Proc. Natl. Acad. Sci. USA Vol. 87, pp. 5523-5527, July 1990 Biochemistry

Isolation and characterization of sulfhydryl and disulfide peptides of human apolipoprotein B-100 (protein structure/low density lipoprotein/peptide purification)

CHAO-YUH YANG*, TAE W. KIM, SHI-AI WENG, BORONG LEE, MANLAN YANG, AND ANTONIO M. GOTTO, JR. Baylor College of Medicine and The Methodist Hospital, Department of Medicine, 6565 Fannin Street, M.S. A-601, Houston, TX 77030

Communicated by Joseph L. Goldstein, April 16, 1990

products from Worthington. The 4-vinylpyridine and tributylphosphine came from Aldrich, and solvents for the automatic gas-phase sequencer were from Applied Biosystems. Other chemicals used were the highest reagent grade available. Isolation of LDL. Human plasma was obtained from The Methodist Hospital Blood Center. Aprotinin (0.055 unit/ml), sodium azide (5.00 Mg/ml), and EDTA (5.08 pg/ml) were added to the plasma, and LDL (density 1.025-1.055 g/ml) was purified by ultracentrifugation in KBr solution (15). Protein purity was verified by electrophoresis on 0.1% SDS/ 5-15% gradient polyacrylamide slab gels. The LDL was dialyzed against 0.1 M ammonium bicarbonate and then carboxymethylated with iodoacetic acid (16). Trypsin Cleavage of apoB-100. One hundred milligrams of apoB-100 obtained from carboxymethylated (CM) LDL was resuspended in 15 ml of 0.1 M ammonium bicarbonate, pH 8.0, and 2 mg ofTPCK-treated trypsin was added and allowed to digest for 5 hr. The peptide mixtures were then subjected to HPLC purification. Pepsin Cleavage of CM-LDL. The CM-LDL (containing 100 mg of apoB-100) in 35 ml of 5% HCOOH was incubated with 2 mg of pepsin at room temperature for 5 hr. The digest was applied to a Sephadex G-50 column (2.6 x 200 cm) to fractionate the surface and core fragment, as described (5). Peptide Purification. The tryptic digest of apoB-100 was fractionated on a Vydac C4 reverse-phase column (9.4 X 200 mm) with a Waters HPLC system equipped with an absorbance detector (model 441) and trifluoroacetic acid buffer system (buffer A: 0.1% trifluoroacetic acid in H20; buffer B: 0.08% trifluoroacetic acid in 95% acetonitrile/5% H20) at 50°C. A Hypersil octadecylsilane reverse-phase column (4.6 x 250 mm) with a phosphate buffer system (buffer A: 0.005 M KH2PO4/K2HPO4, pH 6.0; buffer B: 10% H20/90% acetonitrile) (17) was used for further purification. The separated fractions were subjected to phenylthiocarbamoylamino acid analysis to identify the fractions that contain cysteine residue(s). Identification of Cysteine-Containing Peptides and Amino Acid Analysis. Phenylthiocarbamoyl-amino acid analysis was used to identify peptides that contain cysteine residue(s). The fractions separated from HPLC purification were subjected to phenylthiocarbamoyl-amino acid analysis after oxidation with performic acid (17). Pico-Tag amino acid analysis was done according to the standard method of Waters (18). For analysis of oxidized samples, the Waters Pico-Tag amino acid analysis system was applied, except that flow rate and pH of the buffer system were changed from 1 to 0.8 ml/min and from pH 6.4 to 5.5, respectively.

Twenty-three of the 25 cysteine residues in ABSTRACT apolipoprotein B-100 have been isolated directly from tryptic or peptic peptide mixtures. Sixteen cysteine residues exist in disulfide forms: Cys-1-Cys-3, Cys-2-Cys-4, Cys-5-Cys-6, Cys-7-Cys-8, Cys-9-Cys-10, Cys-11-Cys-12, Cys-13-Cys-14, and Cys-20-Cys-21. All of these except Cys-20-Cys-21 are recently discovered disulfide linkages. In addition to Cys-22 and Cys-24, which have been described as sulfhydryls on low density lipoprotein, Cys-15 to Cys-18 and Cys-23 are in the reduced form. Cys-19 and Cys-25 are not yet confirmed. Our results revealed that all identified disulfide linkages are located in the trypsin-releasable regions and that all except Cys1-Cys-3 and Cys-2-Cys-4 are linked to the neighboring cysteine. We propose a linear model of apolipoprotein B-100 in low density lipoprotein that wraps around the low density lipoprotein molecule.

