FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2024408717> ?p ?o ?g. }

• W2024408717 endingPage "8086" @default.
• W2024408717 startingPage "8081" @default.
• W2024408717 abstract "Lipoprotein lipase (LPL) hydrolyzes chylomicron and very low density lipoprotein (VLDL) triglycerides and potentiates the cellular uptake of lipoproteins. These LPL-lipoprotein associations could involve only protein-lipid interaction, or they could be modulated by apolipoproteins (apo). ApoB is the major protein component of chylomicrons, VLDL, and low density lipoprotein (LDL). ApoB100, a large glycoprotein with a molecular mass of 550 kDa, is composed of several functional domains. A carboxyl-terminal region of the protein is the ligand for the LDL receptor. There are several hydrophobic domains that are believed to be important in lipid binding. The relatively hydrophilic amino-terminal region of apoB, however, has no known function. Using solid phase assays we quantified LPL-lipoprotein complex formation. On a molar basis, severalfold greater amounts of LPL bound to LDL and VLDL than to high density lipoprotein at all the concentrations of LPL tested (0.9-55 nM).To assess the roles of LDL protein versus lipid, we performed competition and ligand blotting experiments. LDL and an amino-terminal fragment of apoB competed better for 125I-LPL binding to LDL than did lipid emulsion particles. Delipidation of LDL-coated plates did not alter LPL binding. On ligand blots, LPL bound to amino-terminal fragments of apoB generated by thrombin digestion but not to apoA1, apoE, or carboxyl-terminal fragments of apoB. Further evidence for LPL interaction with the amino-terminal region of apoB was obtained using anti-apoB monoclonal antibodies. Antibodies directed against the amino-terminal regions of apoB blocked LPL interaction with LDL, whereas those against the carboxyl-terminal region of apoB did not inhibit LPL interaction with LDL. Thus, we conclude that a specific interaction between LPL and the amino-terminal region of apoB may facilitate LPL association with circulating lipoproteins. Lipoprotein lipase (LPL) hydrolyzes chylomicron and very low density lipoprotein (VLDL) triglycerides and potentiates the cellular uptake of lipoproteins. These LPL-lipoprotein associations could involve only protein-lipid interaction, or they could be modulated by apolipoproteins (apo). ApoB is the major protein component of chylomicrons, VLDL, and low density lipoprotein (LDL). ApoB100, a large glycoprotein with a molecular mass of 550 kDa, is composed of several functional domains. A carboxyl-terminal region of the protein is the ligand for the LDL receptor. There are several hydrophobic domains that are believed to be important in lipid binding. The relatively hydrophilic amino-terminal region of apoB, however, has no known function. Using solid phase assays we quantified LPL-lipoprotein complex formation. On a molar basis, severalfold greater amounts of LPL bound to LDL and VLDL than to high density lipoprotein at all the concentrations of LPL tested (0.9-55 nM). To assess the roles of LDL protein versus lipid, we performed competition and ligand blotting experiments. LDL and an amino-terminal fragment of apoB competed better for 125I-LPL binding to LDL than did lipid emulsion particles. Delipidation of LDL-coated plates did not alter LPL binding. On ligand blots, LPL bound to amino-terminal fragments of apoB generated by thrombin digestion but not to apoA1, apoE, or carboxyl-terminal fragments of apoB. Further evidence for LPL interaction with the amino-terminal region of apoB was obtained using anti-apoB monoclonal antibodies. Antibodies directed against the amino-terminal regions of apoB blocked LPL interaction with LDL, whereas those against the carboxyl-terminal region of apoB did not inhibit LPL interaction with LDL. Thus, we conclude that a specific interaction between LPL and the amino-terminal region of apoB may facilitate LPL association with circulating lipoproteins. Lipoprotein lipase (LPL) 1The abbreviations used are: LPLlipoprotein lipaseVLDLvery low density lipoproteinLDLlow density lipoproteinHDLhigh density lipoproteinTGtriglyceridePBSphosphate-buffered salineBSAbovine serum albuminTBSTris-buffered salinemAbmonoclonal antibody. is primarily responsible for hydrolyzing chylomicron and VLDL triglycerides (TG). This enzyme is synthesized in a variety of tissues and cells including adipose, muscle(1Nilsson-Ehle P. Garfinkel A.S. Schotz M.C. Annu. Rev. Biochem. 1980; 49: 667-693Crossref PubMed Scopus (571) Google Scholar), brain(2Eckel R.H. Robbins R.J. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7604-7606Crossref PubMed Scopus (66) Google Scholar), and macrophages(3Khoo J.C. Mahoney E.M. Witztum J.L. J. Biol. Chem. 1981; 256: 7105-7108Abstract Full Text PDF PubMed Google Scholar). After synthesis, the enzyme is transported across the endothelium to its site of activity on the luminal surface of endothelial cells(4Eckel R.H. N. Engl. J. Med. 1989; 320: 1060-1068Crossref PubMed Scopus (857) Google Scholar). The enzyme binds to this site via electrostatic interactions with cell surface proteoglycans (5Saxena U. Klein M.G. Goldberg I.J. J. Biol. Chem. 1991; 266: 17516-17521Abstract Full Text PDF PubMed Google Scholar, 6Shimada K. Lanzillo J.J. Douglas W. Fanburg B.L. Biochim. Biophys. Acta. 1981; 710: 117-121Crossref Scopus (13) Google Scholar) and, perhaps, via protein-protein interaction involving a non-proteoglycan binding protein(7Sivaram P. Klein M.G. Goldberg I.J. J. Biol. Chem. 1992; 267: 16517-16522Abstract Full Text PDF PubMed Google