THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1984 by The American Society of Biological Chemists, Inc.

Vol. 259, No. 8, Issue of April 25, pp. 5495-5499,1984 Printed in U.S.A.

Synthesis, Intracellular Processing, and Signal Peptide of Human Apolipoprotein E*

(Received for publication, August 18, 1983)

Vassilis I. ZannisS, Joseph McPherson, Gabriel Goldberger, Sotirios K. Karathanasis, and Jan L. BreslowS From the Children’s Hospital Corporation and Harvard Medical School, Boston, Massachusetts 02115

Northern blotting analysis has shown apo-E mRNA synthesis by human liver, HepG2 cells, and primary cultures of human monocyte macrophages but not by the macrophage-like cell line U937 and normal or transformed human fibroblasts. Cell-free translation has shown that the primary translation product of apo- E consists of one major and one minor isoprotein of apparent M, = 28,500 and isoelectric points 6.20 and 6.02, respectively. These isoproteins differ by +1 and 0 charges from apo-E3 and have been designated preapo-E. Co-translational treatment of mRNA with dog pancreatic membranes converts both preapo-E is- oproteins to a form which is undistinguishable by two- dimensional gel electrophoresis from plasma apo-E3. The isolation and nucleotide sequence analysis of a full length apo-E cDNA clone has shown that preapo-E contains an 18-amino acid NH2-terminal signal peptide compared to plasma apo-E. The signal peptide sequence is: MetLysValLeuTrpAlaAlaLeuLeuValThrPheLeu- AlaGlyCysGlnAla. Comparison of co-translationally modified apo-E with intracellular, secreted, and plasma forms indicates that after the intracellular cleavage of the signal peptide, the protein is glycosy- lated with carbohydrate chains containing sialic acid, secreted as sialoapo-E (apo-E.), and subsequently de- sialated in plasma. These findings demonstrate that apo-E is synthesized as preprotein and undergoes in- tracellular proteolysis and glycosylation and extracel- lular desialation to attain the major asialoapo-E iso- protein form observed in plasma.

Apolipoprotein E was first identified in 1973 in human very low density lipoprotein (1) and subsequently has been found in every lipoprotein class of all mammalian species thus far studied (2,3). In humans plasma apo-E is a single polypeptide composed of 299 amino acids of known sequence (4). Previous studies have shown that human plasma apo-E has several isoproteins which differ in size and/or charge. This apo-E complexity is the result of both genetic variation in the human population (5-9) and post-translational modification of apo- E with carbohydrate chains containing sialic acid (6, 10). In humans the genetic variation in apo-E is common (5-9) and a strong association has been found between one of the apo-

*This work was supported by Grants HL15895, HL22487, and HL32339 from the National Institutes of Health and by grants from the March of Dimes Birth Defects Foundation. The costs of publi- cation of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertise- ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Established Investigator of the American Heart Association.

E phenotypes, E2/2, and type I11 hyperlipoproteinemia (11- 13). In uitro receptor binding studies have shown that one of the E2/2 phenotype results from a 158 Arg to Cys substitution and binds poorly to apo-B/E receptors (8,9). This diminished apo-E binding affects the catabolism of apo-E containing lipoprotein particles and is consistent with being the cause of the lipoprotein abnormalities and other clinical features of patients with type I11 hyperlipoproteinemia (14). Apo-E syn- thesis has been demonstrated in liver, kidney, adrenal gland, and reticuloendothelial cells (15-20). In uitro experiments show that newly secreted apo-E consists mainly of sialoapo- E isoproteins (18, 20).

In this report we present the relationship between the newly synthesized, intracellularly processed, secreted, and plasma apo-E isoproteins. We also present the DNA sequence of a full length apo-E cDNA clone which specifies the amino acid sequence of the apo-E signal peptide. On the basis of our findings and literature data, we propose a pathway of intra- and extracellular modification of apo-E that may be important for our understanding of human diseases associated with defects in apo-E metabolism.

EXPERIMENTAL PROCEDURES’

RESULTS

Apo-E mRNA Demonstrated in Liver, HepG2 Cells, and 15- day Primary Cultures of Human Peripheral Blood Monocyte Macrophage-Human liver, HepG2 cells, and 15-day primary cultures of human peripheral blood monocyte macrophages all contain mRNA species of approximately 1150 base pairs that hybridize to the pE-301 probe under the conditions of Northern blotting analysis. In contrast, apo-E mRNA was not detected in the human macrophage-like cell line U937 and normal diploid and transformed human fibroblasts (Fig. 1).

