[15] Isolation and characterization of other apolipoproteins

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Acknowledgments This research was supported by Public Health Service Grants P01 HL-22619,R01 HL23019, and General Clinical Research Center and CLINFO Grant NIH RR0-00068.Special thanks go to Ms. Gwen Kraft for preparing the figures and to Ms. Janet Simonsfor preparing the manuscript for publication.

[15] I s o l a t i o n a n d C h a r a c t e r i z a t i o n o f O t h e r A p o l i p o p r o t e i n s

By W. J. MCCONATHY and P. ALAUPOVlC Human plasma lipoproteins constitute a structurally complex and metabolically dynamic system of macromolecules. The complexity of lipoproteins is not only reflected in their marked heterogeneity with respect to hydrated density, size, and electrical charge, but also in a number of specific protein moieties or apolipoproteins. To provide an integrated view of this system, we have proposed that apolipoproteins be used as the specific and distinguishing markers for identification and characterization of discrete lipoprotein particles.1 To express in relatively simple terms the relationship between apolipoproteins, constitutive polypeptides, isomorphic forms, and lipoprotein particles, we have devised a system of nomenclature referred to as the ABC nomenclature) The apolipoproteins are designated by capital letters, the nonidentical polypeptides by Roman numerals, and the polymorphic forms by Arabic numbers. The lipoprotein particles or lipoprotein families are named according to their corresponding apolipoproteins. The purpose of this chapter is to describe the isolation and physicochemical and immunologic properties of apolipoproteins that are not related to apolipoproteins A-I, A-II, B, C-I, C-II, C-III, and E. This includes minor apolipoproteins D, F, G, and H. Apolipoprotein D Apolipoprotein D (apoD) represents a minor protein constituent of the human plasma lipid transport system. Studies in our and other laboratories indicated the presence in H D L of an antigenic determinant that was not related to any of the known apolipoproteins. 2,3 This protein was deP. Alaupovic, Ricerca 12, 3 (1982). 2 W. J. McConathy and P. Alaupovic, FEBS Lett. 37, 178 (1973). 3 W. J. McConathy and P. Alaupovic, Biochemistry 15, 515 (1976).

METHODS IN ENZYMOLOGY, VOL. 128

Copyright © 1986 by Academic Press, inc. All rights of reproduction in any form reserved.

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tected in plasma lipoproteins with both commercial anti-oq-lipoprotein and anti-HDL3 sera. Using these antisera, the following scheme was developed for the isolation and characterization of this antigen.

Preparation of ApoHDL 4 To prepare HDL, 2 liters of pooled fresh or outdated plasma was made 0.01% with respect to sodium azide, EDTA, and thiomerosal. This volume of plasma is necessary in order to obtain sufficient quantities of minor apolipoproteins for further characterization studies. The plasma was adjusted to a solution density of 1.23 g/ml with solid KBr and centrifuged at 45,000 rpm in a Ti60 rotor for 22 hr. The supernate containing the serum lipoproteins was subjected to additional ultracentrifugations with appropriate density adjustments to yield H D L (d 1.073-1.23 g/ml). After several washes at d -- 1.23 g/ml the H D L were usually free of albumin as demonstrated by double diffusion analysis, though its absence does not appear to be essential for the isolation of either apoD or some other minor apolipoproteins. The composition of HDL isolated by this procedure was indistinguishable from that of normal fasting H D L isolated by standard sequential ultracentrifugation. H D L were dialyzed against five changes of redistilled water and lyophilized in a number of 50-ml glass-stoppered centrifuge tubes. Lyophilized H D L (1.2-1.6 g of protein, 150 mg/tube) were mixed with 1 volume of chloroform and shaken by inverting the tube several times. Following dispersion of the lyophilized material, 2 volumes of methanol was added. The tubes were shaken several times while stored at - l 0 ° for 30 min in chloroform: methanol (1/2, v/v). After low-speed centrifugation at 6 °, the solvent was aspirated and the precipitated protein was dispersed in 1 volume of chloroform and 2 volumes of MeOH in the same fashion as the initial step. The residue was extracted four additional times with chloroform: methanol (2/1, v/v) followed by two extractions with peroxide-free diethyl ether. The delipidized H D L (apoHDL) was essentially free of phosphorus and fatty acids.