Scholar). lipoprotein lipase very low density lipoprotein low density lipoprotein high density lipoprotein triglyceride phosphate-buffered saline bovine serum albumin Tris-buffered saline monoclonal antibody. A number of experimental observations suggest that LPL has a greater affinity for LDL than for HDL. LPL is primarily a triacylglycerol hydrolase, and its enzymatic actions are usually assayed using a TG-rich substrate(8Hietanen E. Greenwood M.R.C. J. Lipid Res. 1977; 18: 480-490Abstract Full Text PDF PubMed Google Scholar, 9Nilsson-Ehle P. Schotz M.C. J. Lipid Res. 1976; 17: 536-541Abstract Full Text PDF PubMed Google Scholar). Yet LPL-mediated hydrolysis of LDL, a cholesteryl ester-rich lipoprotein, and VLDL, a TG-rich lipoprotein, had a lower Km than HDL(10Deckelbaum R.J. Eisenberg S. Levy E. Olivecrona T. Bengtsson-Olivecrona G. Circulation. 1985; 72: III93Crossref Scopus (39) Google Scholar). Further evidence that LPL does not bind indiscriminately to all classes of lipoproteins was obtained from studies of human postheparin plasma. Most active LPL in postheparin plasma elutes during gel filtration in a major peak that precedes the peak of LDL cholesterol(11Goldberg I.J. Kandel J.J. Blum C.B. Ginsberg H.N. J. Clin. Invest. 1986; 78: 1523-1528Crossref PubMed Scopus (55) Google Scholar). Although Vilella et al.(12Vilella E. Joven J. Fernandez M. Vilaro S. Brunzell J.D. Olivecrona T. Bengtsson-Olivecrona G. J. Lipid Res. 1993; 34: 1555-1564Abstract Full Text PDF PubMed Google Scholar) reported that some LPL was also associated with HDL, HDL-associated LPL was less active than the LPL on apoB-containing lipoproteins. Thus, active LPL is associated primarily with LDL or buoyant LDL size lipoproteins in the circulation. In addition to its enzymatic actions, LPL can anchor lipoproteins to cell surface and matrix proteoglycans. This molecular bridge has been postulated to increase lipoprotein retention by subendothelial cell matrix and increase cellular lipoprotein uptake. Both Saxena et al.(13Saxena U. Klein M.G. Vanni T.M. Goldberg I.J. J. Clin. Invest. 1992; 89: 373-380Crossref PubMed Scopus (123) Google Scholar) and Eisenberg et al.(14Eisenberg S. Sehayek E. Olivecrona T. Vlodavsky I. J. Clin. Invest. 1992; 90: 2013-2021Crossref PubMed Scopus (195) Google Scholar) showed that LPL anchors apoB-containing lipoproteins to subendothelial cell matrix. Saxena et al.(13Saxena U. Klein M.G. Vanni T.M. Goldberg I.J. J. Clin. Invest. 1992; 89: 373-380Crossref PubMed Scopus (123) Google Scholar) were unable to show any increase in LPL-mediated HDL binding to matrix. In contrast, Eisenberg et al.(14Eisenberg S. Sehayek E. Olivecrona T. Vlodavsky I. J. Clin. Invest. 1992; 90: 2013-2021Crossref PubMed Scopus (195) Google Scholar) reported that LPL caused a small increase in HDL association with matrix heparan sulfate proteoglycans. This increase in HDL binding was almost an order of magnitude less than that found for LDL or VLDL. Saxena et al.(13Saxena U. Klein M.G. Vanni T.M. Goldberg I.J. J. Clin. Invest. 1992; 89: 373-380Crossref PubMed Scopus (123) Google Scholar) performed their experiments in media containing serum lipoproteins that could have competed for LPL binding to HDL. This might explain the disparity between results. Nonetheless, LPL appears to preferentially anchor apoB-containing particles. How does LPL interact with lipoproteins? One hypothesis is that LPL has several hydrophobic regions that bind to lipid molecules on the surface of large particles containing a TG core(15Yang C-Y. Gu Z-W. Yang H-X. Rohde M.F. Gotto A.M. Pownall H.J. J. Biol. Chem. 1989; 264: 16822-16827Abstract Full Text PDF PubMed Google Scholar). LPL, however, does not associate well with protein-free lipid particles. Fielding and Fielding (16Fielding C.J. Fielding P.E. Biochim. Biophys. Acta. 1980; 620: 440-446Crossref PubMed Scopus (20) Google Scholar) reported a method of partial LPL purification by incubating postheparin plasma with 5 mg/ml TG derived from 20% Intralipid in low ionic strength buffer. Even with this protocol in which the TG concentration was severalfold greater than the usual plasma cholesterol or TG concentrations, less than 50% of the LPL activity was recovered with the lipid. Additional studies have shown that LPL associates better with apoB-containing lipoproteins than with Intralipid. In the studies of Rumsey et al.(17Rumsey S.C. Obunike J.C. Arad Y. Deckelbaum R.J. Goldberg I.J. J. Clin. Invest. 1992; 90: 1504-1512Crossref PubMed Scopus (155) Google Scholar), the LPL-mediated increase in emulsion particle binding to the surface of fibroblasts was much less than that found using LDL. Thus, optimal LPL association with lipoproteins may require more than just lipid. In the experiments presented in this paper, we studied LPL interaction with lipoproteins and lipid emulsions. Using a solid phase plate assay, we assessed the interaction between LPL and lipoproteins in a system that was free of lipoprotein receptors and cell surface proteoglycans. Because more LPL associated with LDL than HDL or lipid emulsions, we studied the roles of apoB and lipoprotein lipid in this process. Our data support a role for apoB, specifically the amino-terminal region of apoB, in LPL interaction with lipoproteins. Human LDL (d 1.019-1.063 g/ml) and HDL (d 1.063-1.21 g/ml) were isolated from EDTA-containing plasma by ultracentrifugation(18Havel R.J. Eder H.A. Bragdon J.H. J. Clin. Invest. 1955; 34: 1345-1353Crossref PubMed Scopus (6487) Google Scholar). Apolipoprotein patterns of all the lipoprotein preparations were analyzed by SDS-polyacrylamide gel electrophoresis. Total protein concentration of lipoproteins was determined by the method of Lowry et al.