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Annals of Clinical & Laboratory Science 32:369-376 (2002)
© 2002 Association of Clinical Scientists

Predominant Apolipoprotein J Exists as Lipid-poor Mixtures in Cerebrospinal Fluid

Takefumi Suzuki1,3, Minoru Tozuka1, Kazuyoshi Yamauchi1, Mitsutoshi Sugano1, Tetsuo Nakabayashi1, Nobuo Okumura2, Hiroya Hidaka1, Tsutomu Katsuyama1 and Keiichi Higuchi3
1 Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan
2 Division of Clinical Chemistry, School of Allied Medical Sciences, Shinshu University, Matsumoto, Japan
3 Department of Aging Angiology, School of Medicine, Shinshu University, Matsumoto, Japan

Address correspondence to Minoru Tozuka, Ph.D., Department of Laboratory Medicine, Shinshu University Hospital, 3-1-1 Asahi, Matsumoto 390-8621, Japan; tel 81 263 37 2805; fax 81 263 34 5316; e-mail: mtozuka{at}hsp.md.shinshu-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Apolipoprotein (apo) J, abundant in cerebrospinal fluid (CSF), is known to play a role in the pathogenesis of Alzheimer’s disease (AD); however, the mechanism remains obscure. To characterize the apoJ-containing lipoproteins in CSF, we compared the distribution of apoJ in CSF lipoprotein particles with those of apoE and apoAI. CSF lipoproteins (fractionated by ultracentrifugation, gel-filtration chromatography, and agarose-gel electrophoresis) were characterized by immunoblot analysis using anti-apoJ, anti-apoE, and anti-apoAI antibodies. Immunoprecipitation and immunoabsorption were used to clarify the combinations in which these apolipoproteins exist. All of the apoJ in CSF was in the fraction with density of >= 1.250 g/ml after ultracentrifugation; relatively little apoE and apoAI was in that fraction. In gel-filtration chromatography, the main peak of apoJ-containing lipoprotein particles was clearly distinguishable from those of apoE- and apoAI-containing lipoproteins. Immunoabsorption and agarose-gel electrophoresis indicated that the dominant apoJ-containing lipoprotein particles did not contain apoE. These findings indicate that a significant fraction of the apoJ present in CSF does not co-exist with apoE or apoAI within the same particles. Immunoprecipitation revealed two types of particles: one that contains no apoAI but apoE and another that contains no apoE but apoAI. These results show that several subfractions of lipoprotein particles exist in CSF, differing from each other in their combinations of apoE, apoJ, and apoAI. We concluded that there are at least 9 forms or combinations (including free apolipoproteins) of apoJ, apoE, and apoA1 in the CSF.

(received 15 April 2002; accepted 8 June 2002)

Keywords: apolipoprotein J, cerebrospinal fluid, lipoprotein particles, Alzheimer’s disease


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Apolipoprotein (apo) J, a 70 kDa multifunctional protein normally associated with the high-density lipoproteins (HDL) present in human plasma [1,2], is secreted in the form of lipoparticles by hepatocytes and astrocytes [3]. It consists of two disulfide-linked subunits designated apoJ{alpha} (34–36 kDa) and apoJß(36–39 kDa) [1,2], each of which is a glycoprotein containing nearly 30% of carbohydrate by mass. ApoJ is distributed widely in a variety of tissues of mammals [4]. One characteristic of apoJ is that it is expressed as a result of cellular injury, death, or pathology [5], and one of its functions is to promote the cell interactions that are perturbed in such pathologic settings. ApoJ has been shown to cause cell aggregation and adhesion in vitro, but the molecular mechanism for this effect is obscure [5].

ApoJ is also one of the major apolipoproteins in CSF, together with apoAI, apoAIV, apoD, and apoE [6,7]. A portion of apoE in CSF (as well as in plasma) forms a complex with an apoAII monomer; this is designated as the apoE-AII complex [8]. ApoJ, like apoE, is known to participate in the deposition of amyloid ß (Aß) in the form of senile plaques, a major neuropathological feature of Alzheimer’s disease (AD) [9–11]. Numerous proteins are able to bind synthetic Aß peptides when high concentrations are used; however, apoJ is the major binding protein for Aß in human CSF [12].

