newly Synthesized Apo(a)—Apo(a) folding was confluent 60-mm dishes of cells, which had been in culture for 5–7 days. initially analyzed using a pulse-chase protocol, essentially as Apo(a) isoforms in the baboon are classified into 12 size groups; A, described by Braakman et al. (46). Baboon hepatocytes were the largest, through L, the smallest (40). Hepatocytes from seven dif- ferent animals were analyzed. Two animals expressed an I apo(a) labeled for 10 min with [ S]methionine and cysteine and isoform (plasma Lp(a) levels of 34 (Fig. 1) and 30 mg/dl (Figs. 2 and 7)). chased for 0, 10, 30, or 60 min in unlabeled medium. Cells were Three animals expressed an A isoform (19 mg/dl, Fig. 2C; 20 mg/dl, Fig. lysed in the presence of the alkylating agent, NEM, to preserve 2D; 17 mg/dl, Fig. 3), and one expressed a C isoform (34 mg/dl, Fig. 2B). partially disulfide-bonded apo(a) folding intermediates, and Two animals (Fig. 4) had plasma Lp(a) levels ,2 mg/dl and no apo(a) apo(a) was immunoprecipitated and analyzed, with or without detectable in their plasma by immunoblotting. Northern analysis of prior reduction with 2-mercaptoethanol, by 3–10% SDS-PAGE. hepatic RNA from these null phenotype animals revealed a single apo(a) mRNA species in each case, which gave rise to apo(a) proteins Fig. 1 shows results obtained with hepatocytes expressing a 5050 Apo(a) Folding FIG.2. Folding of apo(a) after post-translational reduction. Hepatocytes expressing the following apo(a) isoforms were analyzed; A 5 I; 35 35 35 B 5 C; C 5 A; D 5 A. Cells were labeled for 1 h with 125 mCi/ml each of [ S]cysteine and Expre S S label. During the last 5 min of incubation, 5mM DTT was added to the culture media. The cells were then washed twice with PBS and chased for up to 60 min (A) or 120 min (B–D) before harvest in the presence of NEM. Apo(a) was immunoprecipitated and analyzed by 3–10% SDS-PAGE, with or without prior reduction with 2-mercaptoethanol. For each experiment, an additional 0 time point was included, and the sample was immunoprecipitated with preimmune goat serum (NS) to control for the specificity of the apo(a) antibody. The positions of the precursor (pr), mature (mt), and complexed (comp.) forms of apo(a) are indicated. Arrowheads indicate the relative electrophoretic mobility of apoB100 (550 kDa). low molecular weight apo(a) isoform (I isoform), the mature mobility also appeared (comp., Fig. 1). Control immunoprecipi- form of which has a molecular mass similar to that of apoB (550 tations and immunoblotting experiments (see below) confirmed kDa). When samples were analyzed after reduction, a single that this band contains apo(a). Since it disappears upon reduc- apo(a) protein, representing the newly synthesized apo(a) pre- tion, it most likely represents a disulfide-linked complex of cursor, could be seen at the 0, 10, and 30 min time points. At 60 apo(a) with itself or with another protein(s). While in this form, min, the fully glycosylated, mature form of apo(a) also ap- apo(a) apparently continued to fold, since the mobility of the peared (Fig. 1). Mature apo(a) contains fully processed complex also increased with time (Fig. 1, and see below). The N-linked and O-linked glycans, and its appearance is a marker mobility of monomeric apo(a) did not reach a maximum until 60 for movement of apo(a) out of the ER and through the Golgi min of chase, concomitant with the appearance of the mature apparatus (42). Densitometric scanning of the autoradiograph protein. Mature apo(a) had a mobility between that of the determined that 13% of apo(a) was in the mature form at 60 precursor’s monomeric and complexed forms (Fig. 1). In com- min, consistent with our previous studies in which we analyzed parison to other characterized secretory proteins, apo(a) has a apo(a) proteins of similar size (21). An almost undetectable greatly prolonged ER residence time. These data demonstrate decrease in the electrophoretic mobility of the reduced apo(a) that the long ER residence time of apo(a) coincides with a precursor was observed with increasing chase times (Fig. 1). prolonged period required for apo(a) to fold. Control experiments established that this was due to decreased Apo(a) Folding after Post-translational Reduction—Due to binding of NEM at later time points as a result of increased the short labeling time required, the above folding assay was intramolecular disulfide formation. NEM presumably in- not sensitive enough to resolve folding intermediates of most creases the electrophoretic mobility of apo(a) by enhancing apo(a) variants. Secretory proteins in the ER of living cells can binding of SDS through electrostatic interaction. be rapidly reduced by addition of the membrane-permeable When analyzed under nonreducing conditions, a single form reducing agent, DTT (48). This effect is reversible; on removal of apo(a) was again seen immediately after the pulse (Fig. 1). of DTT, the reduced proteins fold and are secreted normally Nonreduced apo(a) exhibited an increased mobility in compar- (47–50). To increase the sensitivity of the apo(a) folding assay, ison to the fully reduced protein, indicating some limited co- an alternative protocol was adopted in which hepatocytes were translational disulfide bond formation (46, 47). The electro- labeled for 1 h with [ S]cysteine and -methionine. During the phoretic mobility of nonreduced apo(a) increased at later chase last 5 min of labeling, 5 mM DTT was added to the cultures to times, reflecting the formation of further disulfide bonds. At reduce and unfold radiolabeled apo(a) that had accumulated in the 30-min time point, a form of apo(a) with a greatly decreased the ER. The cells were then washed and chased for times Apo(a) Folding 5051 between 0 and 120 min, and apo(a) folding intermediates were protein appearing at 60 min of chase. (9% of apo(a) had ma- resolved as in Fig. 1. tured at this time point. The absence of radiolabeled mature Fig. 2A shows results obtained with hepatocytes expressing apo(a) after 55 min of labeling may appear contradictory to Fig. the same (I) apo(a) isoform as in Fig. 1. Under reducing condi- 1, where mature apo(a) was easily seen at 60 min. However, the tions, apo(a) folding was seen as a slight decrease in electro- experiment in Fig. 1 also included a 10-min pulse, making the phoretic mobility (“NEM shift”), with the mature form of the 60-min chase time in that experiment effectively a 70-min time point.) Under nonreducing conditions, apo(a) folding was seen as a progressive increase in apo(a) electrophoretic mobility, reaching a maximum at 60 min of chase, coincident with the appearance of the mature protein. The high molecular weight apo(a)-containing complex was also observed, this time as early as 10 min of chase (Fig. 2A). Treatment with 5 mM DTT did not fully unfold apo(a), since the mobility of apo(a) at the start of the chase was greater than that of the fully reduced protein (Fig. 2A). Increasing the concentration of DTT to 20 mM did not cause any further decrease in apo(a) electrophoretic mobility (data not shown). This suggests the presence of a number of disulfide bonds in oxidized apo(a) that are not readily accessi- ble to reduction by DTT. The kinetics and pattern of apo(a) folding observed with the two different protocols were almost identical. The increased sensitivity achieved with the latter assay allowed the folding of a number of apo(a) allelic variants to be compared. Influence of Apo(a) Size on Folding Pattern—Our previous studies demonstrated that large apo(a) isoforms tend to have longer ER residence times than small isoforms (21). The kinet- ics and patterns of folding of large and small apo(a) proteins were therefore compared (Fig. 2). Similar to the I isoform depicted in Fig. 2A, folding of a C (Fig. 2B) and two A (Fig. 2,C FIG.3. Apo(a) folding and maturation times do not always and D) apo(a) isoforms was observed as a barely discernible coincide. Hepatocytes expressing an A isoform of apo(a) were ana- NEM shift in reduced samples. However, in contrast to the I lyzed. A, cells were labeled to steady state as described under “Exper- imental Procedures.” Apo(a) was then immunoprecipitated from the cell isoform, and consistent