Mobility of “HSPG-bound” LPL explains how LPL is able to reach GPIHBP1 on capillaries
2016; Elsevier BV; Volume: 58; Issue: 1 Linguagem: Inglês
10.1194/jlr.m072520
ISSN1539-7262
AutoresChristopher M. Allan, Mikael Larsson, Rachel S. Jung, Michael Ploug, André Bensadoun, Anne P. Beigneux, Loren G. Fong, Stephen G. Young,
Tópico(s)Lipid metabolism and biosynthesis
ResumoIn mice lacking glycosylphosphatidylinositol-anchored high density lipoprotein binding protein 1 (GPIHBP1), the LPL secreted by adipocytes and myocytes remains bound to heparan sulfate proteoglycans (HSPGs) on all cells within tissues. That observation raises a perplexing issue: Why isn't the freshly secreted LPL in wild-type mice captured by the same HSPGs, thereby preventing LPL from reaching GPIHBP1 on capillaries? We hypothesized that LPL–HSPG interactions are transient, allowing the LPL to detach and move to GPIHBP1 on capillaries. Indeed, we found that LPL detaches from HSPGs on cultured cells and moves to: 1) soluble GPIHBP1 in the cell culture medium; 2) GPIHBP1-coated agarose beads; and 3) nearby GPIHBP1-expressing cells. Movement of HSPG-bound LPL to GPIHBP1 did not occur when GPIHBP1 contained a Ly6 domain missense mutation (W109S), but was almost normal when GPIHBP1's acidic domain was mutated. To test the mobility of HSPG-bound LPL in vivo, we injected GPIHBP1-coated agarose beads into the brown adipose tissue of GPIHBP1-deficient mice. LPL moved quickly from HSPGs on adipocytes to GPIHBP1-coated beads, thereby depleting LPL stores on the surface of adipocytes. We conclude that HSPG-bound LPL in the interstitial spaces of tissues is mobile, allowing the LPL to move to GPIHBP1 on endothelial cells. In mice lacking glycosylphosphatidylinositol-anchored high density lipoprotein binding protein 1 (GPIHBP1), the LPL secreted by adipocytes and myocytes remains bound to heparan sulfate proteoglycans (HSPGs) on all cells within tissues. That observation raises a perplexing issue: Why isn't the freshly secreted LPL in wild-type mice captured by the same HSPGs, thereby preventing LPL from reaching GPIHBP1 on capillaries? We hypothesized that LPL–HSPG interactions are transient, allowing the LPL to detach and move to GPIHBP1 on capillaries. Indeed, we found that LPL detaches from HSPGs on cultured cells and moves to: 1) soluble GPIHBP1 in the cell culture medium; 2) GPIHBP1-coated agarose beads; and 3) nearby GPIHBP1-expressing cells. Movement of HSPG-bound LPL to GPIHBP1 did not occur when GPIHBP1 contained a Ly6 domain missense mutation (W109S), but was almost normal when GPIHBP1's acidic domain was mutated. To test the mobility of HSPG-bound LPL in vivo, we injected GPIHBP1-coated agarose beads into the brown adipose tissue of GPIHBP1-deficient mice. LPL moved quickly from HSPGs on adipocytes to GPIHBP1-coated beads, thereby depleting LPL stores on the surface of adipocytes. We conclude that HSPG-bound LPL in the interstitial spaces of tissues is mobile, allowing the LPL to move to GPIHBP1 on endothelial cells. LPL, a triglyceride hydrolase that is synthesized and secreted by myocytes and adipocytes, is crucial for the lipolytic processing of triglyceride-rich lipoproteins (TRLs) inside blood vessels (1Korn E.D. Clearing factor, a heparin-activated lipoprotein lipase. II. Substrate specificity and activation of coconut oil.J. Biol. Chem. 1955; 215: 15-26Abstract Full Text PDF PubMed Google Scholar, 2Korn E.D. Clearing factor, a heparin-activated lipoprotein lipase. I. Isolation and characterization of the enzyme from normal rat heart.J. Biol. Chem. 1955; 215: 1-14Abstract Full Text PDF PubMed Google Scholar, 3Havel R.J. Gordon Jr, R.S. Idiopathic hyperlipemia: metabolic studies in an affected family.J. Clin. Invest. 