(19Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) with bovine serum albumin (BSA) as a standard. A 6507-base pair EcoRI fragment encoding the amino-terminal 46% of human apoB was subcloned from a pCMV5 vector containing the apoB78 cDNA (a gift from Brian Blackhart and Brian McCarthy) into the EcoRI site of pBluescript M13+ (Stratagene, La Jolla, CA) to form pBS-B46. A 2569-base pair BamHI fragment from this vector was then inserted into the BamHI site of the baculovirus transfer vector pAcYM1, yielding pAcB17. This plasmid was cotransfected with the wild-type baculovirus genome (Autographacalifornica nuclear polyhedrosis virus) into Spodoptera frugiperda Sf-21 cells. Recombinant viruses were then selected, plaque-purified, and titered as described(20Summers M.D. Smith G.E. Tex. Agric. Exp. Stn. Bull. 1987; 1555Google Scholar, 21Miller D.W. Safer P. Miller L.K. Genetic Engineering. Plenum Press, New York1986: 277-298Crossref Google Scholar). These viruses encode the 27-amino acid apoB signal peptide as well as the amino-terminal 782 amino acids of mature apoB. Tissue culture supernatants from expressing and nonexpressing cells were harvested, and cells were removed by centrifugation at 1000 × g for 5 min. Phenylmethylsulfonyl fluoride at 0.001% and benzamidine at 0.3 mg/ml were added to the supernatant. Samples were analyzed by 10% SDS-polyacrylamide gel electrophoresis (22Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207231) Google Scholar) followed by either Coomassie Blue staining or immunoblotting (23Burnette W.N. Anal. Biochem. 1981; 112: 195-203Crossref PubMed Scopus (5916) Google Scholar) with anti-human apoB monoclonal antibodies 1D1 and CC3.4 (24Pease R.J. Milne R.W. Jessup W.K. Law A. Provost P. Fruchart J-C. Dean R.T. Marcel Y.L. Scott J. J. Biol. Chem. 1990; 265: 553-568Abstract Full Text PDF PubMed Google Scholar, 25Krul E.S. Kleinman Y. Kinoshita M. Pfleger B. Oida K. Law A. Scott J. Pease R. Schonfeld G. J. Lipid Res. 1988; 29: 937-947Abstract Full Text PDF PubMed Google Scholar) and alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma). In some experiments, amino-terminal fragments of apoB were generated by thrombin digestion of human LDL as described by Cardin et al.(26Cardin A.D. Witt K.R. Chai J. Margolius H.S. Donaldson V.H. Jackson R.L. J. Biol. Chem. 1984; 259: 8522-8528Abstract Full Text PDF PubMed Google Scholar). Cholesteryl ester-rich lipid emulsions were prepared as reported previously (17Rumsey S.C. Obunike J.C. Arad Y. Deckelbaum R.J. Goldberg I.J. J. Clin. Invest. 1992; 90: 1504-1512Crossref PubMed Scopus (155) Google Scholar) by adding 40 mg of triolein (Nu-Check, Elysian, MN) and 40 mg of cholesteryl oleate to 80 mg of egg yolk phosphatidylcholine (Avanti Polar Lipids, Inc., Alabaster, AL). LPL was purified from fresh bovine milk as described previously using the method of Socorro et al.(27Socorro L. Green C.C. Jackson R.L. Prep. Biochem. 1985; 15: 133-143Crossref PubMed Scopus (26) Google Scholar). Protein was determined by the method of Lowry et al.(19Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). When analyzed by SDS-polyacrylamide gel electrophoresis, a single band with a molecular mass of approximately 55 kDa was identified on a Coomassie staining (data not shown). Enzyme activity was assayed using a method described by Nilsson-Ehle and Schotz(9Nilsson-Ehle P. Schotz M.C. J. Lipid Res. 1976; 17: 536-541Abstract Full Text PDF PubMed Google Scholar). The purified enzyme was stored at −70°C. Purified LPL was radioiodinated (7Sivaram P. Klein M.G. Goldberg I.J. J. Biol. Chem. 1992; 267: 16517-16522Abstract Full Text PDF PubMed Google Scholar) using lactoperoxidase and glucose oxidase enzymes (Sigma) as reported(7Sivaram P. Klein M.G. Goldberg I.J. J. Biol. Chem. 1992; 267: 16517-16522Abstract Full Text PDF PubMed Google Scholar). A typical preparation had a specific activity of about 800-1000 cpm/ng LPL, and more than 90% of the counts were precipitable by 10% trichloroacetic acid. LPL was biotinylated as described by Sivaram et al.(28Sivaram P. Wadhwani S.W. Klein M.G. Sasaki A. Goldberg I.J. Anal. Biochem. 1993; 214: 511-516Crossref PubMed Scopus (6) Google Scholar). Briefly, N-hydroxysuccinimide ester of biotin (Vector Laboratories, Burlingame, CA) dissolved in dimethyl sulfoxide was added to purified LPL and incubated for 10 min at 4°C. Biotinylated LDL was then purified from the mixture using a heparin-agarose column, and fractions were characterized by ligand blotting using streptavidin. LPL activity was also measured, and active fractions containing biotinylated LPL were pooled and stored in aliquots at −70°C. Either ascites containing the hybridoma antibodies or purified IgG was used. The monoclonal antibodies mAb 3 and mAb 19 have determinants in the amino-terminal region of apoB(29Curtiss L.K. Edgington T.S. J. Biol. Chem. 1982; 257: 15213-15221Abstract Full Text PDF PubMed Google Scholar). mAb 47 recognizes the LDL receptor binding site of apoB close to the carboxyl terminus(24Pease R.J. Milne R.W. Jessup W.K. Law A. Provost P. Fruchart J-C. Dean R.T. Marcel Y.L. Scott J. J. Biol. Chem. 1990; 265: 553-568Abstract Full Text PDF PubMed Google Scholar). Samples were analyzed using polyacrylamide gels containing 0.5% SDS(22Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207231) Google Scholar). Proteins were then transferred to a nitrocellulose membrane (Schleicher and Schuell) using a Milliblot SDE semidry blotting system (Millipore Corp., Bedford, MA) and then incubated overnight in PBS containing 3% BSA at 4°C. The membrane was then incubated with biotinylated LPL for 2 h at 4°C and then washed six times with PBS containing 0.3% BSA. Proteins bound to biotinylated LPL were visualized by incubation with avidin-conjugated horseradish peroxidase followed by development with 4-chloro-1-naphthol (peroxidase substrate)(7Sivaram P. Klein M.G. Goldberg I.J. J. Biol. Chem. 1992; 267: 16517-16522Abstract Full Text PDF PubMed Google Scholar). Solid phase plate assays were performed as described by Williams et al.