Isolation of ApoD 2 To isolate apoD, the apoHDL was dissolved in 10-20 ml of 8 M urea in 1 mM K 2 H P O 4 , pH 8.0, and the ether was evaporated under a gentle stream of nitrogen. The apoHDL solution was diluted to 2 M with respect to urea by addition of 1 mM K 2 H P O 4 , pH 8.0, and applied to a hydroxylapatite-cellulose column. The column (30 × 2.2 cm) was packed to a 4 S.-O. Olofsson, W. J. McConathy, and P. Alaupovic, Biochemistry 17, 1032 (1978).

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height of 15 cm with a mixture of 2 volumes of settled hydroxylapatite (Bio-Rad Lab., Richmond, CA) and 1 volume of settled microcrystalline cellulose (Baker, Phillipsburg, NJ). The addition of the microcrystalline cellulose improves the flow rate of hydroxylapatite columns. Prior to sample application, the column was washed with 1 M K 2 H P O 4 , pH 8.0, followed by equilibration with 1 mM KEHPO4, pH 8.0. After application of the sample, the column was eluted with 40-50 ml of 1 mM K2HPO4 buffer, pH 8.0. The fraction eluted with 1 mM KEHPO4 w a s rechromatographed under identical conditions on another hydroxylapatite column. After the second chromatography, the eluted fraction was examined by basic polyacrylamide gel electrophoresis (PAGE). The'i-wo successive chromatographic steps on hydroxylapatite-cellulose columns were usually sufficient to yield a protein preparation which was characterized by a single band on 7% PAGE. However, in some cases a minor component was present near the junction of the separating and stacking gels. To remove this minor component and urea, gel filtration on Sephadex G-100 equilibrated with 2 M acetic acid was performed and apoD was eluted as a major symmetrical peak. After lyophilization, this major protein fraction only reacted with an antiserum to apoD. On double diffusion analyses, it gave a negative reaction with antibodies to apolipoproteins A-I, A-II, B, C-I, C-II, C-III, E, F, and albumin. On basic 7% PAGE, the isolated polypeptide exhibited a single band with a mobility midway between the bands corresponding to apoA-II and apoC-II. We concluded on the basis of electrophoretic mobility in 7% PAGE, characteristic amino acid and carbohydrate composition, and immunological properties that the isolated protein is a distinct component of the human plasma lipoprotein system. This protein was named apolipoprotein D.

Isolation of Lipoprotein D

(LP-D) 3

In order to define the lipoprotein nature of this isolated protein moiety and to provide evidence that it indeed represents an apolipoprotein according to the criteria proposed by Alaupovic, 1 the following procedure was developed for the isolation of apoD-containing lipoproteins utilizing immunosorber methodology. 3 Rabbit or goat antiserum specific for apoD was utilized for the isolation of an IgG antibody-containing fraction. The ammonium sulfate-precipitated IgG fraction was dialyzed against 100 mM K 2 H P O 4 , pH 6.5, and coupled to CNBr-activated Sepharose 4B 5 at pH 6.5. After coupling, the immunosorber was washed extensively with an equilibration buffer (150 5 S. March, I. Parikh, and P. Cuatrecasas, Anal. Biochem. 60, 149 (1974).

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mM NaCI, 50 mM Tris-HC1, and 0.01% NAN3, pH 7.5). To avoid prolonged interaction of the dissociating buffer (3 M NaSCN) with lipoproteins eluted from the immunosorber, the chromatography was performed on a column (60 × 2.5 cm) constructed in the following fashion: the column was first packed to a height of 15 cm with medium grade Sephadex G-25 followed by the Sepharose-coupled antibodies (40 cm) and another layer of Sephadex G-25 (4 cm). The G-25 layer at the bottom acts as a molecular sieve and effectively separates the desorbed protein from the dissociation agent, NaSCN. Application of plasma or isolated density classes was followed by extensive washing with the equilibration buffer until the absorbance returned to the baseline. Material bound to the antiapoD-Sepharose column was eluted successively with 50 ml of 3 M NaSCN followed by the equilibration buffer. Approximately 75% of the total bound material was eluted with the equilibration buffer, while the remainder was eluted with NaSCN. Both fractions were first made 1 mM with respect to phosphate by chromatography on a Sephadex G-25 column (60 × 2.5 cm) equilibrated with 1 mM KH2PO4, pH 8.0, and were then applied onto a hydroxylapatite-cellulose column prepared as described for apoD. Fractions eluted with 1 mM KH2PO4, pH 8.0, if not homogeneous, were rechromatographed until a single band was obtained. After purification, the fractions eluted with 1 mM KH2PO4 were desalted by gel filtration on Sephadex G-25 equilibrated with 100 mM (NH4)2CO3 and utilized for characterization studies. Lipoproteins containing apoD, apoA-I, and apoA-II can be eluted from the initial hydroxylapatite column with higher molarities of phosphate buffer.