1960; 39: 1777-1790Crossref PubMed Scopus (129) Google Scholar). For decades, dogma held that the LPL produced by myocytes and adipocytes was bound to negatively charged heparan sulfate proteoglycans (HSPGs) in the glycocalyx coating of blood vessels (4Merkel M. Eckel R.H. Goldberg I.J. Lipoprotein lipase: genetics, lipid uptake, and regulation.J. Lipid Res. 2002; 43: 1997-2006Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar). This model was plausible because LPL contains positively charged heparin-binding domains (5Ma Y. Henderson H.E. Liu M.S. Zhang H. Forsythe I.J. Clarke-Lewis I. Hayden M.R. Brunzell J.D. Mutagenesis in four candidate heparin binding regions (residues 279-282, 291-304, 390-393, and 439-448) and identification of residues affecting heparin binding of human lipoprotein lipase.J. Lipid Res. 1994; 35: 2049-2059Abstract Full Text PDF PubMed Google Scholar, 6Sendak R.A. Melford K. Kao A. Bensadoun A. Identification of the epitope of a monoclonal antibody that inhibits heparin binding of lipoprotein lipase: new evidence for a carboxyl-terminal heparin-binding domain.J. Lipid Res. 1998; 39: 633-646Abstract Full Text Full Text PDF PubMed Google Scholar, 7Lookene A. Nielsen M.S. Gliemann J. Olivecrona G. Contribution of the carboxy-terminal domain of lipoprotein lipase to interaction with heparin and lipoproteins.Biochem. Biophys. Res. Commun. 2000; 271: 15-21Crossref PubMed Scopus (37) Google Scholar, 8Hata A. Ridinger D.N. Sutherland S. Emi M. Shuhua Z. Myers R.L. Ren K. Cheng T. Inoue I. Wilson D.E. et al.Binding of lipoprotein lipase to heparin. Identification of five critical residues in two distinct segments of the amino-terminal domain.J. Biol. Chem. 1993; 268: 8447-8457Abstract Full Text PDF PubMed Google Scholar), and LPL binds avidly to HSPGs on cultured cells and to HSPGs immobilized on 96-well plates (9de Man F.H. de Beer F. van der Laarse A. Smelt A.H. Havekes L.M. Lipolysis of very low density lipoproteins by heparan sulfate proteoglycan-bound lipoprotein lipase.J. Lipid Res. 1997; 38: 2465-2472Abstract Full Text PDF PubMed Google Scholar). Moreover, LPL was known to be released into the plasma with an injection of heparin (1Korn E.D. Clearing factor, a heparin-activated lipoprotein lipase. II. Substrate specificity and activation of coconut oil.J. Biol. Chem. 1955; 215: 15-26Abstract Full Text PDF PubMed Google Scholar, 2Korn E.D. Clearing factor, a heparin-activated lipoprotein lipase. I. Isolation and characterization of the enzyme from normal rat heart.J. Biol. Chem. 1955; 215: 1-14Abstract Full Text PDF PubMed Google Scholar). Over the past 8 years, however, this model for intravascular lipolysis has changed. We now recognize that glycosylphosphatidylinositol-anchored high density lipoprotein binding protein 1 (GPIHBP1), a glycosylphosphatidylinositol-anchored protein of capillary endothelial cells, is the binding site for LPL (10Davies B.S.J. Beigneux A.P. Barnes II, R.H. Tu Y. Gin P. Weinstein M.M. Nobumori C. Nyrén R. Goldberg I.J. Olivecrona G. et al.GPIHBP1 is responsible for the entry of lipoprotein lipase into capillaries.Cell Metab. 2010; 12: 42-52Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). GPIHBP1 captures LPL within the interstitial spaces and shuttles it across endothelial cells to the capillary lumen (10Davies B.S.J. Beigneux A.P. Barnes II, R.H. Tu Y. Gin P. Weinstein M.M. Nobumori C. Nyrén R. Goldberg I.J. Olivecrona G. et al.GPIHBP1 is responsible for the entry of lipoprotein lipase into capillaries.Cell Metab. 