Apolipoprotein B-100 (apoB-100) is the largest (4536 amino acid residues) of the human plasma apolipoproteins and contains the ligand for binding low density lipoprotein (LDL) to LDL receptor on cell surfaces (1). By using a combination of recombinant DNA technology and direct protein sequencing, the primary structure of apoB-100 has been determined (2-5). Through knowledge of the sequence of apoB-100, it has become possible to locate and identify important functional regions, such as the LDL receptor-binding region (3, 4, 6), glycosylation sites (5), heparin-binding sites (7, 8), and other important structure-forming amino acids, such as cysteine (3-5). Clarification of the genetic and evolutionary relationship of apoB-100 to other lipoproteins (5, 9) has also become possible. Based on the differential accessibility of different regions of LDL-apoB-100 to trypsin digestion, a model for the structure and conformation of apoB-100 in LDL has been proposed (5). In this model, certain parts of the protein are buried in lipid. Electron microscopic studies of frozen hydrated LDL (10) and delipidated LDL (11) revealed results similar to those proposed by Yang et al. (4, 5). ApoB-100 is known to be cross-linked to apolipoprotein (a) in lipoprotein (a) through disulfide bridges (12, 13); however, the location of the linkage is still unknown. Two free cysteine residues of apoB-100 on the LDL surface have been localized to positions 3734 and 4190 by use of a fluorescent sulfhydryl probe (14). However, the locations of the remaining sulfhydryl groups and disulfide links are unknown. Herein we report purification and characterization of apoB-100 sulfhydryl groups and disulfide linkages from tryptic and peptic cleavage of the protein. MATERIALS AND METHODS Pepsin and trypsin [treated with L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)] were sequence-grade

Abbreviations: apoB-100, apolipoprotein B-100; CM, carboxymethylated; LDL, low density lipoprotein; >PhNCS, phenylthiohydantoin. *To whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

5523

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Biochemistry: Yang et al.

Sequence Analysis. The sequence of the cysteine-containing peptides was analyzed by an Applied Biosystems gas-phase sequencer model 470A, equipped with a model 120A phenylthiohydantoin (>PhNCS) analyzer. The Applied Biosystems standard method and reagents were used (19). Alkylation of Sulfhydryl Peptides on Glass Filter (20). Cysteine-containing peptide was applied to a precycled gas-phase sequencing filter (21). Details for alkylation of sulfhydryl peptides were as described (22). The resulting data permitted identification of peptides that contained sulfhydryl from those appearing as disulfides. Peptide Labels. T and P represent tryptic and peptic peptide, respectively. Positions of cysteine (C) in apoB-100 were numbered from N to C terminus. There are a total of 25 cysteines. Thus, TC1 represents the tryptic peptide containing the first cysteine residue; TC15 represents the tryptic peptide containing the fifteenth cysteine residue; TC2TC4 represents the tryptic disulfide peptide that connects the second and fourth cysteine; PC5PC6 represents the peptide disulfide peptide connecting the fifth and sixth cysteine, etc.

