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Heterogeneity of Molecular Weight and Apolipoproteins in Low Density Lipoproteins of Healthy Human Males 1 Talwinder S. Kahlona, *, Virgie G. Shore b and Frank T. Lindgren c

awestern Regional Research Center, USDA-ARS, Albany, California 94710, bLawrenceLivermore National Laboratory, Livermore, California 94550 and CDonner Laboratory, University of California, Berkeley, California 94720

The molecular weights of five low density lipoprotein (LDL) subfractions from four normal healthy males were determined by analytic ultracentrifuge sedimentation equilibria. Protein content of each subfraction was determined by elemental CHN analysis, and weights of apoprotein peptides were calculated. Molecular weights in subfractions of increasing density were 2.92 -I- 0.26, 2.94 _+ 0.12, 2.68 _+ 0.09, 2.68 _+ 0.28 and 2.23 +-- 0.22 million Da, and protein weight percentages were 21.05, 21.04, 22.05, 23.10 and 29.10, in subfractions 1, 2, 3, 4 and 5, respectively. Total mean apoprotein weights for respective subfractions were 614 +_ 53, 621 -- 45, 588 + 9, 637 +_ 83 and 645 +-- 62 KDa. In addition to a single apoprotein B-100 (apo B-100) peptide with a mean carbohydrate content of 7.1% and a molecular weight of 550 KDa per LDL particle, there may be one or more apoprotein E peptides of 34 KDa and/or apoprotein C-III of 9 KDa. In addition, subfractions 4 and 5 may contain 3-7% apolipoprotein (a}. There is considerable heterogeneity among LDL subfractions as well as within the same fraction from different individuals. This heterogeneity may relate to differences in origin, metabolism and/or atherogenicity as a result of their content of apoproteins other than apo B-100. L i p i d s 27, 1055-1057 (1992).

Apolipoprotein B-100 (apo B-100) is the major apolipopr~ tein of very low density lipopmteins (VLDL), intermediate density lipoproteins (IDL), and low density lipoproteins (LDL). This apo B-100 is synthesized primarily in the liver and plays a pivotal role in lipoprotein metabolism as the LDL ligand that interacts with the LDL receptor and initiates receptor-mediated endocytosis and LDL catabolisn~ Chen et aL (1), Knott et aL (2), Yang e t al. (3) and Law et aL (4) determined the complete 4536 amino acid sequence of the apo B400 peptide, calculating a molecular weight of apo B-100 of 512,723 to 514,000 Da~ La Belle and Krauss (5) found that 7.1% carbohydrate is associated with LDL apolipoproteins. While LDL contains predominantly apoB, Campos and McConathy (6), Lee and Alaupovic (7) and Chapman e t aL (8) observed the presence of apoprotein E and apoprotein C-III in the LDL density rang~ The present study was undertaken to determine the accurate molecular weight of five LDL subfractions (1.02671.0492 gJmL) and the total mass of the apolipoproteins of these subfractions.

MATERIALS AND METHODS Four healthy normolipoproteinemic male subjects were selected for this study. Subjects were fasted overnight. Blood was drawn into evacuated tubes containing ethylenediaminetetraacetic acid dipotassium salt (EDTA), 130 rag/100 mL, and centrifuged at 1300 • g for 20 rain at 4~ to obtain plasma. The antimicrobial agent garamycin {7.5 mg/100 mL) and the protease inhibitor s-amino caproic acid (130 mg/100 mL) were added. The plasma density was adjusted to 1.019 g/mL by mixing 4 mL plasma with 2 mL of a NaBr solution of density 1.0426 g/mL containing 0.196 moles of NaC1 and 10 rag/100 mL EDTA. The resulting 6-mL solution{s) were centrifuged at 17~ in a 40.3 Beckman {Palo Alto, CA) rotor at 40,000 rpm for 18 h. The top 1 mL VLDL-IDL fraction was removed, and the second 1 mL was taken as background. The subnatant 4 mL was adjusted to 1.0670 g/mL by adding 2 mL of a NaBr solution, 1.1566 g/mL, containing 0.196 moles NaC1 and 10 rag/100 mL EDTA. Again these tubes were similarly centrifuged for 24 h, and the top 1-mL fraction containing total LDL was recovered (9). Five LDL subfractions with densities 1.0267, 1.0306, 1.0358, 1.0422 and 1.0492 g/mL, respectively, were isolated by density gradient preparative ultracentrifugation (Model L8, Beckman) using a Beckman SW 45 Ti rotor (10). LDL subfractions were dialyzed overnight at 4~ against NaC1, 1.0063 g/mL, pH 7.2-7.4. Meniscus depletion equilibrium fringes were obtained after 72 h at 6704 to 9442 rpm in a Beckman Model E ultracentrifuge using a Rayleigh interference optical system for sedimentation equilibrium measurements (11). Air in the samples was displaced by N2 in all the procedures in order to minimize oxidation. Apoprotein mass was determined in delipidated subfractions by CHN analysis. Procedures of the determination of partial specific volumes and CHN mass analysis have been described previously (12). The partial specific volume (~), mL/g, of LDL subfractions was determined according to the equation given by Schachman (13): q = 1/Qo- 1/x{(Q--~o)/~o}

