Apolipoprotein D in urine

Electrophoresis 1990,/ I , 93-94

Leif Holmquist* Division of Medical Chemistry, National Institute of Occupational Health, Solna

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Identification and quantification of apolipoprotein D in normal human urine Apolipoprotein D has been identified in normal human urine, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting with monospecific antibodies. Urinary apolipoprotein D appeared as a main 33 000 u protein together with a minor fraction corresponding to its partially deglycosylated species of lower molecular mass. N o high molecular mass forms of apolipoprotein D naturally occurring in plasma could be detected. The apolipoprotein D mean 5 S D concentration assayed with rocket immunoelectrophoresis, in urine samples from nine apparently healthy normal men, was 1.4 k 1.0 mg/L(range: 0.2-3.0 mg/L). Among the plasma apolipoproteins, apolipoprotein D behaves uniquely as regards its excretion in urine; the other apolipoproteins belonging to the A, B. C and E groups, although of low molecular masses, are present, at most, in trace amounts in normal urine.

Apolipoprotein D was originally discovered and defined by McConathy and Alaupovic I 11 as a lipid-associated protein moiety of human plasma lipoproteins and predominantly resides in the high density lipoprotein (HDL, d=1.063- 1.2 1 kg/L) fraction. Accordingly, apolipoprotein D migrates as an a-protein on agarose gel electrophoresis. This apolipoprotein has been suggested to be functionally involved as a cholesteryl ester transfer protein in the 1ecithin:cholesterol acyltransferase (LCAT) mediated esterification of the plasma cholesterol [ 2 , 3I. However, this roleofthe apolipoprotein has not yet been established [4,5l. Recently its amino acid sequence was obtained from the cloned cDNA sequence (61. The low molecular mass protein unexpectedly demonstrated a high degree of homology to plasma retinol-binding protein and to human a,-microglobulin but no sequence homology to the other A. B. C and E apolipoproteins of the plasma lipoprotein system 161. Although of low molecular masses, ranging from 6500 u to 35 000 u, these latter apolipoproteins are present. at most. in trace amounts in normal urine, reflecting their firm association with the lipid moieties of the lipoprotein complexes. Being associated with the high density lipoproteins, apolipoprotein D might also be expected not to be transferred from plasma to the urine. However, it was further shown that the tissue distribution of apolipoprotein D mRNA differed from that ofthe mRNA coding for the other apolipoproteins 161, most of which are primarily synthesized in the liver. The former mRNA was abundant in kidney tissue. This fact, put together with the sequence homology of apolipoprotein D to retinolbinding protein and a,-microglobulin, which are normal constituents of human urine, raised the question as to whether or not apolipoprotein D is also a component of normal urine: this was investigated in the present study. Samples (2 mL) of urine from healthy males, 22-52 years old, were collected directly after voiding a small portion. The urine samples were promptly chilled to0 "C, centrifuged at 4000 x g for 10 min and concentrated between 5 to 10 times by ultrafiltration on Millipore Ultrafree-PF filter units. 10 000 NMWL regenerated cellulose (Millipore, Bedford, MA, USA). Authentic apolipoprotein D was prepared from isolated human HDL as described by McConathy and Alaupovic [ 71. The preparation yielded amain zone with an apparent moCorrespondence: Dr. Leif Holmquist, Division of Medical Chemistry. National Institute of Occupational Health. S- 17 184 Solna, Sweden Abbreviations: HDL, high density lipoprotein: LCAT, lecithin :cholesterol acyltransferase; SDS, sodium dodecyl sulfate 8VCH Verlagsgesellschaft mbH. D-6940Weinheim, 1990

