Biochimica et Biophysics Elsevier
169
Acta, 1043 (1990) 169-176
BBALIP 53367
Isolation and characterization of human apolipoprotein A-I Fukuoka (110 Glu + Lys). A novel apolipoprotein variant Yoichi Takada
I, Jun Sasaki ‘, Shigenori Ogata 2, Toshiaki and Kikuo Arakawa ’
Nakanishi
3, Yukio Ikehara
2
’ Department of Internal Medicine and ’ Department of Biochemistry and ’ Department of Medicine, Nagasaki
Fukuoka University School of Medicine, Fukuoka University Hospital, Nagasaki (Japan)
(Received 22 September 1989)
Key words: Apolipoprotein A-I; Apolipoprotein variant; Protein characterization; HDL; (Human)
A novel genetic variant of apolipoprotein(apo) A-I Fukuoka, has been identified in a Japanese family. This variant has a relative charge of +2 compared to normal apolipoprotein A-I (A-I,), on the isoelectric focusing gels and the same molecular mass and immunologic characteristics as normal apolipoprotein A-I. This variant, transmitted as an autosomal co-dominant inheritance was purified by preparative Immobiline isoelectric focusing. Sequence analysis after cleavage with lysyl endopeptidase and CNBr, followed by high-performance liquid chromatography revealed a single substitution of lysine at position 110, instead of the usual glutamic acid. This mutant apolipoprotein A-I has much the same potential as to activate lecithin-cholesterol acyltransferase.
Introduction Apolipoprotein A-I (apo A-I) is the major apoprotein of high-density lipoproteins (HDL) [l]. Apolipoprotein A-I and HDL cholesterol are inversely related to the development of premature cardiovascular disease [2]. Apolipoprotein A-I activates lecithin-cholesterol acyltransferase (LCAT) which catalyzes esterification of cholesterol to cholesteryl ester and might participate in the reverse cholesterol transport from tissues to the liver [3]. Mature apolipoprotein A-I is a single polypeptide of 243 amino acids (M, = 28 100) [4]. Newly secreted apolipoprotein A-I appears in plasma as the 249 amino acid pro-apo A-I and is processed to mature apo A-I after cleavage of six amino-terminal amino acids [5]. Apolipoprotein A-I was found to have five isoforms (A-I, to A-I,), on isoelectric focusing gel [6]. Several genetic variants of human apolipoprotein A-I have been reported [1,8-171. These variants have relative charge differences of + 2, + 1 or - 1 compared to major mature apo A-I, apo A-I,. Analysis of the primary structures of these variants revealed a single amino acid substitution, except for one variant with a deletion of
Correspondence: J. Sasaki, Department of Internal Medicine, Fukuoka University Hospital, 45-1, ‘I-chome Nanakuma, Jonan-ku, Fukuoka 81401, Japan. 0005-2760/90/$03.50
single lysine residue (A-I Marburg 1151). During screening programs for the apolipoprotein A-I variant, we identified a novel apolipoprotein A-I variant (A-I Fukuoka), which has a relative charge difference of +2 compared to apo A-I,. We modified the screening method for apolipoprotein A-I and noted the usefulness of lysyl endopeptidase digestion followed by high-performance liquid chromatography for analyzation of the primary structure of the +2 type apo A-I variant. Materials and Methods Patient Proband (T.O.) is a 57-year-old male and was detected as having low HDL cholesterol (HDL-C) level of 13 mg/dl. He was admitted to the Fukuoka University Hospital because of diabetic coma. His HDL-C level increased from 13 mg/dl to 47 mg/dl, at the same time blood glucose level normalized after insulin administration. Materials Serum samples were obtained from the central laboratory of Fukuoka University Hospital. Criteria for sampling included HDL cholesterol levels over than 100 mg/dl or less than 30 mg/dl. Acrylamide, N,N, N ‘, N ‘tetramethylethylenediamine (TEMED), and N, N ‘methylenebisacrylamide were purchased from Bio-Rad. Sodium decyl sulfate was obtained from Eastman
0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
170 Kodak, obtained
ampholytes and Immobiline dry from LKB (Bromma, Sweden).
plates
were
Isoelectric focusing gel electrophoresis Isoelectric focusing gel electrophoresis for screening was performed by either the one-step method [lo] or in combination with ultracentrifugation. For the combination with ultracentrifugation and the one-step methods, 200 ~1 of serum was adjusted to a density (d) of 1.21 g/ml with KBr and centrifuged at 39000 rpm for 5 h in a Hitachi RPL 42T disk rotor at 4°C. 2 pl of the d -C 1.21 g/ml fraction was incubated for 1 h at room temperature with 50 ~1 of 0.01 M Tris-HCl (pH 8.2) containing 1% sodium decyl sulfate and 2% ampholite (pH 4-6) and 5 ~1 of mercaptoethanol. Then 10 ~1 of 80% sucrose and 0.05% Bromophenol blue (BPB) were added and the sample was applied to the gel. The gel solution contained (per liter) 75 g acrylamide, 2.0 g bisacrylamide, 480 g urea, 41 ml ampholite (pH range 4-6) and 0.65 ml TEMED, 0.65% ammonium persulfate. This solution was added to cassettes of a Bio-Rad Protein II vertical slab gel electrophoresis cells with 1.5 mm spacers. A 20-well comb was used to prepare the slots. With this system a maximum of 80 samples can be applied. The upper and lower tray buffers are 0.02 M NaOH and 0.01 M H,PO,, respectively. The gels were electrophoresed for 17 h at 250 V and the voltage was increased to 600 V for 2 h. The gels were stained with a Coomassie blue G-250. 2 g of Coomassie brilliant blue G-250 (Bio-Rad) was dissolved in 150 ml of 80” C water to which 75 ml of 1.5 M H,S04 was added, then the solution was heated for 10 min at 80” C. After filtration, the pH was raised to 5.5 with 5 M KOH and 0.25 g of decylsulfate and 50 g of trichloroacetate were added. The gels were stained for 30 min at 60” C and destained with 20% ethanol at 60°C. Two-dimensional electrophoresis Individual samples from the first isoelectric focusing gels were cut and immersed in 0.002 M ethylmorpholine-HCl (pH 8.5), 0.2% SDS, 0.1% /?-mercaptoethanol, BPB solution and 4% sucrose for 15 min at room temperature and subjected to SDS polyacrylamide gel electrophoresis in an Atto electrophoresis chamber (Atto Corporation, Japan). The SDS system was prepared according to Neville [18]. The stacking gel contained (per liter) 50 g acrylamide, 1.4 g bisacrylamide, 0.375 M Tris-HCl (pH 8.8), 1.0 g SDS, 0.5 ml TEMED and 0.5 g ammonium persulfate. The running gel contained (per liter) 150 g acrylamide, 4 g bisacrylamide, 0.375 M Tris-HCl (pH 8.8), 1.0 g SDS, 0.5 ml TEMED and 0.5 g ammonium persulfate. 1% melted agarose containing 0.06 M Tris-HCl (pH 6.8), 2% SDS and 5% mercaptoethanol was poured on the surface of the upper gel and the first isoelectric focusing gel was immediately placed