[1]

where ~o = solvent d e n s i t y (g/mL); ~ = l i p o p r o t e i n solut i o n density (g/mL); x = mass concentration of LDL solu-

tion (g/mL). Each lipoprotein subfraction together with an aliquot of its corresponding equilibrium gradient background salt solution was dialyzed (3• 24 h each) in a 250-mL cylinder at 4~ against 1.0063 g/mL NaC1 solution (10 mg/dL 1Presented in part at the 78th AOCS Annual Meetingheld in New EDTA, pH 7.2-7.4). The CO2 present in the dialysate was Orleans, LA, May 1987; received best presentation award. displaced by bubbling N2 through it before use. Solvent *To whomcorrespondenceshouldbe addressedat WesternRegional and solution densities were measured to +_ 0.000001 g/mL Research Center, USDA-ARS, 800 Buchanan Street, Albany, CA using an Anton-Paar DMA-60 sixth place density meter 94710. Abbreviations: apo B-100, apo LP, apolipoprotein; apolipoprotein (Anton-Paar, Richmond, VA). The temperature of the denB-100; EDTA, ethylenediaminetetraaceticaciddipotassiumsalt; IDL, sity measuring cell was stabilized at 20 +_ 0.001~ The intermediate density lipoproteins; LDL, low density lipoproteins; reference standards used for the density calibrations were VLDL, very low density lipoproteins. dry air (corrected for barometric pressure) and purest LIPIDS, Vol. 27, no. 12 (1992)

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COMMUNICATION deionized water (boiled for 10 min to remove dissolved C02 and cooled to 20~ The m a s s (g/mL} of each L D L solution was determined to __ 0.5% using a modified C H N analyzer (Model 185, Hewlett-Packard, Palo Alto, CA). E l e m e n t a l C H N determinations were carried out in triplicate, sedimentation equilibrium runs in duplicate~ Considering all experimental uncertainties a molecular weight of 2.68 million D a can be determined with an error of __ 40 K D a (+_ 1.5%). This a m o u n t s to an 8-12 K D a error in the molecular weight of the apolipoprotein depending upon the protein content of a particular subfraction. In reality, the errors m a y be considerably smaller. RESULTS AND DISCUSSION

The molecular weights of five L D L subfractions from four healthy male subjects, as determined by sedimentation equilibrium analysis, ranged from 1.70 to 3.61 million D a {Table 1}. The mean molecular weights for subfractions 1, 2, 3, 4 and 5 were 2.92 _+ 0.26, 2.94 _+ 0.12, 2.68 +_ 0.09, 2.68 + 0.28 and 2.23 +_ 0.22 million Da, respectively. The mean protein content expressed as wt% of L D L was 21.05, 21.04, 22.05, 23.10 and 29.10 for subfractions 1, 2, 3, 4 and 5, respectively (Table 2). Calculated m e a n molecular weights of apoprotein for L D L subfractions 1, 2, 3, 4 and 5 were 614 -!-_53, 621 _+ 45, 588 _ 9, 637 +_ 83 and 645 _ 62 KDa, respectively {Table 3). The m e a n molecular weights of the five L D L subfractions analyzed in this s t u d y (Table 1) tended to decrease with increasing density and decreasing flotation rate of the subfractions. We previously reported t h a t molecular weights of L D L subfractions did not consistently decrease