with their larger size (21), less than 1% lysate and culture medium and analyzed by 3–10% SDS-PAGE after of each of these isoforms had matured by1hof chase. By the reduction with 2-mercaptoethanol. B and C, apo(a) folding was ana- 2-h chase time point, only 5, 4, and 6% (Fig. 2,B–D, respec- lyzed after post-translational reduction with DTT, exactly as described tively) of the proteins were in the mature form. Under nonre- in the legend to Fig. 2, except that an additional chase time of 180 min was included. Samples were analyzed with (B) or without (C) prior ducing conditions, a similar pattern of apo(a) folding interme- reduction with 2-mercaptoethanol, as described under “Experimental diates as that observed for the I isoform was seen for the C and Procedures.” The positions of the precursor (pr apo(a)), mature (apo(a)), A isoforms. In each case, the electrophoretic mobility of apo(a) and complexed (comp.) forms of apo(a) and of apoB100 (550 kDa; ar- increased with time, reaching a maximum by 60 min of chase rowhead) are indicated. NS, preimmune serum immunoprecipitation control. c, cell lysates; m, culture media. (Fig. 2,B–D). A high molecular weight apo(a)-containing com- FIG.4. Null apo(a) proteins show normal folding patterns. Two sets of hepatocytes (I and II), each expressing a single null apo(a) protein were analyzed. A, hepatocytes were labeled to steady state, and apo(a) and apoB (550 kDa) were immunoprecipitated from the cell ly- sates and culture media, as described un- der “Experimental Procedures.” B and C, apo(a) folding was analyzed after post- translational reduction with DTT, exactly as described in the legend to Fig. 2. Sam- ples were analyzed with (B) or without (C) prior reduction with 2-mercaptoethanol. The positions of apoB, the apo(a) precur- sor (pr apo(a)) and the high molecular weight apo(a)-containing complex (comp.) are indicated. X, a protein nonspecifically immunoprecipitated from culture media; NS, preimmune serum immunoprecipita- tion control; c, cell lysates; m, culture media. 5052 Apo(a) Folding FIG.5. Analysis of null apo(a) folding in the absence of DTT and by immunoblotting. Hepatocytes expressing null protein II (Fig. 4) were analyzed. A, apo(a) folding in the absence of DTT was analyzed exactly as described in the legend to Fig. 1. B, unlabeled hepatocytes FIG.6. Role of N-linked glycans in apo(a) secretion. A, sche- were treated for 5 min with 5 mM DTT, washed twice with PBS, and matic representation of N-linked carbohydrate addition and trimming chased for 0, 60, or 120 min before harvest in the presence of NEM. in the ER and the site of action of the inhibitors tunicamycin (TM) and Aliquots of each cell lysate were then resolved by 3–10% SDS-PAGE castanospermine (CST). B, hepatocytes expressing a B apo(a) isoform and immunoblotted for apo(a), as described under “Experimental Pro- were labeled for 30 min and chased for5hinthe presence of 5 mg/ml TM cedures.” In each experiment, samples were analyzed with (reduced)or or1mM CST or deoxymannojirimycin (dNMN), and apo(a) was immu- without (non-reduced) prior reduction with 2-mercaptoethanol. The po- noprecipitated from the cell lysates (c) and culture media (m), as de- sition of the apo(a) precursor (pr apo(a)), the apo(a)-containing complex scribed under “Experimental Procedures.” The positions of the precur- (comp.) and apoB100 (550 kDa; arrowhead) are indicated. ns, preim- sor (pr apo(a)) and mature (apo(a)) forms of apo(a) and of apoB100 (550 mune serum immunoprecipitation control. kDa) are indicated. plex was observed for each isoform, and in most experiments, the complex increased in mobility with time, suggesting that ER and are completely retained and degraded inside the cell apo(a) continued to fold while in this form (Fig. 2, C–D). Thus, (21). To determine whether this phenotype is caused by aber- large and small apo(a) isoforms follow similar folding path- rant folding, the folding of two null apo(a) proteins was ana- ways, and differences in their folding kinetics do not appear to lyzed (Fig. 4). The proteins were of similar size, which, based explain the large variation in their ER residence times. upon electrophoretic mobility, was equivalent to that of an I Analysis of other allelic variants further exemplified the lack apo(a) isoform precursor. Steady-state labeling of hepatocytes of correlation between apo(a) ER residence time and folding expressing these proteins confirmed the null phenotype (Fig. kinetics. We have identified apo(a) variants that take longer 4A). In each case a single apo(a) protein representing the un- than expected to exit the ER. An example of an A isoform with processed null apo(a) precursor (21) was observed in the cell an unusually long ER residence time is shown in Fig. 3. Steady- lysate, and no apo(a) was detectable in the culture medium state labeling confirmed the maturation and secretion of this (Fig. 4A). ApoB was efficiently secreted from each cell type, allelic variant (Fig. 3A). When folding was analyzed, the ex- demonstrating normal functioning of the secretory pathway pected NEM shift was observed in reduced samples (Fig. 3B). (Fig. 4A). However, even after3hof chase, only trace amounts of mature Unexpectedly, the pattern and kinetics of folding of the null apo(a) could be seen (visible on the original autoradiograph, but proteins were essentially indistinguishable from those of se- not sufficient to be reproduced photographically). In nonre- creted apo(a) proteins. Under reducing conditions (Fig. 4B) duced samples (Fig. 3C), the kinetics and pattern of folding for folding was observed as an NEM shift, although no mature this isoform were indistinguishable from those of apo(a) pro- apo(a) was seen, even after2hof chase (Fig. 4B). Under teins with much shorter ER residence times (compare Fig. 3C nonreducing conditions (Fig. 4C), apo(a) folding was observed with Fig. 2A), and apparently completely folded apo(a) was as an increase in electrophoretic mobility with time, reaching a present by 60 min of chase (Fig. 3C). Thus, although the pro- maximum by 60 min of chase. High molecular weight apo(a)- longed ER residence time of apo(a) per se is due to a prolonged containing complexes were also apparent in each case. Thus, period required for folding, differences in apo(a) ER residence despite their inability to exit the ER, these null apo(a) proteins times, at least of the allelic variants studied to date, cannot be follow an apparently normal folding pathway. A gross inability explained by differences in apo(a) folding kinetics. to fold thus does not explain the null apo(a) phenotype. Some small differences in the apo(a) folding patterns were Null apo(a) protein II in Fig. 4 was synthesized at a high observed. The isoforms varied in the proportion of the protein level in comparison to most apo(a) allelic variants. Analysis of that entered the high molecular weight complex. In some cases, the folding of this protein in unperturbed cells (no DTT treat- there was increased heterogeneity in the complex, forming a ment), as described for the secreted apo(a) isoform in Fig. 1, smear toward the top of the gels, which may indicate some also revealed an apparently normal folding pathway (Fig. 5A). aggregate formation. Further experimentation will be required In this case, the high molecular weight apo(a) containing com- to determine whether the extent of aggregate or complex for- plex was apparent as early as the 0 chase time point, suggest- mation correlates with the extent of apo(a) intracellular ing that the complex forms during, or immediately after, trans- degradation. lation. This experiment confirms that the use of DTT did not Folding of Null Apo(a) Proteins—Apo(a) proteins giving rise artificially induce a normal folding pathway for the null pro- to the transcript positive null phenotype are unable to exit the teins. The folding of null apo(a) protein II was also analyzed by Apo(a) Folding 5053 FIG.7. Influence of CST on apo(a) folding. Apo(a) folding in hepatocytes ex- pressing an I apo(a) isoform cultured in the presence or absence of 1 mM CST was analyzed after post-translational reduc- tion with DTT exactly as described in the legend to Fig. 2. Samples were