We previously reported that there is no significant difference in CSF apoE concentration between healthy men and women or among subjects with different apoE phenotypes [13]. In contrast, it has been shown that a significant decrease in the amount of apoJ is associated with the apoE epsilon 4 allele; however, no difference in apoJ levels was detected in CSF samples among AD subjects [9]. Various pieces of evidence suggest that CSF apoJ, like apoE, could play critical roles in the central nervous system (CNS), including the modulation of Aß neurotoxicity and the development of AD; however, the actual roles played by apoJ are obscure.

We have described some characteristics of apoE-containing lipoproteins in CSF [14], but characterization of the apoJ-containing lipoproteins in CSF is needed to help us understand how it participates in AD. In this study, we characterized the apoJ-containing lipoproteins in CSF, and by examining the relationship between apoJ and other apolipoproteins (eg, apoE and apoAI) determined the extent to which they co-exist within the same particles.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Samples.  CSF samples with no red blood-cell contamination, obtained from hospitalized patients without neurological disease, were used in pools for the analysis. Three CSF pools from about 20 patients were used in the present experiments.

Antibodies.  Anti-apoE polyclonal antibody (rabbit) and horseradish-peroxidase-conjugated anti-goat IgG (rabbit) were purchased from DAKO (Japan). Anti-apoJ polyclonal (goat) and monoclonal (murine) antibodies were obtained from Polysciences Inc. (USA) and Quidel Co. (USA), respectively, and anti-apoAI antibody (goat) from Daiichi Pure Chemicals (Japan). Horseradish-peroxidase-conjugated anti-rabbit and anti-mouse IgG (goat) were purchased from MBL Co. (Japan).

Ultracentrifugation.  CSF lipoproteins were isolated by ultracentrifugation (method of David et al [15], with slight modification). Briefly, solid KBr was added to CSF aliquots to adjust the density to 1.006, 1.019, 1.063, 1.125, 1.210, or 1.250 g/ml. The CSF was centrifuged at 541,000 x g for 12 hr (Optima TLX ultracentrifuge, Beckman, USA). The fractions were dialyzed vs phosphate-buffered saline (PBS).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).  Two to 20 µl of lipoprotein fractions were electrophoresed on 8–16 % gradient polyacrylamide gel containing 0.1 % SDS, by the method of Laemmli [16]. Prestained SDS-PAGE protein standards (low range, Bio-Rad, USA) were also electrophoresed to estimate the molecular weights of bands visualized by immunoblot analysis.

Immunoblot analysis.  Immunoblotting was carried out as described previously [17]. Briefly, after electrophoresis the separated proteins were transferred by electrophoresis or diffusion onto nitrocellulose membranes, which were then incubated for 30 min at room temperature with 50 mmol/L Tris-HCl, pH 8.0, containing 20 g/L skim milk (blocking buffer). The membranes were then incubated for 1 hr at room temperature with anti-apoAI, anti-apoE, and anti-apoJ antibodies diluted 500-fold in blocking buffer. After washing, the membranes were incubated for 1 hr at room temperature with peroxidase-conjugated second antibodies diluted 1000-fold in blocking buffer. The bands containing apoAI, apoE, and apoJ were visualized using either 3,3'-diaminobenzidine tetrahydrochloride and hydrogen peroxide or a detection kit (Amersham Life Science, UK) that employs Hyperfilm ECL.

Gel-filtration chromatography.  Two ml of pooled CSF was fractionated by gel-filtration chromatography using a Sepharose CL-6B column (12 x 900 mm), equilibrated with PBS, and calibrated by the separation of plasma lipoproteins. Sixty-five fractions (2.2 mL) were collected, and the fractions extending from small LDL to HDL were subjected to immunoblot analysis using anti-apoJ, anti-apoE, and anti-apoAI antibodies.

