Background We previously showed that levels of prebeta-1 high-density lipoprotein (HDL), the principal acceptor of cholesterol effluxed from cells, including artery wall macrophages, are positively associated with coronary heart disease (CHD) and myocardial infarction (MI) risk. Methods and Results In a multiethnic follow-up cohort of 1249 individuals from University of California-San Francisco clinics, we determined the degree to which prebeta-1 HDL levels, both absolute and percentage of apolipoprotein AI, are associated with CHD and history of MI. Independent, strong, positive associations were found. Meta-analysis revealed for the absolute prebeta-1 HDL for the top tertile versus the lowest, unadjusted odds ratios of 1.90 (95% CI, 1.40-2.58) for CHD and 1.79 (95% CI, 1.35-2.36) for MI. For CHD, adjusting for established risk factors, the top versus bottom tertiles, quintiles, and deciles yielded sizable odds ratios of 2.37 (95% CI, 1.74-3.25, P<0.001), 3.20 (95% CI, 2.07-4.94, P<0.001), and 4.00 (95% CI, 2.11-7.58, P<0.001), respectively. Men and women were analyzed separately in a combined data set of 2507 individuals. The odds ratios for CHD and MI risk were similar. Higher levels of prebeta-1 HDL were associated with all 5 metabolic syndrome features. Addition of prebeta-1 HDL to these 5 features resulted in significant improvements in risk-prediction models. Conclusions Analysis of 2507 subjects showed conclusively that levels of prebeta-1 HDL are strongly associated with a history of CHD or MI, independently of traditional risk factors. Addition of prebeta-1 HDL can significantly improve clinical assessment of risk of CHD and MI.

Prebeta-1 HDL is a molecular species of plasma HDL of approximately 67 kDa mass that contains apolipoprotein A-I, phospholipids, and unesterified cholesterol. It participates in a cyclic process involved in the retrieval of cholesterol from peripheral tissues. In this cycle, unesterified cholesterol from cells is incorporated into prebeta-1 HDL, providing a substrate for esterification of cholesterol by lecithin:cholesterol acyltransferase. Prebeta-1 HDL then becomes incorporated into larger HDL species of alpha mobility as esterification proceeds and is regenerated during the transfer of cholesteryl esters from alpha HDL particles to acceptor lipoproteins. Thus the steady state level of prebeta-1 HDL in plasma reflects the relative efficiencies of the major metabolic processes involved in its generation and removal. We have used an isotope dilution technique to measure prebeta-1 HDL levels in the plasmas of 136 normolipidemic individuals (46 M, 90 F). The mean absolute concentration of prebeta-1 HDL as apolipoprotein A-I was 68 ± 40 μg/ml for women, and 84 ± 49 m/ml for men. Prebeta-1 HDL represented 5.5 ± 3.3% of total apolipoprotein A-I in women, and 7.2 ± 4.0% in men. The distributions of both absolute and percent prebeta-1 HDL are highly asymmetric, with skew toward higher values. However, the skew appears not to be attributable to either plasma cholesterol or triglyceride levels which are also skewed in population samples. The percent prebeta-1 HDL was negatively correlated with HDL cholesterol levels (P < 0.0001), whereas absolute levels of prebeta-1 HDL were positively correlated with apolipoprotein A-I and negatively correlated with HDL cholesterol (P, for both, < 0.0001). Multiple linear regression analysis revealed effects of age and gender, but no association with lipoprotein fractions other than HDL. Lower levels of prebeta-1 HDL were associated with female gender in all models.—O'Connor, P. M., B. R. Zysow, S. A. Schoenhaus, B. Y. Ishida, S. T. Kunitake, J. M. Naya-Vigne, P. N. Duchateau, R. F. Redberg, S. J. Spencer, S. Mark, M. Mazur, D. C. Heilbron, R. B. Jaffe, M. J. Malloy, and J. P. Kane. Prebeta-1 HDL in plasma of normolipidemic individuals: influences of plasma, age, and gender.

Prebeta-1 HDL is a 67-kDa species of plasma high-density lipoproteins (HDL) that contains two copies of apolipoprotein A-I. It functions in a metabolic cycle of cholesterol retrieval and may be formed during lipolysis in plasma. We have found that centrifugal ultrafiltration using a membrane with a permeability limit of 100 kDa discriminates categorically between the 67-kDa species and larger HDL particle species. Thus, the ultrafiltrate samples the pool of prebeta-1 HDL in plasma. We have developed a technique using the dispersal of purified prebeta-1 HDL, labeled covalently with tritium, in plasma samples, to label the prebeta-1 HDL pool. Subsequent determination of the specific activity of prebeta-1 HDL in the ultrafiltrate provides a means of calculating the content of prebeta-1 HDL in plasma by the isotope dilution principle. We employ a modification of an enzyme-linked immunosorbent assay technique for apolipoprotein A-I that allows the equal detection of that protein in prebeta-1 HDL and in other HDL particle species for determination of the fraction of total apolipoprotein A-I that is present in the prebeta-1 HDL particle species. The mean level of prebeta-1 HDL-associated apolipoprotein A-I in plasma samples from 86 normolipidemic adults was 74 ± 43 μg/ml (±SD), representing an average of 6.6% of the total apolipoprotein A-I in plasma.

