Further studies on the subunit structure of human serum low density lipoproteins

BIOCHEMICAL

MEDICINE

Further

19,

Studies Serum CHI-HONG

178-187

(1978)

on the Subunit Structure Low Density Lipoproteins’ CHEN

AND FREDERICK

of Human

ALADJEM”

Received June 21, 1977

There have been numerous studies concerning the subunit structure of LDL” and many conflicting results have been obtained (for a review see ( I)). One of the major difficulties associated with all these studies has been the fact that once lipids are removed from LDL, the resulting apoprotein becomes insoluble in aqueous buffer and exists in high molecular weight forms, even in the presence of strong denaturants, such as 8 M urea. Various means have been employed to overcome this insolubility problem, in particular chemical modification and the use of detergents. We have used the method of Helenius and Simons (2) to prepare material for the study of the subunit structure of LDL (3, 4). A number of components with different molecular weights were obtained on SDS-gel electrophoresis. We suggested that the two components with lowest molecular weight (Bands VIII and IX) represent two fundamental subunits of apoLDL. We now present evidence that both of these materials, as well as the other bands eluted from SDS-gel, have similar amino acid composition. MATERIALS

AND METHODS

Preparation of’ Apopratein

LDL (d I .027- I .043 g/cm:‘) was isolated from pooled human serum by differential ultracentrifugation. It was delipidated with sodium deoxycholate, following essentially the procedure of Helenius and Simons (2) with some modifications. Briefly, the solution containing isolated LDL was 1 Supported by Research Grant No. HL 19084 from the U.S. Public Health Service. Taken in part from the Ph.D. thesis of C.H.C. ” To whom reprint requests should be addressed. ‘I Abbreviations used: LDL, low density lipoproteins; apoLDL. the delipidated apoprotein of LDL; SDS. sodium dodecyl sulfate: NaDOC, sodium deoxycholate. I78 0006-2944/78/019?-0178$09.00/O Copyright 411 rights

0 1978 by Academic Pree. Inc. of reproduction in any form rewrvrd.

SUBUNITSTRUCTUREOFHUMAN

SERUM LDL

179

dialysed for 24 hr against a 0.05 M carbonate buffer (pH 9.7) containing 0.15 M NaCl. Sodium deoxycholate was then added to the dialysed lipoprotein solution at a concentration of 57 mg NaDoc/mg of LDL protein. The LDL protein concentration was usually about 3.5 mgiml. The solution was kept at ambient temperature for one hr and transferred to the cold room overnight. A small amount of insoluble material developed in both the protein-containing sample as well as the non-protein-containing NaDOC control solution, and was removed by low-speed centrifugation. The clear supernatant was applied to a Sephadex G-200 column in the cold room. and the sample was eluted with 0.05 M sodium carbonate buffer (pH 9.7) containing 10 mM NaDOC and 0. I5 M NaCl at a flow rate of 8.8 mlihr. The protein content of fractions collected was monitored by absorption measurement at 280 nm. SDS Polyrctylamide

Gel Electrophoresis

This was carried out essentially according to Fairbands et al. (5). with minor modifications. Details have been described previously (4, 6). N-Termincrl

Determination

These were performed by the procedure of Weiner et (11. (7). The elution of materials from SDS-gel before analysis has previously been described (4). Amino Acid Analysis Amino acid analyses were carried out by the method of Stein et ~1. (8) on stained protein bands sliced from SDS-gels. Blank gels were processed similarly. An analyzer was assembled in our laboratory essentially according to that described by Stein et al. (9), using fluorescamine (Roche Diagnostic) as the fluorogenic reagent. Single-column methodology was employed to elute the amino acids off the column (packed with Durrum DC-4A resin). The buffers used were: buffer I. pH 3.25 citrate buffer, (Nat) = 0.2 M; buffer II, pH 4.25 citrate buffer, (Na’) = 0.2 M; and buffer III, pH 7.9 citrate buffer, (Na+) = 1. I M. Column temperature was started at 49°C and changed to 63°C right after the elution of norleucine, which was added as internal standard in every analysis. Appropriate corrections for the background from blank gel were made in the final calculation of amino acid compositions. Digestion of LDL or ApoLDL with Protetlses Digestion of LDL with trypsin or thermolysin was conducted at 37°C in a shaking water bath in 0.1 M ammonium bicarbonate buffer or 0. I M ammonium acetate buffer containing 5 mM CaCl, (pH 8.5), at an enzyme to protein ratio (w/w) of I to 60, for 16 hr. ApoLDL obtained by NaDOC