2010; 12: 42-52Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 11Beigneux A.P. Davies B.S. Gin P. Weinstein M.M. Farber E. Qiao X. Peale F. Bunting S. Walzem R.L. Wong J.S. et al.Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 plays a critical role in the lipolytic processing of chylomicrons.Cell Metab. 2007; 5: 279-291Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar). The GPIHBP1–LPL complex on the surface of capillaries is also crucial for the margination of TRLs along capillaries, making it possible for LPL-mediated TRL processing to proceed (12Goulbourne C.N. Gin P. Tatar A. Nobumori C. Hoenger A. Jiang H. Grovenor C.R. Adeyo O. Esko J.D. Goldberg I.J. et al.The GPIHBP1-LPL complex is responsible for the margination of triglyceride-rich lipoproteins in capillaries.Cell Metab. 2014; 19: 849-860Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). GPIHBP1 expression is required for TRL processing (11Beigneux A.P. Davies B.S. Gin P. Weinstein M.M. Farber E. Qiao X. Peale F. Bunting S. Walzem R.L. Wong J.S. et al.Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 plays a critical role in the lipolytic processing of chylomicrons.Cell Metab. 2007; 5: 279-291Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar). Several mutations in GPIHBP1's cysteine-rich Ly6 domain have been identified in patients with severe hypertriglyceridemia ("familial chylomicronemia syndrome") (13Franssen R. Young S.G. Peelman F. Hertecant J. Sierts J.A. Schimmel A.W. Bensadoun A. Kastelein J.J. Fong L.G. Dallinga-Thie G.M. et al.Chylomicronemia with low postheparin lipoprotein lipase levels in the setting of GPIHBP1 defects.Circ Cardiovasc Genet. 2010; 3: 169-178Crossref PubMed Scopus (93) Google Scholar, 14Olivecrona G. Ehrenborg E. Semb H. Makoveichuk E. Lindberg A. Hayden M.R. Gin P. Davies B.S. Weinstein M.M. Fong L.G. et al.Mutation of conserved cysteines in the Ly6 domain of GPIHBP1 in familial chylomicronemia.J. Lipid Res. 2010; 51: 1535-1545Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 15Charrière S. Peretti N. Bernard S. Di Filippo M. Sassolas A. Merlin M. Delay M. Debard C. Lefai E. Lachaux A. et al.GPIHBP1 C89F neomutation and hydrophobic C-terminal domain G175R mutation in two pedigrees with severe hyperchylomicronemia.J. Clin. Endocrinol. Metab. 2011; 96: E1675-E1679Crossref PubMed Scopus (53) Google Scholar, 16Yamamoto H. Onishi M. Miyamoto N. Oki R. Ueda H. Ishigami M. Hiraoka H. Matsuzawa Y. Kihara S. Novel combined GPIHBP1 mutations in a patient with hypertriglyceridemia associated with CAD.J. Atheroscler. Thromb. 2013; 20: 777-784Crossref PubMed Scopus (24) Google Scholar, 17Rios J.J. Shastry S. Jasso J. Hauser N. Garg A. Bensadoun A. Cohen J.C. Hobbs H.H. Deletion of GPIHBP1 causing severe chylomicronemia.J. Inherit. Metab. Dis. 2012; 35: 531-540Crossref PubMed Scopus (76) Google Scholar, 18Coca-Prieto I. Kroupa O. Gonzalez-Santos P. Magne J. Olivecrona G. Ehrenborg E. Valdivielso P. Childhood-onset chylomicronaemia with reduced plasma lipoprotein lipase activity and mass: identification of a novel GPIHBP1 mutation.J. Intern. Med. 2011; 270: 224-228Crossref PubMed Scopus (53) Google Scholar, 19Plengpanich W. Young S.G. Khovidhunkit W. Bensadoun A. Karnman H. Ploug M. Gardsvoll H. Leung C.S. Adeyo O. Larsson M. et al.Multimerization of glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) and familial chylomicronemia from a serine-to-cysteine substitution in GPIHBP1 Ly6 domain.J. Biol. Chem. 2014; 289: 19491-19499Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 