into the agarose on the SDS gel. The tray buffer was 0.05 M Tris, 0.384 M glycine and 0.1% SDS (pH 8.4). Electrophoresis was carried out at 20 mA for stacking, and at 40 mA through the running gel. The gels were stained with Coomassie blue R 250. Production of antisera to apolipoprotein A-I Apolipoprotein A-I was purified using an Immobiline gel [19] (see preparative Immobiline isoelectric focusing in Materials and Methods). Purity was checked by amino acid analysis. Male New Zealand white rabbits weighing 2-3 kg were immunized with about 500 pg of pure apolipoprotein A-I which was emulsified with an equal volume of Freund’s complete adjuvant and administered subcutaneously at multiple sites. Booster immunizations were given twice with 250 pg of apolipoprotein A-I, at 2-week intervals. Blood samples were obtained from the ear vein. The purity of antiserum to apolipoprotein A-I was checked using an Ouchterlony plate. As a single precipitin line formed against whole serum and purified apolipoprotein A-I, the antiserum was considered to be monospecific. Immunoblot Identification of two-dimensional electrophoretic spots of apolipoprotein A-I by immunological methods was done by immunoblots (Instruction for the Bio-Rad Immun-Blot (GAR-HRP) Assay Kit). The buffers used for the transfer of SDS gels to nitrocellulose paper were 0.7% acetic acid and 0.25 M Tris-HCl, 0.192 mM glycine, and 20% methanol (pH 8.3). Proteins were transferred to nitrocellulose paper at 40 V for 1 h. After the transfer, the remaining non-detected sites on the nitrocellulose paper were blocked for 1 h by incubation with 0.02 M Tris-HCl and 0.5 M NaCl (pH 7.5) (TBS) containing 3% gelatin. The nitrocellulose paper was then incubated for 16 h in 1% gelatin in TBS containing a monospecific rabbit antibody to apolipoprotein A-I. After incubation, the preparation was washed three times in TBS containing 0.05% Tween-20 (TTBS). The nitrocellulose paper was then incubated for 2 h in 1% gelatin in TBS containing an affinity purified antirabbit IgG horseradish peroxidase reagent (l/3000 dilution, Bio-Rad Lab, Richmond, CA). Then it was washed three times in TTBS. The peroxidase complex was developed with color development reagent (Bio-Rad Lab, Richmond, CA). Preparative Immobiline isoelectric focusing For anlytical purposes, the pure apo A-I isoforms were isolated by preparative Immobiline isoelectric focusing. HDL was isolated by preparative ultracentrifugation [19]. After dialysis against 0.9% NaCl, the HDL were delipidated with ethanol/diethyl ether (3 : 1) and diethyl ether. The delipidated HDL was solubilized in deionized water (20 mg/ml). The Immobiline dry
171 plate (pH 5-6) was rehydrated with 20 ml of 8 M urea for 2 h at room temperature. After a few drops of kerosene had been pipetted onto the cooling plate of the LKB Multiphor Electrophroresis Unit, the gel was placed on the cooling plate. The electrode strips were soaked with 10 mM glutamic acid at the anode and 10 mM NaOH at the cathode and laid along the corresponding edges of the gel. 1 ml of sample solution was applied to the filter paper (10 X 240 mm) laid on the gel surface at 10 mm distance from the anode strip. The gel was electrophoresed overnight at a constant current of 1 mA at 10°C. The focused apoproteins were visualized by soaking the gel in water. Individual visualized bands were excised and eluted with 0.1 M Tris-HCl (pH 7.4) containing 4 M guanidine and 1 mM EDTA. The eluted proteins were exhaustively dialyzed against deionized water and lyophilized. Cleavage and isolation of several peptide fragments of apolipoprotein A-I Purified apo A-I isoforms were digested with lysyl endopeptidase for 6 h at 30°C in 1 ml of 0.01 M Tris-HCl buffer (pH 9.0) at the enzyme-to-substrate ratio of 1 : 100 (w/w) [20]. The digested peptide fragments were separated by reverse-phase HPLC on a Wakosil 5C18-200 column (4.0 X 250 mm) at a flow-rate of 1 ml/mm. The purified apolipoprotein A-I was cleaved with 2.5 M CNBr in 70% (v/v) formic acid for 24 h at room temperature [4]. The sample thus treated was subjected to HPLC through a Superose 12 column (1.0 x 30 cm) with 70% formic acid. Six major peptide peaks were evidenced with monitoring by absorbance at 280 nm. Fractions of each peak were pooled, freezedried, each sample was dissolved in 0.1% trifluoroacetic acid and then subjected to HPLC on a PBondapack Cl8 column (0.4 X 30 cm) with a linear gradient from 0 to 80% acetonitrile in 0.1% trifluoroacetic acid. Fractions of each major peptide peak obtained were freezedried and used for chemical analysis. Determination of amino acid sequences Purified samples of apolipoprotein A-I (30 pg) and the peptide fragments (about 5 pg each) were analyzed for amino acid sequences by an Applied Biosystems Model 477A Gas-Phase Sequencer with an on-line Model 120A phenylthiohydantoin derivative analyzer, based on instructions provided by the manufacturer. Purification of lecithin-cholesterol acyltransferase Lecithin-cholesterol acyltransferase was purified from fresh human plasma, as described [21,22]. The final purification of the enzyme was approx. 20000-fold over the starting plasma. The purified enzyme was stored at 4” C in 0.01% EDTA (pH 74) containing 4 mM 2-mercaptoethanol.
Preparation of phosphatidylcholine (PC)-cholesterol vesicles Egg PC was isolated according to Singleton et al. [23] and further purified by silicic acid column chromatography [22]. Thin-layer chromatography of the preparation on a Silica Gel G plate with chloroform/ methanol/water (165 : 25 : 4, v/v) as the developing solvent gave a single spot of PC. Single bilayer PCcholesterol vesicles were prepared by the method of Batzri and Korn [24]. A typical preparation contained 900 nmol of egg PC and 150 nm of [7or-‘HIcholesterol (specific activity, 4.3 mCi/mmol) per ml. Lecithin-cholesterol
acyltransferase
co-factor
activity
as-
say The enzyme activity was determined as described [22]. The standard assay mixture consisted of 100 ~1 of single bilayer vesicle preparations containing 90 nmol of egg PC and 15 nmol of [7a-‘HIcholesterol, 20 pg of apolipoprotein A-I, 4 mM 2-mercaptoethanol, 0.7 mM EDTA, 2.5 mg of bovine serum albumin and the enzyme solution in a final volume of 250 ~1 of phosphate buffer. The vesicle solution and apolipoprotein A-I were preincubated for 30 min at 37” C under N,, prior to addition to the assay mixture. For the assay, the mixture containing enzyme solution was incubated for 1 h at 37 o C under N,. The rate of cholesterol esterification is given in ng of cholesterol esterified per hour. In all the enzyme assays, we used the labeled PC-cholesterol vesicles prepared within 1 week and the enzyme preparations with over 0.2 units of activity per ng of protein (One unit of enzyme designates the esterification of 1 nmol of unesterified cholesterol per hour at 37 o C under standard assay condition). Protein and lipid analysis Protein was determined by the method of Lowry et al. [25] with bovine serum albumin as the standard. Lipids and apolipoproteins were analyzed as described [26,27]. Results
Fig. 1 shows that the variant on isoelectric focusing gel electrophoresis in a pH gradient of 4 to 6, had a relative charge of + 2 compared to the normal major mature apo A-I,. As determined by two-dimensional electrophoresis, the variant had a molecular weight identical with that of normal apo A-I, (Fig. 2). After two-dimensional electrophoresis, this ‘abnormal’ spot reacted by immunoblot (Fig. 2-C) with a monospecific antibody to apolipoprotein A-I (Fig. 3). Studies of the family revealed two other heterozygotes in the first-degree family members (Fig. 4) thereby suggesting an autosomal co-dominant inheritance. There were no marked differences in the levels of lipids, lipoproteins
Fig. 1. Isoelectric tocusmg gel of serum in a pH range from 4 to 6. Normal serum was applied in lanes A, B; sera from heterozygous apolipoprotein A-I Fukuoka in lane C; d < 1.21 sera separated by RPL-42T rotor in lane D.