Cell-free Synthesis and Co-translational Processing of Hu- man Apo-E-Cell-free translation experiments using mRNA isolated from various cell types corroborated the results of the Northern blotting analysis. In addition, these experiments showed that the primary translation products of apo-E mRNA, derived from tissues having the E3/3 genotype, con- sist of one major (M, = 38,500, PI 6.20) and one minor (Mr = 38,500, PI 6.02) isoprotein designated preapo-E (Fig. 2, A, C,

Portions of this paper (including “Experimental Procedures” and Figs. 3-5) are presented in miniprint ah the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. &quest doc- ument No. 83M-2399, cite the authors, and include a check or money order for $2.80 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

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5496 Synthesis and Modification of Human Apo-E 1

FIG. 1. Northern hybridization of total RNA isolated from human liver and various cell lines of human origin. The RNA was electrophoresed on 1% agarose gels transferred to nitrocellulose filters and hybridized with pE-301 apo-E cDNA probe (25) (labeled with 32P by nick translation). A, lane M, M, markers; lane a, 6 pg of RNA isolated from 15-day cultures of human peripheral blood mon- ocyte macrophages; lane b, 33 pg of RNA isolated from HepG2 cells. B, lane M, M, markers; lane a, 5 pg of RNA isolated from HepG2 cells; lanes b to e, 20 pg of RNA isolated from (b) human liver, (c) transformed human fibroblasts, (d) human macrophage-like U937 cell line, (e) normal human fibroblasts. A and B represent separate experiments.

and D). The major and minor preapo-E isoproteins differ by +1 and 0 charges from apo-E3, respectively. Co-translational treatment of the primary translation product(s) of apo-E mRNA converted the preapo-E isoproteins to a form indistin- guishable on two-dimensional PAGE' from the major plasma asialoapo-E isoprotein (Fig. 2B). Intracellular and newly se- creted apo-E isoprotein forms were assessed by incubating HepG2 cells with [35S]methionine for 1 to 4 h. After incuba- tion, the cells and medium were collected and apo-E immu- noprecipitated and analyzed by two-dimensional PAGE and autoradiography. These experiments showed that sialoapo-E isoprotein forms comprised 42% (average of two experiments) of intracellular apo-E (Fig. 2E), 81 f 11% of newly secreted apo-E (Fig. 2F), and, as previously shown (40), 24 +. 6% of plasma apo-E (Fig. 2H). Fig. 2G shows newly secreted apo-E after treatment with Clostridium perfringens neuraminidase which converts the sialo- to asialoapo-E isoproteins.

DNA Sequence of the Full Length Apo-E cDNA Clone and the Deduced Amino Acid Sequence of the Apo-E Signal Pep- tide-Clone pE-368 has been mapped and sequenced as de- scribed under "Experimental Procedures." Fig. 3 shows a restriction map of this clone and the strategy used to deter- mine its sequence. Fig. 4A shows the complete DNA sequence and corresponding amino acid sequence. Clone pE-368 con- sists of 1146 base pairs including a 5"untranslated region of 60 base pairs, and a 3"untranslated region of 142 base pairs. The clone also contains a poly(A) tail which begins 19 bp downstream of the last A in the polyadenylation signal, AA- TAAA. The DNA sequence shows that the primary transla- tion product of apo-E mRNA (preapo-E) consists of 317 amino acids and contains an 18-amino acid NHZ-terminal extension when compared to plasma apo-E. The amino acid sequence for plasma apo-E specified by this cDNA sequence is identical with the one shown by Rall for the apo-E3 phe- notype (4). Fig. 4, B and C, compares the amino acid and

* The abbreviation used is: PAGE, polyacrylamide gel electropho- resis.