with increasing density (14). The different result is prob a b l y due to the small n u m b e r of subjects involved in these studies. In the current s t u d y the molecular weights of the L D L subfractions of individual subjects did not consistently decrease with increasing density. There was a different subfraction in each of the four subjects showing the highest molecular weight. In general, protein as weight percent of L D L increased as the density of L D L subfractions increased (Table 2). The total apolipoprotein contents of subfractions 1 and 2 were similar, b u t increased by 38% from fraction 2 to fraction 5. The total apoprotein associated with various L D L subfractions had molecular weights ranging from 475 to 841 K D a (Table 3). Since a single molecule of apolipoprotein B-100 with a peptide weight of 514 K D a {1-4} is associated with each L D L particle (15), the two subfractions 6622 No. 1 and 7222 No. 2 with peptide weights of 502 and 475 KDa, respectively, appear to contain only apoprotein B-100. The low molecular weight suggests either t h a t the a p ~ B in these fractions is not glycosylated or, alternatively, t h a t these fractions m a y contain t r u n c a t e d apo B-100. Truncated forms of apo B such as apo B-37, apo B-40, apo B-54.8 and apo B-90 have been reported (16-18}. Since certain fractions contain more apoprotein t h a n could be contributed by apo-B, it is possible t h a t L D L can contain additional apoproteins. Since a single molecule of apo B-100 weighs 550 K D a including its carbohydrate component, a m e a n of 12, 13, 7, 16 and 17 wt% of subfractions 1, 2, 3, 4 and 5, respectively, m a y be comprised of other apoproteins. These other apoproteins could consist of apo E with a molecular weight of 34 K D a and/or apo C - I I I of 9 K D a (8). Since we did not determine the

TABLE 1 Molecular Weight a of Low Density Lipoprotein (LDL~ Subfractions from Four Healthy Human Males Based on Sedimentation Equilibrium U

LDL subfractionc Subject 1 2 3 4 5 6622 2.41 2.98 2.62 3.40 2.54 6769 2.95 3.28 2.45 2.81 2.65 7222 3.61 2.73 2.80 2.09 2.02 7384 2.70 2.78 2.83 2.41 1.70 Mean +_ SEM 2.92 • 0.26 2.94 + 0.12 2.68 +_ 0.09 2.68 _ 0.28 2.23 + 0.22 aMillion daltons. b6704-9442 rpm, 20.0~ CSubfractions 1, 2, 3, 4 and 5 correspond to LDL of density of 1.0267, 1.0306, 1.0358, 1.0422 and 1.0492 g/mL, respectively.

TABLE 2 Weight % Proteln a in Low Density Lipoprotein (LDL) Subfractions in Four Healthy Human Males

LDL subfractionb Subject 1 2 3 4 5 6622 20.84 20.30 22.24 24.73 31.04 6769 21.75 22.83 23.55 24.86 26.65 7222 20.74 21.43 21.95 22.71 27.45 7384 20.86 19.58 20.47 20.09 31.24 Mean + SEM 21.05 + 0.24 21.04 _+ 0.71 22.05 + 0.63 23.10 -- 1.12 29.10 +_ 1.19 aprotein by elemental CHN analysis. bFor density of the subfractions see Table 1. LIPIDS, Vol. 27, no. 12 (1992)

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COMMUNICATION TABLE 3 Total Molecular Weighta of Apolipoproteins in Low Density Lipoprotein (LDL) Subfractions in Four Healthy Human Males

LDL subfraction b Subject 1 2 3 4 6622 502 605 583 841 6769 642 749 577 699 7222 749 585 615 475 7384 563 544 579 532 Mean • SEM 614 • 53 621 • 45 588 • 9 637 • 83 aCalculated ~om the data in Tables 1 and 2 and reported as KDa. bFor density of the subfractions see Table 1.