lecular mass of 33 000 u (u=1.66 x kg) on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by staining with Coomassie Brilliant Blue R-250. and produced a single precipitation arc on double immunodiffusion in Ouchterlony 1 % agarose plates against a reference goat anti-human apolipoprotein D, obtained as a gift from P Alaupovic (Oklahoma City, OK, USA). Monospecific polyclonal antibodies against purified apolipoprotein D were raised in albino rabbits as previously described (81. A rocket immonoelectrophoresis assay of apolipoprotein D and staining with Coomassie Brilliant Blue R-250 was made as described in [91 using purified apolipoprotein D as standard and 10 pL samples. The protein concentrations of the standard solutions were determined according to Lowry et al. [ 101 using bovine serum albumin (Fraction V. Sigma Chemical Company, St. Louis, MO, USA) as protein standard. SDSpolyacrylamide gel electrophoresis was performed according to Laemmli and Favre [ 1 1 I as modified in I 121 using a discontinuoussystemwith a 4 %T,2.7 % C stackinggeland 15 '%IT. 3.7 % C separation gel in a Bio-Rad Protean Dual 12 electrophoresis cell (Bio-Rad Laboratories, Richmond, C A. USA). Electroblotting 1 13 I of SDS-polyacrylamide gels followed by immunologic identification of separated proteins was made in a Bio-Rad Trans-Blot cell using Immobilon membranes (Millipore) and the Bio-Rad goat anti-rabbit (GAR) Gold Conjugate Immun-Blot assay kit and Gold Enhancement kit. SDS-polyacrylamide gel electrophoresis followed by immunoblotting with antibodies monospecific for apolipoprotein D revealed that normal urine contains this apolipoprotein (Fig. 1). The urinary apolipoprotein D showed the same apparent molecular mass of 33 000 u as the main apolipoprotein D molecular form of plasma. In addition, a zone with the same mobility as its partially deglycosylated species with lower molecular mass occurred in a small amount. This form was also detected in the purified apoli'poprotein D preparation at high loads (Fig. 1). N o zones corresponding to the high molecular mass forms seen in plasma could be detected in urine. Rocket immunoelectrophoresis assay of purified apolipoprotein D and of this protein in urine and native plasma yielded rockets of similar shapes (Fig. 2). Using this method for the quantification of apolipoprotein D in urine samples from nine apparently healthy normal men, a mean ? SD concentration of 1.4 +_ 1.O mg/L (range 0.2-3.0 mg/L) was obtained.

*

On leaveofabsence from: King GustafV Research Institute. Karolinska Institute. Solna. Sweden 0173 O ~ ~ S / ~ O / O I O$02so/n I-OO~~

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L. Holmquist

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Electrophoresis 1990, I1,93-94

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Figure 1. Immobilon membrane immunoblot with monospecific antibodies for identification of apolipoprotein D in human urine. Lanes (1) and (2) were obtained with 185ngand375 ng,respectively, of urinary apolipoprotein D, the following corresponding to (3) 75 ng, (4) 375 ng, (5) 0.8 yg, (6) 3.8 pg and (7) 7.5 kg of purified apolipoprotein D from plasma HDL. M, prestained molecular mass marker proteins (Bio-Rad); u = 1.66 x 10-27kg.Anodeatthebottom.

ists in plasma in free form, dissociated from the lipoprotein complexes. A further consequence of the identification of apolipoprotein D in normal human urine is that this well-characterized and stable protein might be used as an alternative or complement to retinol-binding protein, a,-microglobulin and P,-microglobulin in an assay aimed at detecting tubular proteinuria. Received October 9, 1989

References [ 11 McConathy, W.J. and Alaupovic, P.,FEBSLett. 1973.37. 178-182.

Figure 2. Electrophoretogram of rocket immunoelectrophoresis of 10 pL samples of standard solutions of apolipoprotein D ; concentrations in pg/mL. Lane (1) 2.5, (2) 5.0, (3) 7.5, (4) 10 of about 7.5 times concentrated urine; dilutions ( 5 ) 1:lo, (6) 1:4, (7) I :2, (8) 3:4, (9) undiluted. Diluted plasm a ( l 0 ) 1:40,(11) 1:13,(12) 1:10,(13) 1:5.

