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Definitive N-terminal protein sequence and further characterization of the novel apolipoprotein A5 in human serum.

Apolipoprotein A5 (ApoA5) has been proposed to be a key regulator of serum triglyceride concentrations [for a complete review, see Refs. (1, 2)]. The gene for this novel apolipoprotein was originally identified via experiments looking for new open reading frames in the ApoA1ApoC3-ApoA4 gene cluster located on human chromosome 11823 and at the molecular level in animal studies (3, 4). What emerged from this work was a new gene coding for an apolipoprotein with greatest homology to ApoA4; the new protein was named ApoA5 (3,4).

When the human gene for ApoA5 was expressed in transgenic mice, triglyceride concentrations dropped by approximately two thirds (4,5). Likewise, when the mouse ApoA5 gene itself was knocked out, triglyceride concentrations increased ~4-fold (4,5). These data suggested that ApoA5 expression may be highly and inversely correlated with triglyceride concentrations. Our group recently developed the first assay to measure ApoA5 protein concentrations, and we demonstrated that ApoA5 serum concentrations are increased by treatment with a potent and selective peroxisome proliferator-activated receptor-[alpha] (PPAR-[alpha]) agonist (6, 7).

In light of these data, we first sought to characterize further the circulating ApoA5 protein in human serum. On the basis of its amino acid sequence, the protein is predicted to contain a signal peptide that would be expected to be cleaved before secretion into the plasma, but the actual N-terminal sequence of the mature protein is not known. We therefore purified ApoA5 to obtain the definitive N-terminal protein sequence. ApoA5 was purified from 10 mL of pooled human serum by use of 10 txg of anti-ApoA5 antibody covalently coupled to protein A beads in 40 mL of lysis buffer (50 mmol/L HEPES, pH 7.40;150 mmol/L NaCl; 10 mL / L Triton X-100; 5 mmol/L EDTA; 5 mmol/L EGTA; 20 mmol/L NaF; 20 mmol/L [Na.sub.4][P.sub.2][O.sub.7]) supplemented with a mixture of protease inhibitors (Complete, Mini, EDTA-free; Roche Diagnostics) in a 50-mL conical tube. ApoA5 was immunoprecipitated overnight at 4[degrees]C. The beads were then washed twice with lysis buffer, and 20 [micro]L of 2x sample buffer [100 mmol/L Tris, pH 6.80; 40 [micro]g/L sodium dodecyl sulfate (SDS); 200 mL/L glycerol; 20 [micro]g/L bromphenol blue; 15 g/L dithiothreitol)] was added to the tube. The sample was then subjected to electrophoretic separation via 1-dimensional SDS--polyacrylamide gel electrophoresis for 1.5 h at 150 V at room temperature. The gel was transferred to a polyvinylidene difluoride (PVDF) membrane in 0.1 mol/L CAPS transfer buffer, pH 11.0 (Sigma), by use of a Bio-Rad Model 422 ElectroEluter for 1 h at 60 V. The membrane was stained with 0.4 [micro]g/L Coomassie Brilliant Blue G 250 (Sigma) for 1 min and destained with 500 mL/L methanol to reveal several different protein bands, as shown in Fig. 1A.

We performed automated Edman degradation amino acid sequence analysis on the excised bands, using the ABI gas-phase (GP-PVDF-protein) method on a Procise[R] HT 494 protein sequencer (Applied Biosystems). Ten residues were analyzed on a SPHERI-5 PTH (220 x 2.1 mm) ABI Brownlee column at 55[degrees]C with a flow rate of 325 [micro]L/min. We then analyzed the data, using the Model 610A (Applied Biosystems) data analysis program. N-Terminal sequencing definitively identified the ApoA5 band (Fig. 1A), which was expected to be faint based on the low concentration of ApoA5 compared with other apolipoproteins (6), and also provided the actual N-terminal sequence of the protein, which is shown in Fig. 1B. No additional proteins were identified in the region of the ApoA5 band, and the proteins immediately above and below ApoA5 were identified by N-terminal sequencing as IgM-associated peptide and haptoglobin, respectively (Fig. 1A). As shown in Fig. 1B, the actual N-terminal sequence of ApoA5 corresponds to that expected from cleavage of the predicted signal peptide, indicating that the mature protein is an intact molecule with a predicted relative molecular mass ([M.sub.r]) of 39 000.

[FIGURE 1 OMITTED]

ApoA5 is unusual among the apolipoproteins because the amino acid sequence contains a single cysteine residue (1-4). To investigate whether ApoA5 might circulate as a disulfide-linked homodimer or heterodimer, we immunoprecipitated the protein from 100 [micro]L of pooled human serum added to 1 mL of lysis buffer in 1.5-mL Eppendorf tubes. ApoA5 was immunoprecipitated overnight at 4[degrees]C with 1 [micro]g of anti-ApoA5 antibody coupled to protein A beads. The beads were then washed twice with lysis buffer, and 40 [micro]L of 2x sample buffer was added to each tube. For analysis under nonreducing conditions, dithiothreitol was omitted and samples were incubated for 1 h at room temperature.