and apolipoproteins between the carriers and the non-carrier members (Tables I and II). To investigate the amino acid sequence, the variant apolipoprotein A-I was isolated by preparative Immobiline isoelectric focusing gel electrophoresis, using delipidated HDL (Fig. 5). The purified variant isoform was cleaved with lysyl endopeptidase and the digested peptides were separated by reverse-phase HPLC (Fig. 6). The HPLC profile of the lysyl endopeptidase (LE) digest of variant apolipoprotein A-I showed the presence of three peptides, designated LE 3*, LE 5 * and LE 6 *, which were absent in normal apo A-I, (data not shown). Sequence analysis of LE 3 *, LE 5 * and LE 6 * revealed the presence of lysine at position 110 (Table III), at which glutamic acid is identified in the normal apolipoprotein A-I. The results demonstrate the substitution of glutamic acid to lysine at position 110 in the variant form. Sequence analysis of whole LE and CN fragments showed no other mutation in this apolipoprotein A-I variant (Table III, Fig. 7). As this substitution Glu --+ Lys has apparently not been reported, we termed the apolipoprotein A-l variant ‘apo A-I Fukuoka’. The activation of lecithin-cholesterol acyltransferase by this variant and normal apolipoprotein A-I was determined in vitro, using single bilayer vesicles con-
Fig. 2. Two-dimensional gel electrophoresis of normal apolipoprotein A-I, apolipoprotein A-I Fukuoka and immunoblotting. A, normal apo A-I; B, apo AI Fukuoka; C, immunoblot of apo A-I Fukuoka. The mature apo A-I isoproteins are numbered 4 and 5. Monospecific antiserum to apo A-I, was obtained following purification by lmmobiline gel electrophoresis as described in ‘Materials and Methods’. The basic side (cathode) is on the left and acidic side (anode) is on the right.
taining egg PC, cholesterol and apolipoprotein A-I. As shown in Fig. 8, the stimulation of cholesteryl ester synthesis by apolipoprotein A-I Fukuoka was the same
Fig. 3. Ouchterlony double immunodiffusion of purified apolipoprotein A-I and serum against rabbit antiserum to human apolipoprotein A-I. Apolipoprotein A-I was purified by Immobiline gel electrophoresis. Well 1 contained purified apo A-l, well 2 contained anti-apo A-I serum and well 3 human serum.
173 TABLE
I
Serum and lipoprotein lipid levels of the apolipoprotein A-I Fukuoka farnib Name
No.
Carrier
3 4 8
Sex
As
K.O. T.O. Y.O.
59 57 52
F M M
Mean f S.E. Non-carrier
6 11
N.M. E.O.
56 25
F F
Mean f S.E.
TABLE
Cholesterol
(mg/dI)
Triacylglycerol
total
LDL
HDL
(mg/dI)
252 166 208
151 101 95
51.9 47.0 58.7
256 138 269
208 + 20
116+15
52.5 + 2.8
218*33
191 236
42 126
65.2 35.3
100 320
50.3 f 10.6
210+78
214 f 16
s4*30
II
Apolipoprotein
levels of the apolipoprotein A-I Fukuoka farnib No.
3 4 8
Carrier
Sex
4%
K.O. T.O. Y.O.
6 11
Non-wirer
Name
(mg/dl)
A-I
A-II
140 116 113
29.4 17.1 25.3
Mean f SE.
123*7
23.9 f 3.0
56 25
140 107
28.1 21.5
124*12
24.8k2.3
59 57 52
N.M. E.O.
Apolipoproteins
F M M
F F
Mean f S.E.
as that of normal apolipoprotein tions (1 to 20 I-18 apolipoprotein
A-I, at all concentraA-I per assay).
B
CII
CIII
E
6.8 2.9 9.9
16.8 7.5 16.2
6.5 4.8 3.4
86+7
6.5 f 1.7
13.5 +2.5
4.9 f 0.7
66 113
3.0 5.4
5.6 17.5
4.6 5.1
4.2 f 0.8
11.6k4.2
4.8 f 1.2
96 70 93
90*17
(-9
Discussion Apolipoprotein A-I is the major protein constituent of HDL. Because of the importance of HDL levels in predicting the susceptibility to atherosclerosis, screening for apolipoprotein A-I structural variants has been done Pedigree
of the apo A-l Fukuoka
-A- ‘lfukuoka AA3
6
I
7
8
9
0
,propositus
&--“~
q q
A-l Fukuoka normal
heterozygote
A-i
not examined deceased
Fig. 4. Pedigree
of the family with apohpoprotein
A-I Fukuoka.
Fig. 5. ImmobiIine gel electrophoresis of apolipoprotein A-I Fukuoka. HDL was isolated from subject with apolipoprotein A-I Fukuoka heterozygote as described in ‘Materials and Methods’.
LE-IO
60
LE-3.
50 % x 40 8 .E-?‘M
’
’
B B 30 ; I
'LE-23 -11 LE-’ LE.11
I I
20
10
0
1
I
10
20
I
ELUTION
I
I
30
40
TIME
50
I
60
(MIN)
Fig. 6. HPLC elution profile of lysyl endopeptidase treated apolipoprotein A-I Fukuoka. Purified apolipoprotein A-I Fukuoka was treated with lysyl endopeptidase followed separation by reverse phase HPLC on a Wakosil 5C18-200 column (40 x 250 mm). LE 3*, LE 5 * and LE 6 * denote the variant peptides not found in the peptide profile of normal apolipoprotein A-I.
by making use of isoelectric focusing gel electrophoresis. After screening of 500 unrelated individuals, we detected a novel mutant apolipoprotein A-I Fukuoka, with a relative charge difference of +2 compared to normal apo A-I,. To date, accelerated atherosclerosis has not been observed in this family, however, a complete family study remains to be done. There was no difference in the ability to activate LCAT in vitro. Menzel et al. [lo] developed an excellent simple screening method for apolipoprotein A-I variants, but
in this system, sometimes an extra band other than an apolipoprotein A-I isoform appeared, particularly at a point two charge units more basic or acidic than apo A-I,. For this reason, we modified their method using ultracentrifugation. Before applying the serum to the electrofocusing gel, we separated the lipoprotein fraction from 200 ~1 of serum using a Hitachi RPL 42T disk rotor for 5 h. In this modified system, bands hardly distinguishable from the apolipoprotein A-I variants disappeared and the apolipoprotein A-I isoform pattern
TABLE III Amino acid analysis of lysyl endopeptidase Cycle
1 2 3
(LE) treated peptides of apolipoprotein A-I Fukuoka
LE-3*
Cycle yield
amino acid identified
(pmoI)
Trp Gln Lys b
154 355 88
residue a 108 109 110
1 2 3 4 5 6 7 8
LE-6 * amino acid identified
yield
GIu Met Glu Leu Tyr A% Gln Lys
115 12 108 102 83 48 116 37
residue a
(pm@
a The numbers in this column represent the amino acid residue in the primary amino acid sequence of apohpoprotein A-I b Lysine residue in this cycle indicates substitution of glutamic acid for lysine.