In this study, we have identified and characterized a new protein present in human high density lipoprotein that we have designated apolipoprotein L. Using a combination of liquid-phase isoelectrophoresis and high resolution two-dimensional gel electrophoresis, apolipoprotein L was identified and partially sequenced from immunoisolated high density lipoprotein (Lp(A-I)). Expression was only detected in the pancreas. The cDNA sequence encoding the full-length protein was cloned using reverse transcription-polymerase chain reaction. The deduced amino acid sequence contains 383 residues, including a typical signal peptide of 12 amino acids. No significant homology was found with known sequences. The plasma protein is a single chain polypeptide with an apparent molecular mass of 42 kDa. Antibodies raised against this protein detected a truncated form with a molecular mass of 39 kDa. Both forms were predominantly associated with immunoaffinity-isolated apoA-I-containing lipoproteins and detected mainly in the density range 1.123 < d < 1.21 g/ml. Free apoL was not detected in plasma. Anti-apoL immunoaffinity chromatography was used to purify apoL-containing lipoproteins (Lp(L)) directly from plasma. Nondenaturing gel electrophoresis of Lp(L) showed two major molecular species with apparent diameters of 12.2–17 and 10.4–12.2 nm. Moreover, Lp(L) exhibited both pre-β and α electromobility. Apolipoproteins A-I, A-II, A-IV, and C-III were also detected in the apoL-containing lipoprotein particles. In this study, we have identified and characterized a new protein present in human high density lipoprotein that we have designated apolipoprotein L. Using a combination of liquid-phase isoelectrophoresis and high resolution two-dimensional gel electrophoresis, apolipoprotein L was identified and partially sequenced from immunoisolated high density lipoprotein (Lp(A-I)). Expression was only detected in the pancreas. The cDNA sequence encoding the full-length protein was cloned using reverse transcription-polymerase chain reaction. The deduced amino acid sequence contains 383 residues, including a typical signal peptide of 12 amino acids. No significant homology was found with known sequences. The plasma protein is a single chain polypeptide with an apparent molecular mass of 42 kDa. Antibodies raised against this protein detected a truncated form with a molecular mass of 39 kDa. Both forms were predominantly associated with immunoaffinity-isolated apoA-I-containing lipoproteins and detected mainly in the density range 1.123 < d < 1.21 g/ml. Free apoL was not detected in plasma. Anti-apoL immunoaffinity chromatography was used to purify apoL-containing lipoproteins (Lp(L)) directly from plasma. Nondenaturing gel electrophoresis of Lp(L) showed two major molecular species with apparent diameters of 12.2–17 and 10.4–12.2 nm. Moreover, Lp(L) exhibited both pre-β and α electromobility. Apolipoproteins A-I, A-II, A-IV, and C-III were also detected in the apoL-containing lipoprotein particles. Epidemiological studies have demonstrated a strong inverse correlation between the levels of plasma high density lipoproteins (HDL) 1The abbreviations used are: HDL, high density lipoprotein(s); VLDL, very low density lipoprotein(s); IDL, intermediate density lipoprotein(s); LDL, low density lipoprotein(s); PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis. 1The abbreviations used are: HDL, high density lipoprotein(s); VLDL, very low density lipoprotein(s); IDL, intermediate density lipoprotein(s); LDL, low density lipoprotein(s); PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis. and risk of premature coronary heart disease (1Miller G.J. Miller N.E. Lancet. 1975; i: 16-19Abstract Scopus (1988) Google Scholar, 2Gordon T. Castelli W.P. Hjortland M.C. Kannel W.B. Dawber T.R. J. Am. Med. Assoc. 1977; 238: 497-499Crossref PubMed Scopus (208) Google Scholar). However, the mechanisms by which HDL protect against atherosclerosis need further exploration. One proposed protective role of HDL involves reverse cholesterol transport (3Glomset J.A. J. Lipid Res. 1968; 9: 155-167Abstract Full Text PDF PubMed Google Scholar, 4Kunitake S.T. La Sala K.J. Mendel C.M. Chen G.C. Kane J.P. National Institutes of Health Workshop on Lipoprotein Heterogeneity, NIH Publication 87. 2646. National Institutes of Health, Rockville, MD1987: 419-427Google Scholar, 5Fielding C.J. Fielding P.E. J. Lipid Res. 1995; 36: 211-228Abstract Full Text PDF PubMed Google Scholar), a process in which HDL acquire cholesterol from peripheral cells and facilitate its esterification and delivery to the liver. In this process, small, relatively lipid-poor HDL particles, termed pre-β1-HDL, have been postulated to be the first acceptors of cholesterol from the cells (4Kunitake S.T. La Sala K.J. Mendel C.M. Chen G.C. Kane J.P. National Institutes of Health Workshop on Lipoprotein Heterogeneity, NIH Publication 87. 2646. National Institutes of Health, Rockville, MD1987: 419-427Google Scholar, 6Castro G.R. Fielding C.J. Biochemistry. 1988; 27: 25-29Crossref PubMed Scopus (564) Google Scholar, 7Francone O.L. Gurakar A. Fielding C. J. Biol. Chem. 1989; 264: 7066-7072Abstract Full Text PDF PubMed Google Scholar). An additional mechanism may involve the ability of HDL to impede the oxidation of other plasma lipoproteins (8Parthasarathy S. Barnett J. Fong L.G. Biochim. Biophys. Acta. 1990; 1044: 275-283Crossref PubMed Scopus (637) Google Scholar, 9Kunitake S.T. Jarvis M. Hamilton R.L. Kane J.P. Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 6993-6997Crossref PubMed Scopus (133) Google Scholar, 10Ohta T. Takata K. Horiuchi S. Morino Y. Matsuda I. FEBS Lett. 1989; 257: 435-438Crossref PubMed Scopus (121) Google Scholar). A major difficulty in understanding HDL metabolism is the molecular heterogeneity of HDL (11James R.W. Hochstrasser D. Tissot J.-D. Funk M. Appel R. Barja F. Pellegrini C. Muller A.F. Pometta D. J. Lipid Res. 1988; 29: 1557-1571Abstract Full Text PDF PubMed Google Scholar, 12Kunitake S.T. Carilli C.T. Lau K. Protter A.A. Naya-Vigne J. Kane J.P. Biochemistry. 