Characterization of ApoD ApoD, isolated by a procedure combining hydroxylapatite and Sephadex G-100 column chromatography, migrated on 7% PAGE as a single band with a mobility intermediate between the bands corresponding to apoA-II and apoC-II. 2,3,6-9 On double diffusion and immunoelectrophoresis, apoD only reacted with antiserum to apoD. It was characterized by the presence of all common amino acids including half-cystine (Table I). By analytical isoelectric focusing, apoD was shown to exist in at least three isoforms with pls of 5.20, 5.08, and 5.00, with the isoform of pl = 6 S.-O. Olofsson and A. Gustafson, Scand. J. Clin. Lab. Inoest. 33 (Suppl. 137), 57 (1974). 7 I. Chajek and C. J. Fielding, Proc. Natl. Acad. Sci. U.S.A. 75, 3445 (1978). 80. Wiklund, G. Fager, S.-O. Olofsson, C. Wilhelmsson, and G. Bondjers, Atherosclerosis 37, 631 (1980). 9 j. j. Albers, M. C. Cheung, S. L. Ewens, and J. H. Tollefson, Atherosclerosis 39, 395 (1981).

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TABLE I AMINO ACID COMPOSITION OF OTHER APOLIPOPROTEINS Aport (/32-Glycoprotein-I)

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

ApoD ~

ApoF b

ApoG c

Plasma d

Plasma e

65 11 20 106 53 36 100 55 34 49 20 58 10 51 68 28 28 19

33 2 17 70 44 60 100 18 87 90 10 54 12 28 69 31 4 N.D.

65 20 31 96 37 37 100 49 50 66 N.D. 55 Tr. 30 80 19 36 N.D.

109 18 36 105 100 82 100 123 82 64 82 68 14 45 68 54 64 18

106 26 40 116 105 95 100 113 91 79 78 67 14 54 70 50 75 6

W. J. McConathy and P. Alaupovic, Biochemistry 15, 515 (1976). b S. O. Olofsson, W. J. McConathy, and P. Alaupovic, Biochemistry 17, 1032 (1978). c M. Ayrault-Jarrier, J. F. Alix, and J. Polanovski, Biochimie 611,65 (1978). d N. Heimberger, K. Heide, H. Haupt, and H. E. Schultze, Clin. Chim. Acta 10, 293 (1964). e E. Polz, H. Wurm, and G. M. Kostner, Artery 9, 305 (1981). a

5.08 representing the major form. 9 The chemical basis for this heterogeneity remains unknown. The amino terminal acid was blocked) Carbohydrate analysis demonstrated that apoD is a glycoprotein with hexose, glucosamine, and sialic acid accounting for 18% of its dry weight (Table II). By applying similar procedures, several groups have isolated a protein constituent from HDL with similar characteristics.6-9 The major reported discrepancy between these and the initial report 3 is the apparent molecular weight as estimated by SDS-PAGE. Our reevaluation of the molecular weight of apoD by SDS-PAGE now agrees with the recent reports of an estimated molecular weight in the range of 3 2 , 0 0 0 - 3 4 , 0 0 0 . 7,9 The reason for the original discrepancy remains unclear but may be related to the high carbohydrate content of apoD. However, the estimated molecular weight of apoD from the amino acid and carbohydrate composition is 22,100, a

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TABLE II CARBOHYDRATE CONTENT OF APOLIPOPROTEIN D AND APOLIPOPROTEIN H (fl2-GLYCOPROTEIN-I)

Hexose Hexosamine Neurarninic acid

ApoD ~ (% weight)