111 112 113 114 115 116 117 118
175
70 Glv Pro Val Thr Gin Q_p&Jjp --~-----~~~~~~~~~~ 61
80 Glu Thr Glu GJy Leu Arar
Aso v
LE-tzlT--------T-8.120
110
100
91 4Lu
Glu Val Lvs Ala Lvs Val Gh Pro Tvr Ltu rlz----------
Tvr hziin_L~
Val e
~-~~~~~~~ 181
Glu M FlZ
--
Pro Tvr Ser ASJ GLU Leu ArW
-
~~~~~~~-T
190 Glv Ala ArQLeu Ala Glu Tvr His Ala Lvsr ~~~~~~~~~~~~,~~~~_)~~~-T~
200
220
230
211
Val GIu
180
170
160
151
Ara Ala Ar&kHis
90 Lvs u
e
U
-
Ala ArW
Thr Leu Ser Glu &_Ab
Glu
Ala
210 Lvr Pro Ala Iz27 240
e
+e7~~~~~~~%~2-~z~_~2
241 AsnThr ---_)
243 Glo
Fig. 7. Amino acid sequence analysis of apolipoprotein A-I Fukuoka. LE denote lysyl endopeptidase digested peptides and CN denote cyanogen bromide digested peptides. The asterisk indicates the abnormal peptides. The result of sequence analysis demonstrated glutamic acid yielding lysine at residue 110.
became clear (Fig. 1). Since this rotor holds 72 samples, we could use it for daily screening. The apolipoprotein A-I Fukuoka was identified by this modified method. This variant had a charge of + 2 relative to normal apolipoprotein A-I on isoelectric focusing gels. Two different types of apo A-I variants, apo A-I Munster4 [16] (198 Glu + Lys) and apo A-I Norway (136 Glu --, Lys) [15], that have a relative charge of +2 compared to apo A-I, have been reported, both
P
100
I%
o!
10 pg APO
Fig. 8. Activation
20
A-i PER ASSAY
of lecithin-cholesterol acyltransferase protein A-I Fukuoka and apo A-I,.
by apolipo-
result from the substitution Glu + Lys. For the electrophoretic abnormality that shows the +2 charge difference, there has to be a single replacement of an acidic (Glu or Asp) for a basic amino acid (Lys or His). According to combinations of the codons, the possible combinations are the substitution of GAA or GAG for AA4 or AAG for Glu + Lys and GAU or GAC for CAU or CAC for Asp -+ His. To check the former, we treated this variant with lysyl endopeptidase. The HPLC profile of the lysyl endopeptidase digest of variant apolipoprotein A-I showed the presence of peptides designated LE 3 *, LE 5 *, LE 6 * which were absent in the profile of normal apo A-I,. The sequence analysis of these peptides identified the substitution of Glu -+ Lys at position 110, an event which occurs in a segment of the protein sequence that is thought to be one of several repeating 22-residue amphipathic helices. This helix has both a hydrophobic and hydrophilic face, features characteristic of these amphipathic structures. The results demonstrated that the substitution is a point mutation and that this point mutation involves the same nucleotide, though in different positions, as demonstrated in apolipoprotein A-I Munster and apolipoprotein A-I Norway. Apolipoprotein A-I Munster2 [15], (apolipoprotein A-I Marburg), which is a deletion mutant, lacking lysine
176 at position 107 and apolipoprotein A-I Giessen (143 Pro -+ Arg) [14] were reported to be defective in activating lecithin-cholesterol acyltransferase in vitro. However, apolipoprotein A-I Fukuoka (110 Glu + Lys) showed no difference in ability to activate purified LCAT from normal apolipoprotein A-I. In apolipoprotein A-I Munster2 [15] (apolipoprotein A-I Marburg), the deletion of lysine 107 alters the nature and orientation of the hydrophobic and hydrophilic faces. The hydrophobic face in normal apolipoprotein A-I is destroyed and is replaced by a new one that is approximately 90” out of phase with the normal one. The amino acid interchange in apolipoprotein A-I Giessen [15] is located in a putative B-turn that separates two of the amphipathic helices important for protein co-factor properties. Since both glutamic acid and lysine have a polar side chain, the substitution probably does not alter the nature of the hydrophobic or hydrophilic face of the presumed a-helix in apolipoprotein A-I Fukuoka. References MahIey, R.W., Innerarity, T.L., Rail, S.C. and Weisgraber, K.H. (1984) J. Lipid Res. 25, 1277-1294. Caste& W.P., Doyle, J.T., Gordon, T., Hames, C.G., Hjortland, M.C., HuUey, S.B., Kagan, A. and Zukel, W.J. (1977) Circulation 55767-772. Fielding, C.J., Shore, V.G. and Fielding, P.E. (1972) Biochem. Biophys. Res. Commun. 461493-1498. Brewer, H.B., Fairwell, T., La Rue, A., Rona, R., Houser, A. and Bronzert, T.J. (1978) B&hem. Biophys. Res. Commun. 80, 623-630. GhiseBi, G., Gotto, A.M., Tanenbaum, S. and Sherrill, B.C. (1985) Proc. Natl. Acad. Sci. USA 82, 874-878. Zannis, V.I., Breslow, J.L. and Katz, A.J. (1980) J. Biol. Chem. 255, 8612-8617. Franceschini, G., Sirtori, C.R., Capursc, A., Weisgraber, K.H. and MahIey, R.W. (1980) J. CIin. Invest. 66, 892-m.
G., Bersot, T.P., Sirtori, C.R. and 9 Weisgraber, K.H., Franceschini, Mahley, R.W. (1980) J. Clin. Invest. 66, 901-907. R.G. and Assmann, G. (1982) J. Lipid. 10 Menzel, H.J., Kladetzky, Res. 23, 915-922. 11 Utermann, G., Feussner, G., Franceschini, G., Haas, J. and Steinmetz, A. (1982) J. Biol. Chem. 257, 501-507. 12 Schamaun, O., Olaisen, B., Gedde-Dahl, T., Jr. and Teisberg, P. (1983) Hum. Genet. 64, 380-383. 13 Menzel, H.J., Assmann, G., Rall, S.C., Jr., Weisgraber, K.H. and Mahley, R.W. (1984) J. Biol. Chem. 259, 3070-3076. 14 Utermann, G., Haas, J., Steinmetz, A., Paetzold, R., Rail, S.C., Jr., Weisgraber, K.H. and Mahley, R.W. (1984) Eur. J. Biochem. 144, 325-331. 15 Rall, S.C., Jr., Weisgraber, K.H., Mahley, R.W., Ogawa, Y., Fielding, C.J., Utermann, G., Haas, J., Steinmetz, A., Menzel, H.J. and Assmann, G. (1984) J. Biol. Chem. 259, 10063-10070. 16 Weisgraber, K.H., Rall, S.C., MahIey, R.W., Ogawa, Y., Fielding, C.J., Utermann, G., Haas, J., Steinmetz, A., Menzel, H.J. and Assmann, G. (1985) in Proceedings 7th International Symposium on Atherosclerosis (Nostel, P.J., ed.), pp. 113-114, Int. Atherosclerosis Sot., Melbourne, Australia. 17 RaII, S.C., Jr., Weisgraber, K.H., Mahley, R.W., Ehnholm, C., Schamaun, O., Olaisen, B., Blomboff, J.P. and Teisberg, P. (1986) J. Lipid Res. 27, 436-441. 18 Neville, D.M. (1971) J. Biol. Chem. 246, 6328-6334. 19 Weisgraber, K.H., Newhouse, Y.M., Seymour, J.L., Rail, S.C., Jr. and Mahley, R.W. (1985) Anal. B&hem. 151, 455-461. 20 Tsunasawa, S., Sugihara, A., Masaki, T., Sakiyama, F., Takeda, Y., Miwatai, T. and Narita, K. (1987) J. Biochem. 101, 111-121. 21 Doi, Y. and Nishida, T. (1981) Methods Enzymol. 71, 753-767. 22 Nishida, H.I., Nakanishi, T., Yen, E.A., Arai, H., Yen, F.I. and Nishida, T. (1986) J. Biol. Chem. 261, 12028-12035. 23 Singleton, W.S., Gray, MS., Brown, M.L. and White, J.L. (1965) J. Am. Oil Chem. Sot. 42, 53-56. 24 Batzri, S. and Kom, E.D. (1973) Biochim. Biophys. Acta 298, 1015-1019. 25 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 26 Sasaki, J., Kumagae, G., Sata, T., Kuramitsu, M. and Arakawa, K. (1984) Atherosclerosis 51, 163-169. M., Sasaki, J. and Arakawa, K. (1988) Biochim. Bio27 Funakoshi, phys. Acta 963, 98-108.