1 -

FIG. 2. Analysis of ''S-labeled apo-E isoproteins synthe- sized by cell-free translation of total mRNA obtained from HepG2 cells. A shows the analysis by two-dimensional PAGE and autoradiography of proteins immunoprecipitated from the translation mixture with specific anti-apo-E antibodies. An aliquot of 50 pl of the translation mixture of HepG2 mRNA was mixed with 100 pg of human very low density lipoprotein and immunoprecipitated with anti-human apo-E as explained under "Experimental Procedures." The immunoprecipitate was dissolved in lysis buffer and analyzed by two-dimensional PAGE and autoradiography. A shows the autoradi- ogram obtained from this analysis. The position of plasma apo-E4 and apo-E3 indicated by open circles was established by superimpos- ing the autoradiogram on the corresponding two-dimensional slab gel that was stained for protein as explained in previous publications (18, 20). B shows an autoradiogram obtained after similar analysis of the translation products of HepG2 mRNA processed co-translationally with dog pancreatic membranes as explained under "Experimental Procedures." Note the conversion of preapo-E3 to apo-E3. C and D show an autoradiogram obtained after similar analysis of the trans- lation products of total mRNA obtained from human liver and 15- day cultures of human peripheral blood monocyte macrophage, re- spectively. E and F show autoradiograms obtained after two-dimen- sional analysis of intracellular and secreted apo-E which is synthe- sized by HepG2 cells grown in [%]Methionine. The proteins were precipitated from the cell extract or culture medium as explained in A and Ref. 20. G shows secreted apo-E that was treated with C. perfringens neuraminidase prior to the two-dimensional analysis as described in Ref. 20. Note that neuraminidase treatment converts sialoapo-E isoproteins (apo-E3.) to asialoapo-E isoproteins apo-E3. H shows two-dimensional gel electrophoresis of 25 pg of apo-E obtained from normal human subjects. Note the decreased sialation of the plasma apo-E (compare F with H). In all panels only the area of the gel or autoradiogram in the vicinity of apo-E is shown. The cathode is on the left and anode is on the right.

nucleotide sequences, respectively, of the signal peptide region of human apo-E with the corresponding regions of rat apo-E and human apo-A-I. The signal peptide region of human apo- E has 57% and 81% nucleotide sequence homology with the corresponding regions of human apo-A-I and rat apo-E, re- spectively. In addition, the human apo-E signal peptide has 50% and 67% amino acid sequence homology with the human

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Synthesis and Modification of Human Apo-E 5497

apo-A-I and the rat apo-E signal peptides, respectively. The amino acid sequence of human apo-E signal peptide was also compared with 8 human and 10 rat signal peptide sequences compiled in Ref. 41 as well as with the signal peptide sequence with apo-VLDLII of cockerel (42). The homology of the human apo-E signal peptide with the human, rat, and apo- VLDLII signal peptide sequences was 17 to 27%, 6 to 27%, and 17%, respectively.

Determination of the Relationship of Newly Secreted Apo- E3, and Plasma Asialoapo-E3 Isoproteins-HepG2 cells were incubated with [14C]proline to radiolabel the newly secreted apo-E3. Apo-E3. was then isolated and NHz-terminal se- quencing performed as described under “Experimental Pro- cedures.’’ This analysis showed that the newly secreted sialo(apo-E,) isoproteins have proline at amino acid residues 10 and 12. Previous studies (Ref. 4 and Fig. 4A) have shown that proline residues are found in positions 10 and 12 of plasma asialoapo-E. This finding demonstrates that newly secreted apo-E. does not have an NH2-terminal amino acid extension when compared to plasma apo-E.

DISCUSSION

We have shown previously, using organ cultures of human fetal and adult liver, that the secreted form of apo-E consists mainly of sialoapo-E isoproteins, and proposed that these sialoapo-E isoprotein forms must be desialated to attain the major apo-E isoprotein form(s) seen in plasma (17, 20). In the current study, we explore the relationship of the previ- ously described plasma and newly secreted hepatic apo-E isoproteins to those of the primary translation product of apo- E mRNA, the co-translationally modified apo-E, and the intracellular apo-E. In order to perform these studies it was necessary to obtain tissues and cells that synthesize human apo-E both in vivo and in vitro. Northern blotting and cell- free translation analysis experiments with RNA from liver, HepG2 cells, and 15-day cultures of human peripheral blood monocyte macrophage showed that these systems were syn- thesizing apo-E. In other experiments (not shown), we found that apo-E is synthesized by Old World (cynomolgus) monkey liver and adrenal gland and kidney. These findings are in agreement with previous observations about sites of apo-E synthesis (15-20). Utilizing these sources of RNA, we found that the primary translation product of apo-E mRNA, ob- tained from tissues or cells of individuals with the apo-E phenotype E3/3, consists of one major and one minor isopro- tein of apparent M, = 38,500 that are larger than plasma asialoapo-E3 and has been designated preapo-E3. In contrast to preapo-A-I (32), preapo-E could not be detected in the cell- free translation mixture of total mRNA from liver or HepG2 cells without prior immunoprecipitation. This indicates that the abundance or the translation efficiency of apo-E mRNA obtained from liver and HepG2 cells may be lower than that of apo-A-I. In addition, the major and minor preapo-E isopro- teins differ by +1 and 0 charges from plasma apo-E3, respec- tively. Furthermore, co-translational processing experiments revealed that both preapo-E isoproteins are converted to a form indistinguishable, by two-dimensional PAGE, from plasma apo-E3. These observations suggest the presence of a signal peptide sequence in the primary translation product of apo-E mRNA. An apo-E signal peptide has previously been shown for rat apo-E (43-45).