Aport b (% weight)

9.3 4.5 4.8

6.7 5.8 4.4

W. J. McConathy and P. Alaupovic, Biochemistry 15, 15 (1976). b N. Heimburger, K. Heide, H. Haupt, and H. E. Schultze, Clin. Chim. Acta 10, 293 (1964). a

value consistent with the elution volume of apoD when sized by molecular sieve chromatography in the presence of 6 M guanidine-HCl on Sephadex G-100 column chromatography. Lipoprotein D (LP-D) was isolated by a procedure combining chromatography of HDL or whole serum on an immunosorber containing antibodies to apoD, and hydroxylapatite column chromatography. LP-D displayed a single, symmetrical boundary in the analytical ultracentrifuge and a single band on 7% PAGE. When injected into rabbits it produced antisera that only reacted with apoD. LP-D consists of 65-75% protein and 25-35% lipid. The lipid moiety contains cholesterol, cholesteryl ester, triglyceride, and phospholipid (Table III). The phospholipid composition is characterized by a relatively high content of lysolecithin and sphingomyelin and a relatively low content of lecithin. By the same proceTABLE III LIPID COMPOSITION OF LP-D AND LP-F

Triglyceride Cholesterol Cholesteryl ester Phospholipid

LP-D ~

LP-F b

(%)

(%)

8.2 18.1 27.7 46.5

(6.2) (1.0) (7.6) (8.2)

5.2 21.1 63.3 11.5

(1.0) (0.6) (3.2) (2.5)

a W. J. McConathy and P. Alaupovic, Biochemistry 15, 515 (1976). bE. Koren, W. J. McConathy, and P. Alaupovic, Biochemistry 21, 5347 (1982).

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dures, a similar lipoprotein species was isolated from baboon plasma. ~° These results demonstrated that other species contain a plasma apolipoprotein analogous to human apoD.

Distribution and Levels of ApoD Plasma concentrations and distribution of apoD in ultracentrifugally defined lipoprotein density classes (VLDL, LDL, and HDL) were determined by various immunoassays. Studies on plasma levels have shown that apoD represents a minor apolipoprotein with reported levels for normolipidemic subjects ranging from 6 to 10 mg/dl. 8,9,11 Distribution studies indicated that apoD is primarily localized in H D L (60-65%) with only trace amounts present in VLDL and LDL, and the remainder in VHDL. ~l Such analyses are consistent with H D L as the starting material for isolating apoD or LP-D, though apoD can also be isolated from VLDL and LDL. 3 We concluded from these studies that apoD is a unique apolipoprotein that exists in the form of a distinct lipoprotein family with a macromolecular distribution extending from very low-density lipoproteins into very high-density lipoproteins, but with a maximum concentration in highdensity lipoproteins. 3

Functional Aspects of ApoD The functional role of apoD in the metabolism of plasma lipoproteins remains unclear. Several different lines of evidence have linked apoD with lecithin : cholesterol acyltransferase (LCAT), as a component of the same macromolecular complex, H,~2 an activator of LCAT, 13 and a substrate/product lipoprotein. 6 In addition, apoD has been characterized as a transfer protein mediating the movement of cholesterol ester from H D L to L D L or V L D L accompanied by a reciprocal reverse transfer of triglyceride from VLDL and LDL to HDL. 7 This observation has been disputed by other investigators, 9a4 though it seems plausible that there may be several different proteins participating in the movement of lipids between different lipoprotein species. Confirmatory evidence from other 10 D. Bojanovski, P. Alaupovic, W. J. McConathy, and J. L. Kelly, FEBS Lett. 112, 251 (1980). i1 M. D. Curry, W. J. McConathy, and P. Alaupovic, Biochim. Biophys. Acta 491, 232 (1977). 12 p. E. Fielding and C. J. Fielding, Proc. Natl. Acad. Sci. U.S.A. 77, 3327 (1980). 13 G. Kostner, Scand. J. Clin. Lab. Invest. 33 (Suppl. 137), 19 (1974). 14 R. E. Morton and D. B. Zilversmit, Biochim. Biophys. Acta 663, 350 (1981).

