FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2118889971> ?p ?o ?g. }

• W2118889971 endingPage "1704" @default.
• W2118889971 startingPage "1698" @default.
• W2118889971 abstract "Plasma phospholipid transfer protein (PLTP) is an important regulator of plasma HDL levels and HDL particle distribution. PLTP is present in plasma in two forms, one with high and the other with low phospholipid transfer activity. We have used the human hepatoma cell line, HepG2, as a model to study PLTP secreted from hepatic cells. PLTP activity was secreted by the cells into serum-free culture medium as a function of time. However, modification of a previously established ELISA assay to include a denaturing sample pretreatment with the anionic detergent sodium dodecyl sulphate was required for the detection of the secreted PLTP protein. The HepG2 PLTP could be enriched by Heparin-Sepharose affinity chromatography and eluted in size-exclusion chromatography at a position corresponding to the size of 160 kDa. PLTP coeluted with apolipoprotein E (apoE) but not with apoB-100 or apoA-I. A portion of PLTP was retained by an anti-apoE immunoaffinity column together with apoE, suggesting an interaction between these two proteins. Furthermore, antibodies against apoE but not those against apoB-100 or apoA-I were capable of inhibiting PLTP activity.These results show that the HepG2-derived PLTP resembles in several aspects the high-activity form of PLTP found in human plasma. Plasma phospholipid transfer protein (PLTP) is an important regulator of plasma HDL levels and HDL particle distribution. PLTP is present in plasma in two forms, one with high and the other with low phospholipid transfer activity. We have used the human hepatoma cell line, HepG2, as a model to study PLTP secreted from hepatic cells. PLTP activity was secreted by the cells into serum-free culture medium as a function of time. However, modification of a previously established ELISA assay to include a denaturing sample pretreatment with the anionic detergent sodium dodecyl sulphate was required for the detection of the secreted PLTP protein. The HepG2 PLTP could be enriched by Heparin-Sepharose affinity chromatography and eluted in size-exclusion chromatography at a position corresponding to the size of 160 kDa. PLTP coeluted with apolipoprotein E (apoE) but not with apoB-100 or apoA-I. A portion of PLTP was retained by an anti-apoE immunoaffinity column together with apoE, suggesting an interaction between these two proteins. Furthermore, antibodies against apoE but not those against apoB-100 or apoA-I were capable of inhibiting PLTP activity. These results show that the HepG2-derived PLTP resembles in several aspects the high-activity form of PLTP found in human plasma. Both epidemiological and clinical studies provide strong evidence that low plasma HDL cholesterol concentration is a major risk factor for the development of coronary heart disease (CHD) (1Jacobs D. Mebane I. Bangdiwala S. Criqui M. Tyroler H. High density lipoprotein cholesterol as a predictor of cardiovascular disease mortality in men and women: follow-up study of the Lipid Research Clinics Prevalence Study.Am. J. Epidemiol. 1990; 131: 32-47Crossref PubMed Scopus (396) Google Scholar, 2Kannel W. Castelli W. Gordon T. Cholesterol in the prediction of atherosclerosis diseases: new perspectives on the Framingham Study.Ann. Intern. Med. 1979; 90: 85-91Crossref PubMed Scopus (992) Google Scholar, 3Kannel W. High-density lipoproteins: epidemiological profile and risks of coronary artery disease.Am. J. Cardiol. 1983; 52: 9B-12BAbstract Full Text PDF PubMed Scopus (178) Google Scholar). The ability of HDL to protect against the development of CHD has been well documented, and although the exact molecular mechanism(s) behind this finding is still unsolved, it is thought to be due to the role of HDL in the pathway of reverse cholesterol transport, i.e., the transport of cholesterol from peripheral cells to the liver for excretion (4Fielding C. Fielding P. Molecular physiology of reverse cholesterol transport.J. Lipid Res. 1995; 36: 211-228Abstract Full Text PDF PubMed Google Scholar). The HDL in human plasma consists of several subpopulations of particles of distinct structure, function, and composition. This heterogeneity, which is the result of continuous remodelling of HDL by plasma factors, has important implications in terms of the cardioprotective functions of HDL (5Rye K.A. Clay M. Barter P. Remodelling of high density lipoproteins by plasma factors.Atherosclerosis. 