Isolation, characterization and comparative aspects of the major serum apolipoproteins, B-100 and AI, in the common marmoset, Callithrix jacchus

321

Biochimica et Biophysics Acta, 154 (1983) 321-333 Elsevier

BBA 51537

ISOLATION, CHARACTERIZATION AND COMPARATIVE ASPECTS APOLIPOPROTEINS, B-100 AND AI, IN THE COMMON MARMOSET, PATRICIA

FORGEZ

a.*, M. JOHN

CHAPMAN

a and GERVASE

OF THE MAJOR SERUM CALLITHRZX JACCHUS

L. MILLS b

0 UnitP 9 Annexe, INSERM, Equipe de Recherches sur les Lipoprottkes, Pavilion Benjamin Delessert, Hbpital La Pitit!, 83, Boulevard de I’HGpital, 75651 Paris Cedex 13 (France) and b Courtauld Institute of Biochemistry, Middlesex Hospital Medical School, London WIP 7PN (U.K.) (Received

July 4th. 1983)

Key words: LDL; Apolipoprotein

structure; HDL; (Marmoset)

The two major apolipoproteins of marmoset serum have been isolated and characterized, and on the basis of physicochemical and immunological criteria are homologous with the human AI and B-100 proteins. Marmoset apolipoprotein AI was the principal protein of high-density lipoproteins (HDL) and was purified by gel filtration chromatography and electrophoresis in alkaline-urea polyacrylamide gel followed by electrophoretic elution. Purified marmoset apolipoprotein AI displayed an M, of approx. 27000, was polymorphic (five forms) on isoelectric focussing, with pl values in the range 4.8-5.0, and migrated similarly to human apolipoprotein AI in alkaline-urea gels. An overall resemblance was seen in the amino acid composition of marmoset apolipoprotein AI and that of its human counterpart with the notable exception that marmoset AI contained 1 isoleucine residue/mole. An immunological reaction of partial identity between the human and monkey proteins was seen upon immunodiffusion of their HDLs against antiserum to human apolipoprotein AI. Marmoset B-100 was the predominant apoprotein of VLDL and LDL, resembling the human protein in its elution profile on gel filtration chromatography in anionic detergent, and in its high apparent A$ (approx. 520000). The marmoset and human B-100 proteins were alike in amino acid composition and carbohydrate content. Moreover, their immunological behaviour with an antiserum to marmoset apolipoprotein B showed them to share certain antigenic determinant(s). We conclude that the physicochemical properties of the principle apolipoproteins of C&r/& jucchus, a New World primate, markedly resemble those of the human AI and B-100 proteins, suggesting therefore that they may function similarly in lipid transport and metabolism. Counterparts to human apolipoproteins AII, E, CII and CIII have also been tentatively identified.

* To whom correspondence should be addressed. Abbreviations: VLDL, very-low-density lipoproteins, density as defined; LDL, low-density lipoproteins, density as defined; HDL, high-density lipoproteins, density as defined; HDL,, a subclass of HDL, density as defined; Hepes, 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride. For present purposes, the centile nomenclature for B proteins proposed by Kane and colleagues [14] has been adopted, thereby facilitating comparison between the marmoset and human B proteins. 00052760/83/$03.00

0 1983 Elsevier Science Publishers

B.V.

Introduction Investigation of the potential role of the serum low-density lipoprotein (LDL) in the genesis of atherosclerosis in man remains limited, primarily as a result of ethical considerations. Attention has therefore turned increasingly to animal models [l-4], among which the nonhuman primates appear admirably suited on a phylogenetic basis.

322

Our own interest has been focussed on a New World primate (family Cebidae), the common marmoset (Callithrix jacchus). This monkey displays circulating LDL levels (approx. 280 mg/dl) [5,6] which approach those of man rather more closely than those of several Old World species (family Cercopithecoidea) (< 150 mg/ dl) in baboon, Patas, rhesus and cynomolgus monkeys) [2,3,7-lo]. Moreover, several additional qualitative and quantitative similarities are evident between the serum lipoprotein profiles in the two species [5,6]. Apolipoprotein B is the major protein component of the low-density lipoproteins of all mammalian species studied to date [lo-121. Indeed, comparative studies of apolipoprotein B-like proteins in the baboon, rhesus and Patas monkeys have shown them to share certain physicochemical and immunological characteristics with their human counterpart [10,13]. In man, the B protein exists in four forms with unique molecular weights and amino acid compositions [14]. In the centile system of Kane et al. [14], the major species in circulating LDL is termed B-100, a protein of hepatic origin, which may be associated with minor amounts of the B-74 and B-26 forms [14]. Data on the structure and metabolism of the B proteins of non-human primates is lacking, apart from a recent report on African green (Cercopithecus aethiops) and cynomolgus (Macaca fascicularis) monkeys [ 151. Apolipoproteins AI and AI1 typically predominate in the protein moieties of the high-density lipoproteins in non-human primates fed chow diets [12]. The physicochemical properties of these proteins in the chimpanzee, baboon, vervet, patas and rhesus monkeys markedly resemble those of their human counterparts [13,16-201, although minor differences such as the presence of apolipoprotein AI1 in monomeric form in all but the chimpanzee are noteworthy. In order to develop further the potential of C. jacchus as an experimental model for lipoprotein and atherosclerosis research, we here describe the purification and characterization of the two principle vectors of lipid transport in this species, i.e., apolipoproteins B and AI, whose presence had been suggested by our earlier electrophoretic and immunological findings [5,6]. The physicochemical properties of these apolipoproteins are also com-

pared with those of the corresponding Old World monkeys and man.