The existence of an apo-E signal peptide in humans was confirmed and its amino acid sequence deduced through the isolation and characterization of a human apo-E cDNA clone that contained the DNA sequence of the region extending 5’ to the sequence specifying the NH, terminus of plasma apo-

E. The DNA sequence data indicate that preapo-E contains an 18-amino acid NHz-terminal extension compared to plasma apo-E. The signal peptide of apo-E begins with me- thionine, followed by lysine, containing a whole series of hydrophobic residues in the middle segment, and terminates with alanine. Similar features have been observed for the signal peptide of human and rat apo-A-I (32, 44, 46) and rat apo-E (43), and are also common to all other secreted proteins that have been studied (47-50). The %amino acid signal peptide of apo-E has a +1 charge. This is exactly the charge difference between the major preapo-E isoprotein and plasma apo-E3. The origin of the minor preapo-E isoprotein is not clear. It may be generated by removal of the Met-Lys dipep- tide of the signal peptide by a trypsin-like activity present in the rabbit reticulocyte lysate used in the cell-free translation mixture. Alternatively, it may be generated by deamidation of the major preapo-E isoprotein. The signal peptide region of human and rat apo-E have a high degree of amino acid and nucleotide homology. Considerable amino acid and nucleotide homology also exists between human apo-E and apo-A-I signal peptide regions. Such homology has not been observed between human apo-E and numerous nonlipoprotein signal peptides (41) of human or rat proteins as well as between human apo-E and apo-VLDLII signal peptides (42).

Previous experiments suggested that apoliproteins are mod- ified by the addition of carbohydrate chains in the Golgi apparatus (51-53). Similar to other systems, the apo-E mod- ification follows cleavage of the signal peptide and contains several steps. The finding of a higher concentration of si- aloapo-E (apo-E.) in the newly secreted apo-E than in the intracellular apo-E suggests either that sialation may be re- quired for apo-E secretion or that the sialoapo-E (apo-E.) may be preferentially secreted. The finding of lower intracel- lular apo-E. content in HepG2 cells is consistent with recent findings which showed a low degree of sialation of intracellular apo-E synthesized by human kidney and adrenal cortex (19).

As previously reported, newly secreted apo-E has a higher sialo content than plasma apo-E (81 f 11% versus 24 f 6%). Extracellular desialation must therefore occur as a normal step in apo-E metabolism, and we have recently shown that apo-E from the plasma of four Tangier patients contains excessive amounts of sialoapo-E (40). This observation sug- gests that the metabolic defect in Tangier disease, which is believed to be an inability to convert apo-A-I from its secreted form to the mature plasma form (34), disrupts the normal physiological events leading to apo-E desialation in plasma. This finding is also consistent with the hypothesis that apo- E is secreted as sialoapo-E in uivo and that the increased sialation of newly secreted apo-E observed in the organ and cell culture experiments is not an artifact of the culture conditions. The physiological significance of apo-E sialation is not currently known. One possibility is that sialation is required for apo-E secretion, another is that sialation may prevent liver cells from recognizing, internalizing, and ca- tabolizing apo-E just after it has been secreted.

The carbohydrate content of human plasma.apo-E has been reported (10). Preliminary evidence in our laboratory3 showed that the glycosylation of apo-E is not affected by tunicamycin. This finding suggests that the carbohydrate chains are at- tached to apo-E by O-glycosidic linkage. Furthermore, human apo-E has a single asparagine as its penultimate amino acid (4) but lacks the amino acid sequence Asn X Ser (Thr), which is characteristic of N-glycosylation sites.