(30Williams S.E. Ashcom J.D. Argraves W.S. Stricklands D.K. J. Biol. Chem. 1992; 267: 9035-9040Abstract Full Text PDF PubMed Google Scholar). For binding studies, LDL, HDL, or VLDL, diluted in 50 mM Tris, 150 mM NaCl, pH 7.4 (TBS) containing 5 mM Ca2+ was added to 96-well plates and incubated at 4°C overnight. The unbound lipoproteins were removed, and the plates were washed three times with 0.3% BSA in TBS. Then the plates were incubated with 3% BSA in TBS, 5 mM Ca2+ for 1 h at room temperature to block nonspecific binding sites. After washing with 0.3% BSA in TBS, 125I-LPL in TBS, 5 mM Ca2+, 3% BSA was added and incubated for 24 h at 4°C. Again, the plates were washed, and 200 μl of 0.1 N NaOH was added. An aliquot of 150 μl was counted. For competition studies, LDL (50 μg/ml protein, 91 nM) was bound to 96-well plates overnight at 4°C. The experimental protocol used in this experiment was identical to the one described for the binding experiments except that 125I-LPL (5 μg/ml, 45 nM) was added along with competitors. After incubating for 2 h at room temperature, unbound 125I-LPL was removed, and radioactivity bound to the well was measured. Binding to BSA-coated wells was determined in each assay as a control. Competition studies were performed using Intralipid (20%) (Vitrum, Stockholm) and cholesteryl ester-rich emulsions. The molar concentration of Intralipid was calculated using a diameter of 300 nm/lipid particle. Cholesteryl ester-rich lipid emulsion concentration was calculated based on the total neutral lipid concentration (i.e. 30 μg/ul) and assuming that 1 μmol of the particle contains 5 × 106μg. The protein molecular masses of 550 kDa for LDL, 150 kDa for HDL, and 110 kDa for LPL dimers were used to calculate the concentrations of these lipoproteins. For VLDL we estimated that the total molecular mass of VLDL was approximately 1 × 104 kDa and that 10% of this would be the protein mass; thus, we used 1 × 103 kDa as a molecular mass of VLDL. To determine the binding of apoB17 to LPL, enzyme-linked immunosorbent assay was performed using a monoclonal antibody against the amino-terminal region of apoB (mAb 19). Briefly, LPL or BSA at 5 μg/ml was incubated at 4°C overnight in microtiter plates. After washing the plates four times with 0.3% BSA in PBS and blocking with 3% BSA in PBS for 1 h, various concentrations of apoB17 were added. The plates were incubated for 2 h at room temperature, washed four times, and then incubated for another 2 h at room temperature with primary antibody (mAb 19 diluted to 1:500 in PBS). Unbound antibodies were removed, and horseradish peroxidase-conjugated goat anti-mouse IgG (Kirkegaard & Perry Laboratories, Gaithersburg, MD) at 1:500 dilution was added. One and a half hours later, 100 μl of substrate solution containing o-phenylenediamine dihydrochloride (Sigma), 5 μl of 50% H2O2 in 0.1 M citric acid (pH 6) was added to each well. After 20 min at room temperature the absorbance at 492 nm was measured. 125I-LPL binding to LDL-, HDL-, and VLDL-coated plates was determined. We first measured the amounts required to saturate the binding sites of the plate. To do this, 125I-LDL, 125I-HDL, or 125I-VLDL at various protein concentrations (1-100 μg/ml) were incubated overnight at 4°C in microtiter plate wells. After washing, labeled lipoprotein in each well was assessed. Approximately 10 μg/ml LDL or HDL and 25 μg/ml VLDL were required to saturate the plate. At this concentration, 2.3 × 10−4 nmol of LDL protein, 6.1 × 10−4 nmol of HDL protein, and 2.43 × 10−4 nmol of VLDL protein were bound to each well. Thus, severalfold more HDL than LDL or VLDL bound to the plate at all concentrations of lipoproteins. We next assessed 125I-LPL binding to the lipoprotein-coated plates. First, we examined the time course of LPL binding to these lipoproteins. LPL binding reached equilibrium after overnight incubation at 4°C (data not shown). LPL binding to albumin-coated plates was assessed and used as an estimate of nonspecific binding. Data shown in Fig. 1 were obtained after coating the plates overnight at 4°C with 50 μg/ml HDL or LDL. This is a concentration above that required to saturate the plate. The data are expressed in terms of nanomoles of lipoprotein on the plate. 125I-LPL (0.9-55 nM) binding to LDL (opencircles) was significantly greater than to HDL (solidcircles). Using 14 nM LPL, approximately 5-fold more LPL bound to the LDL-coated plates than to the HDL-coated plates. At 14 nM125I-LPL the molar ratio of LPL binding to LDL was 1:1. Although using greater LPL concentrations increased the molar ratio of LPL to LDL (e.g. to 1.6 using 55 nM125I-LPL), the rate of increase of this additional LPL binding was less steep. This suggests that a second, lower affinity process exists. Binding of LPL to VLDL was similar to that of LDL. At each concentration of 125I-LPL used, nonspecific binding to BSA-coated plates was <10% of the total binding, and this was subtracted. The experiments above suggested that LPL has a greater affinity for LDL than for HDL. We next tested whether LPL interaction with LDL was competed by excess unlabeled LDL or Intralipid. In these experiments, we used a short 2-h incubation at room temperature to minimize the hydrolysis of TG in Intralipid. Plates were coated overnight at 4°C with LDL (50 μg/ml), and then 125I-LPL at 45 nM was added alone or with various concentrations of competitors. 