1999; 145: 227-238Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). The regulatory role of phospholipid transfer protein (PLTP) in HDL metabolism is mediated via its two main functions, phospholipid transfer activity (6Jiang X.C. Bruce C. Mar J. Lin M. Ji Y. Francone O.L. Tall A.R. Targeted mutation of plasma phospholipid transfer protein gene markedly reduces high-density lipoprotein levels.J. Clin. Invest. 1999; 103: 907-914Crossref PubMed Scopus (319) Google Scholar, 7Qin S. Kawano K. Bruce C. Lin M. Bisgaier C. Tall A.R. Jiang X. Phospholipid transfer protein gene knock-out mice have low high density lipoprotein levels, due to hypercatabolism, and accumulate apoA-IV-rich lamellar lipoproteins.J. Lipid Res. 2000; 41: 269-276Abstract Full Text Full Text PDF PubMed Google Scholar) and the capability to modulate HDL size and composition in a process called HDL conversion (8Jauhiainen M. Metso J. Pahlman R. Blomqvist S. van Tol A. Ehnholm C. Human plasma phospholipid transfer protein causes high density lipoprotein conversion.J. Biol. Chem. 1993; 268: 4032-4036Abstract Full Text PDF PubMed Google Scholar, 9Tu A-Y. Nishida H. Nishida T. High density lipoprotein conversion mediated by human plasma phospholipid transfer protein.J. Biol. Chem. 1993; 268: 23098-23105Abstract Full Text PDF PubMed Google Scholar, 10Settasatian N. Duong M. Curtiss L. Ehnholm C. Jauhiainen M. Huuskonen J. Rye K.A. The mechanism of the remodeling of high density lipoproteins by phospholipid transfer protein.J. Biol. Chem. 2001; 276: 26898-26905Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). HDL conversion is fully dependent on the phospholipid transfer activity of PLTP (11Huuskonen J. Olkkonen V.M. Ehnholm C. Metso J. Julkunen I. Jauhiainen M. Phospholipid transfer is a prerequisite for PLTP-mediated HDL conversion.Biochemistry. 2000; 39: 16092-16098Crossref PubMed Scopus (47) Google Scholar). PLTP's role in the transfer of surface remnants from triglyceride-rich particles, VLDL, and chylomicrons to HDL during lipolysis is important for the maintenance of serum HDL levels (7Qin S. Kawano K. Bruce C. Lin M. Bisgaier C. Tall A.R. Jiang X. Phospholipid transfer protein gene knock-out mice have low high density lipoprotein levels, due to hypercatabolism, and accumulate apoA-IV-rich lamellar lipoproteins.J. Lipid Res. 2000; 41: 269-276Abstract Full Text Full Text PDF PubMed Google Scholar, 12Tall A. Krumholz S. Olivecrona T. Deckelbaum R. Plasma phospholipid transfer protein enhances transfer and exchange of phospholipids between very low density lipoproteins and high density lipoproteins during lipolysis.J. Lipid Res. 1985; 26: 842-851Abstract Full Text PDF PubMed Google Scholar, 13Jiang X.C. Bruce C. Mar J. Lin M. Ji Y. Francone O. Tall A. Targeted mutation of plasma phospholipid transfer protein gene markedly reduces high-density lipoprotein levels.J. Clin. Invest. 1999; 103: 907-914Crossref PubMed Google Scholar). PLTP-facilitated fusion of HDL particles is accompanied by the release of poorly lipidated apolipoprotein A-I (apoA-I). This release of apoA-I in vivo results in the generation of preβ-HDL particles that are essential as cholesterol/phospholipid acceptors from cells in the reverse cholesterol transport process (4Fielding C. Fielding P. Molecular physiology of reverse cholesterol transport.J. Lipid Res. 1995; 36: 211-228Abstract Full Text PDF PubMed Google Scholar, 14Castro G.R. Fielding C.J. Early incorporation of cell-derived cholesterol into pre-beta-migrating high-density lipoprotein.Biochemistry. 