20Beigneux A.P. Franssen R. Bensadoun A. Gin P. Melford K. Peter J. Walzem R.L. Weinstein M.M. Davies B.S. Kuivenhoven J.A. et al.Chylomicronemia with a mutant GPIHBP1 (Q115P) that cannot bind lipoprotein lipase.Arterioscler. Thromb. Vasc. Biol. 2009; 29: 956-962Crossref PubMed Scopus (135) Google Scholar, 21Gonzaga-Jauregui C. Mir S. Penney S. Jhangiani S. Midgen C. Finegold M. Muzny D.M. Wang M. Bacino C.A. Gibbs R.A. et al.Whole-exome sequencing reveals GPIHBP1 mutations in infantile colitis with severe hypertriglyceridemia.J. Pediatr. Gastroenterol. Nutr. 2014; 59: 17-21Crossref PubMed Scopus (17) Google Scholar, 22Ariza M.J. Martinez-Hernandez P.L. Ibarretxe D. Rabacchi C. Rioja J. Grande-Aragon C. Plana N. Tarugi P. Olivecrona G. Calandra S. et al.Novel mutations in the GPIHBP1 gene identified in 2 patients with recurrent acute pancreatitis.J. Clin. Lipidol. 2016; 10: 92-100.e1Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). All of those mutations block the ability of GPIHBP1 to bind and transport LPL. Recent surface plasmon resonance studies have indicated that GPIHBP1's Ly6 domain is primarily responsible for high-affinity LPL binding, while the acidic domain at GPIHBP1's N terminus simply promotes the formation of an initial LPL–GPIHBP1 complex (23Fong L.G. Young S.G. Beigneux A.P. Bensadoun A. Oberer M. Jiang H. Ploug M. GPIHBP1 and plasma triglyceride metabolism.Trends Endocrinol. Metab. 2016; 27: 455-469Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 24Mysling S. Kristensen K.K. Larsson M. Beigneux A.P. Gardsvoll H. Fong L.G. Bensadouen A. Jorgensen T.J. Young S.G. Ploug M. The acidic domain of the endothelial membrane protein GPIHBP1 stabilizes lipoprotein lipase activity by preventing unfolding of its catalytic domain.eLife. 2016; 5: e12095Crossref PubMed Google Scholar). Immunohistochemistry studies on wild-type and GPIHBP1-deficient (Gpihbp1−/−) mice have provided key insights into LPL and GPIHBP1 physiology. In wild-type mice, the vast majority of the LPL in sections of heart is bound to GPIHBP1 on capillaries (10Davies B.S.J. Beigneux A.P. Barnes II, R.H. Tu Y. Gin P. Weinstein M.M. Nobumori C. Nyrén R. Goldberg I.J. Olivecrona G. et al.GPIHBP1 is responsible for the entry of lipoprotein lipase into capillaries.Cell Metab. 2010; 12: 42-52Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar), revealing that freshly secreted LPL can move efficiently to endothelial cells. The LPL in wild-type mice can be released into the plasma with an injection of heparin (25Weinstein M.M. Yin L. Beigneux A.P. Davies B.S. Gin P. Estrada K. Melford K. Bishop J.R. Esko J.D. Dallinga-Thie G.M. et al.Abnormal patterns of lipoprotein lipase release into the plasma in GPIHBP1-deficient mice.J. Biol. Chem. 2008; 283: 34511-34518Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The immunohistochemistry studies on heart sections from Gpihbp1−/− mice were arguably more intriguing. In the absence of GPIHBP1, the LPL remained attached to HSPGs on the surface of all cells within the tissue. As in wild-type mice, the LPL in Gpihbp1−/− mice could be released into the plasma with an injection of heparin (10Davies B.S.J. Beigneux A.P. Barnes II, R.H. Tu Y. Gin P. Weinstein M.M. Nobumori C. Nyrén R. Goldberg I.J. Olivecrona G. et al.GPIHBP1 is responsible for the entry of lipoprotein lipase into capillaries.Cell Metab. 