species in

Materials and Methods Materials Animals and diets. The marmosets used in this study belong to the breeding colony established by Imperial Chemical Industries, Pharmaceuticals Division, Macclesfield, Cheshire SK10 4TG, U.K. [21]. The animals used were males aged between 4 and 10 years. Blood samples were taken (approx. 1 ml per animal) from unanesthetized animals which had been fasted overnight for 16-18 h. Samples were drawn directly from the femoral vein between 9.30 and 12.00 a.m. Serum was separated by low-speed centrifugation (15 min at 500 x g) at 4°C and individual samples were pooled; 0.01% (w/v) sodium azide and 1 mM EDTA were then added, and the serum was transported to France in frozen form. Lipoprotein separation was commenced immediately. For evaluation in SDS gels of any possible involvement of proteolytic degradation in defining the B protein pattern of marmoset VLDL and of LDL, the proteolytic inhibitor PMSF was added to half of the volume of two separate serum samples (final concentration 1 mM), and VLDL (d < 1.019 g/ml) and LDL (d 1.019-1.055 g/ml) were isolated (see below) in parallel from each. The conditions under which the marmosets were maintained and their diets were identical to those already described by Chapman et al. [5]. Preparative methods Lipoprotein isolation and delipidation. Lipoproteins were isolated by sequential ultracentrifugal flotation according to Have1 et al. [22] in a Beckman L5-50 ultracentrifuge, employing a Beckman 50 Ti rotor. Centrifugation was performed at 50 000 rev/mm (226000 x g) at 10°C. The choice of the density intervals for isolation of the major lipoprotein classes was based on our preceding study of the distribution of the serum lipoproteins in the common marmoset [5]. VLDL were separated as lipoproteins of d < 1.019 g/ml following ultracentrifugation, under the conditions described above, for 24 h. The LDL were isolated in the density interval 1.019-1.055

323

g/ml after centrifugation for 24 h and HDL in the density interval 1.070-l .21 g/ml after centrifugation for 48 h. All lipoprotein classes were washed by an additional centrifugation under the same conditions in 2-3-times the volume of the salt solution corresponding to their higher limiting density. Each lipoprotein fraction was exhaustively dialysed against a solution containing 0.05 M NaCl, 0.04% EDTA, 0.02% NaN, and 0.001% sodium merthiolate at pH 7.4. VLDL and LDL were delipidated with a mixture of ethanol/diethylether (3 : 1, v/v) as described by Brown et al. [23]. HDL were serially delipidated by use of successive mixtures of methanol/chloroform (2 : 3, v/v, and 3 : 2, v/v) according to Olofsson et al. [24]. Apolipoprotein isolation. Apolipoprotein AI was isolated from HDL by gel filtration chromatography followed by electrophoresis. HDL apolipoproteins (approx. 10 mg), solubilized in 6 M urea (Merck) and 2 N acetic acid were loaded onto a Sephadex G-100 column (Wright, 3.2 X 90 cm) equilibrated with 2 N acetic acid. HDL apolipoproteins were separated into two major peaks, II and III (Fig. 2). Fractions of each were pooled and dialysed at 4°C overnight against double-distilled water and lyophilized immediately. Apolipoprotein AI was further purified from peak II material by electrophoretic elution [25]; for this step, we used the alkaline polyacrylamide disc gel electrophoretic system of Davis [26] as modified by Kane [27]. Typically, 4 mg of the lyophilized peak II material containing apolipoprotein AI were solubilized in 3.5-4 ml of a solution containing 6 M urea and 20 mM N-ethylmorpholine (SDS, Vitry-sur-Seine, France) at pH 8.6 and distributed on top of 18 gels (200 pi/gel of dimensions 0.6 cm diameter and 10.5 cm long). After migration at 1.5 mA per gel, one of the gels was rapidly stained in 0.04% Coomassie G 250 (Sigma) in 3.5% perchloric acid [28]. The band corresponding to apolipoprotein AI in the stained gel was then cut out from the unstained gels; these were subsequently crushed and loaded into a small plastic column, to which was tied a dialysis bag (‘Spectrapor’, M, cut-off 3500) [25]. Electrodialysis was then effected in an electrophoresis chamber (model GT14, Hoefer Scientific Instruments, San Francisco, U.S.A.) for 4 h at 4 mA/column and at

15°C. The electrolytic buffers were similar to those used for alkaline polyacrylamide gel electrophoresis [26,27] with the exception that the anodic electrolytic solution contained H,BO, (Merck) at pH 8.9 instead of glycine. On completion, the dialysis bags were removed, tied off, and immediately and exhaustively dialysed against a solution containing 5 mM NH,HCO, at 4°C. Marmoset apolipoprotein B was purified from VLDL and LDL by gel filtration chromatography [29,30], using a Sephadex G-150 column (Wright; 50 x 1.6 cm) equilibrated with a buffer containing 5 mM sodium decyl sulfate, 0.01% (w/v) EDTA and 0.02 M NH,HCO, at pH 7.6. LDL apolipoproteins and VLDL apolipoproteins were each solubilized in 2-3 ml of this solution, to which were added 1% (w/v) SDS and 1% (w/v) pmercaptoethanol. The column effluent was monitored at 280 nm and collected as described for apolipoprotein AI. Analytical methods Analytical isoelectric focussing was performed in glass tubes in a Hoefer electrophoresis unit (model GT14) according to Pagnan et al. [31]. The electrolyte solutions were 0.1 M glycine (upper buffer, pH 7) and 0.01 M Hepes at pH 3.5. Focussing was carried out at a constant voltage (400 V) for 5.5 h at 1O“C. Samples from column chromatographic peaks and purified apolipoproteins were also examined by electrophoresis in the alkaline polyacrylamide disc gel system of Davis [26] as modified by Kane [27]. The molecular weights of apolipoproteins were estimated by electrophoresis in polyacrylamide gels of 3, 4 and 10% monomer using an SDS system as previously described by Weber and Osborn [32] and by Mahley et al. [33]. On completion of electrophoresis, gels were stained with Coomassie brilliant blue R 250 [34]. Calibration curves were constructed from a series of polymerized molecular weight markers ranging in size from 53000 to 265000, and from 14 300 to 71500; (BDH Biochemicals, Poole, U.K.). Amino acid analyses were performed on a Beckman 121 M autoanalyser system. Typically 15-30 pg of lyophilized apoprotein were hydrolysed under conditions detailed by Kane et al. [14], with the exception that acid hydrolysis was performed for 22 h.