apo C - I I I or apo E c o n t e n t of these subfractions, we c a n only speculate o n the relative a m o u n t s of apo E and/or apo C - I I I in t h e s e L D L subfractions. Lee a n d A l a u p o v i c (7) reported apoprotein C - I I I and apo E ratios of 3.67, 1.91 a n d 3.09 for their s u b f r a c t i o n s d e s i g n a t e d 2, 3 a n d 4, respectively, w h i c h a p p e a r to c o r r e s p o n d quite closely to fractions 1, 3 a n d 5 in this study. Only our fraction 1 w i t h a possible apo C - I I I to apo E ratio of 3.0 m a y be considered close to t h e m o l a r ratio of 3.09 r e p o r t e d for their s u b f r a c t i o n 4 (7). C h a p m a n e t al. (8) r e p o r t e d one apo E per 60 L D L particles; however, f r o m our d a t a it is possible to have no apo E per L D L to three apo E per L D L molecule. F r a c t i o n s 4 a n d 5 m a y also c o n t a i n 3 - 7 % as apolipoprotein (a) as previously s u g g e s t e d (8,19). T h e c u r r e n t s t u d y d e m o n s t r a t e s t h a t there is considerable h e t e r o g e n e i t y in molecular w e i g h t and apoprotein c o n t e n t a m o n g L D L s u b f r a c t i o n s as well as a m o n g t h e s a m e subfraction f r o m different individuals. T h e d a t a s t r o n g l y s u g g e s t t h a t in a d d i t i o n to one apo B per L D L molecule there are variable a m o u n t s of smaller apoproteins, p r o b a b l y apo E a n d apo C-III. Subfractions of L D L may have different origins, metabolism and/or atherogenicity based on their content of apoproteins other t h a n apo B-100.

REFERENCES 1. Chen, S~,Yang, C., Chen, P., Setzer, D., Tanimura, M., Li, W., Gotta A.M., and Chan, L. {1986}J. BioL Chem. 261, 12918-12921. 2. Knott, T.J., Pease, R.J., PoweU, L.M., Wallis, S.C., Rall, S.C., 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., and Scott, J. (1986} Nature 323, 734-738. 3. Yang, C., Chen, S., Gianturco, S.H., Bradley, W.A., Sparrow, J.T., Tanimura, M., Li, W., Sparrow, D.A., DeLoof, H., Rosseneu, M., Lee, F., Gu, Z., Gotto, A.M., and Chan, L. (1986} Nature 323, 738-742.

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4. Law, S.W., Grant, S.M., Higuchi, K., Hospattankar, A., Lackner, K., Lee, N., and Brewer, H.B. (1986}Proc. Natl. AcacL Sci. USA 83, 8142-8146. 5. La Belle, M., and Krauss, R.M. (1990)J. LipidRes. 31, 1577-1588. 6. Campos, E., and McConathy, W.J. (1986}Arch. Biochem. Biophys. 249, 455-463. 7. Lee, D.M., and Alaupovic, R (1986) Biochim. Biophys. Acta879, 126-133. 8. Chapman, J.M., Lapland, P.M., Luc, G., Forgez, P., Bruckert, E., Goulinet, S., and Lagrange, D. {1988}J. Lipid Res. 29, 442-458. 9. Havel, R.J., Eder, H.A., and Bragdon, J.H. (1955)J. Clir~ Invest. 34, 1345-1353. 10. Shen, M.M.S~, Krauss, R.M., Lindgren, FT., and Forte, T.M. (1981} J. Lipid Res. 22, 236-244. 11. Kahlon, T.S., Glines, L.A., and Lindgren, FT. (1986}Methods Enzyrnol. 129, 26-45. 12. Kahlon, T.K, Adamson, G.L., Glines, L.A., Orr, J.R., and Lindgren, FT. (1986} Lipids 21, 235-238. 13. Schachman, H.K. {1957} Methods Enzymol. 4, 1-103. 14. Kahlon, T.S., Adamson, G.L., Shen, M.M.S., and Lindgren, FT. (1982} Lipids 17, 323-330. 15. Wiklund, O., Dyer, C.A., Tsao, B.R, and Curtiss, L.K. (1985} J. BioL Chem. 260, 10956-10960. 16. Young, S.G., Northey, ST., and McCarthy, B.J. {1988)Science 241, 591-593. 17. Krul, E.S., Kinoshita, M., Talmud, P., Humphries, S.E., Turner, S., Goldberg, A.C., Cook, K., Boerwinkel, E., and Schonfeld, G. {1989} Arteriosclerosis 9, 856-868. 18. Wagner, R.D., Krnl, E.S., Tang, J., Parhofer, K.G., Garlock, K., Talmud, P., and Schonfeld, G. (1991)J. Lipid Res. 32, 1001-1011. 19. Lindgren, FT., Adamson, G.L., Shore, V.G., Nelson, G.J., and Schmidt, P.C. (1991} Lipids 26, 97-101.

[Received July 1, 1991, and in final revised form October 8, 1992; Revision accepted October 8, 1992]

LIPIDS, Vol. 27, no. 12 (1992)