125I-LPL binding to LDL was blocked by >95% by a 100-fold molar excess of unlabeled LPL (data not shown). This demonstrates the specificity of the interaction between 125I-LPL and LDL. Unlabeled LDL at 9 × 102 nM inhibited LPL association with the plate by 65% (Fig. 2). In contrast, using a similar protocol and molar concentration of Intralipid particles, only about 20% inhibition was observed. Furthermore, inhibition of lipase activity with phenylmethylsulfonyl fluoride did not result in any difference in the competition between LDL and Intralipid for LPL binding (data not shown). These data suggested that LPL interaction with LDL involved more than a simple protein-lipid interaction. To assess the role of LDL lipid in LPL-LDL interactions, we studied the effects of delipidation of LDL on LPL interaction with LDL. Plates were coated with [3H]cholesteryl ester-rich emulsion particles and were delipidated by incubating with an acetone:ethanol (1:1) mixture for 1 h at −20°C. Approximately 80% of the 3H-lipid was removed by this method (data not shown). This protocol was then used to assess 125I-LPL binding to delipidated LDL-coated plates. As shown in Fig. 3(inset), LDL binding to control and LDL binding to delipidated plates were virtually identical. Fig. 3 shows data comparing LDL competition studies performed using control and delipidated LDL-coated plates. 181 nM unlabeled LDL inhibited the binding of 125I-LPL to LDL-coated plates by approximately 50% in both control and delipidated plates. Thus, delipidation did not result in a significant reduction in 125I-LPL binding to LDL nor was its binding to LPL more easily disrupted by competition with LDL in solution. These data further suggest that apoB, not LDL lipid, mediates LPL binding. These experiments were repeated with a method described by Patton et al.(31Patton J.G. Alley M.C. Mao S.J.T. J. Immunol. Methods. 1982; 55: 193-203Crossref PubMed Scopus (23) Google Scholar) using hexane:isopropyl alcohol (3:2, v/v), which was shown to remove 95% of cholesteryl ester and TG. The results were identical to those obtained with acetone:ethanol (1:1). In a separate experiment, we examined the role of phospholipid components of LDL in LPL binding using microtiter plates coated with sphingomyelin, phosphatidylcholine, or lysolecithin. Binding of LPL to these phospholipids was less than 20% of the LPL binding to LDL (data not shown). These data suggest that LDL-LPL interaction is not mediated by interaction between phospholipid and LPL. To test whether LPL would bind to apoB and other apolipoproteins, ligand blotting was performed. Twenty μg of apoA1, apoE, and thrombin-digested human LDL were applied to 3-15% SDS-polyacrylamide gels under nonreducing conditions, transferred to nitrocellulose paper, and probed with biotinylated LPL. Among these proteins, only thrombin-digested fragments of apoB were recognized by LPL (Fig. 4). Furthermore, the positive reaction was confined to amino-terminal parts of apoB, in agreement with the previous data(32Sivaram P. Choi S.Y. Curtiss L.K. Goldberg I.G. J. Biol. Chem. 1994; 269: 9409-9412Abstract Full Text PDF PubMed Google Scholar). Neither apoA1, apoE, nor the carboxyl-terminal regions of apoB were recognized on ligand blots. To further characterize the interaction between LPL and apoB, we used several monoclonal antibodies against apoB. In this experiment microtiter plates were also coated with LPL (90 nM) at 4°C. Following washing and blocking, 125I-LDL (18 nM) was added with antibodies (1:50 dilution of ascites) and incubated for 2 h at 4°C. As shown in Fig. 5, both mAb 3 and mAb 19 inhibited 125I-LPL binding to LDL by 80%, whereas mAb 47 inhibited binding by only 25%. In other experiments we showed that this concentration of mAb 47 inhibited specific LDL binding to fibroblasts by >90%. To further assess the role of the amino-terminal region of apoB versus LDL lipid in LPL-LDL interaction, we performed competition experiments using apoB17 and cholesteryl ester-rich emulsion particles. In these experiments, 125I-LPL binding to LDL-coated plates was competed with apoB17 and cholesteryl ester-rich emulsions. BSA was also used as a negative control, and it did not inhibit the 125I-LPL binding to LDL (data not shown). ApoB17 competed; apoB17 at 1.7 × 103 nM decreased LPL binding to the plates by 74%. In contrast, cholesteryl ester-rich emulsion particles at 1.8 × 103 nM inhibited 125I-LPL binding to LDL-coated plates by approximately 30% (Fig. 6A). We then determined the binding of apoB17 to LPL using LPL-coated plates. The apoB17 was detected by enzyme-linked immunosorbent assay as described under “Materials and Methods.”Fig. 6B shows binding of apoB17 (0-1 μg/ml, 45 mM) to LPL- and BSA-coated plates. At 0.1 μg/ml (4.5 nM) approximately 10-fold more apoB17 bound to LPL-coated plates than to BSA-coated plates. Thus, several types of experiments support the hypothesis that LPL binding to LDL is modulated by apoB and that this process involves a specific interaction with the amino-terminal region of apoB. The present experiments suggest a new role for the amino-terminal region of apoB as a mediator of LPL interaction with lipoproteins. Data supporting this conclusion were obtained by several different methods. When 125I-LPL was added to LDL- or