324

Immunological studies. Double immunodiffusion in 1% agar or 1% agarose was carried out by Ouchterlony’s technique [35]. Antiserum to human apolipoprotein AI was produced in rabbits from an antigen purified by preparative isoelectric focussing [36]. Human apolipoprotein E was purified from the VLDL of hypertriglyceridemic subjects by column chromatography (Sepharose CL6B in 6 M guanidine) [37] followed by preparative isoelectric focussing effected under the same conditions as that for human apolipoprotein AI [36,38]. An antiserum was prepared in rabbits with this antigen. These procedures have been detailed earlier [lo]. Carbohydrate composition. The carbohydrate composition of marmoset LDL was determined by gas-liquid chromatography [39,40] as summarized by Chapman et al. [41]. Protein determination. Lipoprotein protein and purified apolipoproteins were quantitated by the procedure of Lowry et al. 1421, using bovine serum albumin (Sigma) as the working standard.

a

b

c

d

e

f

Fig. 1. Anaiysis of the protein moieties of marmoset HDL (d 1.070-1.21 g/ml) and human HDL, (d 1.125-1.21 g/ml) on the basis of (i) electrophoretic mobility of urea-solubilized apolipoproteins in 7.5% polyacrylamide gels at alkaline pH (271: a, human; b, marmoset. (ii) molecular weight: the total HDL apoproteins were solubilized in SDS solution 1321 and electrophoresed in polyacrytamide gels (10% monomer and 0.1% SDS): c, human: d, marmoset. (iii) isoelectric point: analytical isoelectric focussing was performed in the pH range 4 to 6.5 on the urea-solubilized apoproteins (see ‘Methods’): e, human. f. marmoset.

Results

We approached the purification of the two major marmoset apolipoproteins, AI and B, by first analysing their patterns and relative distributions in three different electrophoretic systems. These systems were chosen so as to provide data on the molecular weights, isoelectric points and mobility at alkaline pH of the various protein components of marmoset VLDL, LDL and HDL on the one hand, and, on the other, to permit comparison with the major human apolipoproteins. Such an approach should then allow evaluation of the validity of the application of procedures typically employed for the isolation of the human AI and B proteins to their marmoset counterparts. HDL apoiipoproteins Some differences between the behaviour of the principle components of human HDL, apolipoprotein and that of marmoset HDL apolipoprotein were evident in the three electrophoretic systems. Thus, in the alkaline urea system (Fig. la, b), the major marmoset band displayed a greater mobility relative to the dye front (0.19) than human apoli-

poprotein AI (0.26). Differences could also be noted between the two species in the mob&ties of components migrating on both the cathodic and anodic sides of apolipoprotein AI, and especially in the region to which human apolipoprotein AIL CII and Cl11 migrate. A greater resemblance was seen, however, in SDS-polyacrylamide gels (Fig. lc, d), in which not only the overall molecular weight profiles but also the sizes of the major human and marmoset proteins were alike (26 000-27 000); nonetheless, in the absence of reducing agents, apolipoprotein AI1 was detected as a dimer of M, = 17000 in human HDL, apolipoprotein, but an equivalent band was absent from marmoset HDL apolipoprotein. Clearly then, apolipoprotein AI1 is not present as a dimeric protein in C. jacchus. The major apolipoprotein bands in marmoset HDL presented as a triplet (bands 4-6) and displayed isoelectric points (5.15 F 0.05, 5.12 + 0.05 and 5.08 + 0.05, respectively) considerably more acidic than those of human apolipoprotein AI (5.56, 5.48 and 5.41) 1361 in the same system (Fig. le, ff. Under nonreducing conditions, a compo-