Agarose-gel electrophoresis.  Profiles of the apolipoproteins included in lipoprotein particles were determined by agarose-gel electrophoresis using a commercial kit (Rep-Lipo, Helena Laboratories, USA). Briefly, 0.5–2.0 µl of CSF and diluted serum were added to sample wells in a 1% agarose gel film. After electrophoresis, separated lipoproteins were transferred onto a nitrocellulose membrane by capillary blotting, and apoJ, apoE, and apoAI were visualized by immunoblotting, as described above.

Immunoabsorption.  Protein A-Sepharose, washed and resuspended with PBS, was distributed into three Eppendorf tubes at 200 µl each. After centrifugation at 500 rpm followed by removal of the supernatant, the Protein A-Sepharose in each tube was incubated for 1 hr at room temperature with 100 µl of anti-apoE, anti-apoJ, or saline. After five washes with PBS, 50 µl of CSF was added to each tube followed by incubation for 2 hr at room temperature with occasional mixing. After centrifugation at 500 rpm, each supernatant was subjected to immunoblot analysis as a sample unabsorbed by a specific antibody.

Immunoprecipitation.  Thirty µl of CSF was incubated overnight at 37°C with 50 µl of anti-apoE and anti-apoJ antibodies. After centrifugation at 14,000 rpm for 5 min and removal of the supernatant, the pellet was quickly washed twice with PBS and resuspended in sample buffer for SDS-PAGE. After electrophoresis of the supernatants and the pellets, apolipoproteins were characterized by immunoblot analysis, as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CSF was ultracentrifuged just once (yielding fractions with densities of <=1.006, <=1.019, <=1.063, <=1.125, <=1.210, <=1.250, >=1.210, and >=1.250 g/ml, respectively), followed by immunoblot analysis using anti-apoJ, anti-apoE, and anti-apoAI antibodies (Fig.1Go). Only small amounts of apoE and apoAI, and no apoJ, were observed in the fractions with density of <=1.063 g/ml. ApoE and apoAI were detected mainly in those fractions with densities between 1.063 and 1.210 g/ml; however, some apoE, especially apoE homodimer, and apoAI were observed in the fractions with density of >=1.210 g/ml. On the other hand, all the apoJ was identified in the fractions with density of >=1.210 g/ml.



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  		Fig. 1. SDS-PAGE followed by immunoblot analysis of CSF lipoprotein subfractions obtained by ultracentrifugation using anti-apoJ (A), anti-apoE (B), and anti-apoAI (C) antibodies. CSF lipoproteins were divided into eight fractions, with densities of <=1.006 g/ml (lane 1), <=1.019 g/ml (lane 2), <=1.063 g/ml (lane 3), <=1.125 g/ml (lane 4), <=1.210 g/ ml (lane 5), <=1.250 g/ml (lane 6), >=1.210 g/ml (lane 7), and >=1.250 g/ml (lane 8). Lane L is the original CSF sample.

 
To be sure that apoJ was not released by ultracentrifugation, we examined CSF by gel-filtration. Some fractions corresponding to the small LDL and HDL of plasma lipoproteins (Fig. 2aGo), were analyzed by SDS-PAGE followed by immunoblot analysis using anti-apoJ, anti-apoE, and anti-apoAI antibodies (Fig. 2bGo). The predominant lipoprotein particles containing apoJ were significantly smaller than those containing apoE and apoAI; however, the extent of the predominant fractions containing apoE was narrower than that containing apoAI.



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  		Fig. 2. Gel-filtration chromatogram for CSF (panel a) and SDS-PAGE followed by immunoblot analysis (panel b) of the specified fractions. Two arrows show the positions corresponding to plasma LDL and HDL, respectively. The fractions were applied to SDS-PAGE followed by immunoblot analysis using anti-apoJ (A), anti-apoE (B), or anti-apoAI (C) antibodies. The number of each lane indicates the fraction number, and lane L represents the original CSF sample.

 
The profiles for the apolipoproteins, including apoJ, apoE, and apoAI, in the CSF lipoproteins were compared with those of serum by agarose-gel electrophoresis followed by immunoblotting (Fig. 3Go). The bands containing apoAI and apoE for CSF showed larger migration distances than those for serum. In contrast, the bands containing apoJ for both CSF and serum showed a similar migration distance. Although the band containing apoAI for CSF overlapped considerably with that containing apoE, the band containing apoJ was completely distinct from those on the anode side.