2010; 12: 42-52Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 25Weinstein M.M. Yin L. Beigneux A.P. Davies B.S. Gin P. Estrada K. Melford K. Bishop J.R. Esko J.D. Dallinga-Thie G.M. et al.Abnormal patterns of lipoprotein lipase release into the plasma in GPIHBP1-deficient mice.J. Biol. Chem. 2008; 283: 34511-34518Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Thus, in the absence of GPIHBP1, the LPL remains bound to HSPGs within the interstitial spaces; that HSPG-bound LPL, which is catalytically active (25Weinstein M.M. Yin L. Beigneux A.P. Davies B.S. Gin P. Estrada K. Melford K. Bishop J.R. Esko J.D. Dallinga-Thie G.M. et al.Abnormal patterns of lipoprotein lipase release into the plasma in GPIHBP1-deficient mice.J. Biol. Chem. 2008; 283: 34511-34518Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), does not simply diffuse away into the lymph and reach the plasma. Indeed, the levels of LPL in the plasma of Gpihbp1−/− mice are far lower than in wild-type mice, implying that the binding of LPL to interstitial HSPGs is quite avid and efficient. The fact that the LPL in Gpihbp1−/− mice accumulates in the interstitium, mainly on the surface of cells, and does not enter the plasma compartment poses a conundrum: Why don't the same interstitial HSPG binding sites in wild-type mice capture newly secreted LPL and thereby impede the movement of LPL to endothelial cells? The answer to that question is not known, but we hypothesize that the binding of LPL to interstitial HSPGs is actually dynamic (with high "on" and "off" rates), such that the LPL is constantly bouncing on and off a large pool of HSPG binding sites. Such a scenario would explain how LPL can move to high-affinity GPIHBP1 binding sites on capillaries. In the current study, we tested, using cell culture and mouse models, the concept that the binding of LPL to HSPGs is dynamic and transient, allowing for rapid transfer of LPL to GPIHBP1. Mammary glands were harvested from lactating wild-type and Gpihbp1−/− mice. Frozen sections (8–10 μm) were incubated with primary antibodies [3 μg/ml for monoclonal antibody 11A12; 1:400 for a hamster anti-CD31 antibody (Millipore, Billerica, MA); 1:1,000 for a rabbit anti-collagen type IV antibody (Cosmo Bio USA, Carlsbad, CA); 10 μg/ml for a goat anti-mouse LPL antibody]. Secondary antibodies (Alexa555- or Alexa647-labeled anti-rat IgG, Alexa488- or Alexa647-labeled anti-hamster IgG, Alexa488- or Alexa647-labeled anti-rabbit IgG, and Alexa549- or Alexa488-labeled anti-goat IgG) were used at a dilution of 1:200 for 1 h at room temperature. Images were obtained with an Axiovert 200 MOT microscope equipped with an Apotome (both from Zeiss, Germany) or by confocal fluorescence microscopy with a Leica SP2 1P-FCS microscope (Heidelberg, Germany). Proteins were size-fractionated on 12% NuPAGE SDS-PAGE gels with MES buffer, followed by transfer to a nitrocellulose membrane. The nitrocellulose membranes were blocked for 1 h at room temperature with Odyssey blocking buffer (LI-COR) and then incubated with a goat polyclonal antibody against mouse LPL (10 μg/ml) (26Page S. Judson A. Melford K. Bensadoun A. Interaction of lipoprotein lipase and receptor-associated protein.J. Biol. Chem. 2006; 281: 13931-13938Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar) followed by an IRDye800-conjugated donkey anti-goat IgG (LI-COR); a mouse monoclonal antibody against the V5 tag (ThermoFisher Scientific; 1:500) followed by an IRDye800-conjugated donkey anti-mouse IgG (LI-COR); a goat polyclonal antibody against the S-protein tag (Abcam; 1:1,000) followed by an IRDye680-conjugated donkey anti-goat IgG (LI-COR); a rabbit polyclonal antibody against β-actin (Novus Biologicals; 1:1,000) followed by an IRDye800-conjugated donkey anti-rabbit IgG (LI-COR); and antibody 11A12 (3 