169
Acta, 1043 (1990) 169-176
BBALIP 53367
Isolation and characterization of human apolipoprotein A-I Fukuoka (110 Glu + Lys). A novel apolipoprotein variant Yoichi Takada
I, Jun Sasaki ‘, Shigenori Ogata 2, Toshiaki and Kikuo Arakawa ’
Nakanishi
3, Yukio Ikehara
2
’ Department of Internal Medicine and ’ Department of Biochemistry and ’ Department of Medicine, Nagasaki
Fukuoka University School of Medicine, Fukuoka University Hospital, Nagasaki (Japan)
(Received 22 September 1989)
Key words: Apolipoprotein A-I; Apolipoprotein variant; Protein characterization; HDL; (Human)
A novel genetic variant of apolipoprotein(apo) A-I Fukuoka, has been identified in a Japanese family. This variant has a relative charge of +2 compared to normal apolipoprotein A-I (A-I,), on the isoelectric focusing gels and the same molecular mass and immunologic characteristics as normal apolipoprotein A-I. This variant, transmitted as an autosomal co-dominant inheritance was purified by preparative Immobiline isoelectric focusing. Sequence analysis after cleavage with lysyl endopeptidase and CNBr, followed by high-performance liquid chromatography revealed a single substitution of lysine at position 110, instead of the usual glutamic acid. This mutant apolipoprotein A-I has much the same potential as to activate lecithin-cholesterol acyltransferase.
Introduction Apolipoprotein A-I (apo A-I) is the major apoprotein of high-density lipoproteins (HDL) [l]. Apolipoprotein A-I and HDL cholesterol are inversely related to the development of premature cardiovascular disease [2]. Apolipoprotein A-I activates lecithin-cholesterol acyltransferase (LCAT) which catalyzes esterification of cholesterol to cholesteryl ester and might participate in the reverse cholesterol transport from tissues to the liver [3]. Mature apolipoprotein A-I is a single polypeptide of 243 amino acids (M, = 28 100) [4]. Newly secreted apolipoprotein A-I appears in plasma as the 249 amino acid pro-apo A-I and is processed to mature apo A-I after cleavage of six amino-terminal amino acids [5]. Apolipoprotein A-I was found to have five isoforms (A-I, to A-I,), on isoelectric focusing gel [6]. Several genetic variants of human apolipoprotein A-I have been reported [1,8-171. These variants have relative charge differences of + 2, + 1 or - 1 compared to major mature apo A-I, apo A-I,. Analysis of the primary structures of these variants revealed a single amino acid substitution, except for one variant with a deletion of
Correspondence: J. Sasaki, Department of Internal Medicine, Fukuoka University Hospital, 45-1, ‘I-chome Nanakuma, Jonan-ku, Fukuoka 81401, Japan. 0005-2760/90/$03.50
single lysine residue (A-I Marburg 1151). During screening programs for the apolipoprotein A-I variant, we identified a novel apolipoprotein A-I variant (A-I Fukuoka), which has a relative charge difference of +2 compared to apo A-I,. We modified the screening method for apolipoprotein A-I and noted the usefulness of lysyl endopeptidase digestion followed by high-performance liquid chromatography for analyzation of the primary structure of the +2 type apo A-I variant. Materials and Methods Patient Proband (T.O.) is a 57-year-old male and was detected as having low HDL cholesterol (HDL-C) level of 13 mg/dl. He was admitted to the Fukuoka University Hospital because of diabetic coma. His HDL-C level increased from 13 mg/dl to 47 mg/dl, at the same time blood glucose level normalized after insulin administration. Materials Serum samples were obtained from the central laboratory of Fukuoka University Hospital. Criteria for sampling included HDL cholesterol levels over than 100 mg/dl or less than 30 mg/dl. Acrylamide, N,N, N ‘, N ‘tetramethylethylenediamine (TEMED), and N, N ‘methylenebisacrylamide were purchased from Bio-Rad. Sodium decyl sulfate was obtained from Eastman
0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
170 Kodak, obtained
ampholytes and Immobiline dry from LKB (Bromma, Sweden).
plates
were
Isoelectric focusing gel electrophoresis Isoelectric focusing gel electrophoresis for screening was performed by either the one-step method [lo] or in combination with ultracentrifugation. For the combination with ultracentrifugation and the one-step methods, 200 ~1 of serum was adjusted to a density (d) of 1.21 g/ml with KBr and centrifuged at 39000 rpm for 5 h in a Hitachi RPL 42T disk rotor at 4°C. 2 pl of the d -C 1.21 g/ml fraction was incubated for 1 h at room temperature with 50 ~1 of 0.01 M Tris-HCl (pH 8.2) containing 1% sodium decyl sulfate and 2% ampholite (pH 4-6) and 5 ~1 of mercaptoethanol. Then 10 ~1 of 80% sucrose and 0.05% Bromophenol blue (BPB) were added and the sample was applied to the gel. The gel solution contained (per liter) 75 g acrylamide, 2.0 g bisacrylamide, 480 g urea, 41 ml ampholite (pH range 4-6) and 0.65 ml TEMED, 0.65% ammonium persulfate. This solution was added to cassettes of a Bio-Rad Protein II vertical slab gel electrophoresis cells with 1.5 mm spacers. A 20-well comb was used to prepare the slots. With this system a maximum of 80 samples can be applied. The upper and lower tray buffers are 0.02 M NaOH and 0.01 M H,PO,, respectively. The gels were electrophoresed for 17 h at 250 V and the voltage was increased to 600 V for 2 h. The gels were stained with a Coomassie blue G-250. 2 g of Coomassie brilliant blue G-250 (Bio-Rad) was dissolved in 150 ml of 80” C water to which 75 ml of 1.5 M H,S04 was added, then the solution was heated for 10 min at 80” C. After filtration, the pH was raised to 5.5 with 5 M KOH and 0.25 g of decylsulfate and 50 g of trichloroacetate were added. The gels were stained for 30 min at 60” C and destained with 20% ethanol at 60°C. Two-dimensional electrophoresis Individual samples from the first isoelectric focusing gels were cut and immersed in 0.002 M ethylmorpholine-HCl (pH 8.5), 0.2% SDS, 0.1% /?-mercaptoethanol, BPB solution and 4% sucrose for 15 min at room temperature and subjected to SDS polyacrylamide gel electrophoresis in an Atto electrophoresis chamber (Atto Corporation, Japan). The SDS system was prepared according to Neville [18]. The stacking gel contained (per liter) 50 g acrylamide, 1.4 g bisacrylamide, 0.375 M Tris-HCl (pH 8.8), 1.0 g SDS, 0.5 ml TEMED and 0.5 g ammonium persulfate. The running gel contained (per liter) 150 g acrylamide, 4 g bisacrylamide, 0.375 M Tris-HCl (pH 8.8), 1.0 g SDS, 0.5 ml TEMED and 0.5 g ammonium persulfate. 1% melted agarose containing 0.06 M Tris-HCl (pH 6.8), 2% SDS and 5% mercaptoethanol was poured on the surface of the upper gel and the first isoelectric focusing gel was immediately placed