180

CHEN AND ALADJEM

delipidation was similarly treated with trypsin at an enzyme to protein ratio of 1 to 100 (w/w) in 0.05 M carbonate buffer (pH 9.7) containing IO mM NaDOC and 0.15 M NaCl for 24 hr. The reaction was stopped by immersing the sample in a boiling water bath for 2-3 min in the presence of 1% SDS. Other Analytical

Procedures

Protein was determined by the modified Lowry procedure of Schacterle and Pollack (IO) using human serum albumin (pentex) as standard. Phosphorus was determined directly on column eluates by the method of Bartlett (I I), and the amount multiplied by 25 to give the amount of phospholipid. Cholesterol was determined by the method of Searcy et a/. (12); triglyceride by a modified procedure of Carlson (13) with triolein (Applied Science Laboratories, Inc.) as standard. RESULTS

Chromatography of NaDOC-treated LDL on Sephadex G-200 yielded two uv-absorbing peaks. The major peak (peak I), which contained most of the protein, was eluted right after the void volume. The minor peak (peak II) contained all the lipids and a small amount of protein. This peak was eluted approximately 60 ml ahead of the total solvent volume on a 2.5 x 90 cm column. As shown previously, when the apoLDL (peak I material) thus obtained was subjected to SDS-polyacrylamide gel electrophoresis. five major and several minor components were obtained (4). The components were designated Band III (MW 77,000), Band IV (MW 66,000), Band V (MW 47,000), Band VI (MW 33,500), Band VII (MW 21,500), Band VIII (MW 13,000) and Band IX (MW 9,500). When the apoLDL had undergone storage at 4°C for about one and a half months, followed by dialysis against a low ionic-strength buffer (0.01 M Tris-HCI, pH 8.0) containing SDS (0. I%), EDTA (2 mM) and dithiothreitol(O.5 mM), bands VIII and IX were the most prominent components, and Band V usually split into three closely-spaced bands. In order to establish the identity or nonidentity of the multiple bands that resolved on SDS-gel, gel fractions containing the bands were cut and amino acid analysis and N-terminal determination were carried out on each. In Table 1 the results are given of amino acid analyses of Bands III through IX isolated from SDS-gel. Also presented is the amino acid composition of whole apoLDL prepared by organic solvent delipidation and determined under the same condition. The contents of proline, halfcrystine and tryptophan were not determined and are not included in the summation of total amino acid residues. Methionine content was estimated directly from the acid hydrolysate and not from material obtained

SUBUNIT

STRUCTURE

OF HUMAN

SERUM

Ial

LDL

after per-formic acid oxidation. The figure given for methionine may therefore be expected to be lower than the true value. Within the limits of experimental error, the data in Table I indicate that the amino acid composition of the material of each band is essentially identical, and identical to that of whole apoLDL. Every fraction contained all the amino acids examined. Good agreement was found between analyses of different preparations (Table 2). Good agreement was also seen between the amino acid composition of apoLDL determined in this study and a previous analysis (14). The technique we used for N-terminal determination was chosen originally for its capability to obtain partial N-terminal sequence on proteins or peptides with unblocked N-terminus at highly sensitive detection level; it requires 0.25 nmole or less of substrate at each step. This would furnish another experimental parameter that could aid in the differentiation of the multiple components isolated on SDS-gel. When the sequence analyses were conducted on each of the components eluted from SDS-gel (Bands II to VII), however, no distinct fluorescent spots other than that of c-lysine were detected after the first degradation cycle. The analysis was therefore not carried out further. AMINO

TABLE 2 ACID COMPOSITION (IN MOLE PERCENT) BANDS VI AND VII OF ApoLDL”,” Band VI (preparation)

Amino

acid

Aspartic acid Threonine Serine Glutamic acid Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine ” In these experiments. determined. h Values are the means

OF

Band VII (preparation)