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  		Fig. 3 Agarose-gel electrophoresis followed by immunoblot analysis of CSF using anti-apoAI (A), anti-apoE (B), and anti-apoJ (C) antibodies. Diluted serum (lane 1) and CSF (lane 2) were applied to agarose-gel and electrophoresed. After blotting onto nitrocellulose, lipoproteins containing apoAI (A), apoE (B), and apoJ (C) were visualized by immunoblot analysis. The lipoprotein containing apoJ in CSF was completely distinguished from those with either apoAI or apoE. Arrows indicate the origin.

 
To determine the co-existence of apoJ, apoE, and apoAI in each particle, CSF incubated with anti-apoJ or anti-apoE bound to Protein A-Sepharose was analyzed by immunoblotting (Fig. 4Go). Almost all the apoE in the CSF was apparently absorbed by anti-apoE bound to Protein A-Sepharose; however, some apoJ and almost all the original amount of apoAI existed in the unabsorbed fraction. On the other hand, a small amount of apoJ and most apoE remained in the unabsorbed fraction after absorption by anti-apoJ bound to Protein A-Sepharose.



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  		Fig. 4. SDS-PAGE followed by immunoblot analysis for supernatants, after absorption of CSF by anti-apoE (lane 2) or anti-apoJ (lane 3) bound to Protein A-Sepharose. Supernatants were analyzed by SDS-PAGE followed by immunoblotting using anti-apoJ (A), anti-apoE (B), or anti-apoAI (C) antibodies. Lane 1 is a control obtained by absorption with Protein A-Sepharose alone.

 
To confirm the co-existence of apoJ and apoE, immunoprecipitation of CSF was performed using anti-apoJ antibody. The apoJ-containing lipoprotein was almost all observed in the precipitated pellet; however, apoE was detected in the supernatant and the pellet with similar intensities (Fig. 5Go). When anti-apoE antibody was used, apoJ-containing lipoprotein was mostly co-precipitated, but some remained in the supernatant.



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  		Fig. 5. Immunoblot analysis for the fractions isolated by immunoprecipitation using anti-apoE (lanes 2 and 3) or anti-apoJ (lanes 4 and 5) antibodies. SDS-PAGE was followed by immunoblot analysis using anti-apoE (A) or anti-apoJ (B) antibodies for supernatants (lanes 2 and 4) and pellets (lanes 3 and 5) obtained by immunoprecipitation.

 
From these results, we concluded that there are at least 9 forms or combinations (including free apolipoproteins) for apoJ, apoE, and apoAI among the lipoproteins in the CSF (Fig. 6Go).



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  		Fig. 6. Scheme of apolipoprotein combinations for lipoproteins existing in CSF. The findings obtained in the present paper support the possibility that the CSF lipoproteins exist in nine forms or combinations, including free apolipoproteins. ApoE includes apoE monomer, apoE-AII complex, and apoE homodimer.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The lipoproteins in the CSF are believed to be important for lipid metabolism in the central nervous system (CNS); however, the lipoprotein profile of the CSF is not entirely clear. ApoE, apoJ, and apoAI, which also exist in the plasma, are known to be major apolipoproteins in CNS [6,7]. In the present study, we clarified the distribution and co-existence of these apolipoproteins among the CSF lipoproteins.

It is well known that release of apolipoproteins from CSF lipoprotein particles is induced during preparative ultracentrifugation [18]. Although we avoided using sequential ultracentrifugation, to minimize the breakaway of apolipoproteins from lipoprotein particles, our finding that almost all the apoJ was observed in the fraction with density of >=1.21 g/ml following ultrcentrifugation, may result from this effect. In contrast, CSF lipoproteins containing apoE and apoAI have the same density as plasma HDL, as described previously [7,14].