1994; 33: 1988-1993Crossref PubMed Scopus (51) Google Scholar). Until recently, ultracentrifugation was the most practical way to purify HDL. This methodology has been the basis for the vast majority of the studies in this field. However, it is now well documented that ultracentrifugation causes protein dissociation and can modify structures of HDL particles (13Kunitake S.T. Kane J.P. J. Lipid Res. 1982; 23: 936-940Abstract Full Text PDF PubMed Google Scholar, 14Cheung M.C. Wolf A.C. J. Lipid Res. 1988; 29: 15-25Abstract Full Text PDF PubMed Google Scholar, 15Cheung M.C. Wolf A.C. Lum K.D. Tollefson J.H. Albers J.J. J. Lipid Res. 1986; 27: 1135-1144Abstract Full Text PDF PubMed Google Scholar). An alternative purification strategy that conserves lipoprotein integrity is immunoaffinity chromatography, which isolates lipoproteins on the basis of their protein content (15Cheung M.C. Wolf A.C. Lum K.D. Tollefson J.H. Albers J.J. J. Lipid Res. 1986; 27: 1135-1144Abstract Full Text PDF PubMed Google Scholar, 16Alaupovic P. Lewis L.A. Opplt J.J. CRC Handbook of Electrophoresis. 1. CRC Press, Inc., Boca Raton, FL1980: 27-46Google Scholar, 17McVicar J.P. Kunitake S.T. Hamilton R.L. Kane J.P. Proc. Natl. Acad. Sci. U.S.A. 1984; 81: 1356-1360Crossref PubMed Google Scholar). The development of the strategy of selected affinity immunosorption is particularly suited to investigation of the protein constituents of lipoprotein complexes because it permits isolation of the lipoproteins under minimally perturbing conditions (17McVicar J.P. Kunitake S.T. Hamilton R.L. Kane J.P. Proc. Natl. Acad. Sci. U.S.A. 1984; 81: 1356-1360Crossref PubMed Google Scholar). For example, functional components such as lecithin:cholesterol acyltransferase and cholesterol ester transfer protein are present in higher concentrations in immunopurified lipoproteins, whereas they are depleted or absent in ultracentrifugally purified lipoproteins (12Kunitake S.T. Carilli C.T. Lau K. Protter A.A. Naya-Vigne J. Kane J.P. Biochemistry. 1994; 33: 1988-1993Crossref PubMed Scopus (51) Google Scholar, 15Cheung M.C. Wolf A.C. Lum K.D. Tollefson J.H. Albers J.J. J. Lipid Res. 1986; 27: 1135-1144Abstract Full Text PDF PubMed Google Scholar, 18Fielding P.E. Fielding C.J. Proc. Natl. Acad. Sci. U.S.A. 1980; 77: 3327-3330Crossref PubMed Scopus (153) Google Scholar). These observations demonstrate the importance of immunoaffinity chromatography in identifying novel HDL-associated proteins of potential physiological significance. In this study, we employed selected affinity immunosorption and two-dimensional gel electrophoresis to identify a new protein we have designated apolipoprotein L (apoL) that is associated with plasma lipoproteins, predominantly with apoA-I-containing lipoproteins (Lp(A-I)). We report here the isolation and plasma lipoprotein distribution of apoL and the cloning and characterization of the cDNA encoding apoL. Blood was drawn from fasting normolipidemic subjects (female and male) and immediately cooled to 4 °C in the presence of preservatives and protease inhibitors (0.04% EDTA, 0.05% NaN3, 1 μg/ml gentamycin, 0.3 mg/ml benzamidine, 1 mm phenylmethylsulfonyl fluoride, 0.13% ε-amino-n-caproic acid, and 10 μg/ml α2-macroglobulin, final concentrations) (12Kunitake S.T. Carilli C.T. Lau K. Protter A.A. Naya-Vigne J. Kane J.P. Biochemistry. 1994; 33: 1988-1993Crossref PubMed Scopus (51) Google Scholar). Plasma was separated by centrifugation at 1000 × g for 45 min at 4 °C. The apoA-I-containing lipoproteins (Lp(A-I)) were isolated by selected affinity immunosorption (17McVicar J.P. Kunitake S.T. Hamilton R.L. Kane J.P. Proc. Natl. Acad. Sci. U.S.A. 1984; 81: 1356-1360Crossref PubMed Google Scholar). Plasma was applied to a selected affinity anti-apoA-I column. The unbound fraction was eluted with Tris-buffered saline (5 mm Tris (pH 7.4), 150 mm NaCl, 0.04% EDTA, and 0.05% NaN3). The Lp(A-I) fraction was eluted with 0.2 m acetic acid (pH 3) and 0.15 m NaCl. The eluate was immediately neutralized to pH 7.4 with 2 mTrizma (Tris base), and preservatives were added as described above. Finally, Lp(A-I) were passed through protein A-Sepharose and anti-albumin columns to remove traces of albumin and immunoglobulins. The apoL-containing lipoproteins (Lp(L)) were isolated similarly. First, 200 μg of apoL was purified by electroelution from two-dimensional gels. The purified protein was used to raise rabbit antisera. Antibodies were adsorbed to protein A-Sepharose, and the IgG fraction was eluted with 0.2 m acetic acid and neutralized with 2 m Tris. The IgG fraction was cross-linked to CNBr-activated Sepharose (Pharmacia, Uppsala) to construct an anti-apoL column. VLDL (d < 1.006 g/ml), IDL (1.006 d> 1.12 g/ml) (Fig. 7 A). Because of the long exposure to the substrate, artifact bands (larger than apoL) were also revealed in IDL and LDL. The quantitative assay for apoL confirmed this by showing an apoL content in HDL of 2 ± 0.7 μg of apoL/mg of total protein versus 0.13 ± 0.13 μg of apoL/mg of total protein in VLDL (Fig. 7 B). ApoL was also detected in the bottom fraction (d > 1.25 g/ml) in trace amounts. To find the subpopulation of Lp(A-I) containing apoL (Lp(A-I:L)), we constructed an anti-apoL column using purified anti-apoL IgG. We isolated apoL-containing lipoproteins (Lp(L)) directly from normolipidemic plasma. Fig. 8 shows an SDS gel comparing the bound fraction (Lp(L)) with the apoA-I-containing lipoproteins. By immunoblotting with specific antiserum, we were able to detect the presence of apolipoproteins A-I, A-II, A-IV, and C-III (data not shown). Fractionation of Lp(A-I) into the Lp(A-I:L) and Lp(A-I w/o L) fractions showed that only ∼10% of Lp(A-I) contained apoL. Nondenaturing PAGE of these particles revealed two major Lp(L) subspecies based on their diameters (Fig.9). ApoL was mainly distributed in large apoA-I-containing lipoproteins (12.2–17 and 10.4–12.2 nm) and was totally absent in the small particles. Moreover, the analysis of Lp(L) lipoproteins by immunoelectrophoresis revealed α- and pre-β-migrating components (Fig.10).Figure 9ApoL-containing lipoproteins exhibit HDL size. The curves represent densitometric scans of 3–35% nondenaturing gradient gel. 40 μg of lipoproteins was loaded, and the gel was electrophoresed for 24 h at 100 V. Proteins were stained with Coomassie Blue R-250. Both Lp(L) and Lp(A-I) particles were isolated from normolipidemic plasma. The calibrator proteins (Pharmacia) included thyroglobulin (17 nm), ferritin (12.2 nm), catalase (10.4 nm), lactate dehydrogenase (8.1 nm), and bovine serum albumin (7.1 nm).