Biochimica et Biophysica Acta, 1127 (1992) 124-130 (0 1992 Elsevier Science Publishers B.V. All rights reserved 0005-2760/92/$05.00
124
BBALIP 53974
Isolation and characterization of two sub-species of Lp(a), one containing apo E and one free of apo E Jean-Marie Bard, Sophie Delattre-Lestavel, V6ronique Clayey, Pascal Pont, Bruno Derudas, Henri-Joseph Parra and Jean-Charles Fruchart SERLIA et INSERM U325, lnstitut Pasteur, Lille (Frame)
(Received 31 December 1991) (Revised manuscript received 19 March 1992)
Key ~vor0s: Apo E; Apolipoprotein (a); Lipoprotein binding
Lipoprotein Lp(a) was isolated by immunoaffinity chromatography using anti apolipoprotcin B and :uiti apolipoprotein (a) immunosorbents. Besides apolipoproteins (a) and B, this fraction was shown to contain apolipoproteins C and E. Therefore, it was decided to further purify this crude Lp(a) into particles containing apolipoprotein E and particles free of ape E, using chromatography with an anti apolipoprotcin E immunosorbent. Lp(a), free of apolipoprotein E was cholesterol ester rich and triaeyl8lycerol poor and was found mainly in the LDL size range. In contrast, Lp(a) containing apolipoprotein E was triacylglycerol rich and was distributed mainly in the VLDL and IDL size range. Binding of these two fractions, one containing ape E and one free of it, to the ape B/E receptor of HeLa cells was studied. Both fractions bound to the receptor but the one containing ape E had a better affinity than the one free of ape E. Further studies are needed to identify the clinical importance of these two different entities.
Introduction Human Lp~a) is a lipoprotein whose high plasma levels have been associated with an increased cardiovascular risk [1-6]. Several studies have shown that the protein moiety of [p(a) contains a single copy of ape 13, cross-linked to aim (a) by one or more disulfide bonds [7-9]. However, species of Lp(a) that have 2 reel of a ~ a ) per reel of ape B have also been found [10]. Although ~:arly reports stated that Lp(a) exhibited a density between LDL and HDL (!.055 kg/I < d < 1.120 ~/I) [11-12], more recent studies indicated that Lp(a) exhibited inter- and intra-individual density heterogeneity [9,13]. Also, apo (a) antigen can be present, not
only in cholesterol ester rich iipoproteins but also in triacylglycerol rich partic!e~ [14]. Results of studies comparing the interaction of Lp(a) with the LDL receptor are rather conflicting. AIthough, in one study [15], the conclusion was reached that L4)(a) creeled fibroblasts independently of the LDL receptor, other investigations [16-18] have concluded that Lp(a) can bind to and be taken up by the same receptor site as LDL, sometimes with a high
affinity [17]. Also the use of drwgs which activate the LDL receptor specific pathway, such as HMG CoA reductase inhibitors has led to mixed results [19-20], most of them showing no change in the Lp(a) level and some of them showing a decrease. We hypothesized that the conflicting results obtained in in vitro studies aimed at the interaction of Lp(a) with the LDL receptor or in pharmacological studies using drugs activating the LDL receptor specific pathway may be explained by the heterogeneity of the Lp(a) particle. Since ape E has been shown to modulate the binding properties of ape B containing lipoproteins to the LDL ,receptor [21-23], we looked first at the ape E content of the Lp(a) particle. Using immunoaffinity chromatography, a method which has been shown to avoid any dissociation of ape E, in contrast with ultracentrifugation [24], we were able to prepare two sub-species of Lp(a) depending on the presence or absence of ape E. Chemical characteristics of these two fractions and their binding properties towards the ape B / E receptor of HeLa cells was determined. Materials and Methods
Isolation of ape (a) containing lipoproteins Corresi~ndence to: J.-M. Bard, SERLIA et INSERM U325. Institut Pasteur, I Rue du Pmfesseur Calmette, 59019 Lill~: C~dex, France.
The isolation of ape (a) containing particles was realized by immunoaffinity chromatography at 4°C
125 monitored at 280 nm, as extensively described in a previous paper [25]. To this aim, polyclonal antibodies against apo B [26] and monoclonal antibodies against apo (a) [27] and apo E [28] were covalently coupled to CNBr activated Sepharose 4B according to the procedure of the manufacturer (Pharmacia Fine Chemicals, Uppsala, Sweden). EDTA plasma was obtained from seven normolipidemic individuals in the morning after an overnight fasting period. Plasma cholesterol concentrations were 1.41 g/l, 1.75 g/l, 1.67 g/l, 1.34 g/l, 1.60 g/l, 1.72 g/! and 1.76 g/l, while plasma triacylglycerol concentrations were 0.88 g/l, 0.77 g/l, 1.45 g/l, 0.78 g/l, 0.78 g/i, 0.53 g/i and 1.61 g / l for samples 1 to 7, respec.. tively. The plasma Lp(a) levels (in total mass) were 0.016 g/I, 0.147 g/l, 0.051 g/I, 0.14 g/l, 0.175 g/l, 0.504 g/i and 0.079 g / l for samples 1 to 7, respectively. As determined by the method developed by Utermann et al. [29], the apo (a) isoforms were S I / S I , $4/$4, Sl/S1 and $2/$4 for samples 2, 3, 5 and 6, respectively. Bands obtained with samples 1, 4 and 7 were too weak to determine precisely the corresponding phenotypes. 40 ml of each sample were submitted first to the anti apo B column. The unretained fraction was eluted with buffer containing 0.01 M Tris-HCi, 0.5 M NaCI, 0.3 mM EDTA, 1.5 mM NaN 3 and 10 ttM PMSF (pH 8.0) at a flow rate of 60 ml/h. The retained fraction was eluted with 3 M sodium thiocyanate (50 mi). A column containing 100 ml of Sephadex G25 (Pharmacia
Fine Chemical, Uppsala, Sweden) was linked to the immunosorbent column in order to separate immediately the lipoproteins from the dissociating agent (sodium thiocyanate). The retained fraction was submitted to the anti apo (a) column and elution was realized as described above. The retained fraction was crude Lp(a):B. Crude Lp(a): B was then submitted to an anti apo E column in the same conditions as above. The retained fraction was Lp(a) containing apo E (Lp(a):B:E) and the unretained fraction was Lp(a) free of apo E (Lp(a):B). Fig. 1 indicates the different separation steps described above.
Chemical characterization of the isolated particles Apolipoproteins A-I, A-II, A-IV, B, C-If, C-Ill and E were quantified by ELISA [30]. Lipoproteins were assumed to contain one single copy of apolipoprotein B per particle [31,32] and molar ratio were calculated assuming molecular weights of 550 000 for apo B, 28 500 for apo A-I, 17000 for apo A-II, 46000 for apo A-IV, 8800 for apo C-II, 8900 for apo C-Ill and 34000 for apo E. Due to its size heterogeneity [29], it is difficult to measure precisely apo (a) and to express its molar concentration. In order to check that the concentration of apo (a) in the isolated particles was compatible with a molar ratio of 1 as expected in a Lp(a) particle, apo (a) was estimated as follows. The isolated particles
Whole plasma
[
I.
AntiApoB
R
UR
I
I Anti Apo (a) UR Crude Lp(a):B
I
Anti Apo E
I
UR Lp(a):B freeofapo E
l.p(a):B:l£
Fig. 1. Flow diagram for immunoaffinity chromatography preparation of crude Lp(a):B, Lp(a):B free of apo E and Lp(a):B:E. R, retained fraction; UR, unretained fraction. The boxes represent affinity columns containing specific antibody against the apolipoprotein indicated.