μg/ml) followed by an IRDye680-conjugated donkey anti-rat IgG). Signals were visualized with an Odyssey scanner (LI-COR). Gpihbp1−/− mice were euthanized and perfused with PBS. The interscapular brown adipose tissue and heart were harvested and embedded in OCT medium on dry ice. Tissue sections of the heart (7 μm) and brown adipose tissue (10 μm) were prepared and placed on glass slides. Tissue sections were then incubated for 1 h at room temperature with PBS alone or PBS containing 10 U/ml of heparinase III (Sigma-Aldrich). The solution was collected and analyzed by Western blotting. We produced secreted versions of human wild-type GPIHBP1, GPIHBP1-W109S, and GPIHBP1Δ(25–50) in Drosophila S2 cells (27Beigneux A.P. Fong L.G. Bensadoun A. Davies B.S. Oberer M. Gardsvoll H. Ploug M. Young S.G. GPIHBP1 missense mutations often cause multimerization of GPIHBP1 and thereby prevent lipoprotein lipase binding.Circ. Res. 2015; 116: 624-632Crossref PubMed Scopus (47) Google Scholar). These GPIHBP1 proteins contained a uPAR tag (27Beigneux A.P. Fong L.G. Bensadoun A. Davies B.S. Oberer M. Gardsvoll H. Ploug M. Young S.G. GPIHBP1 missense mutations often cause multimerization of GPIHBP1 and thereby prevent lipoprotein lipase binding.Circ. Res. 2015; 116: 624-632Crossref PubMed Scopus (47) Google Scholar) and the epitope for the GPIHBP1-specific monoclonal antibody 11A12 (28Gin P. Beigneux A.P. Voss C. Davies B.S. Beckstead J.A. Ryan R.O. Bensadoun A. Fong L.G. Young S.G. Binding preferences for GPIHBP1, a glycosylphosphatidylinositol-anchored protein of capillary endothelial cells.Arterioscler. Thromb. Vasc. Biol. 2011; 31: 176-182Crossref PubMed Scopus (38) Google Scholar). The GPIHBP1 proteins were purified over agarose beads coated with the uPAR-specific monoclonal antibody R24 (29Gårdsvoll H. Hansen L.V. Jørgensen T.J. Ploug M. A new tagging system for production of recombinant proteins in Drosophila S2 cells using the third domain of the urokinase receptor.Protein Expr. Purif. 2007; 52: 384-394Crossref PubMed Scopus (36) Google Scholar). CHO pgsA-745 cells (2 × 106) were electroporated with 2 μg of a plasmid for S-protein–tagged versions of wild-type human GPIHBP1, GPIHBP1-W109S (27Beigneux A.P. Fong L.G. Bensadoun A. Davies B.S. Oberer M. Gardsvoll H. Ploug M. Young S.G. GPIHBP1 missense mutations often cause multimerization of GPIHBP1 and thereby prevent lipoprotein lipase binding.Circ. Res. 2015; 116: 624-632Crossref PubMed Scopus (47) Google Scholar), or GPIHBP1-D,E(25–50)N,Q [a mutant GPIHBP1 in which all acidic amino acids within residues 25–50 were replaced with uncharged amino acids (Asn or Gln)]; the transfected cells were plated on coverslips in 24-well plates. Twenty-four hours later, the transfected cells were incubated with V5-tagged LPL (30Ben-Zeev O. Mao H.Z. Doolittle M.H. Maturation of lipoprotein lipase in the endoplasmic reticulum. Concurrent formation of functional dimers and inactive aggregates.J. Biol. Chem. 2002; 277: 10727-10738Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) for 1 h at 4°C, washed, and then processed for Western blotting. Cell lysates were collected by incubating cells with mammalian protein extraction reagent (M-PER; Thermo Fisher Scientific) with EDTA-free complete protease inhibitor cocktail (Roche) for 5 min at 4°C. The protein extracts were analyzed by Western blotting. HepG2 cells (8 × 105) were plated in a 24-well plate. The next day, the cells were incubated with V5-tagged human LPL for 1 h at 4°C and then washed. Alternatively, the HepG2 cells were incubated with 1 μg bovine LPL with 0.5% BSA (w/v) for 