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 10Apolipoprotein L is present in both pre-β- and α-migrating lipoproteins. Pre-β- and α-migrating lipoproteins were isolated by starch block electrophoresis of Lp(A-I) as described under “Experimental Procedures.” The immunoelectrophoresis of Lp(L) using antisera against apoA-I is shown. Pure pre-β- and α-migrating HDL were used as standards.α, α-migrating Lp(A-I); preβ, pre-β-migrating Lp(A-I).View Large Image Figure ViewerDownload Hi-res image Download (PPT) We have reported here the identification, characterization, and cloning of a new human apolipoprotein that we have designated apolipoprotein L. This new apolipoprotein is mainly associated with the apoA-I-containing lipoproteins of plasma. High density lipoproteins comprise a number of molecular subspecies that differ with respect to protein and lipid composition, particle morphology, and size. The numerous HDL molecular species are not fully apparent when HDL is prepared by ultracentrifugation. Hydrostatic pressure developed in the ultracentrifuge causes the dissociation of a portion of the complement of apolipoproteins (such as apolipoproteins A-I, A-II, C, and E) from HDL and leads to concomitant protein and lipid rearrangements (13Kunitake S.T. Kane J.P. J. Lipid Res. 1982; 23: 936-940Abstract Full Text PDF PubMed Google Scholar, 14Cheung M.C. Wolf A.C. J. Lipid Res. 1988; 29: 15-25Abstract Full Text PDF PubMed Google Scholar, 15Cheung M.C. Wolf A.C. Lum K.D. Tollefson J.H. Albers J.J. J. Lipid Res. 1986; 27: 1135-1144Abstract Full Text PDF PubMed Google Scholar, 33Marcel Y.L. Vezina C. Emond D. Verdery R.B. Milne R.W. J. Lipid Res. 1981; 22: 1198-1205Abstract Full Text PDF PubMed Google Scholar, 34Wilson H.M. Griffin B.A. Watt C. Skinner E.R. Biochem. J. 1992; 284: 477-481Crossref PubMed Scopus (20) Google Scholar, 35Weisgraber K.H. Mahley R.W. J. Lipid. Res. 1980; 21: 316-325Abstract Full Text PDF PubMed Google Scholar). The contents of proteins such as lecithin:cholesterol acyltransferase and cholesterol ester transfer protein shown to interact and to form physical complexes with apoA-I-containing lipoproteins are diminished or totally depleted in HDL altered by ultracentrifugal isolation (15Cheung M.C. Wolf A.C. Lum K.D. Tollefson J.H. Albers J.J. J. Lipid Res. 1986; 27: 1135-1144Abstract Full Text PDF PubMed Google Scholar, 18Fielding P.E. Fielding C.J. Proc. Natl. Acad. Sci. U.S.A. 1980; 77: 3327-3330Crossref PubMed Scopus (153) Google Scholar). Thus, ultracentrifugation hinders identification of the molecular species of HDL and characterization of their constituent proteins. Since first proposed by Alaupovic (16Alaupovic P. Lewis L.A. Opplt J.J. CRC Handbook of Electrophoresis. 1. CRC Press, Inc., Boca Raton, FL1980: 27-46Google Scholar), numerous studies have shown the importance of immunoaffinity fractionation of lipoproteins. The development of the strategy of selected affinity immunosorption permits the isolation of native lipoprotein complexes with minimal perturbation (17McVicar J.P. Kunitake S.T. Hamilton R.L. Kane J.P. Proc. Natl. Acad. Sci. U.S.A. 1984; 81: 1356-1360Crossref PubMed Google Scholar), avoiding the loss of protein constituents that dissociate during isolation by ultracentrifugation (13Kunitake S.T. Kane J.P. J. Lipid Res. 1982; 23: 936-940Abstract Full Text PDF PubMed Google Scholar, 14Cheung M.C. Wolf A.C. J. Lipid Res. 1988; 29: 15-25Abstract Full Text PDF PubMed Google Scholar, 15Cheung M.C. Wolf A.C. Lum K.D. Tollefson J.H. Albers J.J. J. Lipid Res. 1986; 27: 1135-1144Abstract Full Text PDF PubMed Google Scholar, 33Marcel Y.L. Vezina C. Emond D. Verdery R.B. Milne R.W. J. Lipid Res. 1981; 22: 1198-1205Abstract Full Text PDF PubMed Google Scholar, 34Wilson H.M. Griffin B.A. Watt C. Skinner E.R. Biochem. J. 1992; 284: 477-481Crossref PubMed Scopus (20) Google Scholar, 35Weisgraber K.H. Mahley R.W. J. Lipid. Res. 1980; 21: 316-325Abstract Full Text PDF PubMed Google Scholar). In this study, we combined liquid-phase isoelectric focusing and high resolution two-dimensional gel electrophoresis to surmount the problem posed by the predominance of apoA-I in the immunoisolated Lp(A-I) fractions that would otherwise hinder purification of proteins present at lower concentrations. ApoL isolated from the Lp(A-I) particles was observed in two forms: 42 and 39 kDa (minor form) (Fig. 1). The truncated species could represent a proteolytically activated form of the protein, as is the case for several other plasma apolipoproteins (36Edelstein C. Gordon J.I. Toscas K. Sims H.F. Strauss A.W. Scanu A.M. J. Biol. Chem. 1983; 258: 11430-11433Abstract Full Text PDF PubMed Google Scholar, 37Bojanovski D. Gregg R.E. Ghiselli G. Schaefer E.J. Light J.A. Brewer Jr., H.B. J. Lipid Res. 1985; 26: 185-193Abstract Full Text PDF PubMed Google Scholar). If so, the putative precursor form (42 kDa) represents the main constituent. So far, we have not been able to determine if this truncation occurs in vivo or during isolation. Recently, Trofatter et al. (24Trofatter J.A. Long K.R. Murrell J.R. Stotler C.J. Gusella J.F. Buckler A.J. Genome Res. 1995; 5: 214-224Crossref PubMed Scopus (25) Google Scholar) published an expressed sequence tag (clone C22-280, human chromosome 22) that matched the N-terminal sequence we had found for apoL. This sequence revealed 12 residues upstream of the first amino acid of the plasma form of apoL. Since this structure is typical of a signal peptide (38Nielsen H. Engelbrecht J. Brunak S. Von Heijne G. Protein Eng. 1997; 10: 1-6Crossref PubMed Scopus (4928) Google Scholar) and since the cDNA sequence of apoL reveals only one possible open reading frame and encodes a mature protein of 371 amino acids with a molecular mass of 41,041 Da, in agreement with the experimental value, we propose that these 12 residues (starting with a methionine) represent the signal