I

2

I

2

3

I I .73 6.79 8.11 13.99 6.09 7.08 6.01 0.79 6.09 Il.97 2.84 3.56 3.25 8.07 3.66

I I .26 6.72 6.94 14.17 5.15 7.31 6.19 I .27 6.04 12.68 3.06 3.95 2.69 8.95 3.66

II.15 7.20 8.02 12.70 6.63 7.10 6.17 0.62 6.22 Il.56 2.98 4.27 3.45 8.53 3.44

10.90 7.07 7.44 13.17 5.58 7.07 6.33 1.21 5.96 I I .a?. 3.26 4.47 3.07 9.31 4.28

II.69 6.37 7.96 13.63 7.1 I 6.74 5.76 0.88 6.16 I I .69 2.59 4.30 2.64 7.82 3.66

half-cystine

and tryptophan

were

the contents of duplicate

of proline. determination

of each preparation.

not

i5

Met

Val

Ala

Gly

Glu

Set

Thr

Asp

Amino

acid

12.39 (12.41-12.37) 7.06 (7.09-7.03) 9.82 (9.84-9.79) 12.01 (12.28-l 1.75) 6.3 I (6.34-6.28) 6.46 (6.47-6.44) 5.66 (5.78-5.53) 0.89 (0.93-0.84)

Band III

AMINO

Il.49 (I 1.66-l 1.31) 6.08 (6.3 l-5.85) 8.05 (8.13-7.96) 13.74 (13.77-13.71) 5.70 (5.74-5.66) 6.61 (6.71-6.50) 5.51 (5.66-5.35) 0.72 (0.77-0.67)

IV

Band

V

(IN MOLE

II.90 (12.02-11.77) 6.24 (6.32-6.16) 8.24 (8.39-8.09) 13.89 (13.96-13.82) 5.25 (5.26-5.23) 5.78 (5.85-5.70) 5.50 (5.54-5.45) 0.97 ( I .oO-0.94)

COMPOSITIONS

Band

ACID

k 0.34

VI”

1.03 + 0.28

6.10 t 0.22

7.19 -t 0.20

5.62 k 0.55

14.08 2 0.16

7.52 2 0.67

6.75 t 0.13

il.49

Band

-+ 0.37

+ 0.19

VII’

0.91

6.15

7.01

6.29

t 0.30

k 0.23

+ 0.15

2 0.67

13.07 k 0.41

VIII

Band

IX

ON SDS-GEL”

10.92 ( I I .09- 10.75) 6.32 (6.32-6.31) 7.07 (7.14-6.99) 13.61 (13.61-13.60) 8.39 (8.57-8.21) 6.12 (6.24-6.00) 5.60 (5.70-5.49) 0.87 (0.90-0.84)

SEPARATED

II.69 (11.87-11.51) 6.40 (6.62-6.17) 7.76 (7.88-7.64) 13.68 (13.81-13.55) 5.51 (5.57-5.44) 6.45 (6.49-6.4 I) 5.35 (5.54-5.15) 0.95 (I .05-0.84)

Band

OF ApoLDL

7.77 2 0.31

6.98

II.02

Band

TABLE I PERCENT) OF BANDS

1.20

5.40

4.79 0.67

6.10

4.90

Il.90

8.60

6.40

10.50

.4poLDL”

6.73

5.87

12.48

8.39

6.62

11.30

ApoLDL (this study)

5

‘I Eyrent deters ” v (’ \ d-

A%

LYS

His

Phe

Tyr

Leu

Ile

otherwise,

.r referer,

noted

6.08 (6.09-6.06) 12.39 (12.46-12.31) 2.62 (2.62-2.61) 4.77 (4.87-4.66) 2.53 (2.61-2.44) 8.18 (8.30-8.06) 2.85 (2.89-2.81)

values

ard deviations lard deviations

+ 0.11

k 0.58

of duplicate

3.66 ? 0.19

8.51

2.97 + 0.33

4.35

3.77 -c 0.23

_’ 0.64

on two on three

determinations.

two two

determinations determinations

half-cystine

6.16 (6.32-6.00) 12.10 (12.39-11.80) 2.53 (2.58-2.48) 3.93 (4.06-3.80) 2.84 (2.86-2.81) 9.56 (9.62-9.50) 4.01 (4.18-3.83) of proline, preparations, preparations.