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investigative groups will be necessary to substantiate these different views on the role of apoD in lipoprotein metabolism. Apolipoprotein F In addition to the well-characterized apolipoproteins present in HDL, electrophoretic and immunochemical analyses of apoHDL revealed the occurrence of protein constituents with properties differing from those of the known apolipoproteins. In order to clarify the nature of one of these proteins, the following procedures were utilized to isolate and partially characterize one of these constituents which we designated apolipoprotein F (apoF). Isolation o f ApoF 4 The apoHDL, prepared as described under the apoD section, was solubilized in 2 M acetic acid and the diethyl ether was evaporated under a gentle stream of nitrogen. Essentially all of the apoHDL was soluble after the addition of ultrapure urea to approximately 2 M with respect to urea. A p o H D L (1.2-1.6 g) in a volume of 50-60 ml was applied to a Sephadex G-100 column (5.0 x 150 cm) equilibrated with 2 M acetic acid (flow rate, 30 ml/hr). Thirteen-milliliter fractions were collected and monitored at 280 nm. Fractions were combined on the basis of elution pattern and lyophilized. The lyophilized void volume fraction was utilized for the next fractionation step on carboxymethylcellulose (CM52, Whatman). The microcrystalline CM-cellulose was washed with 5 mM phosphoric acid, pH 3.5, until the slurry had a pH of 3.5. The CM-cellulose was poured into a column (1.5 × 25 cm). Prior to use, the ion-exchange bed was equilibrated with the eluting buffer 1 mM KH2PO4, pH 3.5, in 6 M urea. The urea solution was deionized on a mixed bed ion-exchange resin prior to addition of the KH2PO4 and adjustment of pH. The lyophilized void volume fraction was dissolved in 1 mM KHEPO4, pH 3.5, containing 6 M urea, and chromatographed at 6° on carboxymethylcellulose (flow rate, 25 ml/hr) using the solubilizing buffer to elute the unretained fraction. The unretained fraction was eluted in a volume of 60-80 ml. Chromatography of this fraction was repeated under identical conditions until it was free of apoA-I and apoA-II as demonstrated by double diffusion analyses. Two chromatographies over carboxymethylcellulose w e r e sufficient to yield a homogeneous preparation of apoF. This protein preparation gave no reaction with antisera to apolipoproteins A-I, A-II, B, C-I, C-II, C-III, D, or E. The apoF was desalted on a Sephadex G-25 column equilibrated with 2 M acetic acid. This material

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was lyophilized and used for all subsequent analyses. The yield of apoF from 2 liters of plasma was approximately 2-4 mg. Characterization o f A p o F 4

The described isolation procedure yielded a protein which migrated as a single band on basic PAGE in a position similar to apoD. Amino acid analysis demonstrated the presence of all common amino acids except tryptophan (Table I). The molecular weight was estimated to be 28,000. Isoelectric focusing gave a relatively low isoelectric point (pI = 3.7) with no indication of microheterogeneity by this technique. This low isoelectric point suggests that apoF may represent the most acidic apolipoprotein of the human plasma lipoprotein system. A monospecific antiserum to apoF only reacted with apoF. The apolipoprotein nature of this polypeptide was indicated by the uptake of Oil Red O by the precipitin arcs formed when anti-apoF serum was reacted against LDL and HDL. Nonidentity reactions between the known lipoprotein families and the lipoprotein form of apoF were indications that apoF is the protein moiety of a distinct lipoprotein family designated according to the ABC nomenclature lipoprotein F (LP-F). Isolation and Characterization of Lipoprotein F (LP-F) 15

In order to provide more direct evidence for the lipoprotein nature of apoF, the lipoprotein forms of apoF were investigated by the use of immunosorbers. The IgG fractions of antisera to apolipoproteins A-I, AII, F, and apoF-free plasma were isolated, coupled to Sepharose, and the corresponding immunosorbers were constructed as described in the apoD section. Using an anti-apoF immunosorber, the apoF-containing lipoproteins were present in the retained fraction eluted from the immunosorber. Crossed immunoelectrophoretic patterns with an antiserum to apoF showed no difference between the retained fraction and whole plasma. In addition to apoF, only small but constant amounts of apoA-I and apoA-II were always found in the retained fraction. By passing this retained fraction over the immunosorbers constructed with antibodies to apoA-I, apoA-II, and apoF-free plasma, it was possible to demonstrate in the unretained fraction by double diffusion analyses, electroimmunoassay, and basic PAGE a lipoprotein species that only contained a p o F as the protein moiety. The determination of the lipid composition showed the prevalence of cholesteryl esters (Table III). On 15 E. Koren, W. J. McConathy, and P. Alaupovic,

Biochemistry 21, 5347 (1982).