analyzed by 3–10% SDS-PAGE with (reduced)or without (non-reduced) prior reduction with 2-mercaptoethanol. The positions of the precursor (pr), mature (mt), and com- plexed (comp.) forms of apo(a) and of apoB100 (550 kDa; arrowhead) are indicated. immunoblotting (Fig. 5B). A single form of apo(a) was seen small increase in apo(a) molecular weight in CST-treated cells, after DTT treatment in the nonreduced samples, whereas at most notable in the nonreduced samples, was due to the pres- the later time points, proteins corresponding to fully folded ence of the untrimmed glucose residues. The only other differ- apo(a) and the high molecular weight complex were recognized ence between CST and control samples was the absence of by the anti-apo(a) antibody. This confirms the identity of the mature apo(a) in CST-treated cells (Fig. 7). Thus, CST induced intermediates observed in radiolabeled folding experiments as an apo(a) phenotype similar to that of a null apo(a) protein. apo(a). Combined with the results in Figs. 1–4, this suggests that an An interesting observation for each of the null proteins was event that is dependent on the trimming of glucose residues the presence of a minor band below the null apo(a) protein in from N-linked carbohydrate on apo(a) and that occurs after the the cell lysate (Fig. 4). This band was not observed with nor- bulk of apo(a) folding is required for apo(a) secretion. mally secreted apo(a) proteins (Figs. 1 and 2, and data not DISCUSSION shown) and may be a marker for the null phenotype. In addi- tion, a second high molecular weight apo(a)-containing band We have previously demonstrated that newly synthesized was apparent in nonreduced samples from null animal I. Fur- apo(a) has a prolonged residence time in the ER and that allelic ther experimentation will be required to determine the signif- differences in the ability of apo(a) to escape this compartment icance of these observations. are a major determinant of plasma Lp(a) levels (21, 39, 42). Influence of Glycosylation Inhibitors on Apo(a) Maturation Since only correctly folded proteins are permitted to exit the ER and Folding—Apo(a) is highly glycosylated. N-Linked glycosy- (44), we examined the folding of newly synthesized apo(a) and lation occurs co-translationally in the ER lumen (51) and plays the influence of allelic variation on the kinetics and efficiency of a significant role in the folding and secretion of some glycopro- this process. The results revealed a complex and kinetically teins. To begin to investigate the factors that influence the exit extended apo(a) folding pathway. Unexpectedly, no significant of apo(a) from the ER, the role of N-linked glycans in apo(a) differences were found in the kinetics and patterns of folding of maturation and secretion was examined. Two inhibitors were a variety of apo(a) allelic variants. Even null apo(a) proteins, used, tunicamycin (TM), which prevents N-linked glycosylation which fail to escape the ER, appeared to fold normally. These (52), and castanospermine (CST), which inhibits the trimming studies suggest that an event downstream of the folding events of glucose residues from N-linked glycans in the ER (53) (Fig. detectable in our assay must occur for apo(a) to be secreted and 6A). Hepatocytes expressing a B apo(a) isoform were labeled for that allelic differences in the ability of apo(a) to undergo this 30 min and then chased for5hinthe presence of TM or CST, event contribute to the extreme inter-individual variation in and apo(a) in the cell lysates and in the culture media was Lp(a) production rate. analyzed by immunoprecipitation. The effect of deoxyman- The variously disulfide-bonded folding intermediates of nojirimycin, which inhibits the trimming of mannose residues apo(a) were detected in pulse-chase experiments by exploiting from N-linked glycans after transport to the Golgi apparatus differences in their electrophoretic mobility on SDS-PAGE. For (53), was examined as a control. many proteins, folding intermediates are hard to resolve, due TM caused a reduction in the molecular weight of the apo(a) to small differences in electrophoretic mobility or to rapid fold- precursor, due to inhibition of N-linked glycosylation, and com- ing kinetics (46, 49, 54, 55). However, its large size and numer- pletely prevented apo(a) maturation and secretion (Fig. 6B). ous disulfides made apo(a) ideally suited to this type of analy- CST also prevented maturation of apo(a) (Fig. 6B). Thus, both sis. Our experiments revealed that the majority of disulfides in addition and trimming of N-linked carbohydrate are required apo(a) were formed post-translationally and that folding was for apo(a) to exit the ER. In contrast, deoxymannojirimycin did not complete until 30–60 min after synthesis. A unique char- not prevent apo(a) secretion (Fig. 6B). acteristic was the formation of a high molecular weight, disul- The anti-apo(a) antibody was unable to efficiently immuno- fide-linked, apo(a)-containing complex. The complex seems un- precipitate reduced, unglycosylated apo(a), so the effect of TM likely to represent aggregated apo(a), since apo(a) apparently on apo(a) folding could not be determined. The folding pattern continued to fold while in this form. The complex was confined of apo(a) in the presence of CST, however, was almost indistin- to the ER, since only a single form of mature apo(a) was guishable from that in control cells (Fig. 7); in each case, folding observed. Potential roles of the complex could be in targeting was observed as an NEM shift in reduced samples and as a apo(a) for secretion, ER retention, or degradation. Alterna- gradual increase in electrophoretic mobility, reaching a maxi- tively, the complex could be involved in protecting Cys-4057 in mum at 60 min, in nonreduced samples. The high molecular apo(a), which is required for disulfide formation with apoB (56, weight apo(a)-containing complex was also clearly visible. The 57), from inappropriate inter- or intramolecular interactions 5054 Apo(a) Folding during the folding process. Comparison of reduced and nonre- arrangement of disulfide bonds, may be required. duced apo(a) immunoprecipitates did not identify other radio- To begin to dissect the precise requirements for apo(a) to exit labeled proteins that could be a component of the complex (data the ER, we analyzed the role of N-linked carbohydrate in apo(a) not shown). However, ER resident proteins tend to have long secretion. Both addition and proper processing of N-linked glycans was found to be required for apo(a) to escape the ER, half-lives and may not incorporate sufficient radioactivity for detection during the short labeling periods used. Future stud- since both TM, which prevents addition of N-linked glycans (52), and CST, which inhibits the trimming of glucose residues ies will address the composition of the complex and its role in from N-linked glycans in the ER (53), prevented apo(a) matu- apo(a) secretion. ration and secretion. TM causes aggregation of some secretory Similar patterns and kinetics of apo(a) folding were observed proteins (59). We were unable to ascertain the influence of TM whether analyzed in unperturbed cells or after post-transla- on apo(a) folding due to lack of recognition of reduced, ungly- tional reduction of apo(a) with DTT. The latter assay allowed cosylated apo(a) by the anti-apo(a) antibody. This in itself could longer labeling times to be used and permitted comparison of be indicative of aggregate formation, but further studies will be the folding patterns of multiple apo(a) allelic variants. Similar required to address this issue. CST prevents interaction of protocols have been used to study the folding of a number of secretory proteins with the ER chaperone, calnexin, which secretory and transmembrane proteins (47–50). In each case recognizes substrates by binding to monoglucosylated N-linked the characteristic folding pattern is unchanged by DTT carbohydrate side chains (60). Although CST prevented apo(a) treatment. secretion, it had no apparent effect on the pattern or kinetics of Unexpectedly, our studies failed to reveal any significant apo(a) folding. Again, this could reflect our inability to detect differences