1988; 27: 25-29Crossref PubMed Scopus (562) Google Scholar). While the physiological function of PLTP in lipoprotein metabolism is far from resolved, the knowledge gained thus far on the role it plays in HDL remodelling and lipid transfer is substantial. These functions of PLTP are presumably anti-atherogenic. However, studies employing PLTP knockout mice show that complete PLTP deficiency lowers atherosclerosis (15Jiang X-C. Qin S. Qiao C. Kawano K. Lin M. Skold A. Xiao X. Tall A. Apolipoprotein B secretion and atherosclerosis are decreased in mice with phospholipid-transfer protein deficiency.Nat. Med. 2001; 7: 847-852Crossref PubMed Scopus (231) Google Scholar). On the other hand, transgenic mice overexpressing human PLTP display an increased risk for atherosclerosis due to lowered plasma HDL levels (16Van Haperen R. Van Tol A. Van Gent T. Scheek L. Visser P. Van Der Kamp A. Grosveld F. De Crom R. Increased risk of atherosclerosis by elevated plasma levels of phospholipid transfer protein.J. Biol. Chem. 2002; 277: 48938-48943Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Furthermore, with the discovery that PLTP exists in human plasma as two distinct forms, one with high activity and the other with low, it is conceivable that these forms have distinct functions in lipoprotein metabolism (17Oka T. Kujiraoka T. Ito M. Egashira T. Takahashi S. Nanjee M.N. Miller N.E. Metso J. Olkkonen V.M. Ehnholm C. Jauhiainen M. Hattori H. Distribution of phospholipid transfer protein in human plasma: presence of two forms of phospholipid transfer protein, one catalytically active and the other inactive.J. Lipid Res. 2000; 41: 1651-1657Abstract Full Text Full Text PDF PubMed Google Scholar, 18Kärkkäinen M. Oka T. Olkkonen V. Metso J. Hattori H. Jauhiainen M. Ehnholm C. Isolation and partial characterization of the inactive and active forms of human plasma phospholipid transfer protein (PLTP).J. Biol. Chem. 2002; 277: 15413-15418Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Therefore, the relative amount of each form of PLTP in plasma rather than total absence or overexpression will be a crucial determinant when evaluating PLTP's atherogenicity. The association of PLTP with apolipoproteins in vitro (19Pussinen P. Jauhiainen M. Metso J. Pyle L. Marcel Y. Fidge N. Ehnholm C. Binding of phospholipid transfer protein (PLTP) to apolipoproteins A-I and A-II: location of a PLTP binding domain in the amino terminal region of apoA-I.J. Lipid Res. 1998; 39: 152-161Abstract Full Text Full Text PDF PubMed Google Scholar) and in vivo (20Barlage S. Fröhlich D. Böttcher A. Jauhiainen M. Muller H. Noetzel F. Rothe G. Schutt C. Linke R. Lackner K. Ehnholm C. Schmitz G. ApoE-containing high density lipoproteins and phospholipid transfer protein activity increase in patients with a systemic inflammatory response.J. Lipid Res. 2001; 42: 281-290Abstract Full Text Full Text PDF PubMed Google Scholar) has been demonstrated. The low-activity form of PLTP in human plasma preferentially associates with apoA-I, and it is suggested that the high-activity form associates with apoE (18Kärkkäinen M. Oka T. Olkkonen V. Metso J. Hattori H. Jauhiainen M. Ehnholm C. Isolation and partial characterization of the inactive and active forms of human plasma phospholipid transfer protein (PLTP).J. Biol. Chem. 2002; 277: 15413-15418Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Apolipoproteins may be essential for regulating PLTP activity and its ability to modify HDL in vivo. In addition, it has been suggested by Murdoch et al. that the immunoreactivity of the two forms of PLTP from plasma differ and that the use of monoclonal PLTP antibodies in ELISA mass assays may underestimate the mass of the active form (21Murdoch S. Wolfbauer G. Kennedy H. Marcovina S. Carr M. Albers J. Differences in reactivity of antibodies to active versus inactive PLTP significantly impacts PLTP measurement.J. Lipid Res. 2002; 43: 281-289Abstract Full Text Full Text PDF PubMed Google Scholar). To overcome this discrepancy of antibody reactivity, to improve the mass determination of inactive and active PLTP, and to elucidate further the interaction of PLTP with apolipoproteins, we have used the human hepatoma cell line HepG2 as an in vitro model. We now report that PLTP secreted from HepG2 cells resembles in several aspects the high-activity PLTP form in human plasma: it is poorly immunodetectable in its native form, shows affinity for heparin, displays an apparent size of about 160 kDa, and cofractionates with apoE. Furthermore, we show that by pretreating the HepG2-derived PLTP with a strong denaturing anionic detergent, sodium dodecyl sulfate (SDS), we can significantly improve its immunochemical mass quantitation. Human hepatoblastoma-derived cells, HepG2, were obtained from the American Type Culture Collection (ATCC, reference HB-8065). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum, 100 U/ml penicillin/streptomycin, 1 mM sodium pyruvate, and 0.1 mM nonessential amino acids. The cells were cultured in 75 cm2 flasks at 37°C under 5% CO2 and 95% air. For the experiments, the cells were seeded into either 6 cm or 10 cm dishes and grown to 70% confluency in complete culture medium. The medium was then removed, and the monolayers were washed three times with phosphate-buffered saline (PBS) before the addition of serum-free DMEM. Incubation was performed for specific time intervals before the medium was collected and centrifuged at 2,000 rpm for 5 min to remove cells and cell debris. The medium was then stored on ice until used. Polyclonal antibodies R164 (against the N terminus of PLTP, aa 18-144) and R176 (against the carboxyl terminus of PLTP, aa 425-493) as well as the monoclonal anti-PLTP antibody MAb JH59 were produced and characterized as previously described (22Huuskonen J. Jauhiainen M. Ehnholm C. Olkkonen V.M. Biosynthesis and secretion of human plasma phospholipid transfer protein.J. Lipid Res. 1998; 39: 2021-2030Abstract Full Text Full Text PDF PubMed Google Scholar, 23Huuskonen J. Ekström M. Tahvanainen E. Vainio A. Metso J. Pussinen P. Ehnholm C. Olkkonen V. Jauhiainen M. Quantification of human plasma phospholipid transfer protein (PLTP): relationship between PLTP mass and phospholipid transfer activity.Atherosclerosis. 2000; 151: 451-461Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The polyclonal human anti-apoA-I antibody (R261) was produced in New Zealand White rabbits using highly purified apoA-I kindly provided by Dr. Peter Lerch, Swiss Red Cross, Bern, Switzerland. The polyclonal human apoB-100 antibody was raised in sheep using highly purified LDL as antigen. Commercial rabbit anti-human apoB-100 and rabbit anti-human apoE polyclonal antibodies were purchased from DAKO, Denmark. Monoclonal antibody 3D4 against apoA-I was a kind gift from Professor Yves Marcel, Montreal, Canada (24Milthorp P. Weech P.K. Milne R.W. Marcel Y.L. Immunochemical characterization of apolipoprotein A-I from normal human plasma. In vitro modification of apoA-I antigens.Arteriosclerosis. 1986; 6: 285-296Crossref PubMed Google Scholar). The polyclonal human apoE antibody R107 was raised in rabbits with a standard immunization protocol using purified human plasma apoE as antigen. PLTP activity was measured using the radiometric assay described by Damen, Regts, and Scherphof (25Damen J. Regts J. Scherphof G. Transfer of [14C]phosphatidylcholine between liposomes and human plasma high density lipoprotein. Partial purification of a transfer-stimulating plasma factor using a rapid transfer assay.Biochim. Biophys. Acta. 1982; 712: 444-452Crossref PubMed Scopus (131) Google Scholar) with minor modifications (8Jauhiainen M. Metso J. Pahlman R. Blomqvist S. van Tol A. Ehnholm C. Human plasma phospholipid transfer protein causes high density lipoprotein conversion.J. Biol. Chem. 1993; 268: 4032-4036Abstract Full Text PDF PubMed Google Scholar). Human PLTP mass was measured using a modification of the ELISA method of Huuskonen et al. (23Huuskonen J. Ekström M. Tahvanainen E. Vainio A. Metso J. Pussinen P. Ehnholm C. Olkkonen V. Jauhiainen M. Quantification of human plasma phospholipid transfer protein (PLTP): relationship between PLTP mass and phospholipid transfer activity.Atherosclerosis. 2000; 151: 451-461Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The ELISA was modified as follows: samples, standards, and controls were incubated with 0.1% SDS for 30 min at room temperature, and appropriate dilutions (final concentration of SDS 0.02%) were added to the wells. Highly purified human plasma active PLTP (23Huuskonen J. Ekström M. Tahvanainen E. Vainio A. Metso J. Pussinen P. Ehnholm C. Olkkonen V. Jauhiainen M. Quantification of human plasma phospholipid transfer protein (PLTP): relationship between PLTP mass and phospholipid transfer activity.Atherosclerosis. 2000; 151: 451-461Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) was used as the primary standard. Further steps in the ELISA were the same as reported (23Huuskonen J. Ekström M. Tahvanainen E. Vainio A. Metso J. Pussinen P. Ehnholm C. Olkkonen V. Jauhiainen M. Quantification of human plasma phospholipid transfer protein (PLTP): relationship between PLTP mass and phospholipid transfer activity.Atherosclerosis. 