Biochimica et Biophysica Acta, 420 (1976) 342-349

© Elsevier Scientific Publishing Company, Amsterdam - - Printed in The Netherlands BBA 37248 ISOLATION AND C H A R A C T E R I Z A T I O N OF T H E MAJOR APOLIPOP R O T E I N F R O M C H I C K E N H I G H DENSITY LIPOPROTEINS

RICHARD L. JACKSON, HU-Y. U. LIN, LAWRENCE CHAN and ANTHONY R. MEANS Departments of Medicine and Cell Biology, Baylor College of Medicine and The Methodist Hospital, Houston, Texas, 77025 (U.S.A.)

(Received June 9th, 1975) (Revised manuscript received October 13th, 1975)

SUMMARY High density lipoproteins were isolated from plasma of white Leghorn hens by ultracentrifugal flotation between densities 1.063 and 1.210 g/ml. After delipidation, the lipid-free proteins were fractionated by chromatography on Sephadex G-150 in urea; one major apolipoprotein was isolated and characterized. From its chemical, physical and immunochemical properties, the major apoprotein from hen highdensity lipoproteins has characteristics similar to the major apoprotein of human high density lipoproteins, apoA-I. Thus the hen protein has been designated hen apoA-I. Hen apoA-I has a molecular weight of approximately 28 000 as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Its calculated molecular weight from its 234 constituent amino acids is 26 674. Hen apoA-I differed from its human counterpart by containing isoleucine. Treatment of hen apoA-I with carboxypeptidase A yielded a COOH-terminal sequence of Leu-Val-Ala-Gln. Automatic Edman degradation of the apoprotein gave an NHz-terminal sequence of AspGIu-Pro-Gln-Pro-Glu-Leu. Hen apoA-I had a circular dichroic spectrum typical of a-helical structures; the calculated helicity was 90 ~ . Goat antisera prepared to hen apoA-I formed precipitin lines of complete identity to the hen apoprotein but lines of only partial identity to human apoA-I. These studies show that the major apoprotein from hen and human high-density lipoproteins have similar properties to each other suggesting a common physiologic function.

INTRODUCTION In recent years, there has been considerable interest in the chicken as a model for the study of lipoprotein synthesis [1 ]. This model has been particularly useful since the administration of estrogen to cockerels and non-laying hens dramatically increases the synthesis of very low density lipoproteins [2-5]. As a prerequisite to studies on the effects of hormones on high-density lipoprotein synthesis, we now describe the isolation and characterization of the major apoprotein from hen high-density lipoproteins. Since the results of these studies show that the major high-density lipoprotein protein from the hen has many of the same physical, chemical and immunochemical

343 properties as that of its human counterpart, apoA-I [6], we have designated the chicken protein hen apoA-I. MATERIALS AND METHODS