RESULTS Fig. 1 shows our working procedure to isolate sulfhydryl and disulfide peptides of apoB-100 from LDL. Trypsin Digestion. CM-LDL was the starting material used in determining the linkages between cysteine residues in apoB-100. The separation profile of the tryptic peptides on a reverse-phase column is shown in Fig. 2. After Pico-Tag amino acid analysis, 16 fractions containing cysteine were identified. These fractions were collected and rechromatographed to obtain pure peptides. Seven disulfide peptides (TC1TC3, TC2TC4, TC5TC6, TC9TC10, TC11TC12, TC13TC14, and TC20TC21) and six sulfhydryl peptides (TC15, TC16, TC17, TC18, TC23, and TC24) were obtained from this cleavage. Results of the purified disulfide and sulfhydryl peptides are listed in Table 1. Pepsin Digestion. Pepsin-releasable and nonreleasable peptides of CM-LDL were separated by Sephadex G-50 gel filtration. Five fractions were pooled based on their separation profile (data not shown). Four disulfide peptidesPC7PC8, PC9PC10, PC11PC12, and PC13PC14-were isolated from fraction P4, whereas PC5PC6 and PC20PC21 were isolated from fraction P3 and P1 (pepsin nonreleasable fraction), respectively. Sequence information and initial yields of these peptides are listed in Table 2. Disulfide Linkage in apoB-100. Cys-l-Cys-3. Disulfide peptide TC1TC3, which links Cys-1 and Cys-3, was isolated from

Proc. Natl. Acad. Sci. USA 87 (1990) LDL

#

Carboxymethylation

QI-LDL

I

Pepsin digestion

Peptic peptides

Sephadex C-SO

gel

;1

filtration

;2

Pepsin releasable

peptides

Pepssin nonrole easable peptides

P3

1 Delipidation 2 .Trypsin digestion

PS

P4

Tryptic peptides

I1 Delipidation 2. Pepsin digestion PPM

I%

I. WPC Separation using TFA system PlC-AAA for cysteic acid analysis

2.

Fractions containing cys amino acid 1 Rchroatography using phosphate buf fer system

I 2 .PC-AAA for cysteic acid quantitation Pure peptides containing cys amino acid residue

Peptide sequencing

FIG. 1. Scheme for apoB-100 sulfhydryl and disulfide analysis. TFA, trifluoroacetic acid; PTC, phenylthiocarbamoyl. AAA, amino acid analysis.

fractions 9, 10, and 11 of Fig. 2. The rechromatography of fraction 9 on a Hypersil analytical column with a phosphate buffer system to obtain the pure disulfide peptide is shown in Fig. 3A. Based on rechromatography results and N-terminal sequence information, TC1TC3 eluted at 30%o buffer B and revealed two N terminals (glutamic acid and valine). The structure and yield of this peptide are listed in Table 1. Cys-2-Cys4. Disulfide peptide TC2TC4 was purified from fraction 1 of Fig. 2 after rechromatography on a Hypersil octadecylsilane column with a phosphate buffer system. This peptide was eluted at 23% buffer B and had isoleucine and threonine as the N terminus. Amino acid and sequence analysis of this peptide show that this disulfide peptide contains Ile-Asn-Cys-Lys and Thr-Ser-GIn-Cys-Ile-Leu-Lys and is linked by both cysteines. The initial yield of this peptide was 22.4%. Cys-5-Cys-6. The disulfide bridge that linked Cys-5 and Cys-6 was confirmed either by tryptic or peptic cleavage of

12-l

'0 i

11l lti'-'i

1

160

t~ ~ ~ ~ ~ ~ ~ i

0 Tirne (m)i-n)

FIG. 2. Chromatogram of tryptic peptides on a Vydac C4 reverse-phase column (9.4 x 200 mm) with trifluoroacetic acid buffer system. Peaks containing cysteine residues are indicated with numbers.

Biochemistry: Yang et A

Proc. Natl. Acad. Sci. USA 87 (1990)

5525

Table 1. apoB-100 disulfide and sulfhydryl peptides isolated from tryptic cleavage Peptide TC1TC3

Sequence (single-letter code)