peptide of apoL. Therefore, the cDNA presented in this report encodes the full-length apoL protein. The analysis of apoL cDNA (32Appel R.D. Bairoch A. Hochstrasser D.F. Trends Biochem. Sci. 1994; 19: 258-260Abstract Full Text PDF PubMed Scopus (512) Google Scholar) reveals one putative N-glycosylation site (246NISN249) and several candidate serine and threonine residues for O-glycosylation. Post-transcriptional modifications at these sites could explain the charge isoforms of apoL found in plasma (Fig.1). Because we did not find any significant homology between the apoL sequence and any present in SWISS-PROT or GenBank™ (22Bairoch A. Boeckmann B. Nucleic Acids Res. 1991; 19: 2247-2249Crossref PubMed Scopus (376) Google Scholar), it is not yet possible to predict any function of apoL based on homologies. However, the transcription of apoL mRNA by the pancreas suggests a very specific function, possibly enzymatic, in lipid metabolism. Indeed, preliminary data (not shown) seem to indicate a positive correlation between plasma levels of apoL and plasma triglyceride levels. Analysis of the secondary structure of apoL (31Kneller D.G. Cohen F.E. Langridge R. J. Mol. Biol. 1990; 214: 171-182Crossref PubMed Scopus (647) Google Scholar) reveals four possible amphipathic helices (Fig. 5). These would confer a high level of lipophilicity, in agreement with our finding of very little detectable free apoL in plasma. That apoL in plasma is entirely bound to lipoproteins and remains associated with them during exposure to large volumes of buffer during column washing supports the view that it has very high affinity for HDL. Hence, it should be regarded as a true apolipoprotein rather than a plasma protein that exists partially in a lipoprotein-associated form such as haptoglobin. This is the basis of our designating it an apolipoprotein. ApoL, with a mean plasma concentration of 5.9 ± 0.9 μg/ml (n = 5), is a marker for a distinct subpopulation of HDL. Indeed, apoL was found almost exclusively in association with apoA-I in lipoproteins prepared by immunoaffinity chromatography (Fig.6). Moreover, the presence of apoL in plasma lipoproteins isolated by ultracentrifugation and its localization to HDL3 (Fig.7 A) corroborate results obtained by immunoaffinity chromatography. Because of close association between apoA-I and apoL and because it is well known that lipoprotein integrity is better preserved by immunoaffinity isolation, we used the latter methodology to isolate specific lipoprotein subpopulations containing apoL. In agreement with our previous data, the apoL-containing lipoproteins (Lp(L)) contained apoA-I (Fig. 8). Moreover Lp(L) exhibited diameters typical of HDL (Fig. 9). However, it is interesting to note the discordance of the data between HDL purified by ultracentrifugation and the lipoprotein purified by immunoaffinity, showing the protein redistribution occurring during ultracentrifugation (13Kunitake S.T. Kane J.P. J. Lipid Res. 1982; 23: 936-940Abstract Full Text PDF PubMed Google Scholar, 14Cheung M.C. Wolf A.C. J. Lipid Res. 1988; 29: 15-25Abstract Full Text PDF PubMed Google Scholar, 15Cheung M.C. Wolf A.C. Lum K.D. Tollefson J.H. Albers J.J. J. Lipid Res. 1986; 27: 1135-1144Abstract Full Text PDF PubMed Google Scholar, 33Marcel Y.L. Vezina C. Emond D. Verdery R.B. Milne R.W. J. Lipid Res. 1981; 22: 1198-1205Abstract Full Text PDF PubMed Google Scholar, 34Wilson H.M. Griffin B.A. Watt C. Skinner E.R. Biochem. J. 1992; 284: 477-481Crossref PubMed Scopus (20) Google Scholar, 35Weisgraber K.H. Mahley R.W. J. Lipid. Res. 1980; 21: 316-325Abstract Full Text PDF PubMed Google Scholar). We found apoL to be preponderantly in HDL3; however, the Lp(L) particles isolated by selected immunosorption exhibited heterogeneity of size. ApoL was chiefly associated with large HDL particles (Fig. 9). Fig. 9 also shows the existence of a very large apoL-containing lipoprotein corresponding to VLDL. Due to their low content in Lp(L) particles, these minor populations were not detectable by immunoblotting of apoB-containing lipoprotein (Fig. 7), but were only measurable by enzyme-linked immunosorbent assay. Fig.7 B shows the amount of apoL relative to the amount of total protein in the lipoprotein. The apoL content of HDL was >10 times higher than that in VLDL. No apoL was detectable in LDL. Apparently due to protein dissociation during ultracentrifugation, we also found apoL in the fraction of d > 1.25 g/ml. Moreover, apoA-II, apoA-IV, and apoC-III were also detected in Lp(L) (data not show), indicating, as for other subclasses of HDL, the presence of a complex protein complement in Lp(L). Populations of HDL designated as pre-β-HDL have been postulated as serving key roles in reverse cholesterol transport (4Kunitake S.T. La Sala K.J. Mendel C.M. Chen G.C. Kane J.P. National Institutes of Health Workshop on Lipoprotein Heterogeneity, NIH Publication 87. 2646. National Institutes of Health, Rockville, MD1987: 419-427Google Scholar, 6Castro G.R. Fielding C.J. Biochemistry. 1988; 27: 25-29Crossref PubMed Scopus (564) Google Scholar, 39Francone O.L. Fielding C.J. Eur. Heart J. 1990; 11: 218-224Crossref PubMed Google Scholar). One, pre-β1-HDL, appears to act as the initial acceptor of cellular unesterified cholesterol (6Castro G.R. Fielding C.J. Biochemistry. 1988; 27: 25-29Crossref PubMed Scopus (564) Google Scholar). In this study, we showed that a subpopulation of apoL-containing lipoproteins also exhibits pre-β mobility (Fig. 10), possibly belonging to the larger pre-β2- or pre-β3-HDL particle populations. In summary, we have reported in this study the nucleotide and deduced amino acid sequences for a new human apolipoprotein that we have designated apolipoprotein L. This is the first apolipoprotein shown to be secreted by the pancreas. Its origin in that organ may reflect a non-insulin-dependent role of the pancreas in lipid metabolism. This new apolipoprotein is found in plasma, mainly associated with apoA-I-containing lipoproteins. Moreover, apoL-containing lipoproteins clearly define new HDL subspecies. Since no sequence homology was found with any known protein, its function cannot be inferred on a structural basis.