325

nent (band 7) with pZ essentially identical to that of human apolipoprotein AI1 (pZ 5.1 [44]) was identified in the marmoset (pZ 4.95). Again, as seen in alkaline urea gels (Fig. la, b) the profile of the more acidic C peptides (CII and CIII) in the two species was distinct; the differences primarily concerned the various isoforms of CIII which appeared less numerous in the marmoset. Nonetheless a band (denoted 8) migrating with similar pZ (4.9) to that of human apolipoprotein CII (5.0 [45]) was identified in the monkey. The most acidic marmoset apolipoprotein was band 9 (pZ 4.75) which may correspond to a counterpart to human apolipoprotein CIII (pZ of the most acidic form, CIII-3, 4.7 [45]). The more basic components in the monkey HDL (bands l-3) focussed in the pH region 5.3 to 5.8 (5.34 * 0.01, 5.45 f 0.11 and 5.77 f 0.03, respectively) and may represent forms of apolipoprotein E, since the four major isoforms of apolipoprotein E in man (El-E4) display pZ values overlapping this range (i.e., pH 5.4-6.1 [43]). Apolipoprotein AZ In view of the finding that no major HDL protein component was present within the M, range of approx. 8000 to 27000 (Fig. Id), we proceeded to fractionate marmoset HDL apolipoprotein by gel filtration chromatography on Sephadex G-100 (Fig. 2). The elution profile revealed three peaks. Material in peak I corresponded to aggregated protein (gel not shown). Polyacrylamide gel electrophoresis showed peak II to consist mainly of the monkey AI-like protein, the major band in SDS gel displaying an M, of approx. 27 000 and pZ values in the range 5.0-5.2 (Fig. 2, inset B and C). Peak II also contained all the apolipoproteins with M, > 27000 and with more basic pZ than the AI-like protein (Fig. 2, inset gels C and B). By contrast, peak III proteins (approx. seven components in basic urea gels; Fig. 2, inset D) were of low J4, (approx. 8000 or less) (Fig. 2, inset F), and displayed isoelectric points more acidic than those of peak II. The principal apolipoproteins of peak III migrated in alkaline urea gels and upon isoelectric focussing (Fig. 2, inset D and E) with mobilities suggestive of the presence of the AH- and C-like peptides detected in HDL holo-apolipoprotein (Fig. lb, d, f); their low mol. wt. is also consistent with this suggestion (Fig. 2, inset F).

8000

Fig. 2. Elution profile obtained upon gel filtration chromatography of marmoset HDL apolipoprotein (10 mg protein) on a Sephadex G-100 column (3.2X90 cm) in 2 M acetic acid. Insert: polyacrylamide gel electrophoretograms of pooled fractions from peak II (gels A, B and C) and from peak III (D, E and F), in urea-containing gels at alkaline pH (A and D), upon analytical isoelectric focussing at pH 4 to 6.5 (B and E) and in 10% SDS gels (C and F).

Final purification of the marmoset apolipoprotein AI-like component was achieved by electrophoresis in alkaline urea gels, from which it was eluted electrophoretically (see ‘Methods’). The monkey AI-like protein purified in this manner exhibited the following physicochemical characteristics. By the SDS procedure of Weber and Osborn [32]; using gels of 10% monomer, its M, was approx. 27 000 (Fig. 3d). Analytical isoelectric focussing (Fig. 3f) revealed it to be polymorphic, exhibiting five to six bands with isoelectric points of 5.02 k 0.03, 4.97 + 0.02, 4.91 k 0.03, 4.85 k 0.04 and 4.79 k 0.05 (n = 5), respectively; in two experiments, one additional faint band was detected,

326

property distinguishing the monkey apolipoprotein from its human homologue, which typically lacks this hydrophobic amino acid [46]. On the basis of the above findings, we propose to refer to the monkey protein subsequently as marmoset apolipoprotein AI.

a

b

c

d

e

f

Fig. 3. Comparison of the electrophoretic pattern of purified marmoset apolipoprotein AI (b) (d) and (f) with that of marmoset HDL apolipoprotein (a), (c) and (e). (a) and (b), patterns in urea-containing polyacrylamide gels at alkaline pH; (c) and (d), patterns in 10% SDS gels; and (e) and (f). analytical isoelectric focussing patterns in the pH range 4-6.5. All gels were stained with Coomassie brilliant blue R 250.

one with slightly more acidic pl (4.69) and the other with a more basic pl (5.06). These p1 values are slightly more acidic than those found for the AI-like protein when present as a component of marmoset HDL apolipoprotein (Fig. lf), but may possibly be accounted for by the presence of traces of H,BO, employed in the electrophoretic elution procedure. In urea gels, the purified protein presented as a single band (Fig,. 3b), even at high protein loading (approx. 100 pg) of the gel. Note that in all three electrophoretic systems (Fig. 3) the purified marmoset AI-like protein migrated to a position which was essentially indistinguishable from that of the major apolipoprotein band in the HDL apolipoprotein from which it was derived. Marmoset apolipoprotein AI contained relatively large amounts of lysine, aspartic and glutamic acids and leucine (Table I); all of the 16 amino acids commonly present in serum proteins were detected. Cysteine was absent; low levels of tryptophan (1.3 mol/ 100 mol amino acid) were found. These characteristics are shared by human apolipoprotein AI (Table I), with the exception that the marmoset protein possessed 1 isoleucine residue per mole (assuming 239 residues) [46], a

VLDL and LDL apolipoproteins Apolipoprotein B-100. Our earlier studies [5] with the SDS-polyacrylamide gel system (4% monomer) of Weber and Osborn [32] established that the major apolipoproteins of marmoset VLDL (d < 1.019 g/ml) and LDL (d 1.019-1.055 g/ml) were of high M, ( > 250000); such behaviour suggested that they might represent counterparts to the human B proteins. These results were confirmed and

TABLE

I

AMINO ACID COMPOSITION OF APOLIPOPROTEIN FROM MARMOSET AND MAN

AI

Values for marmoset apolipoprotein AI are expressed as moles of amino acid/mole of AI protein and are means* S.D. of duplicate analyses on each of seven separate preparations of apolipoprotein AI. Calculation was based on the assumption that marmoset apolipoprotein AI contains 239 amino acid residues as in man (461, but excluding tryptophan. For human apolipoprotein AI the assumed number of residues is 239 excluding tryptophan [46]. Amino acid

Marmoset apolipoprotein

LYS

20.6+_1.3 2.8 + 0.5 12.6+ 1.2 21.4* 1.8 11.2*0.4 19.9* 1.4 48.5 f 2.5 9.8+ 1.6 13.0+ 2.2 14.1 f 1.1 14.1+ 1.7 2.6 & 0.4 1.1* 1.2 35.9k3.2 6.4kO.8 5.2kO.6 0 3.2 f 1.3

His

A rg Asp Thr Ser Glu Pro Gly Ala Val Met Is0 Leu Tyr Phe cys = Tryptophan a Determined ’ Determined

b

AI

Human apolipoprotein 21 5 16 27 10 15 46 10 10 19 13 3 0 37 I 6 0 4

as cysteic acid [47]. by the procedure of Edelhoch

[48].