126 TABLE !
Lipid and apolipoprotein composition of crude Lp(a) : B particles Results are expressed as numbers of molecules per particle, assuming one apolipoprotein B per particle. N.D., not determined. Sample number TC CE TG PL CE/TC A-I A-ll A-IV C-ll C-ill E
1
2
3
4
5
6
7
mean ± S.D.
2752 1882 1216 459 0.68 0.42 0.38
2198 1436 775 699 0,65 0.37 0,31 N,D, 4,15 2.17 0,42
2116 136~ 910 723 0.64 0,22 0.25 N.D, !.57 !,32 0.84
3557 2432 1248 1112 0.68 0.48 0,27 N.D, traces 2,78 !.23
2130 1377 494 615 0,65 0,37 0,18 N,D. N.D. 1.67 1.00
2833 2183 180 741 0,77 0,38 0,58 N.D. N.D. 1.35 0.53
2337 1557 1438 717 0,67 0.11 0,13 N.D. N.D. 4.6 !,03
2560 + 527 1747 +427 894 +449 723 + 197 0.68 + 0,04 0,34 + 0,13 0,30± 0,15 N.D. N.D. 2.39± 1.16 0.93 ± 0.38
0,08 traces 2,81 1.49
were treated with dithiotreitol (DTT) as previously described [33] to isolate ape (a) from the particle. The remaining iipoprotein particle was separated from ape (a) by ultracentrifugation in a Beckman TL 100 ultracentrifuge at density 1.21 kg/l, in a TLA 100.2 rotor at 4°C for 3 h at 330000 × g. The supernate contained the reduced Lp B. In the bottom fraction, the protein content was estimated by the Lowry method [34], apolipoproteins A-I, A-II, A-IV, B, C-Ill and E were measured by ELISA [30] and albumin was quantified by nephelometry using the urine microalbumine kit from Behring, Germany. The protein content less the total of apolipoproteins and albumin was considered as the ape (a) content. The molar ratio between ape (a) and ape B was approximated, using an arbitrary molecular weight of 500 000 for ape (a). Protein composition was also assessed by sodium dode~lsulphate polyacrylamide gel electrophoresis (SDS-PAGE) [35], followed by immunoblot with anti apo (a), apo B, apo A-IV and apo E [36]. Total cholesterol (TC), free cholesterol (FC), triacylglycerol (TG)and phospholipids (PL) were measured by enzymatic tests (Boehringer-Mannheim, Germany). Esterified cholesterol (EC) was estimated as the difference between TC and FC. Molar razios over apo B were calcule,ted assuming molecular weights of 388, 877 and 775 for cholesterol, triacylglycerol and phospholipids, respectively.
Analysis of panicle size The molecular size distribution of the isolated particles from the first five individuals was determined by gel filtration over a column of Superose 6 HR 10/30 (Plmrmacia Fine Chemicals, Uppsala, Sweden) equilibrated in 0.01 M Tris-HC!, 9.15 M NaCI, 3- 10 -4 M EDTA, 1.5- 10 -3 M NaN 3, 2- 10 -s M PMSF (pH 8). The sample (200 ~!) was eluted at a constant flow rate of 12 ml/h. The absorbance of the eluent was moni-
tored continuously at 280 nm. The eluent was pooled at volumes corresponding to the elution volume of human VLDL (d < 1.006 kg/i: 5.6 to 7.2 ml), IDL (1.006 kg/i < d < 1.019 kg/l: 7.2 to 8 ml), LDL (1.019 kg/! < d < 1.063 kg/!: 8 to 11.2 ml) and HDL (1.063 kg/! < d < 1.21 kg/I: 11.2 to 15.2 ml). Using this procedure, Lp(a) isolated by ultracentrifugation (1.055 kg/! < d < 1.12 kg/I) eluted between 10.4 and 15.2 ml. APO B was measured [30] in each pooled fraction to assess the size distribution of the isolated particles.
Binding studies HeLa cells were grown in Dulbecco's modified Eagle's medium containing penicillin (100 units/ml), streptomycin (100 /.tg/ml) and 10% (v/v) fetal calf serum in 30-ram diameter multidish plates. Nearly confluent cells were preincubated for 48 h in a medium containing 10% (v/v) lipoprotein deficient calf serum (d > 1.21 kg/I). The content of each dish was about 400/~g of cell protein. Isolated particles were assayed in a competitive binding assay against LDL, labele~ ',4th 1251 according to the iodine monochloride method ,)f MacFarlane [37]. The labeled LDL was incubated w;th HeLa cells (5 ~g of LDL apo B / m l incubation mixture or 9 nM as LDL particle) in the presence of variou~ amounts of Lp(a): B particles at 4°C for 2 h. The cells g ere washed nine times and cell associated radioactivity wa~ counted. The amounts of Lp(a): B added to the iabe;ed LDL were calculated on the basis of their apo B t;ontent. Each point was performed in triplicate and experiments were realized on two different Lp(a)" B preparations. Results
In each case, apo (a)/apo B molar ratio was estimated to be close to 1 (0.61 to 0.82) in the three Lp(a)
127 TABLE II
Lipid and apolipoprotein composition of Lp(a) : B, free of apo E Results are expressed as numbers of molecules per particle, assuming one apolipoprotein B per particle. N.D., not determined. Sample number
TC CE TG PL CE/TC A-I A-It A-IV C-I! C-Ill E
1
2
3
4
5
6
7
mean _+S.D.
2028 1352 388 558 0.67 0.47 0.22 0.09 !.00 0.90 0.10
2957 1901 533 920 0.64 0.36 0.23 N.D. traces !.1 0.15
2293 N.D. 443 860 N.D. 0.32 0.18 N.D. N.D. I).83 0.09
2011 1592 407 545 0.79 0.25 0.15 N.D. traces !.1 0.07
1839 1190 279 496 0.65 0.34 0.14 N.D. N.D. 0.32 0. I 0
2942 2146 160 859 0.73 0.10 0.13 N.D. N.D. 3.8 0.09
2432 1619 907 740 0.67 0.21 0.31 N.D. N.D. 1.01 0.04
2357 + 449 1633 + 350 445 + 236 7i I + 176 0.69 + 0.06 0.29+ 0.12 0.19+ 0.06 N.D. N.D. 1.29:t: 1.13 0.09 :!: 0.03
subpopulations indicating that the isolation procedure saves the integrity of the isolated particles. The chemical characterization of crude Lp(a): B particles indicates clearly that apo (a) and apo B are not the only proteic components of this particle (Fig. 2). Considering the number of molecules per particle, calculated assuming one copy of apo B in each, the most prominent proteic component appears to be the apo Cs and E (Table I). Crude Lp(a):B appears cholesterol ester rich and triacyglycerol poor and the size distribution confirms that most of the particles exhibit an LDL size (Table IV and Fig. 3). Fig. 2 and Table It indicate that anti apo E column has removed most of the apo E present in crude Lp(a):B. The lipid composition appears close to that of crude Lp(a): B, i.e., triacyiglycerol poor and cholesterol ester rich. Other apolipoproteins are still present, apo C-Ill being the most important. The size distribu-
tion of Lp(a): B, free of apo E resembles closely that of crude Lp(a):B, i.e., most of the particles exhibit an LDL size (Table IV and Fig. 3). As shown in Fig. 2 and Table Ill, Lp(a):B:E particles differ from Lp(a):B, free of apo E, not only by their proteic composition but also by their lipid composition. These particles are richer in lipids but in comparison with the Lp(a): B free of apo E, the increase in triacylglycerol is much more considerable (about 5fold-higher) than the increase in cholesterol. The number of apo E molecules per particle is about 9 as a mean but may be as high as 22. These particles are also rich in apo Cs, especially apo C-Ill which appears to be the main proteic component, in terms of number of molecules per particle. As it would bc expected from these results the size distribution of Lp(a):B:E appears in net favour of VLDL and IDL (Table IV and Fig. 3). Since only a small percentage of Lp(a): B, free
TABLE !!!