EEEMLENVSLVPPK

VELEVPQLdSFILK

TC2TC4

IN9K TSQ ILK QVLFLDTVYGN9STHFTVK

TC5TC6

DLGQCDR TC9TC10 TC11TC12 TC13TC14 TC20TC21 TC15 TC16 TC17 TC18 TC23 TC24

GLSDEAVTSLLPQLIEVSSPITLQALVQCGQPQCSTHILQWLK TNPTGTQELLDIANYLMEQIQDD9TGDEDYTYLILR _VQSTK QSWSVCKQVFPGLNYCTSGAYSNASSTDSASYYPLTGDTR HSITNPLAVL9EFISQSIK EL TISHIFIPAMGNITYDFSFK ITEVALMGHLSCDTK NTFTLSCDGSLR GTYGLSCQR CSLLVLENELNAELGLSGASMK ADYVEYVLDSTCSSTVQFLEYELNVLGTHK EELCTMFIR

apoB-100. TC5TC6 was purified from peaks 6 and 8 of Fig. 2 on a Hypersil octadecylsilane column and phosphate buffer system and was eluted at 27% buffer B. PC5PC6 was isolated from fraction P3 of peptic cleavage of CM-LDL after Sephadex G-50 gel filtration and was eluted at 34% buffer on a Hypersil octadecylsilane column and phosphate buffer system. The primary structure of TC5TC6 and PCSPC6 was confirmed after amino acid and sequence analyses as listed in Tables 1 and 2, respectively. Cys-7-Cys-8. Disulfide peptide linked between Cys-7 and Cys-8 was isolated from peptide cleavage of fraction P4 (peptic-releasable portion) aftergel filtration. After rechromatography, PC7PC8 was purified. Structural analysis confirmed that peptides Ile-Ser-Ser-Ser-Gln-Ser-Cys-Gln-Tyr-Thr-Leu and Ala-Ile-Cys-Lys-Glu-Gln-His-Leu were connected by disulfide bridges. The initial yield of this peptide was 55.2%. Cys-9-Cys-10. The peptides that contained Cys-9 and Cys-10 were isolated from tryptic (Fig. 2, peak 16) and peptic cleavage. After rechromatography, TC9TC10 and PC9PC10 were obtained in pure form. By using 4-vinylpyridine to alkylate the possible sulihydryl group, PC9PC10 was confirmed as a disulfide peptide connected by Cys-9 and Cys-10. The structures of TC9TC10 and PC9PC10 are listed in Tables 1 and 2, respectively. Cys-11-Cys-12. Disulfide peptides containing Cys-11 and Cys-12 were isolated from peak 14 of Fig. 2 and from peptic cleavage of P4. From sequence and amino acid analysis, the structures of both peptides were confirmed (Tables 1 and 2). Cys-13-Cys-14. The peptides that contained Cys-13 and Cys-14 were found in peak 7 in Fig. 2 and peptic cleavage of P4. After rechromatography, TC13TC14 and PC13PC14 were purified at 27% and 40%o of buffer B, respectively. Both

Position on apoB-100 1-14 53-66 49-52 67-73 148-166 181-187 330-372 428-463 481-486 934-973 3157-3175 3295-3317 1074-1088 1389-1400 1472-1480 1635-1656 3879-3908 4187-4195

Initial yield, % 13.9

22.4 17.8 24.5 16.2

18.5 30.9 15.3 15.3 18.8 9.3 8.7 25.8

peptides were confirmed as disulfide peptides by 4-vinylpyridine treatment. The structure of PC13PC14 is listed in Table 2, and the structure of TC13TC14 is listed in Table 1. Cys-20-Cys-21. The disulfide bridge between Cys-20 and Cys-21 was found either in tryptic or peptic digestion. TC20TC21 was purified from peak 15 of Fig. 2 with a Hypersil octadecylsilane column and phosphate buffer system. TC20TC21 was eluted at 48% buffer B. From the pepsin nonreleasable fraction (P1), PC20PC21 was isolated by delipidation, peptic redigestion, and HPLC purification. The structures of TC20TC21 and PC20PC21 were confirmed by sequence and amino acid analysis (Tables 1 and 2, respectively). Sulfhydryl Group of apoB-100. Peptides containing single cysteine residues were isolated directly from tryptic peptides of apoB-100 after HPLC rechromatography. To confirm that the peptide contained a free sulfhydryl group, S-alkylation of cysteine residues with 4-vinylpyridine was conducted. The sample was then subjected to gas-phase sequence analysis. A >PhNCS-pyridylethyl-cysteine appeared at the cycle, as expected, and eluted between >PhNCS-Val and N,N'diphenylthiourea (23) on an Applied Biosystems 120A >PhNCS analyzer. During gas-phase sequence analysis of unmodified sulfhydryl peptide, a characteristic peak around >PhNCS-Tyr position was also seen at the cycle where a cysteine residue was expected. This characteristic peak results from a side-reaction product of the cysteine residue and indicates whether or not the peptide contained free sulfhydryl. The results confirm the sulfhydryl peptides of apoB-100. Cys-15 and Cys-16. Peptide that contained Cys-15 or Cys-16 was isolated from tryptic cleavage of fraction 3 in Fig.