into the agarose on the SDS gel. The tray buffer was 0.05 M Tris, 0.384 M glycine and 0.1% SDS (pH 8.4). Electrophoresis was carried out at 20 mA for stacking, and at 40 mA through the running gel. The gels were stained with Coomassie blue R 250. Production of antisera to apolipoprotein A-I Apolipoprotein A-I was purified using an Immobiline gel [19] (see preparative Immobiline isoelectric focusing in Materials and Methods). Purity was checked by amino acid analysis. Male New Zealand white rabbits weighing 2-3 kg were immunized with about 500 pg of pure apolipoprotein A-I which was emulsified with an equal volume of Freund’s complete adjuvant and administered subcutaneously at multiple sites. Booster immunizations were given twice with 250 pg of apolipoprotein A-I, at 2-week intervals. Blood samples were obtained from the ear vein. The purity of antiserum to apolipoprotein A-I was checked using an Ouchterlony plate. As a single precipitin line formed against whole serum and purified apolipoprotein A-I, the antiserum was considered to be monospecific. Immunoblot Identification of two-dimensional electrophoretic spots of apolipoprotein A-I by immunological methods was done by immunoblots (Instruction for the Bio-Rad Immun-Blot (GAR-HRP) Assay Kit). The buffers used for the transfer of SDS gels to nitrocellulose paper were 0.7% acetic acid and 0.25 M Tris-HCl, 0.192 mM glycine, and 20% methanol (pH 8.3). Proteins were transferred to nitrocellulose paper at 40 V for 1 h. After the transfer, the remaining non-detected sites on the nitrocellulose paper were blocked for 1 h by incubation with 0.02 M Tris-HCl and 0.5 M NaCl (pH 7.5) (TBS) containing 3% gelatin. The nitrocellulose paper was then incubated for 16 h in 1% gelatin in TBS containing a monospecific rabbit antibody to apolipoprotein A-I. After incubation, the preparation was washed three times in TBS containing 0.05% Tween-20 (TTBS). The nitrocellulose paper was then incubated for 2 h in 1% gelatin in TBS containing an affinity purified antirabbit IgG horseradish peroxidase reagent (l/3000 dilution, Bio-Rad Lab, Richmond, CA). Then it was washed three times in TTBS. The peroxidase complex was developed with color development reagent (Bio-Rad Lab, Richmond, CA). Preparative Immobiline isoelectric focusing For anlytical purposes, the pure apo A-I isoforms were isolated by preparative Immobiline isoelectric focusing. HDL was isolated by preparative ultracentrifugation [19]. After dialysis against 0.9% NaCl, the HDL were delipidated with ethanol/diethyl ether (3 : 1) and diethyl ether. The delipidated HDL was solubilized in deionized water (20 mg/ml). The Immobiline dry
171 plate (pH 5-6) was rehydrated with 20 ml of 8 M urea for 2 h at room temperature. After a few drops of kerosene had been pipetted onto the cooling plate of the LKB Multiphor Electrophroresis Unit, the gel was placed on the cooling plate. The electrode strips were soaked with 10 mM glutamic acid at the anode and 10 mM NaOH at the cathode and laid along the corresponding edges of the gel. 1 ml of sample solution was applied to the filter paper (10 X 240 mm) laid on the gel surface at 10 mm distance from the anode strip. The gel was electrophoresed overnight at a constant current of 1 mA at 10°C. The focused apoproteins were visualized by soaking the gel in water. Individual visualized bands were excised and eluted with 0.1 M Tris-HCl (pH 7.4) containing 4 M guanidine and 1 mM EDTA. The eluted proteins were exhaustively dialyzed against deionized water and lyophilized. Cleavage and isolation of several peptide fragments of apolipoprotein A-I Purified apo A-I isoforms were digested with lysyl endopeptidase for 6 h at 30°C in 1 ml of 0.01 M Tris-HCl buffer (pH 9.0) at the enzyme-to-substrate ratio of 1 : 100 (w/w) [20]. The digested peptide fragments were separated by reverse-phase HPLC on a Wakosil 5C18-200 column (4.0 X 250 mm) at a flow-rate of 1 ml/mm. The purified apolipoprotein A-I was cleaved with 2.5 M CNBr in 70% (v/v) formic acid for 24 h at room temperature [4]. The sample thus treated was subjected to HPLC through a Superose 12 column (1.0 x 30 cm) with 70% formic acid. Six major peptide peaks were evidenced with monitoring by absorbance at 280 nm. Fractions of each peak were pooled, freezedried, each sample was dissolved in 0.1% trifluoroacetic acid and then subjected to HPLC on a PBondapack Cl8 column (0.4 X 30 cm) with a linear gradient from 0 to 80% acetonitrile in 0.1% trifluoroacetic acid. Fractions of each major peptide peak obtained were freezedried and used for chemical analysis. Determination of amino acid sequences Purified samples of apolipoprotein A-I (30 pg) and the peptide fragments (about 5 pg each) were analyzed for amino acid sequences by an Applied Biosystems Model 477A Gas-Phase Sequencer with an on-line Model 120A phenylthiohydantoin derivative analyzer, based on instructions provided by the manufacturer. Purification of lecithin-cholesterol acyltransferase Lecithin-cholesterol acyltransferase was purified from fresh human plasma, as described [21,22]. The final purification of the enzyme was approx. 20000-fold over the starting plasma. The purified enzyme was stored at 4” C in 0.01% EDTA (pH 74) containing 4 mM 2-mercaptoethanol.
Preparation of phosphatidylcholine (PC)-cholesterol vesicles Egg PC was isolated according to Singleton et al. [23] and further purified by silicic acid column chromatography [22]. Thin-layer chromatography of the preparation on a Silica Gel G plate with chloroform/ methanol/water (165 : 25 : 4, v/v) as the developing solvent gave a single spot of PC. Single bilayer PCcholesterol vesicles were prepared by the method of Batzri and Korn [24]. A typical preparation contained 900 nmol of egg PC and 150 nm of [7or-‘HIcholesterol (specific activity, 4.3 mCi/mmol) per ml. Lecithin-cholesterol
acyltransferase
co-factor
activity
as-
say The enzyme activity was determined as described [22]. The standard assay mixture consisted of 100 ~1 of single bilayer vesicle preparations containing 90 nmol of egg PC and 15 nmol of [7a-‘HIcholesterol, 20 pg of apolipoprotein A-I, 4 mM 2-mercaptoethanol, 0.7 mM EDTA, 2.5 mg of bovine serum albumin and the enzyme solution in a final volume of 250 ~1 of phosphate buffer. The vesicle solution and apolipoprotein A-I were preincubated for 30 min at 37” C under N,, prior to addition to the assay mixture. For the assay, the mixture containing enzyme solution was incubated for 1 h at 37 o C under N,. The rate of cholesterol esterification is given in ng of cholesterol esterified per hour. In all the enzyme assays, we used the labeled PC-cholesterol vesicles prepared within 1 week and the enzyme preparations with over 0.2 units of activity per ng of protein (One unit of enzyme designates the esterification of 1 nmol of unesterified cholesterol per hour at 37 o C under standard assay condition). Protein and lipid analysis Protein was determined by the method of Lowry et al. [25] with bovine serum albumin as the standard. Lipids and apolipoproteins were analyzed as described [26,27]. Results
Fig. 1 shows that the variant on isoelectric focusing gel electrophoresis in a pH gradient of 4 to 6, had a relative charge of + 2 compared to the normal major mature apo A-I,. As determined by two-dimensional electrophoresis, the variant had a molecular weight identical with that of normal apo A-I, (Fig. 2). After two-dimensional electrophoresis, this ‘abnormal’ spot reacted by immunoblot (Fig. 2-C) with a monospecific antibody to apolipoprotein A-I (Fig. 3). Studies of the family revealed two other heterozygotes in the first-degree family members (Fig. 4) thereby suggesting an autosomal co-dominant inheritance. There were no marked differences in the levels of lipids, lipoproteins
Fig. 1. Isoelectric tocusmg gel of serum in a pH range from 4 to 6. Normal serum was applied in lanes A, B; sera from heterozygous apolipoprotein A-I Fukuoka in lane C; d < 1.21 sera separated by RPL-42T rotor in lane D.