We have isolated and partially characterized five populations of lipoproteins from the pool of immunoisolated apoA-I-containing lipoproteins obtained from normal human plasma. The first three populations, each containing apoA-I and apoE, were isolated completely by sequential, selected affinity immunosorption against apoA-I and apoE. The lipoproteins isolated by this strategy fall into three morphologic groups; there are discs (LP-AI-E(1)), small spherical lipoproteins (LP-AI-E(2)), and large spherical lipoproteins (LP-AI-E(3)). The LP-AI-E(2) species was sufficiently abundant for detailed characterization. They have slightly larger diameters, and contain more lipid than the bulk of apoA-I-containing lipoproteins and they contain apoA-II:E heterodimers and apoE homodimers. Core lipids are enriched in triglyceride relative to cholesteryl esters. These lipoproteins compete with LDL equally, on a protein mass basis, for binding to human fibroblasts. After removal of apoE-containing lipoproteins from the pool of apoA-I-containing lipoproteins, we discovered two additional subpopulations of lipoproteins that bind to heparin. These lipoproteins, devoid of apoE, occur as populations of small, (LP-AI-HB(1)), and large, spherical lipoproteins, (LP-AI-HB(2). The heparin-binding lipoproteins were separated by gel permeation chromatography. The LP-AI-HB(1) population was of sufficient quantity for detailed study. These lipoproteins also had larger diameters than the bulk of HDL but their core lipids were enriched in cholesteryl esters rather than triglycerides. Three proteins associated with these lipoproteins were found to bind to heparin-Sepharose in the absence of lipid. The approximate molecular weights of these proteins are 40, 70, and 90 kDa. The 70 kDa molecule was found to be the SP 40,40 protein (apoJ).

In this study, we have identified and characterized a new protein present in human high density lipoprotein that we have designated apolipoprotein L. Using a combination of liquid-phase isoelectrophoresis and high resolution two-dimensional gel electrophoresis, apolipoprotein L was identified and partially sequenced from immunoisolated high density lipoprotein (Lp(A-I)). Expression was only detected in the pancreas, The cDNA sequence encoding the full-length protein was cloned using reverse transcription-polymerase chain reaction, The deduced amino acid sequence contains 383 residues, including a typical signal peptide of 12 amino acids, No significant homology was found with known sequences, The plasma protein is a single chain polypeptide with an apparent molecular mass of 42 kDa, Antibodies raised against this protein detected a truncated form with a molecular mass of 39 kDa, Both forms were predominantly associated with immunoaffinity-isolated apoA-I-containing lipoproteins and detected mainly in the density range 1.123 < d < 1.21 g/ml. Free apoL was not detected in plasma. Anti-apoL immunoaffinity chromatography was used to purify apoL-containing lipoproteins (Lp(L)) directly from plasma. Nondenaturing gel electrophoresis of Lp(L) showed two major molecular species with apparent diameters of 12.2-17 and 10.4-12.2 nm. Moreover, Lp(L) exhibited both pre-beta and alpha electromobility. Apolipoproteins A-I, A-II, A-IV, and C-III were also detected in the apoL-containing lipoprotein particles.

Lipoproteins isolated from normal human plasma can bind and neutralize bacterial lipopolysaccharide (LPS) and may represent an important mechanism in host defense against gram-negative septic shock. Recent studies have shown that experimentally elevating the levels of circulating high-density lipoproteins (HDL) provides protection against death in animal models of endotoxic shock. We sought to define the components of HDL that are required for neutralization of LPS. To accomplish this we have studied the functional neutralization of LPS by native and reconstituted HDL using a rapid assay that measures the CD14- dependent activation of leukocyte integrins on human neutrophils. We report here that reconstituted HDL particles (R-HDL), prepared from purified apolipoprotein A-I (apoA-I) combined with phospholipid and free cholesterol, are not sufficient to neutralize the biologic activity of LPS. However, addition of recombinant LPS binding protein (LBP), a protein known to transfer LPS to CD14 and enhance responses of cells to LPS, enabled prompt binding and neutralization of LPS by R- HDL. Thus, LBP appears capable of transferring LPS not only to CD14 but also to lipoprotein particles. In contrast with R-HDL, apoA-I containing lipoproteins (LpA-I) isolated from plasma by selected affinity immunosorption (SAIS) on an anti-apoA-I column, neutralized LPS without addition of exogenous LBP. Several lines of evidence demonstrated that LBP is a constituent of LpA-I in plasma. Passage of plasma over an anti-apoA-I column removed more than 99% of the LBP detectable by ELISA, whereas 31% of the LBP was recovered by elution of the column. Similarly, the ability of plasma to enable activation of neutrophils by LPS (LBP/Septin activity) was depleted and recovered by the same process. Furthermore, an immobilized anti-LBP monoclonal antibody coprecipitated apoA-I. The results described here suggest that in addition to its ability to transfer LPS to CD14, LBP may also transfer LPS to lipoproteins. Since LBP appears to be physically associated with lipoproteins in plasma, it is positioned to play an important role in the neutralization of LPS.