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crossed immunoelectrophoresis against an anti-apoF serum, lipoprotein F showed fl-lipoprotein mobility.

Distribution and Functional Aspects of ApoF There are no published reports on the plasma concentrations of apoF though preliminary results from this laboratory by electroimmunoassay indicate apoF levels of 2.7 mg/100 ml with the major amount occurring in HDL (75-80%) followed by LDL (15-20%) and traces in VLDL and VHDL (d > 1.21 g/ml). Currently, no information is available on the functional role of apoF. Apolipoprotein H (fl2-Glycoprotein-I) In the early 1960s, a previously unrecognized fl2-globulin was isolated from human serum.~6 Further studies led to the designation of this component as fl2-glycoprotein-I which distinguished it from a similar protein of lower carbohydrate content, fl2-glycoprotein-II. ~T Recent investigations by several groups have described the interaction of fl2-glycoprotein-I with lipoproteins, particularly the triglyceride-rich lipoproteins.~8-2° Based on these observations fl2-glycoprotein-I was designated as apolipoprotein H (aport). 2° In the following presentation, fl2-glycoprotein-I will be referred to as aport.

Isolation of Aport Several different variations of similar procedures have been used to isolate aport from both plasma and triglyceride-rich lipoproteins. Such procedures have included precipitation of serum proteins with Rivanol (2ethoxy-6,9-diaminoacridine lactate) or various acids, 21 ultracentrifugation, and a variety of chromatographic steps taking advantage of the size and charge characteristics of a p o r t . 19,2° As a first step in the isolation of aport from human plasma, most procedures have utilized the precipitation of the bulk of plasma proteins with 0.2 N perchloric acid which leaves a substantial quantity of plasma glycoproteins, including aport, in the supernate. As previously outlined, 2~ 200 ml of acid-citrate-dextrose (ACD)-treated plasma is acidified with 25 ~6H. E. Schultze, K. Heide, and H. Haupt, Naturwissenschaften 48, 719 (1961). 17 H. Haupt and K. Heide, Clin. Chim. Acta 12, 419 (1965). ~8 M. Burstein and P. Legmann, Protides Biol. Fluids 25, 407 (1977). ~9 E. Polz, H. Wurm, and G. M. Kostner, Artery 9, 305 (1981). 20 N. S. Lee, H. B. Brewer, Jr., and J. C. Osborne, Jr., J. Biol. Chem. 258, 4765 (1983). 21 j. S. Finlayson and J. F. Muskinski, Biochim. Biophys. Acta 147, 413 (1967).