Table 2. apoB-100 disulfide peptides isolated from peptic cleavage Position on Peptide Sequence (single-letter code) apoB-100 PCSPC6 DTVYGN9STHF 153-163 179-188 ERDLGQ DRF PC7PC8 212-222 ISSSQSCQYTL 232-239 AICKEQHL 356-370 PC9PC1O VQCGQPQCSTHILQW PC11PC12 448-456 IQDD9TGDE K VQSTKPSL 485-494 PC13PC14 937-954 SVCKQVFPGLNYCTSFAY 3164-3169 AVL EF PC20PC21 3297-3303 tTISHIF

Initial

yield, % 37.7

55.2 90.6 17.8 84 45.3

Biochemistry: Yang et al.

5526

Proc. Natl. Acad. Sci. USA 87 (1990)

2 on a Hypersil octadecylsilane column and phosphate buffer system. Results of sequence analysis proved that TC15 and TC16 contained free cysteine residue on cycle 12 and 7, respectively. The structures and the initial yield of these two peptides are listed in Table 1. Cys-17. Cys-17-containing peptide was purified from fraction 2 of Fig. 2. Fig. 3B shows rechromatography of fraction 2. TC17 was eluted at 17% buffer B and proved to be a sulfhydryl peptide covering the residues from 1472 to 1480 (Gly-Thr-Tyr-Gly-Leu-Ser-Cys-Gln-Arg). The initial yield of this peptide was 18.8%. Cys-18. TC18 contained apoB-100 sequence from residues 1635 to 1656 and was purified after rechromatography of fraction 12 of Fig. 2. The structure listed in Table 1 was confirmed by gas-phase sequence and amino acid analysis.

The initial yield was 9.3%. Cys-23. TC23, which was isolated from fraction 13 of Fig. 2, covered the apoB-100 sequence from residues 3879 to 3908. In prior sequence analysis, the peptide was alkylated with 4-vinylpyridine. This fact confirmed that cycle 12 is a free cysteine residue; its structure is listed in Table 1.

'A

t a

10 10

0

0...220 .............

.3.0

0. _

50

30

40

50

Time (min)

DISCUSSION Continuing our studies on the structure ofapoB-100 of human LDL, we have identified the locations of disulfide linkages and sulfhydryl groups of apoB-100. Yields from the enzymatic digestions decreased in this order: peptic disulfide peptides > tryptic disulfide peptides and disulfide peptides > sulfhydryl peptides. These results may be due to peptide size and to the lower stability of sulfhydryl peptides. Although the primary structure of apoB-100 has been determined (3, 4), its conformation on LDL is still unknown. We have worked on this problem by direct sequencing of LDL apoB-100 tryptic peptides. Based on the relative releasability of different regions of the protein-to-tryptic digestion, apoB-100 can be divided into five hypothetical domains (Fig. 4). Domain I, the N-terminal region, contains 14 of the 25 cysteine residues in apoB-100 and has predominantly trypsin-releasable peptides; domain II appears as a transitional region between domains 1 and 3; domain III spans residues 1701-3070 and contains mostly trypsin-nonreleasable peptide regions with occasional trypsin-releasable peptides. Domain IV encompasses 7 of the 19 N-glycosylation sites and contains the putative receptorbinding domains. Lastly, domain V, the C-terminal region, consists almost exclusively of trypsin-nonreleasable peptides. Based on this information, a model of apoB-100 on LDL has been proposed that pictures apoB-100 as a single chain tied about LDL (4). By using electron microscopy to investigate the structure of LDL in the frozen hydrated state, Atkinson (10) has found the LDL particle to be surrounded by a dense halo of protein. Phillips and Schumaker (11) observed a similar result with electron microscopic studies on delipidated LDL after absorption of LDL to a carbon-coated copper grid. They discovered that apoB-100 appeared in an elongated form on the surface of the LDL. In addition, analysis of apoB-100 tryptic peptides by Yang et al. (24) has shown that 13 lipid-binding peptides are distributed throughout the protein molecule, thereby indicating that the lipidbinding characteristics are not confined to a particular region of the protein. A similar conclusion was also obtained by Chen et al. (25). A 2481012 N Nom I0 0 101 .