in the kinetics and patterns of folding of apo(a) more subtle conformational changes in apo(a) caused by CST proteins with very different ER residence times and efficiencies and may suggest a role for calnexin in apo(a) secretion. Alter- of secretion. We had expected that large apo(a) isoforms would natively, CST may inhibit an event downstream of folding, take longer to fold than small isoforms and that folding and which requires carbohydrate trimming, and which is essential maturation times would coincide. However, this was not the for apo(a) secretion. Clearly, further studies will be required to case. A particularly striking example was the A apo(a) isoform address the many outstanding questions regarding the require- analyzed in Fig. 3. Although folding was apparently complete ments for apo(a) exit from the ER and the precise mechanism by 60 min, this protein did not begin to leave the ER until at by which apo(a) allelic variation influences the kinetics and least3hof chase. We had also anticipated that null apo(a) efficiency of this process. proteins would exhibit aberrant folding patterns. However, all null proteins examined to date appear to fold normally, yet are Acknowledgment—We thank Larry Estlack for excellent technical retained and degraded in the ER. assistance. Several hypotheses can be put forward to explain these ob- REFERENCES servations. The bulk of the apo(a) protein consists of a tandem 1. Berg, K. (1963) Acta Pathol. Microbiol. Scand. 59, 369–382 array of identical K4 domains (7), variation in the number of 2. Utermann, G. (1989) Science 246, 904–910 which is responsible for the size polymorphism of apo(a) (3). 3. Lackner, C., Cohen, J. C., and Hobbs, H. H. (1993) Hum. Mol. 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Rainwater, D. L., Manis, G. S., and VandeBerg, J. L. (1989) J. Lipid Res. 30, 60. Bergeron, J. J. M., Brenner, M. B., Thomas, D. Y., and Williams, D. B. (1994) 549–558 Trends Biochem. Sci. 19, 124–128 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Influence of Allelic Variation on Apolipoprotein(a) Folding in the Endoplasmic Reticulum

White, Ann L.; Guerra, Bernadette; Lanford, Robert E.
Journal of Biological Chemistry – Feb 1, 1997
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 8, Issue of February 21, pp. 5048–5055, 1997 © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Influence of Allelic Variation on Apolipoprotein(a) Folding in the Endoplasmic Reticulum* (Received for publication, May 17, 1996, and in revised form, October 29, 1996) Ann L. White‡, Bernadette Guerra, and Robert E. Lanford From the Department of Virology and Immunology, Southwest Foundation for Biomedical Research, San Antonio, Texas 78227 Plasma levels of lipoprotein(a) (Lp(a)) vary over 1000- approximately 12 to 51 (3). Each K4 repeat contains 3 internal fold between individuals and are determined by the disulfide bonds, 1 N-linked, and 6 potential O-linked glycosy- gene for its unique apolipoprotein, apo(a), which has lation sites (7). Thus, a large apo(a) isoform may contain in greater than 100 alleles. Using primary baboon hepato- excess of 150 disulfide bonds, 50 N-linked and 300 O-linked cyte cultures, we previously demonstrated that differ- carbohydrate side chains. ences in the ability of apo(a) allelic variants to escape Lp(a) is only found in the plasma of primates (12) and the the endoplasmic reticulum (ER) are a major determi- hedgehog (13, 14). In humans, plasma levels of Lp(a) vary from nant of Lp(a) production rate. To examine the reason for ,1to .100 mg/dl and are highly heritable (15). High plasma these differences, the folding of newly synthesized levels of Lp(a) are associated with an increased incidence of apo(a) was analyzed in pulse-chase experiments. Sam- cardiovascular diseases (for review, see Ref. 16). Greater than ples were harvested in the presence of N-ethylmaleim- 90% of the inter-individual variation in Lp(a) concentration is ide to preserve disulfide-bonded folding intermediates, attributable to the apo(a) gene locus (17). There is an inverse and apo(a) was analyzed by immunoprecipitation and correlation between apo(a) size and plasma Lp(a) level (5, 6, 10, SDS-polyacrylamide gel electrophoresis. Apo(a) re- 18); however, this relationship is not absolute (6, 10), and a quired a prolonged period (30–60 min) to reach its fully oxidized form. Multiple folding intermediates were re- particular isoform size may be associated with as much as a solved, including a disulfide-linked, apo(a)-containing 200-fold difference in plasma Lp(a) concentrations in different complex. Unexpectedly, all allelic variants examined individuals (19). Thus, sequence variations at the apo(a) locus showed similar patterns and kinetics of folding. Even independent of size also influence Lp(a) levels (20). “Null” “null” apo(a) proteins, which are unable to exit the ER, apo(a) alleles, which do not give rise to any detectable plasma appeared to fold normally. The ER glucosidase inhibi- protein (5, 9), and which are distributed throughout the apo(a) tor, castanospermine, prevented apo(a) secretion, but allele size range (18, 21), are an extreme example of apo(a) did not inhibit folding. This suggests that an event sequence variations that influence Lp(a) levels. A number of which is dependent on trimming of N-linked glucoses, polymorphisms have recently been identified at the apo(a) lo- and which occurs after the folding events detectable in cus (20, 22–31), and it is estimated that there are greater than our assay, is required for apo(a) secretion. Differences 100 apo(a) alleles (20). However, as yet, no identified polymor- in the ability to undergo this event may explain the phism other than K4 number has been demonstrated to di- variable efficiency with which apo(a) allelic variants rectly influence circulating Lp(a) levels. exit the ER. Lp(a) is synthesized by the liver (32–34), and the rate of Lp(a) production determines plasma Lp(a) concentration (35– 37). Due to the lack of a small animal model and the highly Apolipoprotein(a) (apo(a)) is a highly polymorphic, high mo- polymorphic nature of the apo(a) gene, the mechanisms gov- lecular weight glycoprotein that circulates in plasma as a com- erning Lp(a) production rate have remained poorly character- ponent of lipoprotein(a) (Lp(a)). Lp(a) is composed of low den- ized. Some of the variation in Lp(a) production rate is deter- sity lipoprotein in which apoB is attached to apo(a) by disulfide mined by differences in hepatic apo(a) mRNA concentration (9, linkage (1, 2). As many as 34 different isoforms of apo(a) have 38). “Transcript negative null” alleles that do not produce any been identified in human plasma, which vary in size from ,300 detectable hepatic apo(a) mRNA transcript (9, 39) represent an to .800 kDa (3–6). Apo(a) has a highly complex, repetitive extreme example of this type of regulation. Post-transcriptional structure and is homologous with plasminogen (7). Apo(a) con- mechanisms are also important (38). A sequence polymorphism tains an inactive copy of the plasminogen protease domain and in the 59-untranslated region of human apo(a) was recently a single plasminogen kringle 5 (K5) domain, preceded by mul- shown to influence the translation efficiency of apo(a) mRNA in tiple domains with homology to plasminogen K4 (7). The size vitro (31). variation of apo(a) is due to differences in the number of K4 Baboons show very similar characteristics to humans in domains encoded in the apo(a) gene (8–11), which varies from terms of plasma Lp(a) levels and apo(a) isoform sizes (40, 41). We have established primary cultures of baboon hepatocytes as * This research was supported by National Institutes of Health Grant a model system for the analysis of Lp(a) biogenesis (34). Since HL50426. The costs of publication of this article were defrayed in part hepatocytes can be isolated from animals with selected Lp(a) by the payment of page charges. This article must therefore be hereby phenotypes, this provides us with a unique opportunity to marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. examine the influence of apo(a) allelic variation on Lp(a) pro- ‡ To whom correspondence should be addressed. duction