Isolation oflipoproteins. High-density lipoproteins were isolated from white Leghorn laying hens weighing 2-3 kg. The animals were fed a standard chicken diet and were housed in a room that was lighted daily from 07.00 to 19.00. The hens were anesthesized with diethyl ether and killed by decapitation. The blood was collected in 1 ~ EDTA and the red cells were pelleted by low-speed centrifugation. The high density lipoproteins were isolated from plasma by ultracentrifugal flotation procedures [7] in KBr. The density range used for isolation of hen high-density lipoproteins was the same as described by Hillyard et al. [8]. The plasma was adjusted to a density of 1.063 with KBr and then centrifuged for 18 h in a Beckman 60 Ti rotor at 8 °C. The tube was sliced and the infranatant fraction adjusted to a density of 1.210 with solid KBr. The high-density lipoproteins were then obtained by centrifugation for 40 h. To remove traces of albumin, the high-density lipoproteins were resubjected to ultracentrifugation at density 1.210 g per ml. The re-isolated high-density lipoproteins were dialyzed overnight against 0.01 ~ disodium EDTA/0.01 ~o sodium azide, pH 7.5. Lipid-free proteins were obtained by three successive extractions at 4 °C of high density lipoproteins (5 mg) with diethyl ether:ethanol (35 ml, 3:1, by volume) followed by three extractions with diethyl ether [9]; the lipid-free proteins contained less than 1 ~ phospholipid as determined by the method of Bartlett [10]. The apoproteins were fractionated on Sephadex G-150 by methods previously described for the purification of human [9] and pig [11] high-density lipoproteins. Immunological procedures. Antiserum to hen apoA-I was prepared by intramuscular injection of 3 mg of the protein into a goat as described previously [12] for human apoA-I. Activity of the antiserum was determined by immunodiffusion on agar plates [12]. Amino acid sequence procedures. Edman degradation of hen apoA-I was performed automatically with a Beckman Sequencer, Model 890B. N,N-Dimethylbenzylamine (Pierce Chemical) was used as the coupling buffer. The program for the degradation was the same as that described by Hermodson et al. [13]. The phenylthiohydantoin of each amino acid was identified by gas-liquid chromatography [14] on a support of SP-400 with a Beckman GC-65 gas chromatograph apparatus and by thin-layer chromatography [15]. The COOH-terminal amino acid sequence was determined by digestion of hen apoA-I with carboxypeptidase A (COADFP, Worthington). The amino acids released by the enzyme were identified by amino acid analyses. Other methods. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate was performed as described by Weber and Osborn [16]. Circular dichroic spectra were obtained at 23 °C using a Cary 61 spectropolarimeter by the experimental procedures of Lux et al. [17]. Protein was determined by the method of Lowry et al. [18] using bovine serum albumin as the standard protein. RESULTS

Isolation of hen high density lipoproteins. High-density lipoproteins were iso-

344 lated from white Leghorn hen plasma by ultracentrifugal flotation between densities 1.063 and 1.210 g/ml [8]. After delipidation, the apoproteins were subjected to gel filtration on Sephadex G-150 (Fig. 1); one major peak which accounted for 8 5 ~ of the protein applied to the resin was detected. The major protein eluted at approximately the same elution volume as that of apoA-I from human high-density lipoproteins [20]. There was, however, a noticeable absence of a protein in hen high-density lipoproteins which had the same chromatographic properties as that of human apoAII [9, 21, 22] (Fig. 1). The protein eluting in peak B may represent the apoC proteins which are found in human very low density and high-density lipoproteins [9]. The hen apoproteins from the Sephadex Fraction A were desalted on Bio-Gel P-4 in 0.1 M ammonium bicarbonate, lyophilized and further characterized as described below. 2.2 E =

00c)

1.8

I- 1.4 bJ (..) 'o~'nz 1.0

m m l m ~ ,

I

(n 0-6 m ,< 0"2 40

60

80 I00 120 TUBE NUMBER

140

Fig. 1. Gel filtration on Sephadex G-150 of lipid-free hen (e---O) and human ( 0 - - 0 ) high-density lipoproteins. The column (2.5 × 200 cm) was equilibrated with 0.1 M Tris.HCl, pH 8.0, containing 5.4 M urea and 0.01% disodium EDTA. The sample (100 mg protein in 5.0 ml of the equilibrating buffer) was applied to the column and eluted. The flow rate was 30 ml/h and 5 ml fractions were collected. The arrow is used to designate the elution volume of human apolipoprotein A-II which is absent in the hen high-density lipoprotein.

Polyacrylamide gel electrophoresis. The major apoprotein of hen high-density lipoproteins (Fraction A) migrated as a single component on polyacrylamide gel electrophoresis in sodium dodecyl sulfate (Fig. 2). The migration of the hen protein was identical to its human counterpart, thus indicating a molecular weight of approximately 28 000. Amino acid analysis. The amino acid composition of hen apoA-I was very similar to human and pig apoA-I (Table I). From the assumed number of 234 amino acid residues, the calculated molecular weight was 26 674. The most notable difference between hen and human apoA-I was the presence of isoleucine in the hen protein. In addition, there was only one residue ofhistidine compared to five for human apoA-I.