AI

321

extended in the present investigations, in which the preceding SDS gel procedure [32] was modified according to the methodology of Mahley et al. 1331 and according to the recommendations made by Kane et al. [14] for study of the human B proteins by this technique. In gels of 4% acrylamide monomer, the major band in marmoset VLDL apolipoprotein displayed a mean M, of 350000, with a second band of M, 250000 (Fig. 4A, B). This pattern was the same, irrespective of whether VLDL was isolated in the presence or absence of an inhibitor of serine proteinases, 1 mM PMSF. A minor component(s) of M, - 36000 was also identified. The electrophoretic pattern in 4% gels of the major high molecular weight, apolipoprotein B-like components in marmoset LDL apolipoprotein (Fig. 4C, D) closely resembled that in VLDL apolipoprotein. Again, the principle component displayed an M, of 350000; by contrast with VLDL apolipoprotein, three faint bands of 320000, 260 000 and 230000 were also identified. Like VLDL apolipoprotein however, the LDL apolipoprotein electrophoretogram was unaltered by the presence or absence of PMSF during LDL isolation. Despite the reproducibility of the molecular weight obtained for the major marmoset apolipoprotein B-like protein in 4% monomer gels, this system is subject to inaccuracy, since (i) components with such elevated M, typically migrate into the gel to a minimal degree (1.4 to 1.8 cm), and (ii) the portion of the calibration curve involved yields particularly high increments in M, (150000) for small differences in relative mobility (0.01) then minor changes in the mobility of apolipoprotein B correspond to major alterations in its molecular weight. We therefore examined the M, of marmoset B proteins in gels of 3% monomer (Fig. 4E). In this case, the high-Mr band in LDL apolipoprotein (Fig. 4E) migrated twice as far into the gel (3.5-4 cm), displaying a molecular weight of 460 000 when 100 pg protein were loaded, and an M, of approx. 520000 when lo-20 pg protein were used. Clearly then, not only the polyacrylamide concentration but also the quantity of apoprotein loaded onto the gel are of major importance in determining the molecular weights of marmoset apolipoprotein Blike proteins in this SDS system, small amounts of protein ( < 20 pg) and low polyacrylamide con-

.-

F

G

,460

VLDL

LDL

H

420

ape B

Fig. 4. Electrophoretic patterns in SDS-polyacrylamide gel of marmoset VLDL apolipoprotein, LDL apolipoprotein, and the corresponding column fraction 1 following gel filtration chromatography on Sephadex G-150. Pattern A: marmoset VLDL apolipoprotein (d 11.019 g/ml) isolated in the presence of 1 mM (w/v) PMSF. Pattern B: as in A, but in which PMSF was absent throughout. Pattern C: marmoset LDL apolipoprotein (d 1.019-1.055 g/ml) isolated in the presence of 1 mM (w/v) PMSF. Patterns D and E: as in C, but isolated in the absence of PMSF. Pattern F: apolipoprotein B (fraction I) from VLDL apolipoprotein. Patterns G, H: apohpoprotein B (fraction I) from LDL apolipoprotein. Some 100 yg of protein were applied to each gel. The acrylamide monomer concentration in all gels was 4%. with the exception of gels E and H, in which it was 3%. Molecular weights (shown~lO_~) were determined from a series of purified marker proteins which were electrophoresed simultaneously. Gels were stained with Coomassie brilliant blue R 250.

centration (3% or less) favouring higher M, values (500000 or greater) for the major B protein. In all of the experiments, marmoset and human VLDL apolipoprotein and LDL apolipoprotein were electrophoresed in parallel, in which case changes seen in the M, values of the predominant marmoset B protein were also found in those for human B-100. In view of the resemblance in electrophoretic profile in SDS gels of the major B protein(s) of marmoset and human VLDL apolipoprotein and also of LDL apolipoprotein from the two species, we isolated the marmoset apolipoprotein by gel filtration chromatography in anionic detergent [29,49]. The elution profile for marmoset VLDL apolipoprotein (not shown)(d < 1.019 g/ml) on a Sephadex G-150 column showed two major peaks; the

328

first, peak I, eluted at the void volume and showed a marked shoulder corresponding to region II; this was followed by a second major peak, denoted III. Separation between regions II and III was relatively poor. This was not the case for LDL apolipoprotein (d 1.019-1.055 g/ml), the separation of the peak (I) in the excluded volume from that in the included volume (III) being well defined, with minor trailing of material in peak I (region II). Peak I protein accounted for considerably more of LDL apolipoprotein than of VLDL apolipoprotein (approx. 83% and 20% of total protein loaded, respectively). By contrast, material eluted in the included volume as peak III represented only approx 8% of LDL apolipoprotein but considerably more of VLDL apolipoprotein (approx. 50% of total protein). The physicochemical properties of protein from the centre of each peak were then examined. In SDS-polyacrylamide gels, only the component of highest M, in VLDL apolipoprotein and in LDL apolipoprotein was detectable (Fig. 4F, G); in 4% gels; it behaved as a protein of 320 000-400000 molecular weight, whereas in 3% gels this value

TABLE AMINO Expressed duplicate

II ACID

COMPOSITION

OF apolipoprotein

B-100 FROM

MARMOSET

as mol per 100 mol. Values are the mean * S.D. of the number

AND

of preparations

HUMAN

SERUM

in parentheses:

LIPOPROTEINS

each sample was analysed

or triplicate.