2000; 151: 451-461Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). An ELISA-based sandwich quantitation of human apoA-I was also performed. Briefly, the wells were coated with a polyclonal rabbit antibody, R34, against human apoA-I, and the bound protein was detected with a horseradish peroxidase (HRP)-conjugated rabbit anti-human apoA-I immunoglobulin G (IgG), R261. ApoE was also quantitated by ELISA using a polyclonal rabbit capture antibody, R107, to coat the wells and HRP-conjugated anti-human apoE polyclonal rabbit antibody (DAKO) for detection. In the apoB ELISA, the wells were coated with sheep anti-apoB and the antigen was detected with a rabbit anti-human apoB IgG-HRP conjugate. Affinity chromatography was performed using a 5 ml HiTrap Heparin-Sepharose (H-S) column (Amersham Pharmacia Biotech, Uppsala). The column was equilibrated with 25 mM Tris-HCl buffer, pH 7.4, containing 1 mM EDTA, and 20 ml of the HepG2 serum-free culture medium was applied. The material that was bound to the matrix was eluted with 1 M NaCl at a flow rate of 1 ml/min, and 1 ml fractions were collected. The fractions were analyzed for PLTP activity and PLTP mass as well as apolipoprotein mass. The H-S-bound fractions with high PLTP activity were combined and 1 ml was applied to a TSK 4000 size-exclusion (UltroPac Column TSK G4000SW, 7.5 × 600 mm, LKB Bromma, Sweden) silica column. The column was run in TBS buffer containing 0.2% Tween 20 at a flow rate of 0.25 ml/min, and 0.5 ml fractions were collected. The column was calibrated with standard proteins (BioRad Protein Standard No. 151-1901). The H-S-bound fractions with high PLTP activity were combined and dialyzed against 10 mM Tris-HCl, 150 mM NaCl buffer, pH 7.4, containing 1 mM EDTA. The antibodies used were rabbit anti-PLTP (R176), anti-apoE (R107), anti-apoA-I (R34), sheep anti-apoB-100, and nonspecific rabbit IgG. All antibodies (15, 50, and 100 μg of IgG per incubation) were diluted in 10 mM Tris-HCl, 150 mM NaCl buffer, pH 7.4, containing 1 mM EDTA and added in equal volume to the H-S-bound PLTP. The samples (final volume 0.1 ml) were incubated overnight at 4°C, centrifuged, and assayed for PLTP activity. Polyclonal anti-apoE IgG (R107) and a control IgG were coupled to CNBr-activated Sepharose CL-4B according to the manufacturer's instructions (Amersham Pharmacia Biotech). The affinity columns contained 3.7 mg IgG/ml of the matrix. HepG2 culture medium was applied on the columns equilibrated with PBS, pH 7.4. Nonbound fractions were collected (fraction size, 4 ml) and the bound material was eluted with 0.1 M glycine, pH 2.8, into tubes containing 1 M Tris-HCl, pH 8.5, for neutralization. Proteins were resolved by SDS-PAGE according to the method of Laemmli (26Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar), electrotransferred to Hybond-C membranes for Western blotting (27Towbin H. Staehelin T. Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proc. Natl. Acad. Sci. USA. 1979; 76: 4350-4354Crossref PubMed Scopus (44644) Google Scholar), and detected by enhanced chemiluminescence (ECL, Amersham) or conventional HRP color development reagent (BioRad). Nondenaturing polyacrylamide gradient 4–30% gel electrophoresis was performed using the method of Nichols, Krauss, and Musliner (28Nichols A. Krauss R. Musliner T. Nondenaturing polyacrylamide gradient gel electrophoresis.Methods Enzymol. 1986; 128: 417-431Crossref PubMed Scopus (445) Google Scholar). To study the secretion of PLTP from the liver, we used the human hepatoma cell line HepG2 as a model system. PLTP activity and concentrations of apolipoproteins A-I, B-100, and E were measured after 12 h, 24 h, and 48 h of cultivation in serum-free medium. A time-dependent secretion of phospholipid transfer activity into the medium could be observed. Between 12 h and 48 h, PLTP activity increased linearly to a maximum activity of 0.30–0.35 μmol/ml/h at 48 h (Fig. 1). Western blot analysis identified a single immunoreactive band of PLTP protein, the amount of which increased as a function of time. The molecular mass corresponded to ∼80 kDa in SDS-PAGE (Fig. 2), a size similar to that of purified human plasma PLTP.Fig. 2Western blot analysis of PLTP and the apolipoproteins secreted from HepG2 cells. The culture supernatant from each time point (10 μl sample/well) was electrophoresed in 5% (apoB-100) or 12.5% (PLTP, apoE, and apoA-I) SDS-PAGE gels and then electrotransferred to Hybond-C membranes. Proteins were detected with commercial anti-apoB and anti-apoE antisera, and anti-PLTP (MAb JH59) or anti-apoA-I (MAb 3D4) antibodies, and the bound antibodies were visualized by enhanced chemiluminescence (ECL). Molecular masses of the analyzed proteins are displayed.