HDL-coated plates and incubated at 4°C overnight to achieve equilibrium, severalfold more LPL bound to LDL- than to the HDL-coated plates (e.g. approximately 6-fold with 55 nM125I-LPL). Binding of LPL to VLDL was similar to that of LDL, indicating that protein-protein interaction between LPL and apoB is more important than protein-lipid interaction. In these experiments, we did not calculate the kinetic parameters using a method such as Scatchard analysis (33Scatchard G. Ann. N. Y. Acad. Sci. 1949; 51: 660-672Crossref Scopus (17806) Google Scholar) for several reasons. First, LPL aggregates at higher concentrations. Second, LPL dimers dissociate over time. Thus, it is likely that monomerization and denaturation of the LPL occurred during the overnight incubation. Because active dimeric LPL is more likely to associate with LDL and inactive LPL associates with HDL(12Vilella E. Joven J. Fernandez M. Vilaro S. Brunzell J.D. Olivecrona T. Bengtsson-Olivecrona G. J. Lipid Res. 1993; 34: 1555-1564Abstract Full Text PDF PubMed Google Scholar), the Kd for active LPL binding to LDL might be lower and the Kd for HDL higher. Nonetheless, we conclude that LPL interaction with LDL is greater than with HDL and hypothesize that this is due to the presence of apoB on LDL. Further support for this hypothesis was obtained using ligand blots. LPL bound to amino-terminal fragments of apoB generated by thrombin digestion but not to apoA1, apoE, or carboxyl-terminal fragments of apoB. Thus, the reason LPL preferentially associates with LDL in plasma and on cell surfaces might result from LPL association with apoB. Apolipoproteins other than apoB may interact with LPL. Both apoCIII (34Brown W.V. Baginsky M.L. Biochem. Biophys. Res. Commun. 1972; 46: 375-382Crossref PubMed Scopus (344) Google Scholar) and apoE (35Ekman R. Nilsson-Ehle P. Clin. Chim. Acta. 1975; 63: 29-35Crossref PubMed Scopus (66) Google Scholar) have been reported to decrease LPL activity when added to some in vitro assay systems. ApoE, however, did not interact with LPL on ligand blots (Fig. 4). ApoE shares two properties with apoB that make it unique among plasma apolipoproteins; both bind to LDL receptors and both interact with a variety of glycosaminoglycans including heparin(36Weisgraber K.H. Rall Jr., S.C. Mahley R.W. Milne R.W. Marcel Y.V. Sparrow J.T. J. Biol. Chem. 1986; 261: 2068-2076Abstract Full Text PDF PubMed Google Scholar, 37Hui D.Y. Innerarity T.L. Mahley R.W. J. Biol. Chem. 1980; 255: 1804-1807Abstract Full Text PDF PubMed Google Scholar). Because the homologous areas of apoB and apoE are near the carboxyl-terminal region of apoB (38Milne R. Theolis Jr., R. Maurice R. Pease R.J. Weech P.K. Rassart E. Fruchart J.C. Scott J. Marcel Y.L. J. Biol. Chem. 1989; 264: 19754-19760Abstract Full Text PDF PubMed Google Scholar) and LPL binds to the amino-terminal portion of apoB, it is not surprising that apoE did not directly bind to LPL on ligand blots. ApoCII is the obligatory activator for LPL hydrolysis of lipoprotein TG, but the mechanism of apoCII-LPL interaction is uncertain. Surprisingly, Shirai et al.(39Shirai K. Matsuoka N. Jackson R.L. Biochim. Biophys. Acta. 1981; 665: 504-510Crossref PubMed Scopus (23) Google Scholar) showed that addition of apoCII to phospholipid emulsion particles decreased LPL association with the emulsion. Thus, LPL interaction with apoB appears to differ from its interaction with other apolipoproteins. A second lipoprotein component that might affect LPL-lipoprotein interaction is lipid. Lipoproteins contain both core and surface lipid. In addition to affecting the size and geometry of the lipoprotein surface, some hydrophobic core lipids (TG and cholesteryl ester) are thought to be exposed on the lipoprotein surface(41Deckelbaum R.J. Shipley G.G. Small D.M. J. Biol. Chem. 1977; 252: 744-754Abstract Full Text PDF PubMed Google Scholar). To assess the role of lipids in LPL binding to particles, LPL binding to LDL was competed with Intralipid (a TG-rich, protein-deficient emulsion) and a cholesteryl ester-rich emulsion. LPL binding to LDL was inhibited by excess LDL and by the amino-terminal fragment of apoB. Less inhibition was observed with the same or greater molar concentrations of lipid emulsion particles. Further evidence that lipid is not critical for LPL-LDL interaction was obtained by comparing LPL binding to LDL- and delipidated LDL-coated plates. Binding and competition studies using these two LDL-coated plates were indistinguishable. Thus, neither the core nor surface lipids interacted with LPL in the same manner as apoB. Active LPL is thought to be a noncovalently linked homodimer, and each subunit molecular mass is approximately 55 kDa. This enzyme contains five functional sites (15Yang C-Y. Gu Z-W. Yang H-X. Rohde M.F. Gotto A.M. Pownall H.J. J. Biol. Chem. 1989; 264: 16822-16827Abstract Full Text PDF PubMed Google Scholar, 42Enerback S. Semb H. Bengtsson-Olivecrona G. Carlsson P. Hermansson M.-L. Olivecrona T. Bjursell G. Gene (Amst.). 1987; 58: 1-12Crossref PubMed Scopus (69) Google Scholar) including (a) a catalytic site containing an active serine residue, (b) an interfacial substrate recognition site consisting of hydrophobic basic residues, (c) a heparin binding site, (d) an apolipoprotein CII binding site, and (e) a site for subunit-subunit interaction. Although it has been suggested that LPL interaction with lipoproteins involves the hydrophobic lipid recognition sites, there are several studies that do not support this hypothesis. Hydrophobic interactions are potentiated by high ionic strength solutions; however, LPL is dissociated from lipoproteins and lipid particles in high salt solutions(11Goldberg I.J. Kandel J.J. Blum C.B. Ginsberg H.N. J. Clin. Invest. 