345

m

B

iI"m

II

j A

B

C

Fig. 2. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of purified hen apoproteins from Fig. 1. A, 20#g of peak A; B, 20/~g of peak B; C, standard proteins of known molecular weight as follows: Top arrow, bovine serum albumin, molecular weight 69 000; middle, human high density apolipoprotein apoA-I, molecular weight 28 331 [20]; and bottom, human high density apolipoprotein apoA-II, molecular weight 17 360 [21, 22]. Each gel was stained with Coomassie brilliant blue. TABLE I AMINO ACID COMPOSITION OF HEN APOA-I The values are expressed as mol of amino acid per mol of protein asuming a subunit molecular weight of 26 674. The results represent averages of duplicate analyses of three different samples. Amino acid

Hen apoA-I

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine • Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophan Total Mol. wt.

19.7 (20) 11.8 c (12) 12.8 c (13) 50.5 (50) 8.1 (8) 4.8 (5) 21.0 (21) _ 12.5 a (13) 4.1 (4) 4.9 a (5) 33.0 a (33) 4.9 (5) 4.0 a (4) 20.3 (20) 1.0 (1) 16.0 (16) 3.8 r (4) 234 26 674

Human apoA-I a 21 10 14 47 10 10 19 13 3 -39 7 6 21 5 16 4 245 28 331

Pig apoA-I b 24 7 12 51 10 12 30 13 4 3 35 7 8 22 3 16 4 261 29 800

" Baker et al. [20]. b Jackson et al. [11]. c Extrapolated to zero time from 24-, 48-, 96-h hydrolyzates. d 96-h hydrolyzates. e Determined after performic acid oxidation by the method of Hirs [23]. t Determined by the method of Liu and Chang [24].

346 T MS STEP I (X3200)

StEp Z

Glu

(x 3zoo} + BSA

+ BSA

q

|

STEP s

|

{x3zoo)

STEP 4 (X3200)

+ BSA

1

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6

8

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6

8

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2

4

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{xisoo} + BSA

6

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6

4

6

8

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Leu

STEP 7 (x isoo)

STEP 6

(xl6oo)

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ELUTION TIME (Min Fig. 3. Gas-liquid chromatography recorder tracings for seven steps of the Edman degradation of hen apoA-I. The protein (200 nmol) was degraded automatically with a Beckman sequencer using the N,N-dimethylbenzylamine system described by Hermodson et al. [13]. The peak at approximately 7 rain corresponds to the internal standard norleucine (100 nmol). Bovine serum albumin (BSA) is the silylating reagent [N,O-bis(trimethyl-silylacetamine)]. The yields of each step of the degradation were 48, 75, 80, 30, 42, 30, and 52%, respectively. Each step was confirmed by thin-layer chromatography procedures [15].

Amino- and carboxy-terminal sequence. A u t o m a t e d E d m a n degradation o f hen a p o A - I gave an N H z - t e r m i n a l sequence as f o l l o w s : A s p - G l u - P r o - G l n - P r o - G l u - L e u . The recorder tracings for each step o f the E d m a n degradation are s h o w n in Fig. 3. The reported N H z - t e r m i n a l sequence [25] for h u m a n a p o A - I is A s p - G l u - P r o - P r o Gln-Ser-Pro. Treatment o f hen a p o A - I with carboxypeptidase A gave a C O O H - t e r m i n a l sequence o f L e u - V a l - A l a - G l n (Table ]I). This c o m p a r e s with L e u - A s n - T h r - G l n for h u m a n a p o A - I [26].