Amino acid

Lys His A% Asp Thr Ser Glu Pro GIY Ala Val Met Is0 Leu Tyr Phe Cys

increased to fall in the range 420000-520000. Such size estimates correspond well to those determined for the equivalent components in the holo-apoprotein (Fig. 4A, B, C, D, E). The amino acid compositions of the peak I proteins are presented in Table II, and are compared to those of the form of the human B protein of highest molecular weight, i.e., B-100 [14]. The amino acid profiles of human B-100 and of the marmoset peak I protein isolated from the respective serum LDLs are remarkably alike, and may only be distinguished by minor differences (approx. 1 mol%) in lysine, glycine and glutamic acid contents. A resemblance in amino acid composition was also evident between the same proteins isolated from the respective serum VLDL of the two species, although this was not as marked as that for LDL apolipoprotein B. Indeed, differences of the order of l-2 mol% were seen for lysine, histidine, serine, glutamic acid, isoleucine and leucine; the larger disparity for glycine (approx. 4 mol%) may reflect an inadvertent contamination. The marked similarity between the peak I proteins of marmoset VLDL apolipoprotein

Marmoset

Human

B-100

B-100

VLDL (2)

LDL (3)

VLDL

LDL

6.0+ 0.8 1.2&0.2 3.8 f 0.2 10.1 kO.1 6.4kO.2 9.4 f 0.5 13.OkO.6 3.8 f 0.3 8.6 f 0.4 6.8 + 0.5 5.6 f 0.6 1.4+1.2 4.7 i 0.3 10.8&0.3 2.9 f 0.1 4.5 f 0.1 0.9 * 0.2

7.0 f 0.4 2.0*0.3 3.2 f 0.2 10.3 + 0.1 6.7 * 0.1 8.9+.1.0 12.750.4 3.8 k 0.2 6.2+ 1.2 6.6 * 0.1 5.2kO.4 1.8*0.1 5.4kO.3 11.7*0.4 3.1+0.1 4.9 + 0.1 0.6 k 0.1

8.1 2.6 3.5 10.6 6.6 8.4 11.7 3.8 4.8 6.1 5.5 1.7 6.1 11.8 3.3 5.0 0.4

8.0 2.6 3.4 10.7 6.6 8.6 11.6 3.8 4.7 6.1 5.6 1.6 6.0 11.8 3.4 5.1 0.4

in

329

TABLE

III

CARBOHYDRATE HUMAN LDL

COMPOSITION

OF MARMOSET

AND

Results for marmoset are the means of duplicate determinations on LDL (d 1.019-1.055 g/ml) isolated from the pooled serum of several animals, and are expressed as percent by weight of monosaccharide relative to protein content, the latter determined by the procedure of Lowry et al. 142). Data for human LDL (d 1.024-1.050 g/ml) are taken from Ref. 41. n.d. not detectable. Monosaccharide

% by weight Marmoset

LDL

O.lO_ 0.69 1.58 2.45 0.70

~

0.96 1.61 1.95 0.21 4.79

5.41

n.d.

Galactose Mannose Glucosamine Sialic acid Total

TABLE

LDL

Human

Fucose

and LDL apolipoprotein suggestss that they correspond to the same apolipoprotein. Carbohydrate content. Marmoset LDL apolipoprotein (some 83% of which is apolipoprotein B) contained all of the major monosaccharides typical of human LDL apolipoprotein (Table III). The total carbohydrate content of the monkey protein appeared slightly lower, this decrease being principally accounted for by reduction in the levels of mannose and sialic acid. Immunological studies. In order to evaluate the immunological cross-reactivity of marmoset and human apolipoproteins, immunodiffusion studies were conducted with monospecific antibodies to human apolipoprotein AI, human apolipoprotein E and marmoset apolipoprotein B. Upon immunodiffusion against antiserum to human apolipoprotein AI, marmoset whole serum and the homologous antigen gave a reaction of partial identity with a spur in favour of human AI;

IV

COMPARISON MAN

OF THE

AMINO

ACID

COMPOSITIONS

OF apolipoprotein

AI FROM

NON-HUMAN

PRIMATES

Data are expressed

as moles of amino acid/mole

Amino acid

Marmoset

Rhesus

Baboon

Vervet

Patas

Chimpanzee

Man

Ref.:

WI

1171

1181

[131

1161

141

Lys His A% Asp Thr Ser Glu Pro GIY Ala Val Met Is0 Leu Tyr Phe Cys Trp Number residues a Adapted

of AI protein.

21 3 13 21 11 20 48 10 13 14 14 3 1 36 6 5 0 3

20 6 13 18 10 13 44 10 10 17 14 2 0 36 6 5 0 5

20.3 7.3 15.5 19.5 11 15.9 46.1 10.2 11 18.1 14.9 2.9 0 37.1 6.5 5.1 0 3.2

24 7 14 16 8 12 38 12 11 23 15 1 0 34 6 5 0 5

16.3 7.2 12.7 20.8 9.1 17.2 47.1 9.3 11.0 19.4 15.8 2.2 0 38.5 1.2 5.5 0

20 5 14 19 9 14 42 10 9 17 11 3 0 35 6 5 0 5

21 5 16 21 10 15 46 10 10 19 13 3 0 37 7 6 0 4

242

229

244.6

231

239 a

224

243

of

from Mahley

et al. [13].