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ml of 1.8 N perchloric acid at - 2 ° followed by low-speed centrifugation at 4° to remove the precipitated proteins. The turbid supernate is adjusted to pH 7 by dropwise addition of 12 N NaOH which clarifies the solution. To the supernate is added solid (NH4)2SO4 (380 g/liter) with stirring. Following the solubilization of ammonium sulfate, the solution is allowed to stand for 30 min at 4° followed by collection of the precipitate by lowspeed centrifugation. The precipitate is solubilized and dialyzed against 50 mM Tris-HC1, pH 8.0. The dialyzed fraction is applied to a DEAEcellulose column (Whatman DE52, 1.5 x 30 cm) equilibrated with the same buffer. After application of the sample and elution of the unretained fraction, the remaining proteins are eluted with a linear salt gradient from 0 (200 ml) to 300 mM NaCI (200 ml) in 50 mM Tris-HCl, pH 8.0. The aport is eluted in the unretained fraction and at the beginning of the gradient using commercially available antisera to/32-glycoprotein-I as the monitoring tool. The fractions containing aport are pooled, dialyzed exhaustively against distilled water, and lyophilized. As a final step in the purification procedure, the lyophilized fraction is solubilized in 10 mM Tris-HCl buffer, pH 8.0, and applied to a heparin-Sepharose column (1.5 x 25 cm) as previously described. 2° The column is eluted with 100 ml of l0 mM Tris-HCl, pH 8, followed by a linear gradient consisting of 300 ml solutions of the 10 mM buffer and the same buffer containing 400 mM NaC1. The major portion of retained aport is eluted as a symmetrical peak and its presence is detected by the use of commercially available anti-fl2glycoprotein-I. Fractions reacting with anti-/32-glycoprotein-I serum are monitored by basic PAGE and those appearing homogeneous are dialyzed and lyophilized. The purity of aport is tested by both basic PAGE and reactivity with antisera to apolipoproteins and other serum proteins, including the commercial antiserum specific for/32-glycoprotein-I. As an alternative procedure, 19 serum (100 ml) is mixed with an equal volume of a 1.68% (w/v) Rivanol solution (Serva). The pH is adjusted to 8.0 by adding 10% Na2CO3, followed by stirring for 10 min at room temperature. This mixture is centrifuged and to the supernate is added solid NaC1 to a final concentration of 5 g/100 ml. Addition of 1 M HCI to give a pH of 7.0 is followed by stirring for 10 min and then low-speed centrifugation to remove the precipitate. The supernate is cooled to - 2 ° and 3 ml of 70% (w/v) perchloric acid is added per 100 ml. After stirring for l0 min at - 2 ° , the material is centrifuged in the cold and the precipitate is discarded. The supernate is immediately neutralized with 10% Na2CO3, dialyzed exhaustively against distilled water, and lyophilized. Solubilization of the lyophilized crude aport preparation in l0 mM Tris-HCl, pH 8.0, is followed by chromatography on a heparin-Sepharose (Pharmacia) column (25 x 1 cm). The column is eluted in steps with the initial buffer (10 mM

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Tris) containing 200 mM NaCl, 300 mM NaC1, and 1 M NaC1. The majority of aport is eluted in the 300 mM NaC1 fraction and is judged homogeneous on the basis of basic PAGE and double diffusion analyses. Differences in the molarity of NaC1 required to elute aport from heparin-Sepharose have been observed. For this reason, the molarity of NaC1 solutions for elution of aport may require adjustments, depending on the source of the heparin-Sepharose column. Procedures for isolating aport from VLDL have been previously outlined and have included heparin-Sepharose and gel permeation column chromatography.19,2° Analyses of aport isolated from plasma and VLDL have demonstrated a charge heterogeneity by both DEAE-cellulose column chromatography and isoelectric focusing.~9,2~

Characterization of Aport On basic PAGE, the isolated aport has a mobility similar to that of acid analyses of aport isolated from plasma and VLDL demonstrated the occurrence of all common amino acids, with a relative enrichment in proline and half-cystine, and similar amino acid compositions for serum (Table I) and the isomorphic forms isolated from VLDL.19 Aport is a glycoprotein with hexoses, hexosamine, and sialic acid accounting for approximately 16% of the dry weight (Table II). The estimated molecular weight of aport is 54,000 by SDS-PAGE, ~9 while the weight average molecular weight by sedimentation equilibrium has been shown to be 43,000 in the presence and absence of denaturing solvents.E° The difference between the two methods is probably due to the anomalous behavior of glycoproteins on SDS-PAGE. Studies on the isomorphic forms of aport (pI range, 5 . 6 - 6 . 4 ) 22 have suggested that the polymorphism is due to the oligosaccharide side chains rather than to a variation in the sialic acid content in each of these various f o r m s . 17,21,22 a p o E . 19 Amino

Distribution o f

A p o H 23

The identification of aport (/32-glycoprotein-I) as a constituent of plasma lipoproteins has led to studies on its concentration and distribution in lipoprotein density classes. Determinations were performed by radial immunodiffusion using commercially available antisera and 1,1,3,3tetramethyl urea-treated lipoprotein fractions. Plasma levels were in the range of 16-30 rag/I00 ml with 70-75% in the 1.21 g/ml infranate, 15-18% 22 I. Schousboe, Int. J. Biochem. 15, 35 (1983). 23 E. Polz and G. M. Kostner, FEBS Lett. 102, 183 (1979).

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in HDL, 8-10% in VLDL, and 1% in LDL. Five hours after a heavy fat load, aport can be detected in chylomicrons.