TABLE II RELEASE OF AMINO ACIDS FROM HEN APOA-I WITH CARBOXYPEPTIDASE A Each reaction mixture contained 20 nmol of hen apoA-I in 0.10 ml of N-ethylmorpholine buffer, pH 8.5, and 0.01 ml of carboxypeptidase A (8.5 mg/ml). The incubations were performed at 30 °C. At the indicated times, 0.1 ml of glacial acetic acid was added and the samples were evaporated to dryness with nitrogen; amino acids were determined by amino acid analyses. The values shown are the residues per tool of apoprotein based on a molecular weight of 26 674. Amino acid

1 rain

5 rain

10 min

20 min

Glutamine Alanine Valine Leucine

0.34 0.26 0.17 0.10

0.65 0.53 0.32 0.20

0.84 0.71 0.51 0.26

0.67 0.98 0.68 0.40

347

63

Fig. 4. Immunodiffusion of hen and human apoA-I against anti-hen apoA-I. The center well contained 8/d of goat antiserum prepared against hen apoA-I. The outer wells contained 10 Fg in 8/~1 of the following apoproteins: A, hen apoA-I; B, human apoA-I.

Immunochemical studies. Goat antiserum prepared against hen apoA-I formed single precipitin lines of complete identity to the hen apoprotein (Fig. 4). However, only lines of partial identity were found with human apoA-I. Circular dichroic spectrum. The circular dichroic spectra for human and hen apoA-I are shown in Fig. 5. Each apoprotein exhibited negative troughs at 222 and 208 nm, characteristic of a-helical structure. The calculated a-helical content [17] was 9 0 ~ for hen apoA-I and 65 ~o for the human apoprotein.

0 o-s K

~ -I0

~¢~fl_l-15

//'

ttl

~ -20

/'

~-Z5

$ -35

~ 200

= 220 240 WAVELENGTH (nm)

260

Fig. 5. The far ultraviolet circular dichroic spectra for hen (

--) and human ( The actual tracings, calculated for mean residue ellipticity, are shown.

.

.

.

.

.

.

.

.

.

) apoA-I.

348 DISCUSSION The results o f the present study establish the similarities between the m a j o r a p o p r o t e i n o f hen high-density l i p o p r o t e i n s and h u m a n a p o A - I . The overall a m i n o acid c o m p o s i t i o n o f hen a p o A - I was similar to b o t h h u m a n a n d pig a p o A - I . All three a p o p r o t e i n s have C O O H - t e r m i n a l g l u t a m i n e a n d m o l e c u l a r weights o f a p p r o x i m a t e l y 28 000. H i l l y a r d et al. [8] have also isolated hen high density l i p o p r o t e i n s between density 1.063 and 1.210. By electrophoresis o f d e l i p i d a t e d high density l i p o p r o t e i n s in a p h e n o l / u r e a / a c e t i c acid system, these a u t h o r s detected one m a j o r a n d several m i n o r a p o p r o t e i n s . The m a j o r c o m p o n e n t h a d a m o l e c u l a r weight o f a p p r o x i m a t e l y 30 000 a n d is no d o u b t the same p r o t e i n as in the present study. However, since H i l l y a r d et al. [8] did n o t fractionate their a p o p r o t e i n s , a direct c o m p a r i s o n o f the physicalchemical p r o p e r t i e s is not possible. It is o f considerable interest that hen a p o A - I h a d a significantly greater quantity o f a-helical structure than h u m a n a p o A - I , 90 ~ c o m p a r e d to 65 ~o. It is also greater t h a n t h a t r e p o r t e d for the pig [11], rat [27], rhesus m o n k e y [28] a n d s a l m o n [29]. ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d by Health, E d u c a t i o n a n d Welfare Research G r a n t s HL-05435/34 and HL-16512-01. The a u t h o r s are i n d e b t e d to Ms. D e b b i e M a s o n for her assistance in the p r e p a r a t i o n o f the manuscript. The a u t h o r s also wish to express their a p p r e c i a t i o n to Drs. A. M. G o t t o and B. O ' M a l l e y for their helpful suggestions. R.L.J. and L.C. are Established Investigators o f the A m e r i c a n H e a r t Association. A . R . M . is the recipient o f a F a c u l t y Research A w a r d o f the A m e r i c a n Cancer Society.

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