rate. Using this system, we have demonstrated that, in The abbreviations used are: apo, apolipoprotein; CST, castanosper- comparison to other secretory proteins, newly synthesized mine; PBS, phosphate-buffered saline; DTT, dithiothreitol; ER, endo- apo(a) has a prolonged residence time in the endoplasmic re- plasmic reticulum; Lp, lipoprotein; NEM, N-ethylmaleimide; PAGE, polyacrylamide gel electrophoresis; TM, tunicamycin. ticulum (ER) before it is processed to its mature form and 5048 This paper is available on line at http://www-jbc.stanford.edu/jbc/ This is an Open Access article under the CC BY license. Apo(a) Folding 5049 FIG.1. Folding of newly synthesized apo(a). Hepatocytes expressing an I isoform of apo(a) were labeled for 10 min with 125 mCi/ml each of 35 35 35 [ S]cysteine and Expre S S label and then chased for 0, 10, 30, or 60 min in the presence of 0.5 mM cycloheximide. Cells were then harvested in the presence of 20 mM NEM, and apo(a) was immunoprecipitated. Samples were analyzed by 3–10% SDS-PAGE, with (reduced) or without (non-reduced) prior reduction with 2-mercaptoethanol, as described under “Experimental Procedures.” The positions of the precursor (pr) and mature (mt) forms of apo(a) are indicated, as well as a high molecular weight apo(a)-containing complex (comp.) and nonspecifically immunopre- cipitated proteins (ns) in the nonreduced samples. approximately the size of an I isoform. transported to the cell surface (21, 39, 42, 43). Differences in Steady-state Labeling—For steady-state labeling experiments, hepa- the efficiency with which apo(a) allelic variants undergo post- tocytes were incubated for 20 h in SFM containing 0.1 3 the normal translational processing accounts, at least partially, for the concentration of methionine and cysteine plus 125 mCi/ml each of inverse correlation between apo(a) size and plasma Lp(a) level 35 35 35 [ S]cysteine and Expre S S label. Culture media were clarified at (21). In addition, “transcript positive null” apo(a) phenotypes, 2000 3 g for 10 min and adjusted to 1% Nonidet P-40. Cells were in which there is no detectable plasma Lp(a), but which are washed with PBS and lysed in 2 ml of extraction buffer (50 mM Tris, 150 mM NaCl, 1% Nonidet P-40, pH 9.0). associated with substantial levels of apo(a) mRNA in the liver Effect of Glycosylation Inhibitors—To analyze the influence of glyco- (9, 39), are explained by the production of defective apo(a) sylation inhibitors on apo(a) maturation and secretion, hepatocytes proteins that are unable to exit the ER and are retained and were preincubated for1hin methionine and cysteine-free SFM in the degraded inside the cell (21). Thus, the efficiency with which presence or absence of tunicamycin (5 mg/ml), castanospermine, or apo(a) leaves the ER is a major determinant of plasma Lp(a) deoxymannojirimycin (1 mM each). The cells were then labeled for 30 levels. min in the same media containing 125 mCi/ml each of [ S]cysteine and 35 35 Expre S S label and then chased in complete SFM plus drugs for 5 h. The ER lumen provides a specialized environment to pro- Culture media and cells were harvested as described above. mote the folding and assembly of newly synthesized transmem- Analysis of Apo(a) Folding—Apo(a) folding was initially analyzed brane and secretory proteins. Misfolded and unassembled pro- essentially as described by Braakman et al. (46). Hepatocytes were teins are retained and degraded in the ER (44). Considering the preincubated for1hin methionine and cysteine-free SFM and then complex structure of the apo(a) glycoprotein, we hypothesized labeled for 10 min in the same medium containing 125 mCi/ml each of 35 35 35 that the long ER residence time of apo(a) may be due to the [ S]cysteine and Expre S S label. The cells were then either har- vested immediately or were washed once with PBS and chased for extended time required to reach its correct conformation. Dif- various periods in complete SFM containing 0.5 mM cycloheximide. To ferences in the proficiency with which apo(a) allelic variants harvest, the cells were placed on ice, washed twice with ice-cold phos- fold may account for their varying abilities to escape the ER. To phate-buffered saline (PBS) containing 20 mM N-ethylmaleimide test this hypothesis, we analyzed the folding of a number of (NEM), and then lysed in 1 ml of ice-cold extraction buffer containing 20 apo(a) allelic variants in the ER. mM NEM. In later experiments, this protocol was modified to optimize detection of apo(a) folding intermediates. The experiments were per- EXPERIMENTAL PROCEDURES formed as described above, except that the cells were labeled for 1 h. 35 35 35 Materials—[ S]Cysteine and Expre S S label were from DuPont Five minutes before the end of labeling, 5 mM dithiothreitol (DTT) was NEN. Protein A-agarose was from Repligen Corp. (Cambridge, MA). added to the cultures. The hepatocytes were then washed twice with Sheep anti-human apoB was from Boehringer Mannheim, and goat PBS, chased, and harvested, as above. anti-human Lp(a) was from Biodesign (Kennebunkport, ME). Methio- Immunoprecipitation and SDS-PAGE—Samples were immunopre- nine and cysteine-free Williams medium E was purchased from Life cipitated exactly as described previously (42). Prior to electrophoresis, Technologies, Inc. Tunicamycin was from Sigma, and castanospermine immunoprecipitates were heated to 100 °C for 5 min in gel sample and deoxymannojirimycin were from Genzyme Corp. (Cambridge, MA). buffer with (reduced samples) or without (nonreduced samples) 10% All other chemicals were of analytical grade. 2-mercaptoethanol. Proteins were resolved by 3–10% SDS-PAGE and Hepatocyte Isolation and Culture—Hepatocytes were isolated as de- fluorography (42). scribed previously (45). Lobectomy was performed according to institu- Immunoblotting—Aliquots of cell lysates were resolved by 3–10% tional guidelines under general anesthesia; ketamine hydrochloride SDS-PAGE, either with or without prior reduction with 2-mercaptoeth- was used as an immobilizing agent, and anesthesia was maintained anol, and immunoblotted for apo(a), exactly as described previously with sodium pentobarbital. Analgesics were provided 48 h post-opera- (42). tively. Cells were cultured in a serum-free medium (SFM) formulation (formula III) as described previously (43), except for the omission of RESULTS thyrotropin releasing factor. All experiments were performed using Folding of Newly Synthesized Apo(a)—Apo(a) folding was confluent 60-mm dishes of cells, which had been in culture for 5–7 days. initially analyzed using a pulse-chase protocol, essentially as Apo(a) isoforms in the baboon are classified into 12 size groups; A, described by Braakman et al. (46). Baboon hepatocytes were the largest, through L, the smallest (40). Hepatocytes from seven dif- ferent animals were analyzed. Two animals expressed an I apo(a) labeled for 10 min with [ S]methionine and cysteine and isoform (plasma Lp(a) levels of 34 (Fig. 1) and 30 mg/dl (Figs. 2 and 7)). chased for 0, 10, 30, or 60 min in unlabeled medium. Cells were Three animals expressed an A isoform (19 mg/dl, Fig. 2C; 20 mg/dl, Fig. lysed in the presence of the alkylating agent, NEM, to preserve 2D; 17 mg/dl, Fig. 3), and one expressed a C isoform (34 mg/dl, Fig. 2B). partially disulfide-bonded apo(a) folding intermediates, and Two animals (Fig. 4) had plasma Lp(a) levels ,2 mg/dl and no apo(a) apo(a) was immunoprecipitated and analyzed, with or without detectable in their plasma by immunoblotting. Northern analysis of prior reduction with 2-mercaptoethanol, by 3–10% SDS-PAGE. hepatic RNA from these null phenotype animals revealed a single apo(a) mRNA species in each case, which gave rise to apo(a) proteins Fig. 1 shows results obtained with hepatocytes expressing a 5050 Apo(a) Folding FIG.2. Folding of apo(a) after post-translational reduction. Hepatocytes expressing the following apo(a) isoforms were analyzed; A 5 I; 35 35 35 B 5 C; C 5 A; D 5 A. Cells were labeled for 1 h with 125 mCi/ml each of [ S]cysteine and Expre S S label. During the last 5 min of incubation, 5mM DTT was added to the culture media. The cells were then washed twice with PBS and chased for up to 60 min (A) or 