1986; 78: 1523-1528Crossref PubMed Scopus (55) Google Scholar). Although LPL appears to interact with polar lipid, i.e. phospholipid, our studies using phospholipid-coated plates (data not shown) and previously published data (17Rumsey S.C. Obunike J.C. Arad Y. Deckelbaum R.J. Goldberg I.J. J. Clin. Invest. 1992; 90: 1504-1512Crossref PubMed Scopus (155) Google Scholar) demonstrate that such an interaction is much weaker than LPL association with lipoproteins. It is not clear whether LPL interaction with apoB is required for lipolysis of TG-rich lipoproteins. In our preliminary experiments, a monoclonal antibody against apoB, mAb 19, did not inhibit LPL hydrolysis of VLDL, suggesting that this interaction is not required for lipolysis (data not shown). However, Connelly et al.(40Connelly P.W. Maguire G.F. Vezina C. Hegele R.A. Kuksis A. J. Biol. Chem. 1994; 269: 20554-20560Abstract Full Text PDF PubMed Google Scholar) reported that lipolysis of VLDL by LPL was noncompetitively inhibited by LDL and intermediate density lipoproteins. Thus, LPL may associate with LDL or intermediate density lipoproteins, and this in turn prevents the association of LPL with VLDL. ApoB100, a large glycoprotein with a molecular mass of 550 kDa, is virtually the only protein component of LDL particles. The carboxyl-terminal region of apoB is involved in binding to LDL receptors, and this region also contains five of seven apoB heparin binding sites(43Weisgraber K.H. Rall Jr., S.C. J. Biol. Chem. 1987; 262: 11097-11103Abstract Full Text PDF PubMed Google Scholar). Under physiologic ionic conditions, however, LDL binds weakly to heparin(44Iverius P.-H. J. Biol. Chem. 1972; 247: 2607-2613Abstract Full Text PDF PubMed Google Scholar), demonstrating the importance of tertiary structure in apoB interaction with other molecules. ApoB also contains numerous hydrophobic domains throughout its length that are believed to be important in lipid binding (45Olofsson S.O. Bjursell G. Bostrom K. Carlsson P. Elovson J. Protter A.A. Reuben M.A. Bondjers G. Atherosclerosis. 1987; 68: 1-17Abstract Full Text PDF PubMed Scopus (126) Google Scholar) and multiple proline-rich sequences predicted to form amphipathic β-sheets and β-turns that are thought to have high lipid binding potential(46Knott T.J. Pease R.J. Powell L.M. Wallis S.C. Rall Jr., S.C. Innerarity T.L. Blackhart B. Taylor W.H. Marcel Y. Milne R. Johnson D. Fuller M. Lusis A.J. McCarthy B.J. Mahly R.W. Levy-Wilson B. Scott J. Nature. 1986; 323: 734-738Crossref PubMed Scopus (402) Google Scholar, 47De Loof H. Rosseneu M. Yang C.Y. Li W.H. Gotto Jr., A.M. Chan L. J. Lipid Res. 1987; 28: 1455-1465Abstract Full Text PDF PubMed Google Scholar). These sequences, however, are not found in the amino-terminal 1000 amino acids. In addition, apoB contains 25 cysteine residues of which 12, in disulfide form, are located in the first 500 amino acids. Thus, the amino-terminal region of apoB is thought to be a globular structure that extends away from the lipid core of lipoproteins(48Chan L. J. Biol. Chem. 1992; 267: 25621-25624Abstract Full Text PDF PubMed Google Scholar). The tertiary structure of apoB varies as a function of the size, lipid composition, and, perhaps, apolipoprotein content of lipoproteins. Galeano et al.(49Galeano N.F. Milne R. Marcel Y.L. Walsh M.T. Levy E. Ngu'yen T. Gleeson A. Arad Y. Witte L. Al-Haideri M. Rumsey S.C. Deckelbaum R.J. J. Biol. Chem. 1994; 269: 511-519Abstract Full Text PDF PubMed Google Scholar) recently demonstrated that LDL particle size rather than core lipid composition was the major determinant of LDL interaction with the LDL receptor. Kunitake et al.(50Kunitake S.T. Young S.G. Chen G.C. Pullinger C.R. Zhu S. Pease R.J. Scott J. Phillip H. Schilling J.S. Kane J.P. J. Biol. Chem. 1990; 265: 20739-20746Abstract Full Text PDF PubMed Google Scholar) reported that (a) the type of lipoproteins on which apoB resides, i.e. VLDL, intermediate density lipoprotein, or LDL, and (b) the core lipid composition, i.e. TG:cholesterol ratio, affect the conformation of apoB in the amino-terminal region. Similarly, McKeone et al.(51McKeone B.J. Patsch J.R. Pownall H.J. J. Clin. Invest. 1993; 91: 1926-1933Crossref PubMed Scopus (73) Google Scholar) demonstrated that TG induced structural changes in apoB remote from the receptor binding region. These changes included differences in immunoreaction with antibody mAb 3 and Staphylococcus aureus V8 protease cleavage at approximately the first 100 kDa of apoB. Whether such differences in the conformation of the amino-terminal region of apoB alter LPL-lipoprotein association remains to be established. LPL-apoB interactions could play a role in efficient catabolism of circulating lipoproteins. The initial event in lipoprotein TG hydrolysis may be apoE binding to heparan sulfate proteoglycans, a process that anchors circulating lipoproteins to the endothelial cell surface. Data supporting this function of apoE have been reported by several laboratories(52Ji Z.-S. Brecht W.J. Miranda R.D. Hussain M.M. Innerarity T.L. Mahley R.W. J. Biol. Chem. 1992; 268: 10160-10167Abstract Full Text PDF Google Scholar, 53Mahley R.W. Weisgraber K.H. Innerarity T.L. Biochim. Biophys. Acta. 1979; 575: 81-89Crossref PubMed Scopus (102) Google Scholar). We postulate that a secondary interaction that further approximates the lipoprotein and LPL is LPL interaction with the amino-terminal region of apoB. We thank Dr. Joseph Obunike for purifying bovine LPL." @default.