The isolation of apolipoprotein A-I-containing lipoproteins [Lp(A-I)] by selected-affinity immunosorption minimizes the loss of associated proteins that occurs during the isolation of high-density lipoproteins (HDL) by sequential ultracentrifugation. We have used two-dimensional gel electrophoretic analysis to separate the proteins associated with Lp(A-I). Using a combination of amino acid sequencing of transblotted proteins and Western blotting with specific antisera, we have identified a number of associated proteins. The positions of the apolipoproteins (apo) A-I, A-II, A-IV, C-III, D, and E were located on the gels. Lecithin-cholesterol acyltransferase and cholesteryl ester transfer protein were identified in association with Lp(A-I) to a greater extent than found associated with HDL after centrifugation. In addition to those proteins previously identified in association with HDL, we detected a number of plasma proteins associated with Lp(A-I), namely, fibrinogen, haptoglobin, proline-rich protein (C4b-binding protein), and apolipoprotein J (SP40,40 sulfated glycoprotein). The co-isolation of these proteins with Lp(A-I) does not appear to be an artifact in that they have very low affinity for a sham column containing covalently bound preimmune goat IgG in place of the anti-apoA-I IgG. These findings suggest that in addition to apolipoproteins that exist largely in association with lipoproteins there is another class of proteins which exist in lipoprotein-associated form and in the dispersed state. Detection and identification of these lipoprotein-associated proteins may aid in the mechanistic determination of a number of observed functions attributed to HDL.

We isolated and characterized immunoreactive apolipoprotein B (apoB)-containing lipoproteins from human atherosclerotic plaque and plasma to determine whether very-low-density lipoprotein (VLDL) can enter and become incorporated into the atherosclerotic lesion and how plaque apoB-containing lipoproteins differ from apoB-containing lipoproteins isolated from plasma. Atherosclerotic plaques were obtained during aortic surgery and processed immediately. Lipoproteins were extracted from minced plaque in a buffered saline solution (extract A). In selected cases a second extraction was done after plaque was incubated with collagenase (extract B). Lipoproteins were then isolated from the extracts by anti-apoB immunosorption and separated into VLDL + intermediate-density lipoprotein (IDL) (d < 1.019 g/mL) and low-density lipoprotein (LDL) (1.019 < d < 1.070 g/mL) fractions by ultracentrifugation. The VLDL + IDL fractions from plaque contained more than one third of the total apoB-associated lipoprotein cholesterol in both extracts A and B. The lipid composition of VLDL + IDL in both extracts was related to that of plasma VLDL + IDL. By electron microscopy mean particle diameters of VLDL + IDL from extracts A and B were 9% and 23%, respectively, greater than VLDL + IDL diameters from plasma. Mean diameters of LDL from extracts A and B were 11% and 31% greater than LDL diameters from plasma. The apoE-apoB ratio of extract A VLDL + IDL was nearly twice that of plasma VLDL + IDL and severalfold higher than that of extract A LDL. Immunoblots of both VLDL + IDL and LDL from extract A demonstrated minimal fragmentation of apoB.(ABSTRACT TRUNCATED AT 250 WORDS)

Infection and inflammation induce alterations in hepatic synthesis and plasma concentrations of the acute phase proteins. Our results show that apolipoprotein (apo) J is a positive acute phase protein. Endotoxin (LPS), tumor necrosis factor (TNF), and interleukin (IL)-1 increased hepatic mRNA and serum protein levels of apo J in Syrian hamsters. Hepatic apo J mRNA levels increased 10- to 15-fold with doses of LPS from 0.1 to 100 micrograms/100 g body weight within 4 h and were elevated for > or = 24 h. Serum apo J concentrations were significantly increased by 16 h and further elevated to 3.3 times that of control, 24 h after LPS administration. Serum apo J was associated with high density lipoprotein and increased fivefold in this fraction, after LPS administration. Hepatic apo J mRNA levels increased 3.5- and 4.6-fold, with TNF and IL-1, respectively, and 8.2-fold with a combination of TNF and IL-1. Serum apo J concentrations were increased 2.3-fold by TNF, 79% by IL-1, and 2.9-fold with a combination of TNF and IL-1. These results demonstrate that apo J is a positive acute phase protein.

Apolipoprotein A-I-(apoA-I-) containing lipoproteins isolated by immunoaffinity chromatography can be divided into two general subfractions on the basis of the presence [Lp(AI + AII)] or absence [Lp(AI - AII)] of apoA-II. The Lp(AI - AII) subfraction can be further subfractionated into two subgroups with pre-beta mobility as well as those of alpha mobility. We have characterized the Lp(AI - AII) and Lp(AI + AII) subfractions after the removal of pre-beta high-density lipoproteins (pre-beta-HDL) to compare only the two subfractions with alpha mobility. The Lp(AI - AII) and Lp(AI + AII) of alpha mobility, while both heterogeneous subfractions, share many gross features in common. Both subfractions were predominantly spherical in shape, had similar conformation of apoA-I as investigated by circular dichroism and specific endoproteases, and had similar contents of phospholipids, phospholipid species, triglycerides, and cholesterol ester. However, there was significantly less protein (-10%) and more free cholesterol (+46%) in the Lp(AI - AII) subfraction than in the Lp(AI + AII) subfraction. We investigated the generation of pre-beta-HDL from both the Lp(AI - AII) and Lp(AI + AII) subfractions during incubation with low-density lipoproteins and cholesteryl ester transfer protein. We found that both Lp(AI - AII) and Lp(AI + AII) subfractions were capable of generating pre-beta-HDL-like particles. Our results suggest that the formation of pre-beta-HDL involves dissociation of apoA-I from both Lp(AI - AII) and Lp(AI + AII) subfractions. These results refine a model describing the cycling of apoA-I between pre-beta-HDL and alpha-HDL linked to the movement of cholesteryl esters through HDL.