Contents

6.56 (6.62-6.49) 12.27 (12.29-12.24) 2.99 (3.05-2.93) 4.51 (4.52-4.50) 3.25 (3.45-3.05) 8.96 (9.03-8.89) 3.72 (3.87-3.57)

different different

3.82 + 0.49

8.70

3.13 + 0.39

+ 0.13

3.01 -t 0.28

11.69 -’ 0.20

6.10 t 0.17

2.95 ” 0.14

12.33 -t 0.43

6.06

of four analyses performed of six analyses performed

and ranges

7.06 (7.08-7.04) 12.58 (12.71-12.45) 3.34 (3.42-3.26) 4.75 (4.79-4.70) 3.22 (3.42-3.01) 8.18 (8.27-8.09) 3.12 (3.30-2.94)

are the means

6.42 (6.52-6.3 I) 12.59 (12.85- 12.33) 2.78 (2.87-2.68) 4.84 (4.88-4.79) 3.16 (3.35-2.97) 8.52 (8.63-8.4 I) 3.83 (3.93-3.73)

each. each.

and tryptophan

are not

3.50

8.10

8.13 3.88

2.50

4.04

3.30

3.55

5.10

II.10

12.96

5.17

5.70

5.87

184

CHEN AND ALADJEM

Digestion of LDL with either trypsin or thermolysin resulted in products that yielded 8-12 discrete bands upon electrophoresis in SDS-gel (Figs. IA and B). About half of them had a molecular weight less than 50,000. The band patterns of LDL produced after the action of these two enzymes were somewhat similar to each other, but quite different from

FIG. I: Electrophoresis of enzyme-treated LDL and apoLDL in SDS-polyacrylamide gel. (A) LDL digested with thermolysin. (B) LDL digested with trypsin. (C) NaDOCddipidated apoLDL digested with trypsin. The anode is toward the bottom. For experiental details, see text.

SUBUNIT

STRUCTURE

OF

HUMAN

SERUM

LDL

185

that of NaDOC-delipidated apoLDL. Digestion of NaDOC-delipidated apoLDL with trypsin yielded products which show as one major diffuse zone with molecular weight approximately 10,000 under the same conditions (Fig. IC). The nature of these digestion products was not investigated further. DISCUSSION

Before we discuss these results, we want to point out that LDL has been reported to contain protease-like activity (15). We have searched for such activity but we could find no evidence for it (4). Absence of this activity has now been confirmed (16). Further corroborative evidence that the multiple bands observed on SDS-gels are not artifacts generated by the action of protease activity comes from the protease digestion experiments of LDL and apoLDL (see Results and Fig. I). The data show that the band pattern of apoLDL on SDS-gel is quite different from that of apoLDL treated with trypsin, and different from that of LDL treated with either trypsin or thermolysin. The possibility that the multiple bands formed on SDS-gel were due to differential SDS-binding by a single component has been ruled out by the previously reported results of the Ferguson plot experiment (4. 6). One significant finding of the present study is the observation that the amino acid composition of each of the multiple bands is nearly identical and identical to that of whole LDL (Tables I and 2). We interpret this to mean that the multiple bands which we observe are an oligomeric series and that each of the bands represents a different state of aggregation of one or of a small number of essentially similar, fundamental subunit(s) of the protein moiety of LDL. Using the highly sensitive dansylation technique, we have been unable to detect any N-terminal amino acid in the various fractions. eluted from SDS-gel. This suggests that the N-termini of the polypeptide(s) present in these fractions are all either highly unreactive to the reagent or are blocked. While it is generally assumed that glutamic acid is the N-terminus of LDL apoprotein, it should be noted that only one mole of glutamic acid has been demonstrated per 500.000 g of peptide (17). Quantitative N-terminal analysis such as that of Stark (18) might resolve this problem. The present results then show that apoLDL can be dissociated into fractions of differing molecular weights and that these fractions have similar amino acid composition (Table I). Since only very small quantities of these fractions were available, the usual physicochemical methods for molecular weight measurements could not be used and the data in Table 1 are given as mole percent. The results of the amino acid analyses are compatible with either the presence of one fundamental subunit of