• W2024408717 created "2016-06-24" @default.
• W2024408717 creator A5013807783 @default.
• W2024408717 creator A5023841024 @default.
• W2024408717 creator A5025548638 @default.
• W2024408717 creator A5027370958 @default.
• W2024408717 creator A5059089783 @default.
• W2024408717 creator A5066239059 @default.
• W2024408717 creator A5066344931 @default.
• W2024408717 creator A5066507748 @default.
• W2024408717 creator A5069227122 @default.
• W2024408717 date "1995-04-01" @default.
• W2024408717 modified "2023-09-27" @default.
• W2024408717 title "Lipoprotein Lipase Association with Lipoproteins Involves Protein-Protein Interaction with Apolipoprotein B" @default.
• W2024408717 cites W1485292742 @default.
• W2024408717 cites W1489542806 @default.
• W2024408717 cites W1502732154 @default.
• W2024408717 cites W1505230657 @default.
• W2024408717 cites W1508962214 @default.
• W2024408717 cites W1516552294 @default.
• W2024408717 cites W1524608424 @default.
• W2024408717 cites W1531031204 @default.
• W2024408717 cites W1534056652 @default.
• W2024408717 cites W1539895365 @default.
• W2024408717 cites W1552903954 @default.
• W2024408717 cites W1553982335 @default.
• W2024408717 cites W1558242213 @default.
• W2024408717 cites W1558433859 @default.
• W2024408717 cites W1604410629 @default.
• W2024408717 cites W1605483534 @default.
• W2024408717 cites W1775749144 @default.
• W2024408717 cites W1874360846 @default.
• W2024408717 cites W1900087291 @default.
• W2024408717 cites W1952404770 @default.
• W2024408717 cites W1983649348 @default.
• W2024408717 cites W2020885344 @default.
• W2024408717 cites W2021983069 @default.
• W2024408717 cites W2027773820 @default.
• W2024408717 cites W2028640832 @default.
• W2024408717 cites W2044021360 @default.
• W2024408717 cites W2044512420 @default.
• W2024408717 cites W2049556830 @default.
• W2024408717 cites W2055195672 @default.
• W2024408717 cites W2060324183 @default.
• W2024408717 cites W2078198093 @default.
• W2024408717 cites W2080408033 @default.
• W2024408717 cites W2081010186 @default.
• W2024408717 cites W2088843957 @default.
• W2024408717 cites W2092359033 @default.
• W2024408717 cites W2100837269 @default.
• W2024408717 cites W2103933115 @default.
• W2024408717 cites W2111817213 @default.
• W2024408717 cites W2122887823 @default.
• W2024408717 cites W2123035356 @default.
• W2024408717 cites W2123163977 @default.
• W2024408717 cites W2129209158 @default.
• W2024408717 cites W2135503869 @default.
• W2024408717 cites W2144320579 @default.
• W2024408717 cites W2155759352 @default.
• W2024408717 cites W2160129472 @default.
• W2024408717 cites W2173345687 @default.
• W2024408717 cites W2183619190 @default.
• W2024408717 cites W2336692995 @default.
• W2024408717 cites W4240237349 @default.
• W2024408717 doi "https://doi.org/10.1074/jbc.270.14.8081" @default.
• W2024408717 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/7713910" @default.
• W2024408717 hasPublicationYear "1995" @default.
• W2024408717 type Work @default.
• W2024408717 sameAs 2024408717 @default.
• W2024408717 citedByCount "50" @default.
• W2024408717 countsByYear W20244087172012 @default.
• W2024408717 crossrefType "journal-article" @default.
• W2024408717 hasAuthorship W2024408717A5013807783 @default.
• W2024408717 hasAuthorship W2024408717A5023841024 @default.
• W2024408717 hasAuthorship W2024408717A5025548638 @default.
• W2024408717 hasAuthorship W2024408717A5027370958 @default.
• W2024408717 hasAuthorship W2024408717A5059089783 @default.
• W2024408717 hasAuthorship W2024408717A5066239059 @default.
• W2024408717 hasAuthorship W2024408717A5066344931 @default.
• W2024408717 hasAuthorship W2024408717A5066507748 @default.
• W2024408717 hasAuthorship W2024408717A5069227122 @default.
• W2024408717 hasBestOaLocation W20244087171 @default.
• W2024408717 hasConcept C126322002 @default.
• W2024408717 hasConcept C174782155 @default.
• W2024408717 hasConcept C181199279 @default.
• W2024408717 hasConcept C185592680 @default.
• W2024408717 hasConcept C2778163477 @default.
• W2024408717 hasConcept C2779134260 @default.
• W2024408717 hasConcept C2779697368 @default.
• W2024408717 hasConcept C2780072125 @default.
• W2024408717 hasConcept C2780213950 @default.
• W2024408717 hasConcept C55493867 @default.
• W2024408717 hasConcept C56623246 @default.
• W2024408717 hasConcept C57089818 @default.
• W2024408717 hasConcept C62746215 @default.
• W2024408717 hasConcept C71924100 @default.
• W2024408717 hasConcept C8243546 @default.
• W2024408717 hasConcept C94879076 @default.

• next