AND

330

a similar pattern of precipitin arcs was seen with marmoset HDL (not shown). Reaction of antiserum to human apolipoprotein E against either whole serum, VLDL or HDL from marmoset and from man resulted in a precipitin pattern indicative of partial identity in each instance. Immunological heterogeneity in either apolipoprotein E itself or in the lipoprotein particles onto which it was bound, or both, was suggested by the double lines seen between the antiserum and antigen wells for marmoset VLDL and HDL, respectively. Finally, rabbit antiserum to marmoset apolipoprotein B produced a reaction of partial immunological identity between marmoset and human whole serum, with a spur in favour of the marmoset antigen. Discussion We have described the isolation and characterization of the two major apolipoproteins of marmoset serum, one of which is the principal protein component of the HDL class, the other of LDL and VLDL. Indeed, comparison of these two apolipoproteins with their human counterparts, AI and B-100, revealed marked similarities in all of the physical, chemical and immunological properties examined. Moreover, the high degree of homology between these apolipoproteins in the two species, considered together with the qualitative and quantitative resemblance in serum lipoprotein profile (51, prompts us to suggest that the common marmoset may constitute a valuable model for studies of the biosynthesis, intravascular metabolism and catabolic fate of the B-100 and AI proteins, and in addition, of certain factors (e.g., hormones and nutritional elements) which may modulate these processes. The single most abundant protein in marmoset HDL (50-708 of the protein moiety) is, on the basis of its physicochemical properties and immunological cross-reactivity, analogous to human apolipoprotein AI. The apparent molecular weight of marmoset apolipoprotein AI (approx. 27000) by SDS-gel electrophoresis approximated that determined for the human protein (approx. 27000) by this procedure. Some minor differences in the proportions of histidine, arginine, serine, glutamic

acid, glycine and alanine were evident upon comparison of the amino acid composition of marmoset and human apolipoprotein AI, in addition to the presence in the monkey protein of one isoleucine residue per mole; nonetheless, a close overall resemblance was seen in the amino acid profile of apolipoprotein AI in the two species. Indeed, this similarity extended to the AI proteins from the rhesus, baboon, vervet and Patas monkeys and to the chimpanzee (Table IV). Marmoset apolipoprotein AI was, however, unique in possessing isoleucine; it also displayed the highest glycine and serine contents and lowest proportions of alanine and histidine among the seven species. The appearance of an isoleucine residue in marmoset apolipoprotein AI is consistent with the hypothesis that the New World monkeys diversified before the appearance of the Old World group; further data on AI from other New World species may confirm this thesis. It is also of interest to note that even though normal human apolipoprotein AI lacks this amino acid, a mutant form, AI Milano, has recently been identified which possesses 1 isoleucine residue/mole [51]. Rhesus, baboon and chimpanzee AIs appeared most closely related to that of man, with the vervet apolipoprotein exhibiting some notable differences (elevated lysine and diminished serine, glutamic acid and methionine levels). Further interpretation of the unique chemical characteristics of the marmoset and vervet apolipoproteins awaits determination of their covalent structure, but it may be suggested that such knowledge may further understanding of the relationship between the role of certain amphiphilic segments in the function of apolipoprotein AI [50]. Clearly, apolipoprotein AI may be considered a rather stable protein on a phylogenetic basis, and thus a primordial element of lipid transport. The more acidic isoelectric points of marmoset as compared to human apolipoprotein AI (ranges pH 4.79-5.02 and 5.27-5.41 [36], respectively) may be largely explained by the higher proportion of acidic residues (69 residues/mole versus 67 in man) and diminished level of basic amino acids in the former (37 residues/mole versus 42 in man). The charge heterogeneity of marmoset AI is shared by that of man [36,44,45], baboon [17], rhesus [lo] and vervet monkeys [18] and chimpanzee [16]; the

331

possibility that this property is derived at least in part from structural alteration during purification cannot be excluded, since, as noted by others [18], the number of charge forms in pure apolipoprotein AI (5 to 6) was greater than that seen in the apolipoprotein AI region in gels of HDL apolipoprotein (approx. 3 bands) (Figs. 1 and 3). An apparent reaction of complete immunological identity occurs between human apolipoprotein AI and the homologous protein from the chimpanzee [ 161 and rhesus [19] and vervet monkeys [18]. The partial immunological identity of the marmoset and human AI proteins may therefore be taken to indicate the earlier evolutionary diversification of marmoset apolipoprotein AI. The second major marmoset apolipoprotein was homologous with the human B-100 protein [14] on the basis of its molecular weight, amino acid composition, carbohydrate content, elution profile on gel filtration chromatography, and predominance in the protein moieties of marmoset VLDL and LDL; in addition, a monospecific antiserum to the marmoset protein cross-reacted immunologically

TABLE

with human B-100, revealing a partial identity between these proteins as components of whole serum. In consequence, we shall henceforth refer to the monkey protein as marmoset B-100. The molecular weight of marmoset B-100 corresponded well to that of its human counterpart (approx. 520000 and 549000 [14], respectively). Such results were observed irrespective of whether the marmoset protein was present in the form of VLDL apolipoprotein or LDL apolipoprotein, or after chromatographic purification. A second component of high M, (approx. 250000) was seen in SDS gels of apolipoprotein VLDL (Fig. 4A, B), but was detected in LDL apolipoprotein in only trace amounts (Fig. 4C, D). Its presence was independent of proteolysis, since PMSF addition during lipoprotein isolation was without effect. Moreover, this protein was absent from B-100 isolated as peak I upon chromatographic separation from either VLDL apolipoprotein or LDL apolipoprotein, possibly because it would elute in the region (II) behind B-100. We