Immunoprecipitated proteins were separated for 1 h at 175 V at room temperature and transferred to ECL nitrocellulose paper (Amersham Biosciences) for 1 h (100 V, 4[degrees]C). Blots were blocked for 1 h at room temperature in Tris-buffered saline (TBS)-casein blocking buffer (Pierce) containing 1 mL/L Tween 20. The blots were then probed with horseradish peroxidase-labeled anti-ApoA5 antibody in blocking buffer for 1 h at room temperature. Blots were washed 3 times (10 min each) with TBS-Tween (10 mmol/L Tris, pH 7.40;150 mmol/L NaCl; 1 mL/L Tween 20) followed by a final wash in TBS. Blots were developed with ECL reagent (Amersham Biosciences) and exposed to Bio-Max x-ray film (Kodak). Consistent with previous observations, and as shown in Fig. 1C, the protein migrated as a band at [M.sub.r] ~39 000 under reducing conditions. ApoA5 also migrated mainly as a band at [M.sub.r] ~39 000 under nonreducing conditions, confirming that the protein circulates in the plasma mainly as a monomer and not a homodisulfide- or heterodisulfide-linked dimer. If ApoA5 were disulfide-linked to another protein, it would be expected that such a nonreduced complex would be detected at a higher molecular mass on the blot by the polyclonal ApoA5 antibody (which would detect ApoA5 covalently linked to another protein). Also consistent with ApoA5 circulating as a monomer, no increased relative yield of ApoA5 was obtained when disulfide bonds were reduced before immunoprecipitation (data not shown).

In light of previous observations that ApoA5 was present in chylomicrons, HDL, and VLDL but not LDL (6), we analyzed lipoprotein particle fractions, including IDL and plasma infranatant, for the presence of ApoA5. To do this, we used sequential flotation ultracentrifugation for stepwise isolation of highly purified lipoprotein fractions (Athens Research and Technology), as described previously (6), with the exception that we also prepared IDL and lipoprotein particle-free infranatant fractions. VLDL was collected from 0.950 to 1.006 kg/L, IDL from 1.006 to 1.020 kg/L, and LDL from 1.020 to 1.063 kg/L. Interestingly, as shown in Fig. 1D, ApoA5 was present in chylomicrons, HDL, and VLDL but not in IDL, LDL, or plasma infranatant (lipoprotein particle-free plasma). These data indicated that ApoA5 circulates mostly in lipoprotein particles and not in a free form, as ApoA4 does, and that ApoA5 is not present in either LDL or IDL, indicating that conversion of VLDL to IDL is accompanied by loss of particle-associated ApoA5.

Together, these data further characterize ApoA5 by determining the definitive N-terminal sequence of the circulating protein, showing that ApoA5 circulates mainly as a monomer and not as a homodisulfide- or heterodisulfide-linked dimer, and further detailing its lipoprotein particle distribution. The fact that ApoA5 was found in VLDL, but not IDL or LDL, suggests that VLDL particles lose ApoA5 as they are converted first to IDL and then LDL.

Interest in ApoA5 has been increased by reports that PPAR-[alpha] agonists increase its mRNA expression and circulating protein concentrations (7-9). The absence of a dramatic inverse correlation between ApoA5 concentrations and serum triglycerides, however, makes elucidating the role of ApoA5 in lipid metabolism complex (6,10,11). The mechanism by which ApoA5 may possibly lower triglyceride concentrations is unclear, particularly if serum ApoA5 concentrations are not highly inversely correlated with triglycerides. Although ApoA5 has been demonstrated recently to stimulate lipoprotein lipase--mediated VLDL-triglyceride hydrolysis (12-16), it seems difficult to reconcile this property with the miniscule serum concentrations of ApoA5 compared with those of ApoC3. When differences in the molecular masses of the 2 proteins are taken into account, there is ~4000-fold more ApoC3 than ApoA5 in serum on a molar basis (6).

Another explanation may be that ApoA5 acts primarily intracellularly in the liver to decrease VLDL synthesis and release (17). If this is indeed the case, then the ApoA5 present in serum lipoprotein particles may represent leakage of the apolipoprotein into the circulation. In this case, serum ApoA5 concentrations would still be useful biomarkers for PPAR-a agonists, which might partly decrease triglycerides by increasing ApoA5 synthesis within the liver, where ApoA5 could act intracellularly to decrease assembly and secretion of VLDL particles.

There continues to be interest in better understanding the role of ApoA5 in the regulation of serum triglycerides, with additional recent reports of ApoA5 polymorphisms associated with hypertriglyceridemia and with hyperlipidemia in patients with the ApoE 2/2 phenotype (18-20). One important step will clearly be to correlate ApoA5 protein concentrations with ApoA5 gene polymorphisms. Another step will undoubtedly be the injection of recombinant ApoA5 protein into animal models of dyslipidemia to determine whether the protein itself can directly lower triglycerides when administered at physiologic concentrations.

We thank Dr. Holger Schilske, Dr. David Robbins, Nancy Hale, Paula Santa, and Jayne Talbot for their support.

References

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(20.) Schaefer JR, Sattler AM, Hackler B, Kurt B, Hackler R, Maisch B, et al. Hyperlipidemia in patients with apolipoprotein E 2/2 phenotype: apolipoprotein A5 S19W mutation as a cofactor [Letter]. Clin Chem 2004;50:2214.

DOI: 10.1373/clinchem.2005.061374

William E. Alborn, Melvin G. Johnson, Melvin J. Prince, and Robert J. Konrad * (Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN; * address correspondence to this author at: Eli Lilly Corporate Center, Bldg. 88-358C, Indianapolis, IN 46285; fax 317-276-5281, e-mail konrad_robert@lilly.com)
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Title Annotation:Technical Briefs
Author:Alborn, William E.; Johnson, Melvin G.; Prince, Melvin J.; Konrad, Robert J.
Publication:Clinical Chemistry
Date:Mar 1, 2006
Words:2317
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