Apolipoprotein (apo) A-I-containing lipoproteins can be separated into two subfractions, pre-beta HDL and alpha HDL (high density lipoproteins), based on differences in their electrophoretic mobility. In this report we present results indicating that these two subfractions are metabolically linked. When plasma was incubated for 2 h at 37 degrees C, apoA-I mass with pre-beta electrophoretic mobility disappeared. This shift in apoA-I mass to alpha electrophoretic mobility was blocked by the addition of either 1.4 mM DTNB or 10 mM menthol to the plasma prior to incubation, suggesting that lecithin:cholesterol acyltransferase (LCAT) activity was involved. There was no change in the electrophoretic mobility of either pre-beta HDL or alpha HDL when they were incubated with cholesterol-loaded fibroblasts. However, after exposure to the fibroblasts, the cholesterol content of the pre-beta HDL did increase approximately sixfold, suggesting that pre-beta HDL can associate with appreciable amounts of cellular cholesterol. Pre-beta HDL-like particles appear to be generated by the incubation of alpha HDL with cholesteryl ester transfer protein (CETP) and either very low density lipoproteins (VLDL) or low density lipoproteins (LDL). This generation of pre-beta HDL-like particles was documented both by immunoelectrophoresis and by molecular sieve chromatography. Based on these findings, we propose a cyclical model in which 1) apoA-I mass moves from pre-beta HDL to alpha HDL in connection with the action of LCAT and the generation of cholesteryl esters within the HDL, and 2) apoA-I moves from alpha HDL to pre-beta HDL in connection with the action of CETP and the movement of cholesteryl esters out of the HDL. Additionally, we propose that the relative plasma concentrations of pre-beta HDL and alpha HDL reflect the movement of cholesteryl esters through the HDL. Conditions that result in the accumulation of HDL cholesteryl esters will be associated with low concentrations of pre-beta HDL, whereas conditions that result in the depletion of HDL cholesteryl esters will be associated with elevated concentrations of pre-beta HDL. This postulate is consistent with published findings in patients with hypertriglyceridemia and LCAT deficiency.

We have found transition metals tightly bound to apolipoprotein A-I-containing lipoproteins [Lp(A-I)] isolated by selected affinity immunosorption from human serum. Prominent among the metal ions detected were iron and copper. By immunoblotting the proteins of Lp(A-I), we detected both transferrin and ceruloplasmin. The transferrin-containing Lp(A-I) particles, isolated by selected affinity immunosorption against transferrin, were larger (mean diameter of 14.2 nm) and had a higher protein content than most high density lipoproteins (HDL). Ultracentrifugally isolated HDL were found to contain much less transferrin, whereas transferrin was found associated with apolipoprotein A-I from the greater than 1.21-g/ml ultracentrifugal fraction. This suggests that the complex is not recovered in the classic HDL density interval because of its very high density. HDL inhibit copper-catalyzed oxidation of low density lipoproteins (LDL) in vitro. We have found that immunoisolated Lp(A-I) are an order of magnitude more effective in inhibiting the oxidation of LDL than ultracentrifugally isolated HDL, on the basis of protein mass. When the Lp(A-I) particles containing transferrin and ceruloplasmin were removed from the bulk of Lp(A-I), inhibition of the in vitro oxidation of LDL was significantly decreased.

The low density lipoproteins (LDL) from patients with Tangier disease are enriched in triglycerides, 27% of LDL mass versus 7% for normal LDL. To study whether this unique LDL core lipid composition affects the surface disposition of apolipoprotein (apo) B-100, we analyzed the LDL by protease digestion and in competitive radioimmunoassays. Limited proteolytic digestion of Tangier LDL by Staphylococcus aureus V8 protease generated a prominent fragment of 120 kDa (cleavage site at residue 1076), which was not visible in similarly digested normal LDL. In competitive radioimmunoassay, Tangier LDL bound weakly to the apoB-specific monoclonal antibody MB20, compared with control LDL. We localized the MB20 epitope between residues 1031 and 1084 of apoB-100, probably very near residue 1076. DNA sequencing of exon 21 of apoB genomic clones (coding for residues 1014-1084) from a Tangier patient revealed no difference from the normal DNA sequence, thus eliminating a protein polymorphism as a basis for the altered protease sensitivity and antibody binding. When the triglyceride contents of Tangier LDL were reduced to 10% of mass by incubation with normal high density lipoproteins, production of the 120-kDa fragment by proteolysis decreased and MB20 binding increased in affinity, implying a change toward normal conformation of apoB-100. Thus, using two independent techniques, proteolytic digestion and binding of monoclonal antibodies, we have demonstrated an alternative conformation of apoB-100 in the vicinity of residue 1076, which reflects the content of triglycerides in the LDL particle.

Apolipoprotein A-I-containing lipoproteins (high density lipoproteins, HDL) can be separated into two subfractions, which have pre-beta and alpha electrophoretic mobilities, respectively. These fractions differ in both composition and structure. Some preparations of pre-beta-migrating HDL, but not alpha-migrating HDL, were found to contain two polypeptides with Mr of approximately 26 and 14 kDa, which are scission products of apolipoprotein (apo) A-I. They are recognized by monospecific antibodies to apo A-I and have N-terminal sequences identical to those of mature apo A-I. This proteolytic scission of apo A-I occurs primarily after venipuncture. Immediate addition of protease inhibitors minimized the appearance of the fragments in plasma. To study the relative susceptibilities of pre-beta and alpha HDL to proteolysis, the lipoproteins were incubated in vitro with plasmin. The apo A-I in pre-beta HDL was extensively degraded, but that in alpha-migrating HDL was degraded to a much lesser extent, indicating that the appearance of apo A-I fragments in pre-beta HDL was due to enhanced sensitivity to proteolysis. To varying degrees, thrombin, kallikrein, elastase, arginine C endoprotease, and chymotrypsin also appear to cleave pre-beta HDL faster than alpha HDL. Most of the proteases generated a 12 to 14 kDa peptide fragment under conditions of limited cleavage. These results suggest that the conformational state of apo A-I in pre-beta-migrating HDL or its spatial relationship to lipids is significantly different from that of apo A-I in alpha-migrating HDL.(ABSTRACT TRUNCATED AT 250 WORDS)