186

CHEN AND ALADJEM

apoLDL, or of a small number of fundamental subunits, all with very similar amino acid composition. The results of immunochemical studies (19-23), and the reports of differences with respect to amino acid composition (24-26) provide evidence for heterogeneity of LDL and apoLDL. The present observations taken together with the results just cited suggest that the protein moiety of LDL is probably composed of a small number of fundamental subunits of similar primary structure, differing in only a few amino acids, similar perhaps to the amino acid differences which exist among the constant regions of immunoglobulin chains. The presence of small quantities of subunits with different amino acid composition or of subunits with different carbohydrate structure can not be excluded. Since no covalent bonds were broken in our separation procedure, we conclude that LDL apoprotein subunits form non-covalently bonded aggregates and that such aggregates constitute the protein moiety of LDL. SUMMARY

The apoprotein moiety of human serum low density lipoprotein was separated by SDS-gel electrophoresis into several components ranging in molecular weight between approximately 80,000 and 10,000 daltons. Amino acid analyses and N-terminal determination were done on each fraction. Every fraction was found to have essentially identical amino acid composition. We interpret the data to indicate that apoLDL is an aggregate composed of a small number of fundamental subunits of similar amino acid composition. ACKNOWLEDGMENTS The authors are grateful to Dr. John J. Albers, University of Washington, Seattle, for carrying out the cholesterol and triglyceride analyses, and to Dr. N. Swaminathan for help in the set-up of the fluorescamine system.

REFERENCES I. Morrisett. J. D.. Jackson, R. L.. and Gotto, A. M.. Jr., Ann. Rev. B&hem. 44, 183 (1975). 2. Helenius, A.. and Simons, K., Biochem. 10, 2542 (1971). 3. Chen, C.-H., and Aladjem, F., Fed. Proc. 33, 229a (1974). 4. Chen, C.-H., and Aladjem, F., Biochem. Biophys. Rrs. Comm. 60, 549 (1974). 5. Fairbanks, G., Steck, T. L., and Wallach, D. F. H., Biochemistry 10, 2606 ( 197 I). 6. Chen, C.-H., Ph.D. Thesis, University of Southern California (1975). 7. Weiner, A. M., Platt, T.. and Weber, K., .I. Biol. Chem. 247, 3242 (I 972). 8. Stein, S., Chang, C. H., Bohlen, P., lmai. K., and Udenfriend, S.. Ancrl. Biochem. 60, 272 (1974). 9. Stein, S., Bohlen, P., Dairman, W., and Udenfriend. S.. Arch. Biochem. Biophys. 155, 202 (1973). 10. Schacterle. G. R.. and Pollack. R. L.. Ant//. Biwhem. 51, 654 (1973). 1I. Bartlett. G. R., J. Biol. Chrm. 234, 466 (1959).

SUBUNIT 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

25. 26.

STRUCTURE

OF HUMAN

SERUM

LDL

187

Searcy. R. L.. Bergquist, L. M., and Jung, R. C.. J. Lipid Res. 1, 349 (1960). Carlson, L. A., Acrcl Med. SW. Ups. 64, 208 (1959). Bersot, T. P.. Ph.D. Thesis, Vanderbilt University, p. 25 (1972). Krishnaiah. K. V., and Wiegandt. H., FEBS Left. 40, 265 (1974). Chapman. M. J., and Kane. J. P.. Biochem. Biophys. Res. Comm. 66, 1030 (1975). Margolis, S. in “Structural and Functional Aspects of Lipoproteins in Living System.” (E. Tria and A. M. Scanu. Eds.), pp. 386-388. .Academic Press. New York, 1969. Stark. G. R.. Methods Enzymol. 25, 103 (1972). Aladjem, F.. Lieberman. M.. and Gofman, J. W.. J. E. Erp. Med. 105, 49 (1957). Aladjem, F.. and Campbell, D. H.. Nclture 179, 203 (19.57). Aladjem. F., Naturr 209, 1003 (1966). Middleton. E.. Amer. .I. Physiol. 185, 309 (1956). Lee, D. M.. and Alaupovic. P., Biochemistry 9, 2244 (1970). Shore, B.. and Shore, V., Biochemistry 8, 4510 (1969). Shore, B., and Shore, V., in “Die Lipoproteine des Blutes.” Wissenschaftliche Veroeffentlichungen der Deutschen. Gesellschaft fuer Ernaehrung, Band 23. 1973. Kane, J. P.. Richards, E., and Havel. R. J.. Proc. Nnrl. Acnd. SG., U. S. 66, 1075 ( 1970).