Lipid and apolipoprotein composition of Lp(a) : B : E particles Results are expressed as numbers of molecules per particle, assuming one apolipoprotein B per parlicle. N.D., not determined.
TC CE TG PL CE/TG A-I A-I! A-IV C-I! C-Ill E
Sample number 1 2
3
4
5
6
7
mean + S.D.
4065 3{)16 2143 1245 0.74 0.18 0.56 0.19 6.9 16.8 10.1
3197 N.D. 3042 1721 N.D. 0.07 0.26 N.D. 5.6 7.7 4.3
2934 1834 1824 1376 0.62 0.07 0.23 N.D. 0.7 8.4 5.8
3309 2336 1505 876 0.71 0.22 0.44 N.D. N.D. 6.5 5.3
3708 2882 543 1400 0.78 0.16 1.00 N.D. N.D. 5.0 12.7
4752 2942 1422 1527 0.62 0.18 0.80 N.D. N,D. 9.90 22.3
3566 :1:658 2532 + 484 1686 + 775 1290 :t: 316 0.70::1: 0.07 0.14::t: 0.06 0.56 ± 0.28 N.D. 5.1 ::t: 3.0 9.3 5:: 3.8 9.2 5: 6.6
3003 2184 1326 887 0.73 0.13 0.62 N.D. 7.2 10.5 3.9
128 14
..... A
e
............ c
.....
v
' --~a
.P,
12
'°
/'X
8
1 i~,¢"
' ~ , . ,~
-"°..°°°.°.°°
° . ,°
2 O
t 6,4
I~
~6
1112 ' l ~ s|
I 14.4
I 16
Fig. 3, A chanlcteristic elution profile of crude Lp(a):B ~ , L ~ a ) : B free of apo E . . . . . . , Lp(a):B:E . . . . . . obtained by FPLC chromatography over a Superose 6 gel filtration column. In this system, VLDL (d < !.006 kg/i) are eluted between 5.6 ml and 7.2 ml, IDL (I.006 kg/I < d < 1,019 kg/I) between 7.2 ml and 8 ml, LDL (I.019 kg/I < d < !.063 kg/I) between 8 ml and 1!.2 ml and HDL (I.063 k g / l < d < !.21 kg/I) between 11,2 ml and 15.2 ml. Lpla) isolated by ultracentrifugation 11,055 kg/I < d < 1,12 kg/l) . . . . . elutes between 10.4 ml and 15.2 ml.
Fig, 2. SDS PAGE analysis of crude Lp(a): B (lane B), Lp(a): B free of apo E (lane C), Lp(a):B:E (lane D). in lane A, the foliow!ng molecular weight calibration proteins were run: phosphorylase b (94000), bovine serum albumin (67000), ovalbumin (43000), carbonic anhydrase (30000), ,soybean trypsin inhibitor (20100), alpha lactalbumin (14400). Ape Ca). ape B. ape E and ape A-IV, as checked by immunoblot are indicated by arrows.
15.6%, 9.0% and 6.4% of total Lp(a): B in samples I to 7, respectively, Fig. 4 shows the competition curve between labeled LDL and unlab~eled LDL, Lp(a):B and Lp B resulting from the DTI' reduction of Lp(a): B. These Lp(a):B preparations were free of apo E. No significant difference could be observed between the three competition curves when results were expressed in mol of apo B, i.e., in terms of molar concentration of each particle. Competition curves between labeled LDL and Lp(a):B free of apo E, or Lp(a):B:E are shown in
of apo E was found in VLDL and LDL, most but not all Lp(a) particles in these density ranges contain apo E. As grossly estimated by the percentage of apo B from crude Lp(a):B recovered in Lp(a):B:E, this latter particle represents 20.5%, 18.1%, 22.6%, 47.8%, T A B L E IV
l~vnt~e si:e dislriln,timt of ,qndiln~nnt'in (a) comain#,R mniclt.s Aim(a) phenotypc
S,mplc,s
Size VLDL
IDL
LDL
HDL
N.D, 2.6 N,D.
N.D. 95.4 N.D.
N.D. 2.0 N.D.
No, !
U,D,
Crude Lp(a): B Lp(a): B free of E Lp(a): B: E
N,D, traces N,D,
No, 2
SI/SI
Crude Lp(a): B Lp(a): B free of E Lp(a):B:E
1,0 0.6 18.4
5.9 3,7 29,9
93.1 94.4 51.7
traces 1.3 traces
No, 3
$4/$4
Crude Lp(a): B Lp(a): B free of E Lp(a): B: E
3. I traces 51.8
7,0 3. I 48.2
89. I 95.3 traces
0.8 1.6 traces
No. 4
U,D.
Crude Lp(a): B Lp(ah B free of E Lp(aI:B:E
3.3 1.0 31.8
5.9 7.6 36.6
90.0 86.8 31.6
0.8 4.6 traces
No, 5
SI/Si
Crude Lp(a): B Lp(a): B free of E Lp(a):B:E
6.5 6.2 45.8
6.3 9.6 31.2
85.6 82.2 23.0
1.6 2.0 traces
Mean ± S,D,, N,D,, not determined; U,D., undetectable.
1 7 ,I6
129 curve, suggesting that the LDL receptor affinity depends on the ape E content of the particle.
"-'~ 1201
lOO¢ o u
"6
8o.
o c-
60-
o ,',
40-
Discussion
_1 Q
"|
20.
,-
0
8
--_....._.._& o
lb
2b nmol
3'o
4'o
sb
opoBII
Fig. 4. Competition between LDL and ape B containing particles for binding to the LDL receptur of HeLa cells. After 48 I! preincubation with LPDS, cells were incubated for 2 h at 4°C with 5 ~tg/ml of t2Sl.labeled LDL (9.1 nmol ape B/I) in the presence of various concentrations of unlabeled particles: A, LDL; El, Lp(a):B free of ape E; @, Lp B obtained after DTT reduction of L.n(a):B free of aim E. Each experimental point represents the averageof a triplicate assay. Results are expressed as percentages of the maximum ~2Sllabeled LDL binding,obtained in the absence of any competitor. Fig. 5. Presence of ape E in the particle enhanced the interaction of Lp(a): B: E with the LDL receptor. Lp(a):B: E appears to be a better competitor for LDL binding than Lp(a): B free of ape E, or LDL itself. 4.5 p g / m l of LDL ape B or 3.7 ~tg/ml of Lp(a): B ape B were needed to inhibit 50% of the labeled LDL (5 ttg/ml) binding, while only 1.05/.tg of Lp(a): B : E ape B were necessary to get the same effect. In the latter, the ape E / a p e B molar ratio was 22.3. Binding curve obtained with a L p ( a ) : B : E particle for which the ape E / ape B molar ratio was 12, was intermediate between the Lp(a): B curve and the previous Lp(a): B: E ,-,~ 120]
1o
"s
eo. 1
x~ c o n ..i Q .J,
60- I 40201
oo
1~
2b
3'o
,~o
sb
nmol apoBII
Fig. 5. Competition between LDL and Lp(a): B free of ape E or Lp(a):B:E. After 48 h.preincubation with LPDS, cells were incubated for 2 h at 4"C with 5 ~g/ml of I251-1abeled LDL (9.1 nmoi ape B/I) in the presence of various concentrations of unlabeled particles: 13, Lp(a): B free of ape E; II, Lp(a): B: E (22 molecules of ape E per particle). Each experimental point represents the average of a triplicate assay. Results are expressed as percentages of the maximum t251-1abeled LDL binding, obtained in the absence of any competitor.