Apolipoprotein D is a single glycopeptide chain of unknown function composed of 169 amino acid residues 161. On denaturing SDS-electrophoresis the puriiied protein appears as a 33 000 u molecular mass species [41. It shows 3 major isoforms on isoelectric focusing with isodectric points ranging from 5.0-5.2 (41. In plasma and in samples of delipidated HDL, minor amounts of species with higher and lower molecular masses, the latter representing partially deglycosylated protein, have been reported [14, 151. The cconcentration of apolipoprotein D in normal plasmais around 120mg/L [ 161. The present study, to our knowledge, for the first time clearly shows that apolipoprotein D is a component of normal urine, occurring in the mg/L concentration. This is in contrast to the low molecular mass apolipoproteins belonging to the A, B, C and E groups, which in normal urine are not detectable [ 171or present in only minute amounts [8, 181. With respect to excretion, apolipoprotein D thus behaves similarily to the plasma proteins of reported sequence homology. The association of apolipoprotein D with the plasma high density lipoproteins might be explained by its P-sheet structure favoring the formation of complexes between protein and phospholipids. However, the appearance of this protein in normal urine indicates that apolipoprotein D to a significant extent also ex-

121 Chajec, T. and Fielding, C. J., Proc. Natl. Acad. Sci. USA 1978. 75. 3445-3449. [3 I Fielding, P. E. and Fielding, C. J., Proc. Natl. Acad. Sci. USA 1980. 77,3227-3330. 141 Albers, J. J., Cheung, M. C., Ewens, S. L. and Tollefson. J . H.. Atherosclerosis 1981,39, 395-409. 151 Morton, R. E. and Zilversmit, D. B., Biochim. Biophys. Acta 198 1. 663,350-355. [61 Drayna, D., Fielding, C., McLean, J., Baer, B.,Castro, G.. Chen.E.. Comstock, L., Henzel, W., Kohr, W., Rhee, L., Wion, K. and Lawn. R., J. Biol. Chem. 1986,261, 16 535-16 539. [71 McConathy, W. J. and Alaupovic, P., Methods Enzymol. 1986.128. 297-3 10. I81 Holmquist, L., J . Immunol. Methods 1980,34,243-251. 191 Weeke, B., in: Axelsen, N. H., KroII, J. and Weeke, B. (Eds.), A Manual of Quantitative Immunoelectrophoresis, Universitetsforlaget, Oslo 1973, pp. 15-46. [ 101 Lowry, 0. H., Rosebrough, N. J., Farr, A. C. and Randell, R. J.. J . Biol. Chem. 1951,193,226-275. [ I l l Laemmli, U. K. and Favre, M.,J. Mol. Bid. 1973,80,575-599. 1121 Polyacrylamide Gel Electrophoresis, Laboratory Techniques, Pharmacia Fine Chemicals, Uppsala 1980. 1 131 Towbin, H., Staehelin, T. and Gordon,J.,Proc. Natl. Acad. Sci. U S A 1979,76,4350-4354. 141 Weech, P. K., Camato, R., Milne, R. W. and Marcel, Y. L.?J . Biol. Chem. 1986,261,7941-7951. 151 Camato, R. Marcel, Y. L., Milne, R. W., Lussier-Cacan. S. and Weech, P. K., J. Lipid Res. 1989,30,865-875. 161 Curry, M. D., McConathy, W. J . and Alaupovic, P.. Biochim. Biophys. Acta 1977,491,232-241. 171 Kashyap, M. L., Ooi, B. S., Hynd, B. A., Glueck, C. J., Pollak. V. E. and Robinson, K.,Artery 1979,6, 108-121. I81 Kashyap, M. L., Srivastava, L. S., Hynd, B. A , ; Brady, D.. Perisatti. G.,Glueck,C.J. andGartside,P. S.,Atherosclerosis 1980,35.29-40.

Identification and quantification of apolipoprotein D in normal human urine.

Apolipoprotein D has been identified in normal human urine, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblottin...
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