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because HepG2 cells have been reported to secrete a number of apolipoproteins (29Knowles B. Howe C. Aden D. Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen.Science. 1980; 209: 497-499Crossref PubMed Scopus (1491) Google Scholar, 30Wang S. Pessah M. Infante J. Catala D. Salvat C. Infante R. Lipid and lipoprotein metabolism in HepG2 cells.Biochim. Biophys. Acta. 1988; 961: 351-363Crossref PubMed Scopus (48) Google Scholar, 31Thrift R. Forte T. Cahoon B. Shore V. Characterization of lipoproteins produced by the human liver cell line, HepG2, under defined conditions.J. Lipid Res. 1986; 27: 236-250Abstract Full Text PDF PubMed Google Scholar), the secretion of apoA-I, apoE, and apoB-100 was analyzed by ELISA assays. The concentration of apoA-I, apoB-100, and apoE in the growth medium increased as a function of time, and at 48 h reached 8 μg/ml, 6.6 μg/ml, and 0.8 μg/ml, respectively (Figs. 1, 2). Western blot analysis of the growth medium demonstrated single protein bands for apoA-I, apoB-100, and apoE with the anticipated molecular masses (Fig. 2). The previously described ELISA assay for PLTP only detected trace amounts of the HepG2 PLTP protein in the culture supernatant, a result that was discrepant with the strong immunoreactivity observed upon Western blot analysis (Fig. 2). Murdoch and colleagues recently reported that the reactivity of different antibodies with the two PLTP forms varies considerably (21Murdoch S. Wolfbauer G. Kennedy H. Marcovina S. Carr M. Albers J. Differences in reactivity of antibodies to active versus inactive PLTP significantly impacts PLTP measurement.J. Lipid Res. 2002; 43: 281-289Abstract Full Text Full Text PDF PubMed Google Scholar). This implies that the epitope regions on PLTP may be differentially exposed in the two forms. Given the observation that the current ELISA for PLTP failed to detect mass for fractions containing abundant PLTP activity and protein (Figs. 1, 2), we considered the possibility that the HepG2-derived PLTP could be in a conformation poorly recognized by our antibodies and/or could participate in a complex in which epitopes are masked. We therefore subjected the HepG2 PLTP to a pretreatment with 0.1% SDS to unmask potentially hidden epitopes. To quantify the mass using the SDS-ELISA protocol, calibration was performed. For the standard, we used highly purified human plasma active PLTP (23Huuskonen J. Ekström M. Tahvanainen E. Vainio A. Metso J. Pussinen P. Ehnholm C. Olkkonen V. Jauhiainen M. Quantification of human plasma phospholipid transfer protein (PLTP): relationship between PLTP mass and phospholipid transfer activity.Atherosclerosis. 2000; 151: 451-461Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). A standard curve was established after SDS pretreatment of the calibrator. A linear response was obtained in the concentration range of 1–100 ng/ml. This pretreatment step enabled us to reproducibly measure PLTP mass in the HepG2 cell supernatant (Fig. 3). The PLTP mass values of the HepG2 culture supernatant that were determined by the SDS-ELISA assay increased as a function of time and correlated well with the activity measured. At 48 h, the specific PLTP activity (ratio of PLTP activity and mass) was 2.8–3.9 μmol/μg PLTP protein/h. The specific activity for the high-activity form in human plasma isolated by H-S affinity chromatography (18Kärkkäinen M. Oka T. Olkkonen V. Metso J. Hattori H. Jauhiainen M. Ehnholm C. Isolation and partial characterization of the inactive and active forms of human plasma phospholipid transfer protein (PLTP).J. Biol. Chem. 2002; 277: 15413-15418Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) and assayed by SDS-ELISA is 2.5–3.3 μmol/μg PLTP protein/h. As human plasma contains both high- and low-activity forms of PLTP (18Kärkkäinen M. Oka