120 min (B–D) before harvest in the presence of NEM. Apo(a) was immunoprecipitated and analyzed by 3–10% SDS-PAGE, with or without prior reduction with 2-mercaptoethanol. For each experiment, an additional 0 time point was included, and the sample was immunoprecipitated with preimmune goat serum (NS) to control for the specificity of the apo(a) antibody. The positions of the precursor (pr), mature (mt), and complexed (comp.) forms of apo(a) are indicated. Arrowheads indicate the relative electrophoretic mobility of apoB100 (550 kDa). low molecular weight apo(a) isoform (I isoform), the mature mobility also appeared (comp., Fig. 1). Control immunoprecipi- form of which has a molecular mass similar to that of apoB (550 tations and immunoblotting experiments (see below) confirmed kDa). When samples were analyzed after reduction, a single that this band contains apo(a). Since it disappears upon reduc- apo(a) protein, representing the newly synthesized apo(a) pre- tion, it most likely represents a disulfide-linked complex of cursor, could be seen at the 0, 10, and 30 min time points. At 60 apo(a) with itself or with another protein(s). While in this form, min, the fully glycosylated, mature form of apo(a) also ap- apo(a) apparently continued to fold, since the mobility of the peared (Fig. 1). Mature apo(a) contains fully processed complex also increased with time (Fig. 1, and see below). The N-linked and O-linked glycans, and its appearance is a marker mobility of monomeric apo(a) did not reach a maximum until 60 for movement of apo(a) out of the ER and through the Golgi min of chase, concomitant with the appearance of the mature apparatus (42). Densitometric scanning of the autoradiograph protein. Mature apo(a) had a mobility between that of the determined that 13% of apo(a) was in the mature form at 60 precursor’s monomeric and complexed forms (Fig. 1). In com- min, consistent with our previous studies in which we analyzed parison to other characterized secretory proteins, apo(a) has a apo(a) proteins of similar size (21). An almost undetectable greatly prolonged ER residence time. These data demonstrate decrease in the electrophoretic mobility of the reduced apo(a) that the long ER residence time of apo(a) coincides with a precursor was observed with increasing chase times (Fig. 1). prolonged period required for apo(a) to fold. Control experiments established that this was due to decreased Apo(a) Folding after Post-translational Reduction—Due to binding of NEM at later time points as a result of increased the short labeling time required, the above folding assay was intramolecular disulfide formation. NEM presumably in- not sensitive enough to resolve folding intermediates of most creases the electrophoretic mobility of apo(a) by enhancing apo(a) variants. Secretory proteins in the ER of living cells can binding of SDS through electrostatic interaction. be rapidly reduced by addition of the membrane-permeable When analyzed under nonreducing conditions, a single form reducing agent, DTT (48). This effect is reversible; on removal of apo(a) was again seen immediately after the pulse (Fig. 1). of DTT, the reduced proteins fold and are secreted normally Nonreduced apo(a) exhibited an increased mobility in compar- (47–50). To increase the sensitivity of the apo(a) folding assay, ison to the fully reduced protein, indicating some limited co- an alternative protocol was adopted in which hepatocytes were translational disulfide bond formation (46, 47). The electro- labeled for 1 h with [ S]cysteine and -methionine. During the phoretic mobility of nonreduced apo(a) increased at later chase last 5 min of labeling, 5 mM DTT was added to the cultures to times, reflecting the formation of further disulfide bonds. At reduce and unfold radiolabeled apo(a) that had accumulated in the 30-min time point, a form of apo(a) with a greatly decreased the ER. The cells were then washed and chased for times Apo(a) Folding 5051 between 0 and 120 min, and apo(a) folding intermediates were protein appearing at 60 min of chase. (9% of apo(a) had ma- resolved as in Fig. 1. tured at this time point. The absence of radiolabeled mature Fig. 2A shows results obtained with hepatocytes expressing apo(a) after 55 min of labeling may appear contradictory to Fig. the same (I) apo(a) isoform as in Fig. 1. Under reducing condi- 1, where mature apo(a) was easily seen at 60 min. However, the tions, apo(a) folding was seen as a slight decrease in electro- experiment in Fig. 1 also included a 10-min pulse, making the phoretic mobility (“NEM shift”), with the mature form of the 60-min chase time in that experiment effectively a 70-min time point.) Under nonreducing conditions, apo(a) folding was seen as a progressive increase in apo(a) electrophoretic mobility, reaching a maximum at 60 min of chase, coincident with the appearance of the mature protein. The high molecular weight apo(a)-containing complex was also observed, this time as early as 10 min of chase (Fig. 2A). Treatment with 5 mM DTT did not fully unfold apo(a), since the mobility of apo(a) at the start of the chase was greater than that of the fully reduced protein (Fig. 2A). Increasing the concentration of DTT to 20 mM did not cause any further decrease in apo(a) electrophoretic mobility (data not shown). This suggests the presence of a number of disulfide bonds in oxidized apo(a) that are not readily accessi- ble to reduction by DTT. The kinetics and pattern of apo(a) folding observed with the two different protocols were almost identical. The increased sensitivity achieved with the latter assay allowed the folding of a number of apo(a) allelic variants to be compared. Influence of Apo(a) Size on Folding Pattern—Our previous studies demonstrated that large apo(a) isoforms tend to have longer ER residence times than small isoforms (21). The kinet- ics and patterns of folding of large and small apo(a) proteins were therefore compared (Fig. 2). Similar to the I isoform depicted in Fig. 2A, folding of a C (Fig. 2B) and two A (Fig. 2,C FIG.3. Apo(a) folding and maturation times do not always and D) apo(a) isoforms was observed as a barely discernible coincide. Hepatocytes expressing an A isoform of apo(a) were ana- NEM shift in reduced samples. However, in contrast to the I lyzed. A, cells were labeled to steady state as described under “Exper- imental Procedures.” Apo(a) was then immunoprecipitated from the cell isoform, and consistent with their larger size (21), less than 1% lysate and culture medium and analyzed by 3–10% SDS-PAGE after of each of these isoforms had matured by1hof chase. By the reduction with 2-mercaptoethanol. B and C, apo(a) folding was ana- 2-h chase time point, only 5, 4, and 6% (Fig. 2,B–D, respec- lyzed after post-translational reduction with DTT, exactly as described tively) of the proteins were in the mature form. Under nonre- in the legend to Fig. 2, except that an additional chase time of 180 min was included. Samples were analyzed with (B) or without (C) prior ducing conditions, a similar pattern of apo(a) folding interme- reduction with 2-mercaptoethanol, as described under “Experimental diates as that observed for the I isoform was seen for the C and Procedures.” The positions of the precursor (pr apo(a)), mature (apo(a)), A isoforms. In each case, the electrophoretic mobility of apo(a) and complexed (comp.) forms of apo(a) and of apoB100 (550 kDa; ar- increased with time, reaching a maximum by 60 min of chase rowhead) are indicated. NS, preimmune serum immunoprecipitation control. c, cell lysates; m, culture media. (Fig. 2,B–D). A high molecular weight apo(a)-containing com- FIG.4. Null apo(a) proteins show normal folding patterns. Two sets of hepatocytes (I and II), each expressing a single null apo(a) protein were analyzed. A, hepatocytes were labeled to steady state, and apo(a) and apoB (550 kDa) were immunoprecipitated from the cell ly- sates and culture media, as described un- der “Experimental Procedures.” B and C, apo(a) folding was analyzed after post- translational reduction with DTT, exactly as described in the legend to Fig. 2. Sam- ples were analyzed with (B) or without (C) prior reduction with 2-mercaptoethanol. The positions of apoB, the apo(a) precur- sor (pr apo(a)) and the high molecular weight apo(a)-containing complex (comp.) are indicated. X, a protein nonspecifically immunoprecipitated from culture media; NS, preimmune serum immunoprecipita- tion control; c, cell lysates; m, culture media. 