This work demonstrates clearly that ape (a) and ape B are not the only proteic components of Lp(a) particles. It appears that Lp(a) represents a mixture of particles with different apolipoprotein composition. The use of immunoaffinity chromatography avoids any ultracentrifugation step which may artificially remove apolipoproteins from the particles [24]. Furthermore, it allows the isolation of particles from the whole size spectrum. These two reasons probably explain why this was not demonstrated earlier. Actually, most of the isolation procedures start with a material enriched in Lp(a) by ultracentrifugation at density between 1.055 kg/I and 1.12 kg/I. Our results indicate that most of the L p ( a ) : B : E particles are found in VLDL and IDL. Thus, it is logical that Lp(a) prepared after such an ultracentrifugation step does not contain significant amounts of apolipoproteins C and E. It has been shown that ape (a) is not only found at densities between 1.055 k g / l and 1.12 k g / l but can be associated with chylomicrons and chylomicron remnants [14]. It was recently established [38] that in the fasted state, 0 to 17% of total plasma ape (a) are associated with triacylglycerol rich lipoproteins, while in the fed state, this percentage increases to 0 to 83%, this increase being correlated with the increase in plasma triacylglycerol. All our subjects were in the fasted state, as may be confirmed by their plasma triacyiglycerol concentrations (from 0.53 g / ! to 1.61 g/I) as well as the absence of ape B 48 in SDS-PAGE and immunoblot analysis. As shown in Table IV, the percentage of Lp(a): B found in VLDL fall within the range observed previously in the fasted state [38]. However, our data indicate that a very small percentage of Lp(a): B, free of ape E, is found in the VLDL and IDL size ranges. Therefore, we may say that most, but not all, the Lp(a) particles present in these size ranges contain ape E. Due to the size heterogeneity of ape (a) [29], it is not possible to measure precisely the molar composition of the isolated particles in ape (a). However, the use of an arbitrary molecular weight of 500000 was used to roughly estimate the ape (a) content. These results obtained by this method agree with the expected ape (a)/apo B molar ratio of one, expected in a Lp(a) particle [7-9]. The presence of two populations of ape (a) containing lipoproteins differing by their protein and lipid composition may indicate that there are at least two distinct synthetic pathways. However, it was recently described [39-40] that Lp(a) may bind to other ape B containing lipoproteins with LDL as well as VLDL
130
densities. This binding implicates the kringle-4-1ike domains of apo (a) and may be due to hydrophobic forces [41]. Our results do not exclude this possibility of an interaction of Lp(a): B with other apo B containing lipoproteins within the blood stream. Since apo E has been shown to modulate the binding prope~ies of apo B containing lipoproteins towards the LDL re~ptor [21-23], these results suggest that the two sub~pulations may also have different catabolic pathways. Our binding study clearly indicates that Lp(a):B represents a good competitor for the LDL binding to its receptor, one Lp(a): B particle being apparently equivalent to one LDL particle. Lp(a): B: E particles represent better competitors for the LDL binding towards the apo B / E receptor of HeLa cells. These results fit completely with our previous study on apo B containing particles [23], which has demonstrated that, when apo E is present on an apo B containing particle, its affinity for the apo B / E receptor is increased. However, it is not possible to exclude, from our data, an influence of the particle size on the receptor binding, because the presence of apo E on an apo (a) particle is associated with an increase in size. Nevertheless, the discovery of two subclasses of apo (a) containing particles, one containing apo E and one free of it, with different binding capabilities to the apo B / E receptor raises new questions on the metabolism of this intriguing particle. We may grossly estimate from our experiments, that about 20% of the apo (a) containing particles contain apo E. Further studies are needed to determine the clinical importance of this distribution. In particular, it should be determined if this amount is stable or different between individuals and how drugs affecting lipid metabolism may influence this concentration.
&cknowled8ements The authors wish to thank Mr. Paul Kelly for his help in polishing the English of the manuscript and Mrs. Corinne Copin for her skillful technical assistance.
References I Kostner, G,M., Avogaro, P., Cazzolato, G., Marth, E., BittolonBon, G. and Qunici, G.B. (1981) Atherosclerosis 38, 51-60. 2 Dahlen, G.H., Attar, M. and Guyton, J.R (1983) Arteriosclerosis 3, 478a(abstract), 3 Koltdnger, P,G. and Jurgens, G. (1985) Athemsclerosis 58, 187198. 4 Armstrong, VOW,, Cremer, P., Eberle, E., Manke, A., Schulze, F., Wieland, H., lfzeuzer, H. and Seidel, D. (1986) Atherosclerosis 62, 249-257. 5 Dahlen, G.H., Guyton, J.R., Attar, M., Farmer, J.A., Kautz, J.A. and Gotto, A.M. (1986) Circulation 74, 758-765. 6 Rhoads, G.G., Dahlen, G.H., Bets, K., Morton, N.E. and Dannenbers, A.I... (1986) J. Am. Med. Ass. 256, 2540-2544.