B

. 1-

14

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11 I

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I

I I

.-

---.

?

I I --

--

1

171 C

- -

region

I Cl

t

6 0

a)

-Core region

m

0

10

20

30

Time (min)

FIG. 3. (A) Rechromatography of peak 9 of Fig. 2 on a Hypersil octadecylsilane column with phosphate buffer system. Disulfide peptide TC1TC3 was obtained. (B) Rechromatography of peak 2 of Fig. 2 on a Hypersil octadecylsilane column with phosphate buffer system. Sulfhydryl peptide TC17 was purified.

FIG. 4. (A) Location of apoB-100 disulfide and sulfhydryl peptides. Positions of cysteine amino acids on apoB-100 were numbered from N to C terminus. There are a total of 25 cysteines. Cys-19 and Cys-25 are still to be confirmed. (B) Schematic representation of apoB-100 structure in LDL based on disulfide and sulfhydryl information. o, Location of N-glycosylated carbohydrates; e, cysteine residues; =, disulfide linkages.

Biochemistry: Yang et al. Thornton (26) has studied the effect of distance between cysteine residues on the formation of the disulfide bridges in proteins of known sequence and connectivity in order to search for common features. Several general patterns appear to influence the formation of disulfide bridges. For example, half-cysteines that are close together in sequence preferentially form disulfide bridges. Likewise, disulfide bonds that extend over >150 residues are rarely seen (26). Our data confirm some of these observations. The half-cysteines that are known to form disulfide bridges (Cys-1 to -14 and Cys-20 and -21) in apoB-100 are never >50 residues apart. The only exception is the disulfide bridge formed by Cys-20 and -21, which are separated by 131 residues. The remaining cysteine residues (Cys-15 to -19 and Cys-22 to -25) are separated from the closest cysteine by >138 residues. Only Cys-15 and -16 are closer together (separated by 85 residues) and are known to exist in their free sulfhydryl form. Our data show that 16 of the 25 cysteine residues in apoB-100 existed in disulfide forms. All 14 cysteine residues in domain 1 (9) are linked in disulfide forms, and all except Cys-1-Cys-3 and Cys-2-Cys-4 are linked to their neighboring cysteines. All six isolated single cysteine-containing peptides-namely, Cys-15 through -18, -23, and -24-have proven to be sulfhydryl peptides. Furthermore, two of the sulfhydryl groups-namely, Cys-22 and Cys-24-were exposed on the LDL surface (14). Peptide containing Cys-24 was also found in fractions 4 and 5 of Fig. 2. Locations of disulfide and sulfhydryl groups on apoB-100 are presented in Fig. 4A, which shows the structure of apoB-100 in elongated form despite the eight disulfide bridges. Cys-19 and Cys-25 remain to be confirmed. A refined structure of apoB-100 in LDL based on our information is shown in Fig. 4B. Although apoB-100 has been reported to contain thioester linkages (27), our studies found none. Lipoprotein (a) is a lipoprotein that consists of LDL and apolipoprotein (a). LDL is linked to apolipoprotein (a) through disulfide bonds (12, 13). By using a sensitive sulfhydryl probe, Coleman et al. (14) located Cys-22 and Cys-24 as the free cysteines exposed on the LDL surface. Cys-24 was alsoisolatedfromapoB-100trypticpeptides aftercarboxymethylation and delipidation of LDL, whereas Cys-22 was not found. Those results indicate that Cys-24 might have less exposure on the LDL surface than Cys-22. Armstrong et al. (28) have reported that lipoprotein (a) does not bind to the LDL receptor, but that reduction and removal of apolipoprotein (a) restores the affinity of the remaining LDL for the receptor to normal levels. Because Cys-22 is located more closely to the putative receptor-binding domain of apoB-100 as opposed to Cys-24, we speculate that Cys-22 of apoB-100 may be the cysteine residue linked to the cysteine of apolipoprotein (a). In conclusion, based on the published lipid-binding peptides (24, 25), electron microscopic data (10, 11), free sulfhydryl on LDL surface (14), and this sulfhydryl and disulfide information, we propose that the structure of apoB-100 in LDL is likely an elongated form that wraps around the LDL molecule. We thank Billy Touchstone, Zi-Wei Gu, and Hui-Xin Yang for excellent technical assistance, Susan Kelly for the artwork, Marjorie Needham for preparation of the manuscript, and Anita Cecchin for editorial assistance. Special appreciation is extended to Dr. Henry Pownall for helpful discussions and for reading the manuscript. This