5052 Apo(a) Folding FIG.5. Analysis of null apo(a) folding in the absence of DTT and by immunoblotting. Hepatocytes expressing null protein II (Fig. 4) were analyzed. A, apo(a) folding in the absence of DTT was analyzed exactly as described in the legend to Fig. 1. B, unlabeled hepatocytes FIG.6. Role of N-linked glycans in apo(a) secretion. A, sche- were treated for 5 min with 5 mM DTT, washed twice with PBS, and matic representation of N-linked carbohydrate addition and trimming chased for 0, 60, or 120 min before harvest in the presence of NEM. in the ER and the site of action of the inhibitors tunicamycin (TM) and Aliquots of each cell lysate were then resolved by 3–10% SDS-PAGE castanospermine (CST). B, hepatocytes expressing a B apo(a) isoform and immunoblotted for apo(a), as described under “Experimental Pro- were labeled for 30 min and chased for5hinthe presence of 5 mg/ml TM cedures.” In each experiment, samples were analyzed with (reduced)or or1mM CST or deoxymannojirimycin (dNMN), and apo(a) was immu- without (non-reduced) prior reduction with 2-mercaptoethanol. The po- noprecipitated from the cell lysates (c) and culture media (m), as de- sition of the apo(a) precursor (pr apo(a)), the apo(a)-containing complex scribed under “Experimental Procedures.” The positions of the precur- (comp.) and apoB100 (550 kDa; arrowhead) are indicated. ns, preim- sor (pr apo(a)) and mature (apo(a)) forms of apo(a) and of apoB100 (550 mune serum immunoprecipitation control. kDa) are indicated. plex was observed for each isoform, and in most experiments, the complex increased in mobility with time, suggesting that ER and are completely retained and degraded inside the cell apo(a) continued to fold while in this form (Fig. 2, C–D). Thus, (21). To determine whether this phenotype is caused by aber- large and small apo(a) isoforms follow similar folding path- rant folding, the folding of two null apo(a) proteins was ana- ways, and differences in their folding kinetics do not appear to lyzed (Fig. 4). The proteins were of similar size, which, based explain the large variation in their ER residence times. upon electrophoretic mobility, was equivalent to that of an I Analysis of other allelic variants further exemplified the lack apo(a) isoform precursor. Steady-state labeling of hepatocytes of correlation between apo(a) ER residence time and folding expressing these proteins confirmed the null phenotype (Fig. kinetics. We have identified apo(a) variants that take longer 4A). In each case a single apo(a) protein representing the un- than expected to exit the ER. An example of an A isoform with processed null apo(a) precursor (21) was observed in the cell an unusually long ER residence time is shown in Fig. 3. Steady- lysate, and no apo(a) was detectable in the culture medium state labeling confirmed the maturation and secretion of this (Fig. 4A). ApoB was efficiently secreted from each cell type, allelic variant (Fig. 3A). When folding was analyzed, the ex- demonstrating normal functioning of the secretory pathway pected NEM shift was observed in reduced samples (Fig. 3B). (Fig. 4A). However, even after3hof chase, only trace amounts of mature Unexpectedly, the pattern and kinetics of folding of the null apo(a) could be seen (visible on the original autoradiograph, but proteins were essentially indistinguishable from those of se- not sufficient to be reproduced photographically). In nonre- creted apo(a) proteins. Under reducing conditions (Fig. 4B) duced samples (Fig. 3C), the kinetics and pattern of folding for folding was observed as an NEM shift, although no mature this isoform were indistinguishable from those of apo(a) pro- apo(a) was seen, even after2hof chase (Fig. 4B). Under teins with much shorter ER residence times (compare Fig. 3C nonreducing conditions (Fig. 4C), apo(a) folding was observed with Fig. 2A), and apparently completely folded apo(a) was as an increase in electrophoretic mobility with time, reaching a present by 60 min of chase (Fig. 3C). Thus, although the pro- maximum by 60 min of chase. High molecular weight apo(a)- longed ER residence time of apo(a) per se is due to a prolonged containing complexes were also apparent in each case. Thus, period required for folding, differences in apo(a) ER residence despite their inability to exit the ER, these null apo(a) proteins times, at least of the allelic variants studied to date, cannot be follow an apparently normal folding pathway. A gross inability explained by differences in apo(a) folding kinetics. to fold thus does not explain the null apo(a) phenotype. Some small differences in the apo(a) folding patterns were Null apo(a) protein II in Fig. 4 was synthesized at a high observed. The isoforms varied in the proportion of the protein level in comparison to most apo(a) allelic variants. Analysis of that entered the high molecular weight complex. In some cases, the folding of this protein in unperturbed cells (no DTT treat- there was increased heterogeneity in the complex, forming a ment), as described for the secreted apo(a) isoform in Fig. 1, smear toward the top of the gels, which may indicate some also revealed an apparently normal folding pathway (Fig. 5A). aggregate formation. Further experimentation will be required In this case, the high molecular weight apo(a) containing com- to determine whether the extent of aggregate or complex for- plex was apparent as early as the 0 chase time point, suggest- mation correlates with the extent of apo(a) intracellular ing that the complex forms during, or immediately after, trans- degradation. lation. This experiment confirms that the use of DTT did not Folding of Null Apo(a) Proteins—Apo(a) proteins giving rise artificially induce a normal folding pathway for the null pro- to the transcript positive null phenotype are unable to exit the teins. The folding of null apo(a) protein II was also analyzed by Apo(a) Folding 5053 FIG.7. Influence of CST on apo(a) folding. Apo(a) folding in hepatocytes ex- pressing an I apo(a) isoform cultured in the presence or absence of 1 mM CST was analyzed after post-translational reduc- tion with DTT exactly as described in the legend to Fig. 2. Samples were analyzed by 3–10% SDS-PAGE with (reduced)or without (non-reduced) prior reduction with 2-mercaptoethanol. The positions of the precursor (pr), mature (mt), and com- plexed (comp.) forms of apo(a) and of apoB100 (550 kDa; arrowhead) are indicated. immunoblotting (Fig. 5B). A single form of apo(a) was seen small increase in apo(a) molecular weight in CST-treated cells, after DTT treatment in the nonreduced samples, whereas at most notable in the nonreduced samples, was due to the pres- the later time points, proteins corresponding to fully folded ence of the untrimmed glucose residues. The only other differ- apo(a) and the high molecular weight complex were recognized ence between CST and control samples was the absence of by the anti-apo(a) antibody. This confirms the identity of the mature apo(a) in CST-treated cells (Fig. 7). Thus, CST induced intermediates observed in radiolabeled folding experiments as an apo(a) phenotype similar to that of a null apo(a) protein. apo(a). Combined with the results in Figs. 1–4, this suggests that an An interesting observation for each of the null proteins was event that is dependent on the trimming of glucose residues the presence of a minor band below the null apo(a) protein in from N-linked carbohydrate on apo(a) and that occurs after the the cell lysate (Fig. 4). This band was not observed with nor- bulk of apo(a) folding is required for apo(a) secretion. mally secreted apo(a) proteins (Figs. 1 and 2, and data not DISCUSSION shown) and may be a marker for the null phenotype. In addi- tion, a second high molecular weight apo(a)-containing band We have previously demonstrated that newly synthesized was apparent in nonreduced samples from null animal I. Fur- apo(a) has a prolonged residence time in the ER and that allelic