7 Gaubatz, J.W., Heideman, C., Gotto, A.M. Jr, Morrissett, ,I.D. and Dahlen, G.H. (1983) J. Biol. Chem. 258, 4582-4589. 8 Utermann, G. and Weber, W. (1983) FEBS Lett. 154, 357-361. 9 Fless, G.M., Rolih, C.A. and Scanu, A.M. (1984) J. Biol. Chem. 259, 11470-11478. I0 Fless, G.M,, ZumMallen, M.E. and Scanu, A.M. (1986) J. Biol. Chem. 261, 8712-8718. 11 Berg, K. (1963) Acta Pathol. Microbiol. Scand. 59, 369-382. 12 Ehnholm, C., Garoff, H,, Renkonen, O, and Simons, K, (1972) Biochemistry II, 3229-3232. 13 Harvie, N.R. and Schultz, J.S. (1973) Biochem, Genet. 9, 235-245. 14 Bersot, T.P., Innerarity, R,W., Pitas, R.E., Rail, S.C., Weisgraber, LJ. and Mahley, R,W. (1986) J. Clin. Invest. 77, 622-630. 15 Martmann-Moe, K. and Berg, K. (1981) Clin. Genet, 20, 352-362. 16 Havekes, L., Vermeer, B.J., Brugman, T. and Emeis, J. (1981) FEBS Lett. 132, 169-173. 17 Floren, C.H., Albers, JJ. and Bierman, E.L. (1981) Biochem. Biophys. Res. Commun. 102, 636-637. 18 Krempler, F., Kostner, G.M., Roscher, A., Haslauer, F., Bolzano, K. and Sandhofer, F. (1983)J. Clin. Invest. 71, 1431-1441. 19 Thiery, J., Armstrong, W.W., Schleef, J,, Creutzfeldt, C., Creutzfeldt, W. and Seidel, D. (1988} KUn. Wochenshr. 66, 462463. 20 Leren, T.P., Hjermann, !., Berg, K., Leren, P., loss, O.P. and Viksmoen, L. (1988) Atherosclerosis 73, 135-141. 21 Gianturco, S.H., Brown, F.B., Gotto, A.M. and Bradley, W.A. (1982) J. Lipid Res. 23, 981-983. 22 Yamada, N., Shames, D.M., Stoudemie, J.D. and Havel, R.J. (1986) Proc. Natl. Acad. Sci. USA 83, 3479-3483. 23 Agnani, G., Bard, J.M., Candelier, L., Delattre, S., Fruchart, J.C. and Clayey, V. (1989) Arteriosclerosis, 1!, 1021-1029. 24 Castro, G.R. and Fielding, CJ. (1984) J. Lipid Res. 25, 58-67. 25 Bard, J.M., Candelier, L., Agnani, G., Clayey, V., Torpier, G., Steinmetz, A. and Fruchart, J.C. (1991) Biochim. Biophys. Acta, 1082, 170-176. 26 Fruchart, J.C., Kora, !., Cachera, C., Clayey, V., Duthilleul, P. and Moschetto, Y. (1982) Clin. Chem. 28, 59-62. 27 Vu-Dac, N., Mezdour, H., Parra, H.J., Luc, G., Luyeye, i. and Fruchart, J.C. (1989) J. Lipid Res. 30, 1437-1444. 28 Mezdour, H., Clayey, V., Kora, I., Koffigan, M., Barkia, A. and Fruchart, J.C. (1987)J. Chromatogr. 414, 35-45. 29 Utermann, G., Menzel, H.J., Kraft, H.G., Duba, H.C., Kemmler, H.G. and Seitz, C. (1987) J. Clin. Invest. 80, 458-465. 30 Fruchart, J.C., Fievet, C. and Pu~itois, P. (1985) Methods of Enzym. Anal. 126-138. 31 Knott, TJ., Pease, RJ., Powell, L.M., Wailis, S.C., Rail, T.L. Jr, lnnerarity, T.L., Blackhart, B., Taylor, W.H., Marcel, Y., MUne, R., Johnson, D., Fuller, M., Lusis, A.J., McCarthy, B.J., Mahley, R.W., Lew-Wilson, B. and Scott, J. (1986) Nature 323, 734-738. 32 Elovson, J., Chatterton, J.E., Bell, G.T., Schumaker, V.N., Reuben, M.A., Puppione, D.L., Reeve, J.R. Jr and Young, N.L. (1988) J. Lipid Res. 29, 1461-.;471. 33 Fless, G.M., ZumMallen, M.E. and Scanu, A.M. (1985) J. Lipid Res. 26, 1224-1229. 34 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 35 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412. 36 Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350-4354. 37 MaeFarlane, A.S. (1958) Nature, 182, 53-58. 38 Cohn, J.S., Lain, C.W.K., Sullivan, D.R. and Hensley, W.J. (1991) Atherosclerosis 90, 59-66. 39 Ye, S.Q., Trieu, V.N, Stiers, D.L. and McConathy, W.J. (1988) J. Biol. Chem. 263, 6337-6343. 40 Trieu, V.N. and McConathy, W.J. (1990) Biochemistry 29, 59195924. 41 Trieu, V.N., Zioncheck, T.F., Lawn, R.M. and McC,mathy, WJ. (1991) J. Biol. Chem. 266, 5480-5485.
124
BBALIP 53974
Isolation and characterization of two sub-species of Lp(a), one containing apo E and one free of apo E Jean-Marie Bard, Sophie Delattre-Lestavel, V6ronique Clayey, Pascal Pont, Bruno Derudas, Henri-Joseph Parra and Jean-Charles Fruchart SERLIA et INSERM U325, lnstitut Pasteur, Lille (Frame)
(Received 31 December 1991) (Revised manuscript received 19 March 1992)
Key ~vor0s: Apo E; Apolipoprotein (a); Lipoprotein binding
Lipoprotein Lp(a) was isolated by immunoaffinity chromatography using anti apolipoprotcin B and :uiti apolipoprotein (a) immunosorbents. Besides apolipoproteins (a) and B, this fraction was shown to contain apolipoproteins C and E. Therefore, it was decided to further purify this crude Lp(a) into particles containing apolipoprotein E and particles free of ape E, using chromatography with an anti apolipoprotcin E immunosorbent. Lp(a), free of apolipoprotein E was cholesterol ester rich and triaeyl8lycerol poor and was found mainly in the LDL size range. In contrast, Lp(a) containing apolipoprotein E was triacylglycerol rich and was distributed mainly in the VLDL and IDL size range. Binding of these two fractions, one containing ape E and one free of it, to the ape B/E receptor of HeLa cells was studied. Both fractions bound to the receptor but the one containing ape E had a better affinity than the one free of ape E. Further studies are needed to identify the clinical importance of these two different entities.
Introduction Human Lp~a) is a lipoprotein whose high plasma levels have been associated with an increased cardiovascular risk [1-6]. Several studies have shown that the protein moiety of [p(a) contains a single copy of ape 13, cross-linked to aim (a) by one or more disulfide bonds [7-9]. However, species of Lp(a) that have 2 reel of a ~ a ) per reel of ape B have also been found [10]. Although ~:arly reports stated that Lp(a) exhibited a density between LDL and HDL (!.055 kg/I < d < 1.120 ~/I) [11-12], more recent studies indicated that Lp(a) exhibited inter- and intra-individual density heterogeneity [9,13]. Also, apo (a) antigen can be present, not
only in cholesterol ester rich iipoproteins but also in triacylglycerol rich partic!e~ [14]. Results of studies comparing the interaction of Lp(a) with the LDL receptor are rather conflicting. AIthough, in one study [15], the conclusion was reached that L4)(a) creeled fibroblasts independently of the LDL receptor, other investigations [16-18] have concluded that Lp(a) can bind to and be taken up by the same receptor site as LDL, sometimes with a high
affinity [17]. Also the use of drwgs which activate the LDL receptor specific pathway, such as HMG CoA reductase inhibitors has led to mixed results [19-20], most of them showing no change in the Lp(a) level and some of them showing a decrease. We hypothesized that the conflicting results obtained in in vitro studies aimed at the interaction of Lp(a) with the LDL receptor or in pharmacological studies using drugs activating the LDL receptor specific pathway may be explained by the heterogeneity of the Lp(a) particle. Since ape E has been shown to modulate the binding properties of ape B containing lipoproteins to the LDL ,receptor [21-23], we looked first at the ape E content of the Lp(a) particle. Using immunoaffinity chromatography, a method which has been shown to avoid any dissociation of ape E, in contrast with ultracentrifugation [24], we were able to prepare two sub-species of Lp(a) depending on the presence or absence of ape E. Chemical characteristics of these two fractions and their binding properties towards the ape B / E receptor of HeLa cells was determined. Materials and Methods
Isolation of ape (a) containing lipoproteins Corresi~ndence to: J.-M. Bard, SERLIA et INSERM U325. Institut Pasteur, I Rue du Pmfesseur Calmette, 59019 Lill~: C~dex, France.