and apolipoproteins between the carriers and the non-carrier members (Tables I and II). To investigate the amino acid sequence, the variant apolipoprotein A-I was isolated by preparative Immobiline isoelectric focusing gel electrophoresis, using delipidated HDL (Fig. 5). The purified variant isoform was cleaved with lysyl endopeptidase and the digested peptides were separated by reverse-phase HPLC (Fig. 6). The HPLC profile of the lysyl endopeptidase (LE) digest of variant apolipoprotein A-I showed the presence of three peptides, designated LE 3*, LE 5 * and LE 6 *, which were absent in normal apo A-I, (data not shown). Sequence analysis of LE 3 *, LE 5 * and LE 6 * revealed the presence of lysine at position 110 (Table III), at which glutamic acid is identified in the normal apolipoprotein A-I. The results demonstrate the substitution of glutamic acid to lysine at position 110 in the variant form. Sequence analysis of whole LE and CN fragments showed no other mutation in this apolipoprotein A-I variant (Table III, Fig. 7). As this substitution Glu --+ Lys has apparently not been reported, we termed the apolipoprotein A-l variant ‘apo A-I Fukuoka’. The activation of lecithin-cholesterol acyltransferase by this variant and normal apolipoprotein A-I was determined in vitro, using single bilayer vesicles con-
Fig. 2. Two-dimensional gel electrophoresis of normal apolipoprotein A-I, apolipoprotein A-I Fukuoka and immunoblotting. A, normal apo A-I; B, apo AI Fukuoka; C, immunoblot of apo A-I Fukuoka. The mature apo A-I isoproteins are numbered 4 and 5. Monospecific antiserum to apo A-I, was obtained following purification by lmmobiline gel electrophoresis as described in ‘Materials and Methods’. The basic side (cathode) is on the left and acidic side (anode) is on the right.
taining egg PC, cholesterol and apolipoprotein A-I. As shown in Fig. 8, the stimulation of cholesteryl ester synthesis by apolipoprotein A-I Fukuoka was the same
Fig. 3. Ouchterlony double immunodiffusion of purified apolipoprotein A-I and serum against rabbit antiserum to human apolipoprotein A-I. Apolipoprotein A-I was purified by Immobiline gel electrophoresis. Well 1 contained purified apo A-l, well 2 contained anti-apo A-I serum and well 3 human serum.
173 TABLE
I
Serum and lipoprotein lipid levels of the apolipoprotein A-I Fukuoka farnib Name
No.
Carrier
3 4 8
Sex
As
K.O. T.O. Y.O.
59 57 52
F M M
Mean f S.E. Non-carrier
6 11
N.M. E.O.
56 25
F F
Mean f S.E.
TABLE
Cholesterol
(mg/dI)
Triacylglycerol
total
LDL
HDL
(mg/dI)
252 166 208
151 101 95
51.9 47.0 58.7
256 138 269
208 + 20
116+15
52.5 + 2.8
218*33
191 236
42 126
65.2 35.3
100 320
50.3 f 10.6
210+78
214 f 16
s4*30
II
Apolipoprotein
levels of the apolipoprotein A-I Fukuoka farnib No.
3 4 8
Carrier
Sex
4%
K.O. T.O. Y.O.
6 11
Non-wirer
Name
(mg/dl)
A-I
A-II
140 116 113
29.4 17.1 25.3
Mean f SE.
123*7
23.9 f 3.0
56 25
140 107
28.1 21.5
124*12
24.8k2.3
59 57 52
N.M. E.O.
Apolipoproteins
F M M
F F
Mean f S.E.
as that of normal apolipoprotein tions (1 to 20 I-18 apolipoprotein
A-I, at all concentraA-I per assay).
B
CII
CIII
E
6.8 2.9 9.9
16.8 7.5 16.2
6.5 4.8 3.4
86+7
6.5 f 1.7
13.5 +2.5
4.9 f 0.7
66 113
3.0 5.4
5.6 17.5
4.6 5.1
4.2 f 0.8
11.6k4.2
4.8 f 1.2
96 70 93
90*17
(-9
Discussion Apolipoprotein A-I is the major protein constituent of HDL. Because of the importance of HDL levels in predicting the susceptibility to atherosclerosis, screening for apolipoprotein A-I structural variants has been done Pedigree
of the apo A-l Fukuoka
-A- ‘lfukuoka AA3
6
I
7
8
9
0
,propositus
&--“~
q q
A-l Fukuoka normal
heterozygote
A-i
not examined deceased
Fig. 4. Pedigree
of the family with apohpoprotein
A-I Fukuoka.
Fig. 5. ImmobiIine gel electrophoresis of apolipoprotein A-I Fukuoka. HDL was isolated from subject with apolipoprotein A-I Fukuoka heterozygote as described in ‘Materials and Methods’.
LE-IO
60
LE-3.
50 % x 40 8 .E-?‘M
’
’
B B 30 ; I
'LE-23 -11 LE-’ LE.11
I I
20
10
0
1
I
10
20
I
ELUTION
I
I
30
40
TIME
50
I
60
(MIN)
Fig. 6. HPLC elution profile of lysyl endopeptidase treated apolipoprotein A-I Fukuoka. Purified apolipoprotein A-I Fukuoka was treated with lysyl endopeptidase followed separation by reverse phase HPLC on a Wakosil 5C18-200 column (40 x 250 mm). LE 3*, LE 5 * and LE 6 * denote the variant peptides not found in the peptide profile of normal apolipoprotein A-I.
by making use of isoelectric focusing gel electrophoresis. After screening of 500 unrelated individuals, we detected a novel mutant apolipoprotein A-I Fukuoka, with a relative charge difference of +2 compared to normal apo A-I,. To date, accelerated atherosclerosis has not been observed in this family, however, a complete family study remains to be done. There was no difference in the ability to activate LCAT in vitro. Menzel et al. [lo] developed an excellent simple screening method for apolipoprotein A-I variants, but
in this system, sometimes an extra band other than an apolipoprotein A-I isoform appeared, particularly at a point two charge units more basic or acidic than apo A-I,. For this reason, we modified their method using ultracentrifugation. Before applying the serum to the electrofocusing gel, we separated the lipoprotein fraction from 200 ~1 of serum using a Hitachi RPL 42T disk rotor for 5 h. In this modified system, bands hardly distinguishable from the apolipoprotein A-I variants disappeared and the apolipoprotein A-I isoform pattern
TABLE III Amino acid analysis of lysyl endopeptidase Cycle
1 2 3
(LE) treated peptides of apolipoprotein A-I Fukuoka
LE-3*
Cycle yield
amino acid identified
(pmoI)
Trp Gln Lys b
154 355 88
residue a 108 109 110
1 2 3 4 5 6 7 8
LE-6 * amino acid identified
yield
GIu Met Glu Leu Tyr A% Gln Lys
115 12 108 102 83 48 116 37
residue a
(pm@
a The numbers in this column represent the amino acid residue in the primary amino acid sequence of apohpoprotein A-I b Lysine residue in this cycle indicates substitution of glutamic acid for lysine.
111 112 113 114 115 116 117 118
175
70 Glv Pro Val Thr Gin Q_p&Jjp --~-----~~~~~~~~~~ 61
80 Glu Thr Glu GJy Leu Arar
Aso v
LE-tzlT--------T-8.120
110
100
91 4Lu
Glu Val Lvs Ala Lvs Val Gh Pro Tvr Ltu rlz----------
Tvr hziin_L~
Val e
~-~~~~~~~ 181
Glu M FlZ
--
Pro Tvr Ser ASJ GLU Leu ArW
-
~~~~~~~-T
190 Glv Ala ArQLeu Ala Glu Tvr His Ala Lvsr ~~~~~~~~~~~~,~~~~_)~~~-T~
200
220
230
211
Val GIu
180
170
160
151
Ara Ala Ar&kHis
90 Lvs u
e
U
-
Ala ArW
Thr Leu Ser Glu &_Ab
Glu
Ala
210 Lvr Pro Ala Iz27 240
e
+e7~~~~~~~%~2-~z~_~2
241 AsnThr ---_)
243 Glo
Fig. 7. Amino acid sequence analysis of apolipoprotein A-I Fukuoka. LE denote lysyl endopeptidase digested peptides and CN denote cyanogen bromide digested peptides. The asterisk indicates the abnormal peptides. The result of sequence analysis demonstrated glutamic acid yielding lysine at residue 110.
became clear (Fig. 1). Since this rotor holds 72 samples, we could use it for daily screening. The apolipoprotein A-I Fukuoka was identified by this modified method. This variant had a charge of + 2 relative to normal apolipoprotein A-I on isoelectric focusing gels. Two different types of apo A-I variants, apo A-I Munster4 [16] (198 Glu + Lys) and apo A-I Norway (136 Glu --, Lys) [15], that have a relative charge of +2 compared to apo A-I, have been reported, both
P
100
I%
o!