T. Olkkonen V. Metso J. Hattori H. Jauhiainen M. Ehnholm C. Isolation and partial characterization of the inactive and active forms of human plasma phospholipid transfer protein (PLTP).J. Biol. Chem. 2002; 277: 15413-15418Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 32Oka T. Kujiraoka T. Ito M. Nagano M. Ishihara M. Iwasaki T. Egashira T. Miller N. Hattori H. Measurement of human plasma phospholipid transfer protein by sandwich ELISA.Clin. Chem. 2000; 46: 1357-1364Crossref PubMed Scopus (38) Google Scholar), it appears that the secreted HepG2 PLTP resembles the high-activity form. To monitor the size of the nascent PLTP complexes secreted from HepG2 cells, we used nondenaturing gradient gel electrophoresis of HepG2 cell culture medium collected at 48 h. Western blot analysis of the gels revealed that PLTP migrated corresponding to the molecular mass range of 150–450 kDa (Fig. 4). This observation supports the notion that PLTP in the growth medium is not a monomer but part of a large complex. PLTP in human plasma associates with apolipoproteins, forming different complexes of defined size, and has previously been shown to display affinity for heparin (18Kärkkäinen M. Oka T. Olkkonen V. Metso J. Hattori H. Jauhiainen M. Ehnholm C. Isolation and partial characterization of the inactive and active forms of human plasma phospholipid transfer protein (PLTP).J. Biol. Chem. 2002; 277: 15413-15418Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Therefore, HepG2 cell culture medium collected at 48 h was subjected to H-S affinity chromatography (Fig. 5). Of the PLTP activity and mass applied, >90% was recovered as heparin-bound material that could be eluted with 1 M NaCl. Gradient elution (0–1 M NaCl) did not resolve two forms of PLTP, as both the activity and mass eluted together. Significant portions of apoB-100, apoE, and apoA-I coeluted with PLTP from the H-S column. The H-S elution fractions with PLTP activity and mass were combined and contained 0.8 μg/ml of PLTP, 1.2 μg/ml of apoE, 0.25 μg/ml of apoA-I, and a major portion of apoB-100. To further characterize the size and composition of the PLTP-containing complex in the HepG2 cell growth medium, size-exclusion chromatography and Western blot analysis were used. H-S elution fractions exhibiting high PLTP activity (1.0–1.5 μmol/ml/h) were combined, dialyzed against TBS, adjusted to 0.2% Tween 20 and applied to a TSK 4000 column. Detergent was used in this chromatography step to avoid nonspecific adsorption of proteins to the matrix. In the absence of detergent, PLTP activity was retarded by the matrix (data not shown). A major portion (80%) of the PLTP activity and all of the detectable PLTP mass applied were recovered in a position corresponding to a molecular mass of about 160 kDa (Fig. 6). To assess the particle composition of PLTP eluting at this 160 kDa position, the apolipoprotein constituents in the elution fractions were analyzed with ELISA and Western blotting. ApoA-I and apoB-100 did not coelute with PLTP, as the fractions containing apoA-I were located within the 44 kDa region, corresponding to poorly lipidated apoA-I (33Nanjee M.N. Brinton E.A. Very small apolipoprotein A-I-containing particles from human plasma: isolation and quantification by high-performance size-exclusion chromatography.Clin. Chem. 2000; 46: 207-223Crossref PubMed Scopus (53) Google Scholar), and fractions containing apoB-100 were within the size region of 670 kDa (data not sho" @default.
• W2118889971 created "2016-06-24" @default.
• W2118889971 creator A5005591393 @default.
• W2118889971 creator A5016356740 @default.
• W2118889971 creator A5043534256 @default.
• W2118889971 creator A5050115479 @default.
• W2118889971 creator A5075525029 @default.
• W2118889971 date "2003-09-01" @default.
• W2118889971 modified "2023-09-30" @default.
• W2118889971 title "PLTP secreted by HepG2 cells resembles the high-activity PLTP form in human plasma" @default.
• W2118889971 cites W1486434785 @default.
• W2118889971 cites W1536933520 @default.
• W2118889971 cites W1551851929 @default.
• W2118889971 cites W1567784298 @default.
• W2118889971 cites W1577273492 @default.
• W2118889971 cites W1930168886 @default.
• W2118889971 cites W1947043095 @default.
• W2118889971 cites W1963337827 @default.
• W2118889971 cites W1965707506 @default.