The isolation of ape (a) containing particles was realized by immunoaffinity chromatography at 4°C
125 monitored at 280 nm, as extensively described in a previous paper [25]. To this aim, polyclonal antibodies against apo B [26] and monoclonal antibodies against apo (a) [27] and apo E [28] were covalently coupled to CNBr activated Sepharose 4B according to the procedure of the manufacturer (Pharmacia Fine Chemicals, Uppsala, Sweden). EDTA plasma was obtained from seven normolipidemic individuals in the morning after an overnight fasting period. Plasma cholesterol concentrations were 1.41 g/l, 1.75 g/l, 1.67 g/l, 1.34 g/l, 1.60 g/l, 1.72 g/! and 1.76 g/l, while plasma triacylglycerol concentrations were 0.88 g/l, 0.77 g/l, 1.45 g/l, 0.78 g/l, 0.78 g/i, 0.53 g/i and 1.61 g / l for samples 1 to 7, respec.. tively. The plasma Lp(a) levels (in total mass) were 0.016 g/I, 0.147 g/l, 0.051 g/I, 0.14 g/l, 0.175 g/l, 0.504 g/i and 0.079 g / l for samples 1 to 7, respectively. As determined by the method developed by Utermann et al. [29], the apo (a) isoforms were S I / S I , $4/$4, Sl/S1 and $2/$4 for samples 2, 3, 5 and 6, respectively. Bands obtained with samples 1, 4 and 7 were too weak to determine precisely the corresponding phenotypes. 40 ml of each sample were submitted first to the anti apo B column. The unretained fraction was eluted with buffer containing 0.01 M Tris-HCi, 0.5 M NaCI, 0.3 mM EDTA, 1.5 mM NaN 3 and 10 ttM PMSF (pH 8.0) at a flow rate of 60 ml/h. The retained fraction was eluted with 3 M sodium thiocyanate (50 mi). A column containing 100 ml of Sephadex G25 (Pharmacia
Fine Chemical, Uppsala, Sweden) was linked to the immunosorbent column in order to separate immediately the lipoproteins from the dissociating agent (sodium thiocyanate). The retained fraction was submitted to the anti apo (a) column and elution was realized as described above. The retained fraction was crude Lp(a):B. Crude Lp(a): B was then submitted to an anti apo E column in the same conditions as above. The retained fraction was Lp(a) containing apo E (Lp(a):B:E) and the unretained fraction was Lp(a) free of apo E (Lp(a):B). Fig. 1 indicates the different separation steps described above.
Chemical characterization of the isolated particles Apolipoproteins A-I, A-II, A-IV, B, C-If, C-Ill and E were quantified by ELISA [30]. Lipoproteins were assumed to contain one single copy of apolipoprotein B per particle [31,32] and molar ratio were calculated assuming molecular weights of 550 000 for apo B, 28 500 for apo A-I, 17000 for apo A-II, 46000 for apo A-IV, 8800 for apo C-II, 8900 for apo C-Ill and 34000 for apo E. Due to its size heterogeneity [29], it is difficult to measure precisely apo (a) and to express its molar concentration. In order to check that the concentration of apo (a) in the isolated particles was compatible with a molar ratio of 1 as expected in a Lp(a) particle, apo (a) was estimated as follows. The isolated particles
Whole plasma
[
I.
AntiApoB
R
UR
I
I Anti Apo (a) UR Crude Lp(a):B
I
Anti Apo E
I
UR Lp(a):B freeofapo E
l.p(a):B:l£
Fig. 1. Flow diagram for immunoaffinity chromatography preparation of crude Lp(a):B, Lp(a):B free of apo E and Lp(a):B:E. R, retained fraction; UR, unretained fraction. The boxes represent affinity columns containing specific antibody against the apolipoprotein indicated.
126 TABLE !
Lipid and apolipoprotein composition of crude Lp(a) : B particles Results are expressed as numbers of molecules per particle, assuming one apolipoprotein B per particle. N.D., not determined. Sample number TC CE TG PL CE/TC A-I A-ll A-IV C-ll C-ill E
1
2
3
4
5
6
7
mean ± S.D.
2752 1882 1216 459 0.68 0.42 0.38
2198 1436 775 699 0,65 0.37 0,31 N,D, 4,15 2.17 0,42
2116 136~ 910 723 0.64 0,22 0.25 N.D, !.57 !,32 0.84
3557 2432 1248 1112 0.68 0.48 0,27 N.D, traces 2,78 !.23
2130 1377 494 615 0,65 0,37 0,18 N,D. N.D. 1.67 1.00
2833 2183 180 741 0,77 0,38 0,58 N.D. N.D. 1.35 0.53
2337 1557 1438 717 0,67 0.11 0,13 N.D. N.D. 4.6 !,03
2560 + 527 1747 +427 894 +449 723 + 197 0.68 + 0,04 0,34 + 0,13 0,30± 0,15 N.D. N.D. 2.39± 1.16 0.93 ± 0.38
0,08 traces 2,81 1.49
were treated with dithiotreitol (DTT) as previously described [33] to isolate ape (a) from the particle. The remaining iipoprotein particle was separated from ape (a) by ultracentrifugation in a Beckman TL 100 ultracentrifuge at density 1.21 kg/l, in a TLA 100.2 rotor at 4°C for 3 h at 330000 × g. The supernate contained the reduced Lp B. In the bottom fraction, the protein content was estimated by the Lowry method [34], apolipoproteins A-I, A-II, A-IV, B, C-Ill and E were measured by ELISA [30] and albumin was quantified by nephelometry using the urine microalbumine kit from Behring, Germany. The protein content less the total of apolipoproteins and albumin was considered as the ape (a) content. The molar ratio between ape (a) and ape B was approximated, using an arbitrary molecular weight of 500 000 for ape (a). Protein composition was also assessed by sodium dode~lsulphate polyacrylamide gel electrophoresis (SDS-PAGE) [35], followed by immunoblot with anti apo (a), apo B, apo A-IV and apo E [36]. Total cholesterol (TC), free cholesterol (FC), triacylglycerol (TG)and phospholipids (PL) were measured by enzymatic tests (Boehringer-Mannheim, Germany). Esterified cholesterol (EC) was estimated as the difference between TC and FC. Molar razios over apo B were calcule,ted assuming molecular weights of 388, 877 and 775 for cholesterol, triacylglycerol and phospholipids, respectively.
Analysis of panicle size The molecular size distribution of the isolated particles from the first five individuals was determined by gel filtration over a column of Superose 6 HR 10/30 (Plmrmacia Fine Chemicals, Uppsala, Sweden) equilibrated in 0.01 M Tris-HC!, 9.15 M NaCI, 3- 10 -4 M EDTA, 1.5- 10 -3 M NaN 3, 2- 10 -s M PMSF (pH 8). The sample (200 ~!) was eluted at a constant flow rate of 12 ml/h. The absorbance of the eluent was moni-
tored continuously at 280 nm. The eluent was pooled at volumes corresponding to the elution volume of human VLDL (d < 1.006 kg/i: 5.6 to 7.2 ml), IDL (1.006 kg/i < d < 1.019 kg/l: 7.2 to 8 ml), LDL (1.019 kg/! < d < 1.063 kg/!: 8 to 11.2 ml) and HDL (1.063 kg/! < d < 1.21 kg/I: 11.2 to 15.2 ml). Using this procedure, Lp(a) isolated by ultracentrifugation (1.055 kg/! < d < 1.12 kg/I) eluted between 10.4 and 15.2 ml. APO B was measured [30] in each pooled fraction to assess the size distribution of the isolated particles.