10 pg APO
Fig. 8. Activation
20
A-i PER ASSAY
of lecithin-cholesterol acyltransferase protein A-I Fukuoka and apo A-I,.
by apolipo-
result from the substitution Glu + Lys. For the electrophoretic abnormality that shows the +2 charge difference, there has to be a single replacement of an acidic (Glu or Asp) for a basic amino acid (Lys or His). According to combinations of the codons, the possible combinations are the substitution of GAA or GAG for AA4 or AAG for Glu + Lys and GAU or GAC for CAU or CAC for Asp -+ His. To check the former, we treated this variant with lysyl endopeptidase. The HPLC profile of the lysyl endopeptidase digest of variant apolipoprotein A-I showed the presence of peptides designated LE 3 *, LE 5 *, LE 6 * which were absent in the profile of normal apo A-I,. The sequence analysis of these peptides identified the substitution of Glu -+ Lys at position 110, an event which occurs in a segment of the protein sequence that is thought to be one of several repeating 22-residue amphipathic helices. This helix has both a hydrophobic and hydrophilic face, features characteristic of these amphipathic structures. The results demonstrated that the substitution is a point mutation and that this point mutation involves the same nucleotide, though in different positions, as demonstrated in apolipoprotein A-I Munster and apolipoprotein A-I Norway. Apolipoprotein A-I Munster2 [15], (apolipoprotein A-I Marburg), which is a deletion mutant, lacking lysine
176 at position 107 and apolipoprotein A-I Giessen (143 Pro -+ Arg) [14] were reported to be defective in activating lecithin-cholesterol acyltransferase in vitro. However, apolipoprotein A-I Fukuoka (110 Glu + Lys) showed no difference in ability to activate purified LCAT from normal apolipoprotein A-I. In apolipoprotein A-I Munster2 [15] (apolipoprotein A-I Marburg), the deletion of lysine 107 alters the nature and orientation of the hydrophobic and hydrophilic faces. The hydrophobic face in normal apolipoprotein A-I is destroyed and is replaced by a new one that is approximately 90” out of phase with the normal one. The amino acid interchange in apolipoprotein A-I Giessen [15] is located in a putative B-turn that separates two of the amphipathic helices important for protein co-factor properties. Since both glutamic acid and lysine have a polar side chain, the substitution probably does not alter the nature of the hydrophobic or hydrophilic face of the presumed a-helix in apolipoprotein A-I Fukuoka. References MahIey, R.W., Innerarity, T.L., Rail, S.C. and Weisgraber, K.H. (1984) J. Lipid Res. 25, 1277-1294. Caste& W.P., Doyle, J.T., Gordon, T., Hames, C.G., Hjortland, M.C., HuUey, S.B., Kagan, A. and Zukel, W.J. (1977) Circulation 55767-772. Fielding, C.J., Shore, V.G. and Fielding, P.E. (1972) Biochem. Biophys. Res. Commun. 461493-1498. Brewer, H.B., Fairwell, T., La Rue, A., Rona, R., Houser, A. and Bronzert, T.J. (1978) B&hem. Biophys. Res. Commun. 80, 623-630. GhiseBi, G., Gotto, A.M., Tanenbaum, S. and Sherrill, B.C. (1985) Proc. Natl. Acad. Sci. USA 82, 874-878. Zannis, V.I., Breslow, J.L. and Katz, A.J. (1980) J. Biol. Chem. 255, 8612-8617. Franceschini, G., Sirtori, C.R., Capursc, A., Weisgraber, K.H. and MahIey, R.W. (1980) J. CIin. Invest. 66, 892-m.
G., Bersot, T.P., Sirtori, C.R. and 9 Weisgraber, K.H., Franceschini, Mahley, R.W. (1980) J. Clin. Invest. 66, 901-907. R.G. and Assmann, G. (1982) J. Lipid. 10 Menzel, H.J., Kladetzky, Res. 23, 915-922. 11 Utermann, G., Feussner, G., Franceschini, G., Haas, J. and Steinmetz, A. (1982) J. Biol. Chem. 257, 501-507. 12 Schamaun, O., Olaisen, B., Gedde-Dahl, T., Jr. and Teisberg, P. (1983) Hum. Genet. 64, 380-383. 13 Menzel, H.J., Assmann, G., Rall, S.C., Jr., Weisgraber, K.H. and Mahley, R.W. (1984) J. Biol. Chem. 259, 3070-3076. 14 Utermann, G., Haas, J., Steinmetz, A., Paetzold, R., Rail, S.C., Jr., Weisgraber, K.H. and Mahley, R.W. (1984) Eur. J. Biochem. 144, 325-331. 15 Rall, S.C., Jr., Weisgraber, K.H., Mahley, R.W., Ogawa, Y., Fielding, C.J., Utermann, G., Haas, J., Steinmetz, A., Menzel, H.J. and Assmann, G. (1984) J. Biol. Chem. 259, 10063-10070. 16 Weisgraber, K.H., Rall, S.C., MahIey, R.W., Ogawa, Y., Fielding, C.J., Utermann, G., Haas, J., Steinmetz, A., Menzel, H.J. and Assmann, G. (1985) in Proceedings 7th International Symposium on Atherosclerosis (Nostel, P.J., ed.), pp. 113-114, Int. Atherosclerosis Sot., Melbourne, Australia. 17 RaII, S.C., Jr., Weisgraber, K.H., Mahley, R.W., Ehnholm, C., Schamaun, O., Olaisen, B., Blomboff, J.P. and Teisberg, P. (1986) J. Lipid Res. 27, 436-441. 18 Neville, D.M. (1971) J. Biol. Chem. 246, 6328-6334. 19 Weisgraber, K.H., Newhouse, Y.M., Seymour, J.L., Rail, S.C., Jr. and Mahley, R.W. (1985) Anal. B&hem. 151, 455-461. 20 Tsunasawa, S., Sugihara, A., Masaki, T., Sakiyama, F., Takeda, Y., Miwatai, T. and Narita, K. (1987) J. Biochem. 101, 111-121. 21 Doi, Y. and Nishida, T. (1981) Methods Enzymol. 71, 753-767. 22 Nishida, H.I., Nakanishi, T., Yen, E.A., Arai, H., Yen, F.I. and Nishida, T. (1986) J. Biol. Chem. 261, 12028-12035. 23 Singleton, W.S., Gray, MS., Brown, M.L. and White, J.L. (1965) J. Am. Oil Chem. Sot. 42, 53-56. 24 Batzri, S. and Kom, E.D. (1973) Biochim. Biophys. Acta 298, 1015-1019. 25 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 26 Sasaki, J., Kumagae, G., Sata, T., Kuramitsu, M. and Arakawa, K. (1984) Atherosclerosis 51, 163-169. M., Sasaki, J. and Arakawa, K. (1988) Biochim. Bio27 Funakoshi, phys. Acta 963, 98-108.