ther experimentation will be required to determine the signif- differences in the ability of apo(a) to escape this compartment icance of these observations. are a major determinant of plasma Lp(a) levels (21, 39, 42). Influence of Glycosylation Inhibitors on Apo(a) Maturation Since only correctly folded proteins are permitted to exit the ER and Folding—Apo(a) is highly glycosylated. N-Linked glycosy- (44), we examined the folding of newly synthesized apo(a) and lation occurs co-translationally in the ER lumen (51) and plays the influence of allelic variation on the kinetics and efficiency of a significant role in the folding and secretion of some glycopro- this process. The results revealed a complex and kinetically teins. To begin to investigate the factors that influence the exit extended apo(a) folding pathway. Unexpectedly, no significant of apo(a) from the ER, the role of N-linked glycans in apo(a) differences were found in the kinetics and patterns of folding of maturation and secretion was examined. Two inhibitors were a variety of apo(a) allelic variants. Even null apo(a) proteins, used, tunicamycin (TM), which prevents N-linked glycosylation which fail to escape the ER, appeared to fold normally. These (52), and castanospermine (CST), which inhibits the trimming studies suggest that an event downstream of the folding events of glucose residues from N-linked glycans in the ER (53) (Fig. detectable in our assay must occur for apo(a) to be secreted and 6A). Hepatocytes expressing a B apo(a) isoform were labeled for that allelic differences in the ability of apo(a) to undergo this 30 min and then chased for5hinthe presence of TM or CST, event contribute to the extreme inter-individual variation in and apo(a) in the cell lysates and in the culture media was Lp(a) production rate. analyzed by immunoprecipitation. The effect of deoxyman- The variously disulfide-bonded folding intermediates of nojirimycin, which inhibits the trimming of mannose residues apo(a) were detected in pulse-chase experiments by exploiting from N-linked glycans after transport to the Golgi apparatus differences in their electrophoretic mobility on SDS-PAGE. For (53), was examined as a control. many proteins, folding intermediates are hard to resolve, due TM caused a reduction in the molecular weight of the apo(a) to small differences in electrophoretic mobility or to rapid fold- precursor, due to inhibition of N-linked glycosylation, and com- ing kinetics (46, 49, 54, 55). However, its large size and numer- pletely prevented apo(a) maturation and secretion (Fig. 6B). ous disulfides made apo(a) ideally suited to this type of analy- CST also prevented maturation of apo(a) (Fig. 6B). Thus, both sis. Our experiments revealed that the majority of disulfides in addition and trimming of N-linked carbohydrate are required apo(a) were formed post-translationally and that folding was for apo(a) to exit the ER. In contrast, deoxymannojirimycin did not complete until 30–60 min after synthesis. A unique char- not prevent apo(a) secretion (Fig. 6B). acteristic was the formation of a high molecular weight, disul- The anti-apo(a) antibody was unable to efficiently immuno- fide-linked, apo(a)-containing complex. The complex seems un- precipitate reduced, unglycosylated apo(a), so the effect of TM likely to represent aggregated apo(a), since apo(a) apparently on apo(a) folding could not be determined. The folding pattern continued to fold while in this form. The complex was confined of apo(a) in the presence of CST, however, was almost indistin- to the ER, since only a single form of mature apo(a) was guishable from that in control cells (Fig. 7); in each case, folding observed. Potential roles of the complex could be in targeting was observed as an NEM shift in reduced samples and as a apo(a) for secretion, ER retention, or degradation. Alterna- gradual increase in electrophoretic mobility, reaching a maxi- tively, the complex could be involved in protecting Cys-4057 in mum at 60 min, in nonreduced samples. The high molecular apo(a), which is required for disulfide formation with apoB (56, weight apo(a)-containing complex was also clearly visible. The 57), from inappropriate inter- or intramolecular interactions 5054 Apo(a) Folding during the folding process. Comparison of reduced and nonre- arrangement of disulfide bonds, may be required. duced apo(a) immunoprecipitates did not identify other radio- To begin to dissect the precise requirements for apo(a) to exit labeled proteins that could be a component of the complex (data the ER, we analyzed the role of N-linked carbohydrate in apo(a) not shown). However, ER resident proteins tend to have long secretion. Both addition and proper processing of N-linked glycans was found to be required for apo(a) to escape the ER, half-lives and may not incorporate sufficient radioactivity for detection during the short labeling periods used. Future stud- since both TM, which prevents addition of N-linked glycans (52), and CST, which inhibits the trimming of glucose residues ies will address the composition of the complex and its role in from N-linked glycans in the ER (53), prevented apo(a) matu- apo(a) secretion. ration and secretion. TM causes aggregation of some secretory Similar patterns and kinetics of apo(a) folding were observed proteins (59). We were unable to ascertain the influence of TM whether analyzed in unperturbed cells or after post-transla- on apo(a) folding due to lack of recognition of reduced, ungly- tional reduction of apo(a) with DTT. The latter assay allowed cosylated apo(a) by the anti-apo(a) antibody. This in itself could longer labeling times to be used and permitted comparison of be indicative of aggregate formation, but further studies will be the folding patterns of multiple apo(a) allelic variants. Similar required to address this issue. CST prevents interaction of protocols have been used to study the folding of a number of secretory proteins with the ER chaperone, calnexin, which secretory and transmembrane proteins (47–50). In each case recognizes substrates by binding to monoglucosylated N-linked the characteristic folding pattern is unchanged by DTT carbohydrate side chains (60). Although CST prevented apo(a) treatment. secretion, it had no apparent effect on the pattern or kinetics of Unexpectedly, our studies failed to reveal any significant apo(a) folding. Again, this could reflect our inability to detect differences in the kinetics and patterns of folding of apo(a) more subtle conformational changes in apo(a) caused by CST proteins with very different ER residence times and efficiencies and may suggest a role for calnexin in apo(a) secretion. Alter- of secretion. We had expected that large apo(a) isoforms would natively, CST may inhibit an event downstream of folding, take longer to fold than small isoforms and that folding and which requires carbohydrate trimming, and which is essential maturation times would coincide. However, this was not the for apo(a) secretion. Clearly, further studies will be required to case. A particularly striking example was the A apo(a) isoform address the many outstanding questions regarding the require- analyzed in Fig. 3. Although folding was apparently complete ments for apo(a) exit from the ER and the precise mechanism by 60 min, this protein did not begin to leave the ER until at by which apo(a) allelic variation influences the kinetics and least3hof chase. We had also anticipated that null apo(a) efficiency of this process. proteins would exhibit aberrant folding patterns. However, all null proteins examined to date appear to fold normally, yet are Acknowledgment—We thank Larry Estlack for excellent technical retained and degraded in the ER. assistance. Several hypotheses can be put forward to explain these ob- REFERENCES servations. The bulk of the apo(a) protein consists of a tandem 1. Berg, K. (1963) Acta Pathol. Microbiol. Scand. 59, 369–382 array of identical K4 domains (7), variation in the number of 2. Utermann, G. (1989) Science 246, 904–910 which is responsible for the size polymorphism of apo(a) (3). 3. Lackner, C., Cohen, J. C., and Hobbs, H. H. (1993) Hum. Mol. 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