Proc. Natl. Acad. Sci. USA 87 (1990)

5527

work was supported by a National Institutes of Health Grant HL-27341 (National Research and Demonstration Center in Arteriosclerosis), Grant-in-Aid 870863 from the American Heart Association, and a grant from The Methodist Hospital.

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Chem. 261, 12918-12921. 3. Knott, T. J., Pease, R. J., Powell, L. M., Wallis, S. C., Rall, S. C., Jr., Innerarity, T. L., Blackhart, B., Taylor, W. H., Marcel, Y., Milne, R., Johnson, D., Fuller, M., Lusis, A. J., McCarthy, B. J., Mahley, R. W., Levy-Wilson, B. & Scott, J. (1986) Nature (London) 323, 734-738. 4. Yang, C. Y., Chen, S. H., Gianturco, S. H., Bradley, W. A., Sparrow, J. T., Tanimura, M., Li, W. H., Sparrow, D. A., Deloof, H., Rossencu, M., Lee, F. S., Gu, Z. W., Gotto, A. M., Jr., & Chan, L. (1986) Nature (London) 323, 738-742. 5. Yang, C. Y., Gu, Z. W., Weng, S. A., Kim, T. W., Chen, S. H., Pownall, H. J., Sharp, P. M., Liu, S. W., Li, W. S., Gotto, A. M., Jr., & Chan, L. (1989) Arteriosclerosis 9, 96-108. 6. Milne, R., Theolis, R., Jr., Maurice, R., Pease, R. J., Weech, P. K., Rassart, E., Fruchart, J. C., Scott, J. & Marcel, Y. L. (1989) J. Biol. Chem. 264, 19754-19760. 7. Weisgraber, K. H. & Rall, S. C., Jr. (1987) J. Biol. Chem. 262, 11097-11103. 8. Hirose, N., Blankenship, D. T., Krivanek, M. A., Jackson, R. L. & Cardin, A. D. (1987) Biochemistry 26, 5505-5512. 9. Li, W. S., Tanimura, M., Luo, C. C., Datta, S. & Chan, L. (1988) J. Lipid Res. 29, 245-272. 10. Atkinson, D. (1988) Arteriosclerosis 8, 598a-599a. 11. Phillips, M. L. & Schumaker, V. N. (1989) J. Lipid Res. 30, 415-422. 12. Utermann, G. & Weber, W. (1983) FEBS Lett. 154, 357-361. 13. Gaubatz, J. W., Heideman, C., Gotto, A. M., Jr., Morrisett, J. D. & Dahlen, G. H. (1983) J. Biol. Chem. 258, 4582-4589. 14. Coleman, R. D., Kim, T. W., Gotto, A. M., Jr., & Yang, C. Y.

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