In Fig. 5 we show a schematic representation of the series

V. I. Zannis, and G. B. Forbes, unpublished observations.

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5498 Synthesis and Modification of Human Apo-E

of events in intra- and extracellular apo-E modification. This scheme is based on the findings of Fig. 2 and Refs. 4, 10, 17, 18, and 20. Each of these events could play a significant role in the regulation of apo-E-mediated events in lipoprotein metabolism. Structural mutations in apo-E or the apo-E mod- ifying enzymes which may affect any of these processes could result in human disease. Similar types of mutations have been recently described for the human low density lipoprotein receptor (54). Recently, patients with the type I11 hyperlipo- proteinemia phenotype have been described who lack plasma apo-E (55 ) . It will be important to assess whether these patients have a mutation in the apo-E gene itself or in genes coding for proteins that modify apo-E. In conclusion, the present work highlights the steps in intra- and extracellular apo-E modification which may be important for our under- standing of the structure and function of this apolipoprotein.

Acknowledgments-We would like to thank Dr. H. T. Keutmann for performing the microsequence analysis of human apo-E., Dr. F. Sessions Cole for providing total RNA of 15-day cultures of human peripheral blood monocyte macrophage, and Gayle Forbes, Miriam Ritz, and Lorraine Duda for their expert assistance.

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Synthesis and Modification of Human Apo-E 5499

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V I Zannis, J McPherson, G Goldberger, S K Karathanasis and J L BreslowSynthesis, intracellular processing, and signal peptide of human apolipoprotein E.

1984, 259:5495-5499.J. Biol. Chem. 

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Additions and Corrections

V O ~ . 259 (1984) (5797-5802) V O ~ . 259 (1984) 3612-3615

Isolation and characterization of UDP-ga1actose:N- acetylglucosamine 4/3-galactosyltransferase activity induced in rat parotid glands treated with isoproter- enol.

Labeling the adenine nucleotide binding domain of the sarcoplasmic reticulum Ca,Mg-ATPase with photoaf- finity analogs of ATP.

Michael B . Cable and F. Norman Briggs Michael G. Humphreys-Beher

The author line should include four additional authors, with their affiliations, as shown below:

Michael G. Humphreys-Beher$§, Miriam Immel?II, Neil Jentoft**, Michael Gleasonq, and Don M. Carlsonll

From the $Department of Biological Sciences and the Toe- partment of Biochemistry, Purdue University, West Lafayette, Indinna 47907 and the **Department of Pediatrics, Case West- ern Reserve University, Cleveland, Ohio 44106

The footnote including grant support should have the following addition:

This work was supported in part by United States Public Health Service Grant AM 19175. This is Journal Paper 10030 from the Purdue University Agricultural Experiment Station.

Other footnotes include the following:

(1 Deceased. $ Present address: Department of Microbiology, University

of Alabama in Birmingham, University Station, Birmingham, AL 35294.

Vol. 259 (1984) 5495-5499

Page 3613, last paragraph: The calculations of the concentrations of 8-azido-ATP were incorrect. We state that we used a A,,, of 260 nm and an extinction coefficient of 15,400. We used a X,,, of 281 nm and an extinction coefficient of 15,400. The correct extinction coefficient is 13,300 (Bailey, B., and Hoffman, J., (1974) Proc. Natl. Acad. Sei. U. S. A. 74, 4375). The extinction coefficient for BzATP was also incorrect. We have recently determined that the cor- rect extinction coefficient for the compound is about 40,700 by determination of total phosphate in pure BzATP. This value is in agreement with that deter- mined by R. Yount (personal communication). The rea- son that the extinction coefficient for BzATP differs greatly from that for ATP is unclear. All concentra- tions of BzATP reported in the paper must be decreased accordingly. The most significant effect of the new concentration for BzATP is on the interpretation of the results shown in Figs. 5 and 7 on page 3614. The actual specific activity of the [a-32P]BzATP was higher than originally calculated, and the amount of analog incorporated was therefore lower than reported. The amount of analog incorporated was thus insufficient to account for all the inhibition of ATPase activity ob- served. We are currently determining the true stoichi- ometry of incorporation and inhibition. We will report the true relation incorporation and inhibition when the studies are complete.

Page 3614, last line:

The term K[a-32P]” should read “[y-32P].” The corrected sentence should read:

Synthesis, intracellular processing, and signal peptide of human apolipoprotein E.

The maximum incorporation achieved in this experiment was 1.3 nmol of [y3’P]BzATP per mg of protein.

Vassilis I. Zannis, Joseph McPherson, Gabriel Goldberger, Sotirios K. Karathanasis, and J a n L. Breslow

Page 5495, summary, line 8:

“apparent M, = 28,500” should be “apparent M, = 38,500.” The correct line is:

“apparent M, = 38,500 and isoelectric points 6.20 and . . .”

14315