• W2118889971 cites W1973447588 @default.
• W2118889971 cites W2008788533 @default.
• W2118889971 cites W2009026141 @default.
• W2118889971 cites W2009789367 @default.
• W2118889971 cites W2011656212 @default.
• W2118889971 cites W2036700661 @default.
• W2118889971 cites W2040206216 @default.
• W2118889971 cites W2046201364 @default.
• W2118889971 cites W2072585523 @default.
• W2118889971 cites W2078679613 @default.
• W2118889971 cites W2079627410 @default.
• W2118889971 cites W2083392018 @default.
• W2118889971 cites W2084240983 @default.
• W2118889971 cites W2100225528 @default.
• W2118889971 cites W2100837269 @default.
• W2118889971 cites W2101108802 @default.
• W2118889971 cites W2107473291 @default.
• W2118889971 cites W2116432245 @default.
• W2118889971 cites W2122729979 @default.
• W2118889971 cites W2126704108 @default.
• W2118889971 cites W2127055642 @default.
• W2118889971 cites W2145525423 @default.
• W2118889971 cites W2155466774 @default.
• W2118889971 cites W2158055619 @default.
• W2118889971 cites W2163518354 @default.
• W2118889971 cites W2182812454 @default.
• W2118889971 cites W2184191366 @default.
• W2118889971 cites W2200554475 @default.
• W2118889971 cites W2331843979 @default.
• W2118889971 cites W2338151611 @default.
• W2118889971 cites W2347061855 @default.
• W2118889971 doi "https://doi.org/10.1194/jlr.m300059-jlr200" @default.
• W2118889971 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12810820" @default.
• W2118889971 hasPublicationYear "2003" @default.
• W2118889971 type Work @default.
• W2118889971 sameAs 2118889971 @default.
• W2118889971 citedByCount "64" @default.
• W2118889971 countsByYear W21188899712012 @default.
• W2118889971 countsByYear W21188899712013 @default.
• W2118889971 countsByYear W21188899712014 @default.
• W2118889971 countsByYear W21188899712015 @default.
• W2118889971 countsByYear W21188899712016 @default.
• W2118889971 countsByYear W21188899712017 @default.
• W2118889971 countsByYear W21188899712018 @default.
• W2118889971 countsByYear W21188899712021 @default.
• W2118889971 countsByYear W21188899712022 @default.
• W2118889971 crossrefType "journal-article" @default.
• W2118889971 hasAuthorship W2118889971A5005591393 @default.
• W2118889971 hasAuthorship W2118889971A5016356740 @default.
• W2118889971 hasAuthorship W2118889971A5043534256 @default.
• W2118889971 hasAuthorship W2118889971A5050115479 @default.
• W2118889971 hasAuthorship W2118889971A5075525029 @default.
• W2118889971 hasBestOaLocation W21188899711 @default.
• W2118889971 hasConcept C185592680 @default.
• W2118889971 hasConcept C3020134792 @default.
• W2118889971 hasConcept C43617362 @default.
• W2118889971 hasConcept C86803240 @default.
• W2118889971 hasConcept C95444343 @default.
• W2118889971 hasConceptScore W2118889971C185592680 @default.
• W2118889971 hasConceptScore W2118889971C3020134792 @default.
• W2118889971 hasConceptScore W2118889971C43617362 @default.
• W2118889971 hasConceptScore W2118889971C86803240 @default.
• W2118889971 hasConceptScore W2118889971C95444343 @default.
• W2118889971 hasIssue "9" @default.
• W2118889971 hasLocation W21188899711 @default.
• W2118889971 hasLocation W21188899712 @default.
• W2118889971 hasOpenAccess W2118889971 @default.
• W2118889971 hasPrimaryLocation W21188899711 @default.
• W2118889971 hasRelatedWork W1531601525 @default.
• W2118889971 hasRelatedWork W2319480705 @default.
• W2118889971 hasRelatedWork W2384464875 @default.
• W2118889971 hasRelatedWork W2398689458 @default.
• W2118889971 hasRelatedWork W2606230654 @default.
• W2118889971 hasRelatedWork W2607424097 @default.
• W2118889971 hasRelatedWork W2748952813 @default.
• W2118889971 hasRelatedWork W2899084033 @default.
• W2118889971 hasRelatedWork W2948807893 @default.
• W2118889971 hasRelatedWork W2778153218 @default.
• W2118889971 hasVolume "44" @default.

• next