V

COMPARISON

OF THE AMINO

ACID

COMPOSITIONS

OF B-100 FROM

NON-HUMAN

PRIMATES

AND

MAN

Data are expresed as mo1/100 mol amino acid residues. B-100 was isolated from serum LDL of d 1.024-1.050 g/ml or of d 1.024-1.045 g/ml by gel filtration chromatography in anionic detergent (sodium decyl sulfate) for rhesus and baboon. For human B-100 the protein was prepared from serum LDL (d 1.024-1.050 g/ml) by preparative electrophoresis in SDS-polyacrylamide gel. Amino LYS His Arg Asp Thr Ser Glu Pro GIY Ala VaI Met Is0 Leu Tyr Phe CYS

acid

Marmoset Ref.:

Rhesus

Baboon

Man

PO1

UOI

1141

7.0 2.0 3.2 10.3 6.7 8.9 12.7 3.8 6.2 6.6 5.2 1.8 5.4 11.7 3.1 4.9 0.6

9.0 2.3 3.8 10.7 6.4 8.1 12.0 3.1 6.2 5.9 6.2 0.9 5.4 11.4 3.4 5.0 _

9.1 2.4 3.4 10.9 6.1 8.0 12.2 4.0 5.3 6.4 5.2 1.6 5.2 11.6 3.0 5.3 _

8.0 2.6 3.4 10.7 6.6 8.6 11.6 3.8 4.7 6.1 5.6 1.6 6.0 11.8 3.4 5.1 0.4

332

conclude that the approx. 250000 M, component of VLDL apoIipoprotein may either correspond to the B-48 form (M, in man 264000) [14], or to a non-B related protein. Clearly resolution of this question awaits its isolation and characterization. We have not detected marmoset counterparts to the human B-74 and B-26 proteins in either VLDL or LDL. In this context, it is relevant that Kane et al. [14] found about half of their normal human donors to lack these proteins, which appear to be fragments of B-100 1141. The amino acid profiles of the marmoset and human B-100 proteins were essentially indistinguishable from each other, and from those of the baboon and rhesus monkey (Table V). All of these proteins were characterized by elevated contents of lysine, aspartic and glutamic acids, and leucine. Further information on the phylogenetic relationship between these B proteins again awaits determination of their primary sequence. Resemblances in chemical composition of B-100 from marmoset and man included their carbohydrate content (Table III). Moreover, the monosaccharide profile of marmoset LDL apoIipoprotein and that of LDL apolipoprotein-2 (d 1.019-1.063 g/ ml) from chimpanzee, patas, baboon, rhesus and spider monkeys [52] were alike, although the total carbohydrate contents of both the human and monkey LDL apolipoproteins reported by the latter authors were up to 50% lower than those we have found (Table III). By contrast, Fless and Scanu 1531 found a significantly higher sugar content in rhesus LDL apolipoprotein (6.57 pg/lOO pg protein), with a dist~bution markedly similar to that in marmoset, except for an &fold elevation in sialic acid (2.13 and 0.27% by weight in rhesus and marmoset, respectively). It will be of interest to determine whether sialic acid and mannose contribute to the antigenicity of the non-human primate LDLs, as indeed they appear to in man [54]. The remaining apolipoproteins in the marmoset were distinct from the B proteins in solubility characteristics and mainly with &f, value in the 8~-50000 (Fig. Id and Fig. range approx. 4A-D). One of these, as judged by the presence in HDL apolipoprotein of a band of M, = 40000 on SDS gel electrophoresis (Fig. Id), by components

with pl in the range of 5.3-5.8 upon isoelectric focussing (Fig. lf), and by the presence of a common antigenic determinant(s) in marmoset and human HDL when reacted upon immunodiffusion with a monospecific antiserum to human apolipoprotein E, was a protein analogous to human apolipoprotein E (pl 5.4-6.1, mol. wt. 38 000) [43]. A second was analogous to human apolipoprotein AI1 on the basis of its migration in alkalineurea polyacrylamide gel (Fig. lb), the presence upon isoelectric focussing of a component (band 7) of pI= 5.0 (Fig. lf), and its elution position on gel filtration chromatography (Fig. 2 and insets D, E and F). The absence of a band corresponding to human apolipoprotein AII (M, = 17000) in SDS gels under nonreducing conditions (Fig. lc, d) suggests that marmoset apolipoprotein AIL like that of other non-human primates except chimpanzee [12], is a monomeric protein. Whether the pi 4.9 (band 8) and 4.75 (band 9) proteins of marmoset HDL are counterparts to human apolipoprotein CII and apolipoprotein CIII, respectivefy, remains speculative, Certainly isoforms of CIII-like peptides appear less numerous in marmoset than in man, a finding already corroborated in the vervet monkey [18]. The isolation and characterization of these low molecular weight apolipoproteins from C. jucchus will be the subject of further investigation in our laboratory. Acknowledgements P.F. gratefully acknowledges the award of a fellowship from ICI-Pharma Ltd. The material presented is included in a thesis for ‘Doctorat de 3eme Cycle’ submitted to the University of Paris XII in 1982 by P.F.; the experimental work described was performed in Unit 35, INSERM, Groupe de Recherches sur le Metabolisme des Lipides, Hopital Henri Mondor, 94010 Creteil, France. It is a pleasure to acknowledge the continued support and encouragement of Drs. D.C.N. Earl and A. Rossi. We are indebted to Dr. J.P. Kane and Mrs. K. Tornitch for assistance in amino acid analysis, and to Mr. W. Hiddleston and Dr. F. McTaggart for supplying serum samples. Our colleagues Drs. S. Goldstein, S.C. Rall, Jr., and PK. Weech gave valuable counsel.

333

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