Binding studies HeLa cells were grown in Dulbecco's modified Eagle's medium containing penicillin (100 units/ml), streptomycin (100 /.tg/ml) and 10% (v/v) fetal calf serum in 30-ram diameter multidish plates. Nearly confluent cells were preincubated for 48 h in a medium containing 10% (v/v) lipoprotein deficient calf serum (d > 1.21 kg/I). The content of each dish was about 400/~g of cell protein. Isolated particles were assayed in a competitive binding assay against LDL, labele~ ',4th 1251 according to the iodine monochloride method ,)f MacFarlane [37]. The labeled LDL was incubated w;th HeLa cells (5 ~g of LDL apo B / m l incubation mixture or 9 nM as LDL particle) in the presence of variou~ amounts of Lp(a): B particles at 4°C for 2 h. The cells g ere washed nine times and cell associated radioactivity wa~ counted. The amounts of Lp(a): B added to the iabe;ed LDL were calculated on the basis of their apo B t;ontent. Each point was performed in triplicate and experiments were realized on two different Lp(a)" B preparations. Results
In each case, apo (a)/apo B molar ratio was estimated to be close to 1 (0.61 to 0.82) in the three Lp(a)
127 TABLE II
Lipid and apolipoprotein composition of Lp(a) : B, free of apo E Results are expressed as numbers of molecules per particle, assuming one apolipoprotein B per particle. N.D., not determined. Sample number
TC CE TG PL CE/TC A-I A-It A-IV C-I! C-Ill E
1
2
3
4
5
6
7
mean _+S.D.
2028 1352 388 558 0.67 0.47 0.22 0.09 !.00 0.90 0.10
2957 1901 533 920 0.64 0.36 0.23 N.D. traces !.1 0.15
2293 N.D. 443 860 N.D. 0.32 0.18 N.D. N.D. I).83 0.09
2011 1592 407 545 0.79 0.25 0.15 N.D. traces !.1 0.07
1839 1190 279 496 0.65 0.34 0.14 N.D. N.D. 0.32 0. I 0
2942 2146 160 859 0.73 0.10 0.13 N.D. N.D. 3.8 0.09
2432 1619 907 740 0.67 0.21 0.31 N.D. N.D. 1.01 0.04
2357 + 449 1633 + 350 445 + 236 7i I + 176 0.69 + 0.06 0.29+ 0.12 0.19+ 0.06 N.D. N.D. 1.29:t: 1.13 0.09 :!: 0.03
subpopulations indicating that the isolation procedure saves the integrity of the isolated particles. The chemical characterization of crude Lp(a): B particles indicates clearly that apo (a) and apo B are not the only proteic components of this particle (Fig. 2). Considering the number of molecules per particle, calculated assuming one copy of apo B in each, the most prominent proteic component appears to be the apo Cs and E (Table I). Crude Lp(a):B appears cholesterol ester rich and triacyglycerol poor and the size distribution confirms that most of the particles exhibit an LDL size (Table IV and Fig. 3). Fig. 2 and Table It indicate that anti apo E column has removed most of the apo E present in crude Lp(a):B. The lipid composition appears close to that of crude Lp(a): B, i.e., triacyiglycerol poor and cholesterol ester rich. Other apolipoproteins are still present, apo C-Ill being the most important. The size distribu-
tion of Lp(a): B, free of apo E resembles closely that of crude Lp(a):B, i.e., most of the particles exhibit an LDL size (Table IV and Fig. 3). As shown in Fig. 2 and Table Ill, Lp(a):B:E particles differ from Lp(a):B, free of apo E, not only by their proteic composition but also by their lipid composition. These particles are richer in lipids but in comparison with the Lp(a): B free of apo E, the increase in triacylglycerol is much more considerable (about 5fold-higher) than the increase in cholesterol. The number of apo E molecules per particle is about 9 as a mean but may be as high as 22. These particles are also rich in apo Cs, especially apo C-Ill which appears to be the main proteic component, in terms of number of molecules per particle. As it would bc expected from these results the size distribution of Lp(a):B:E appears in net favour of VLDL and IDL (Table IV and Fig. 3). Since only a small percentage of Lp(a): B, free
TABLE !!!
Lipid and apolipoprotein composition of Lp(a) : B : E particles Results are expressed as numbers of molecules per particle, assuming one apolipoprotein B per parlicle. N.D., not determined.
TC CE TG PL CE/TG A-I A-I! A-IV C-I! C-Ill E
Sample number 1 2
3
4
5
6
7
mean + S.D.
4065 3{)16 2143 1245 0.74 0.18 0.56 0.19 6.9 16.8 10.1
3197 N.D. 3042 1721 N.D. 0.07 0.26 N.D. 5.6 7.7 4.3
2934 1834 1824 1376 0.62 0.07 0.23 N.D. 0.7 8.4 5.8
3309 2336 1505 876 0.71 0.22 0.44 N.D. N.D. 6.5 5.3
3708 2882 543 1400 0.78 0.16 1.00 N.D. N.D. 5.0 12.7
4752 2942 1422 1527 0.62 0.18 0.80 N.D. N,D. 9.90 22.3
3566 :1:658 2532 + 484 1686 + 775 1290 :t: 316 0.70::1: 0.07 0.14::t: 0.06 0.56 ± 0.28 N.D. 5.1 ::t: 3.0 9.3 5:: 3.8 9.2 5: 6.6
3003 2184 1326 887 0.73 0.13 0.62 N.D. 7.2 10.5 3.9
128 14
..... A
e
............ c
.....
v
' --~a
.P,
12
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8
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' ~ , . ,~
-"°..°°°.°.°°
° . ,°
2 O
t 6,4
I~
~6
1112 ' l ~ s|
I 14.4
I 16
Fig. 3, A chanlcteristic elution profile of crude Lp(a):B ~ , L ~ a ) : B free of apo E . . . . . . , Lp(a):B:E . . . . . . obtained by FPLC chromatography over a Superose 6 gel filtration column. In this system, VLDL (d < !.006 kg/i) are eluted between 5.6 ml and 7.2 ml, IDL (I.006 kg/I < d < 1,019 kg/I) between 7.2 ml and 8 ml, LDL (I.019 kg/I < d < !.063 kg/I) between 8 ml and 1!.2 ml and HDL (I.063 k g / l < d < !.21 kg/I) between 11,2 ml and 15.2 ml. Lpla) isolated by ultracentrifugation 11,055 kg/I < d < 1,12 kg/l) . . . . . elutes between 10.4 ml and 15.2 ml.
Fig, 2. SDS PAGE analysis of crude Lp(a): B (lane B), Lp(a): B free of apo E (lane C), Lp(a):B:E (lane D). in lane A, the foliow!ng molecular weight calibration proteins were run: phosphorylase b (94000), bovine serum albumin (67000), ovalbumin (43000), carbonic anhydrase (30000), ,soybean trypsin inhibitor (20100), alpha lactalbumin (14400). Ape Ca). ape B. ape E and ape A-IV, as checked by immunoblot are indicated by arrows.
15.6%, 9.0% and 6.4% of total Lp(a): B in samples I to 7, respectively, Fig. 4 shows the competition curve between labeled LDL and unlab~eled LDL, Lp(a):B and Lp B resulting from the DTI' reduction of Lp(a): B. These Lp(a):B preparations were free of apo E. No significant difference could be observed between the three competition curves when results were expressed in mol of apo B, i.e., in terms of molar concentration of each particle. Competition curves between labeled LDL and Lp(a):B free of apo E, or Lp(a):B:E are shown in
of apo E was found in VLDL and LDL, most but not all Lp(a) particles in these density ranges contain apo E. As grossly estimated by the percentage of apo B from crude Lp(a):B recovered in Lp(a):B:E, this latter particle represents 20.5%, 18.1%, 22.6%, 47.8%, T A B L E IV
l~vnt~e si:e dislriln,timt of ,qndiln~nnt'in (a) comain#,R mniclt.s Aim(a) phenotypc
S,mplc,s
Size VLDL
IDL
LDL
HDL
N.D, 2.6 N,D.