The interesting finding from our agarose-gel electrophoresis was that a large amount of apoJ, if not all, was found in lipoprotein particles distinguishable from the main particles containing apoE. In addition, gel-filtration chromatography revealed that the main peak for apoJ-containing particles was smaller than those for either apoE- or apoAI-containing particles, but slightly larger than that for albumin (data not shown). This raises the possibility that apoJ forms a lipid-poor complex rather than being contained in spherical lipoprotein particles. These findings suggest that some, if not all, of the apoJ observed in the fraction with density of >=1.210 g/ml following ultracentrifugation may not be remnants released from the lipoprotein particles observed in the fraction with density of <=1.210 g/ml.

Recently, LaDu et al [7,19] reported that apoJ secreted by primary astrocytes was distributed across the particle-size range in gel-filtration chromatography, and that the apoJ peak coincided with those for apoE and apoAI. However, our results, obtained by immunoblot analysis, differed from theirs in that the main particle containing apoJ was clearly smaller than those containing apoE and apoAI.

A significant amount of apoJ may be contained in particles without apoE, since some of the apoJ existed in the unabsorbed fraction despite the almost complete absorption of apoE by anti-apoE bound to Protein A-Sepharose. Although anti-apoJ bound to Protein A-Sepharose failed to absorb apoJ completely, it was obvious that most apoE was not co-absorbed by anti-apoJ bound to Protein A-Sepharose. Immunoprecipitation pointed to essentially similar conclusions: first, the existence of two kinds of particles containing apoJ, one that was and one that was not co-immunoprecipitated by anti-apoE antibody, and second, the existence of two kinds of particles containing apoE, one that was and one that was not co-immunoprecipitated by anti-apoJ antibody. There appear to be three kinds of lipoprotein particles containing apoJ and/or apoE: these consist of apoJ alone, apoE alone, and apoJ and apoE together.

Pitas et al [20], employing immunoaffinity and heparin-Sepharose column chromatography, reported that apoE and apoAI were present in distinct lipoprotein particles in CSF. Our use of the immunoabsorption method indicated that predominant apoAI also existed alone in lipoprotein particles without apoE or apoJ. There appear therefore to be three kinds of lipoprotein particles containing apoAI and/or apoE: namely, particles containing apoAI alone, apoE alone, and apoAI and apoE together.

Although Koch et al [21] observed four lipoprotein classes in CSF, our data suggest that the predominant portions of each apolipoprotein, apoJ, apoE, and apoAI, do not co-exist in the same particles, and that only a minor part of them form complexes.

In humans, apoE exists in three major isoforms [E2 (Cys112, Cys158), E3 (Cys112, Arg158), and E4 (Arg112, Arg158)], which are the products of three independent alleles at a single locus [22,23]. ApoE2 and apoE3, but not apoE4, forms the apo(E-AII) complex [8], and only apoE2 forms apo(AII-E-AII) complex [17]. It depends on the number of cysteine residues in the apoE molecules. Although apoE4 is known to be a risk factor for AD [24,25], we generally observe many AD patients with apoE phenotypes including apoE3 and apoE2 but not apoE4. This discrepancy raises the possibility that, in addition to the apoE genotype, differences in the distribution and concentration of apoJ in CSF lipoprotein particles are critical factors in the pathogenesis of AD. Further studies are needed to address this question.

ApoAI and apoAII in CSF are derived from the plasma through the blood-brain barrier (BBB) [19]. On the other hand, apoE in the CSF is synthesized within the CNS, based on the fact that the apoE phenotype in the CSF is not changed following liver transplantation from a donor with a different apoE phenotype [26]. The apoJ in the CSF is also synthesized within the CNS; however, the contribution (if any) made by plasma apoJ is not known. Recently, glycoprotein 330 has been identified as a receptor that plays a role in receptor-mediated transport of apoJ and apoJ-Aß complex at the blood-brain and blood-cerebrospinal fluid barriers [27]. This raises the possibility that some of the apoJ in the CSF may be derived from the plasma. In contrast, apoAI is believed to derive entirely from the plasma, since its mRNA has not been found to be expressed within the CNS [28]. Although most of these apolipoproteins in the CSF are distributed in the fraction with density of >=1.063 g/ml, which is a narrower range than in the plasma, our results suggest that several combinations of apolipoproteins exist among the lipoproteins in the CSF. Thus, in terms of both composition and role, the lipoproteins in CSF are more complicated than those in plasma.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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