Function o f Aport Several different lines of evidence have indicated a role of aport in the metabolism of triglyceride-rich lipoproteins. One includes the detection of flz-glycoprotein-I as a constituent of both chylomicrons and VLDL. 18-z° Other studies have shown that the addition of aport increases the enzymatic activity of lipoprotein lipase in the presence of apoC-II, suggesting that the enzymatic activity of LPL in triglyceride metabolism may be modulated by aport, z4 Additional experiments using the rat as an in vivo model have shown that the infusion of aport increases the clearance rate of intralipid triglycerides. 25 In addition to its possible role in the metabolism of triglyceride-rich lipoproteins, aport has been shown to have an affinity for both mitochondria and platelets. 22 Other Constituents One additional protein of human plasma has been designated as an apolipoprotein. This component was isolated from VHDL (d 1.21-1.25 g/ ml) by a column chromatographic procedure similar to that outlined for apoD using hydroxylapatite. 26This protein, however, was eluted with 100 mM K2HPO4, pH 8.0. It had an apparent molecular weight of 72,000, exhibited no reactivity with antisera directed against known apolipoproteins, and had a distinct amino acid composition (Table I). Based on these observations and its reactivity with anti-apoHDL serum, this protein component was designated apolipoprotein G (apoG). However, it has not yet been established whether this apolipoprotein occurs in lipoprotein particles as a sole protein or in combination with other apolipoproteins. No information is available either on the lipoprotein forms or the function of apoG. In addition to the protein components discussed in this and other chapters of this volume, a number of other protein components have been reported to be associated with human plasma lipoproteins. The amino acid analyses of some of these proteins isolated from HDL are shown in Table IV. Due to insufficient information related to the chemical, physical, and immunologic properties of these various protein components. 24 y. Nakaya, E. J. Schaefer, and H. B. Brewer, Jr., Biochem. Biophys. Res. Commun. 95, 1168 (1980). 2~ H. Wurm, E. Beubler, E. Polz, A. Holasek, and G. Kostner, Metabolism 31, 484 (1982). 26 M. Ayrault-Jarrier, J.-F. Alix, and J. Polonovski, Biochimie 60, 65 (1978).

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PLASMAAPOLIPOPROTEINS

[ 15]

TABLE IV MINOR POLYPEPTIDE CHAINS PRESENT IN APoHDL OR ApoVHDL Threonine-poor serum amyloid A (SAA) polypeptidesa

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

Glycine-serine-rich polypeptide~

D-2 b

Proline-rich polypeptidec

p l = 6.5

p l = 6.0

29 15 17 54 29 125 100 28 117 54 9 25 0 16 38 13 15 0

59 1 3 24 32 38 100 27 25 34 8 35 5 7 52 23 25 6

46 21 40 71 57 72 100 70 68 32 27 36 5 32 50 30 30 N.D.

41 23 86 144 5 85 100 42 110 154 N.D. 10 11 22 42 43 75 33

40 20 70 116 9 77 100 35 90 123 N.D. 17 17 18 40 38 60 25

S. O. Olofsson, G. Fager, and A. Gustafson, Scand. J. Clin. Lab. Invest. 37, 749 (1977). b C. T. Lim, J. Chung, H. J. Kayden, and A. M. Scanu, Biochim. Biophys. Acta 420, 332 (1976). c T. Sata, R. J. Havel, L. Kotite, and J. P. Kane, Proc. Natl. Acad. Sci. U.S.A. 73, 1063 (1976). d V. G. Shore, B. Shore, and S. B. Lewis, Biochemistry 17, 2174 (1978).

a

including capacity to form distinct lipoproteins and circumstances of their appearance within the lipoprotein density spectrum, it is still not possible to recognize these proteins as integral components of the plasma lipoprotein system. The common characteristic of all of these proteins is an affinity for some portion of the lipoprotein molecule. It is not known what function they may play in the transport or metabolism of lipids. Although there is no available evidence to indicate a direct role for these proteins in the transport of lipids, they may have some auxiliary structural or metabolic functions. We have suggested, therefore, that some of these proteins, if not recognized eventually as apolipoproteins, may be considered and classified as auxiliary proteins of the lipid transport system. 1