N.D. 95.4 N.D.
N.D. 2.0 N.D.
No, !
U,D,
Crude Lp(a): B Lp(a): B free of E Lp(a): B: E
N,D, traces N,D,
No, 2
SI/SI
Crude Lp(a): B Lp(a): B free of E Lp(a):B:E
1,0 0.6 18.4
5.9 3,7 29,9
93.1 94.4 51.7
traces 1.3 traces
No, 3
$4/$4
Crude Lp(a): B Lp(a): B free of E Lp(a): B: E
3. I traces 51.8
7,0 3. I 48.2
89. I 95.3 traces
0.8 1.6 traces
No. 4
U,D.
Crude Lp(a): B Lp(ah B free of E Lp(aI:B:E
3.3 1.0 31.8
5.9 7.6 36.6
90.0 86.8 31.6
0.8 4.6 traces
No, 5
SI/Si
Crude Lp(a): B Lp(a): B free of E Lp(a):B:E
6.5 6.2 45.8
6.3 9.6 31.2
85.6 82.2 23.0
1.6 2.0 traces
Mean ± S,D,, N,D,, not determined; U,D., undetectable.
1 7 ,I6
129 curve, suggesting that the LDL receptor affinity depends on the ape E content of the particle.
"-'~ 1201
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40-
Discussion
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opoBII
Fig. 4. Competition between LDL and ape B containing particles for binding to the LDL receptur of HeLa cells. After 48 I! preincubation with LPDS, cells were incubated for 2 h at 4°C with 5 ~tg/ml of t2Sl.labeled LDL (9.1 nmol ape B/I) in the presence of various concentrations of unlabeled particles: A, LDL; El, Lp(a):B free of ape E; @, Lp B obtained after DTT reduction of L.n(a):B free of aim E. Each experimental point represents the averageof a triplicate assay. Results are expressed as percentages of the maximum ~2Sllabeled LDL binding,obtained in the absence of any competitor. Fig. 5. Presence of ape E in the particle enhanced the interaction of Lp(a): B: E with the LDL receptor. Lp(a):B: E appears to be a better competitor for LDL binding than Lp(a): B free of ape E, or LDL itself. 4.5 p g / m l of LDL ape B or 3.7 ~tg/ml of Lp(a): B ape B were needed to inhibit 50% of the labeled LDL (5 ttg/ml) binding, while only 1.05/.tg of Lp(a): B : E ape B were necessary to get the same effect. In the latter, the ape E / a p e B molar ratio was 22.3. Binding curve obtained with a L p ( a ) : B : E particle for which the ape E / ape B molar ratio was 12, was intermediate between the Lp(a): B curve and the previous Lp(a): B: E ,-,~ 120]
1o
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Fig. 5. Competition between LDL and Lp(a): B free of ape E or Lp(a):B:E. After 48 h.preincubation with LPDS, cells were incubated for 2 h at 4"C with 5 ~g/ml of I251-1abeled LDL (9.1 nmoi ape B/I) in the presence of various concentrations of unlabeled particles: 13, Lp(a): B free of ape E; II, Lp(a): B: E (22 molecules of ape E per particle). Each experimental point represents the average of a triplicate assay. Results are expressed as percentages of the maximum t251-1abeled LDL binding, obtained in the absence of any competitor.
This work demonstrates clearly that ape (a) and ape B are not the only proteic components of Lp(a) particles. It appears that Lp(a) represents a mixture of particles with different apolipoprotein composition. The use of immunoaffinity chromatography avoids any ultracentrifugation step which may artificially remove apolipoproteins from the particles [24]. Furthermore, it allows the isolation of particles from the whole size spectrum. These two reasons probably explain why this was not demonstrated earlier. Actually, most of the isolation procedures start with a material enriched in Lp(a) by ultracentrifugation at density between 1.055 kg/I and 1.12 kg/I. Our results indicate that most of the L p ( a ) : B : E particles are found in VLDL and IDL. Thus, it is logical that Lp(a) prepared after such an ultracentrifugation step does not contain significant amounts of apolipoproteins C and E. It has been shown that ape (a) is not only found at densities between 1.055 k g / l and 1.12 k g / l but can be associated with chylomicrons and chylomicron remnants [14]. It was recently established [38] that in the fasted state, 0 to 17% of total plasma ape (a) are associated with triacylglycerol rich lipoproteins, while in the fed state, this percentage increases to 0 to 83%, this increase being correlated with the increase in plasma triacylglycerol. All our subjects were in the fasted state, as may be confirmed by their plasma triacyiglycerol concentrations (from 0.53 g / ! to 1.61 g/I) as well as the absence of ape B 48 in SDS-PAGE and immunoblot analysis. As shown in Table IV, the percentage of Lp(a): B found in VLDL fall within the range observed previously in the fasted state [38]. However, our data indicate that a very small percentage of Lp(a): B, free of ape E, is found in the VLDL and IDL size ranges. Therefore, we may say that most, but not all, the Lp(a) particles present in these size ranges contain ape E. Due to the size heterogeneity of ape (a) [29], it is not possible to measure precisely the molar composition of the isolated particles in ape (a). However, the use of an arbitrary molecular weight of 500000 was used to roughly estimate the ape (a) content. These results obtained by this method agree with the expected ape (a)/apo B molar ratio of one, expected in a Lp(a) particle [7-9]. The presence of two populations of ape (a) containing lipoproteins differing by their protein and lipid composition may indicate that there are at least two distinct synthetic pathways. However, it was recently described [39-40] that Lp(a) may bind to other ape B containing lipoproteins with LDL as well as VLDL
130
densities. This binding implicates the kringle-4-1ike domains of apo (a) and may be due to hydrophobic forces [41]. Our results do not exclude this possibility of an interaction of Lp(a): B with other apo B containing lipoproteins within the blood stream. Since apo E has been shown to modulate the binding prope~ies of apo B containing lipoproteins towards the LDL re~ptor [21-23], these results suggest that the two sub~pulations may also have different catabolic pathways. Our binding study clearly indicates that Lp(a):B represents a good competitor for the LDL binding to its receptor, one Lp(a): B particle being apparently equivalent to one LDL particle. Lp(a): B: E particles represent better competitors for the LDL binding towards the apo B / E receptor of HeLa cells. These results fit completely with our previous study on apo B containing particles [23], which has demonstrated that, when apo E is present on an apo B containing particle, its affinity for the apo B / E receptor is increased. However, it is not possible to exclude, from our data, an influence of the particle size on the receptor binding, because the presence of apo E on an apo (a) particle is associated with an increase in size. Nevertheless, the discovery of two subclasses of apo (a) containing particles, one containing apo E and one free of it, with different binding capabilities to the apo B / E receptor raises new questions on the metabolism of this intriguing particle. We may grossly estimate from our experiments, that about 20% of the apo (a) containing particles contain apo E. Further studies are needed to determine the clinical importance of this distribution. In particular, it should be determined if this amount is stable or different between individuals and how drugs affecting lipid metabolism may influence this concentration.
&cknowled8ements The authors wish to thank Mr. Paul Kelly for his help in polishing the English of the manuscript and Mrs. Corinne Copin for her skillful technical assistance.
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