1 h at 4°C and then washed. HepG2 cells are known to express large amounts of HSPGs on their cell surface (31Zaiss A.K. Lawrence R. Elashoff D. Esko J.D. Herschman H.R. Differential effects of murine and human factor X on adenovirus transduction via cell-surface heparan sulfate.J. Biol. Chem. 2011; 286: 24535-24543Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). To determine whether the LPL on the surface of HepG2 cells was bound to HSPGs, the cells were incubated for 20 min at room temperature with PBS alone, heparin (10–100 U/ml), or heparinase III (15–30 U/ml; Sigma-Aldrich). The supernatant fluid was collected and aliquots were analyzed by Western blotting. HepG2 cells (8 × 105) were loaded with V5-tagged human LPL for 1 h at 4°C and then washed. Secreted versions of human wild-type GPIHBP1 or buffer alone were added to the cells in a volume of 200 μl for 30 min at 4°C. The supernatant fluid was collected and the cells were washed and incubated with 200 μl heparin (500 U/ml) for 20 min at 4°C. Both the initial supernatant fluid and the heparin-released material were analyzed by Western blotting. Bands were visualized and quantified with an Odyssey scanner (LI-COR). In some experiments, LPL-loaded HepG2 cells were incubated with 10 μg/ml of wild-type GPIHBP1 in 200 μl at 4°C for 5, 10, 20, or 30 min, or buffer only for 30 min. Supernatant fluids were analyzed for GPIHBP1 and LPL by Western blotting. CHO pgsA-745 cells (2 × 106) were electroporated with 2 μg of a plasmid for S-protein–tagged versions of wild-type human GPIHBP1, GPIHBP1-W109S, or GPIHBP1-D,E(25–50)N,Q; the transfected cells were plated on coverslips in 24-well plates. After 24 h, the coverslips containing the transfected CHO pgsA-745 cells were placed face-down onto the HepG2 cells that had been loaded with V5-tagged human LPL or bovine LPL in PBS/Ca/Mg buffer and incubated at 37°C for 20 min. The pgsA-745 cells were then washed and processed for Western blotting, immunocytochemistry, or LPL activity measurements. In a separate experiment, HepG2 cells were plated on coverslips and placed face-down onto LPL-loaded CHO pgsA-745 cells transfected with an S-protein–tagged version of wild-type human GPIHBP1. For Western blotting, cell lysates were collected as described earlier. For immunocytochemistry studies, the transfected cells were fixed in 3% paraformaldehyde for 15 min and blocked with 10% donkey serum in PBS/Mg/Ca. The cells were then incubated overnight at 4°C with a mouse monoclonal antibody against the V5 tag (Thermo Fisher Scientific; 1:100) and a goat polyclonal antibody against the S-protein tag (Abcam; 1:800), followed by a 30 min incubation with an Alexa568-conjugated donkey anti-goat IgG (Thermo Fisher Scientific; 1:800) and an Alexa647-conjugated donkey anti-mouse IgG (Thermo Fisher Scientific; 1:800). After washing, the cells were fixed with 3% paraformaldehyde for 15 min and stained with 4′,6-diamidino-2-phenylindole (DAPI) to visualize DNA. Images were recorded with an Axiovert 200M microscope and processed with Zen 2010 software (all from Zeiss). Within each experiment, the exposure conditions for each construct were identical. LPL activity on the surface of transfected cells was measured with [3H]triolein–Intralipid (0.5 μCi [3H]triolein per milligram Intralipid triglyceride). Coverslips were placed in 500 μl incubations containing 50 units of heparin per milliliter, 2 mg of triglyceride per milliliter in PBS/Mg/Ca containing 6% BSA (w/v, pH 7.4), and 5% (v/v) heat-inactivated rat serum (as a source of apo-CII). Samples were incubated at room temperature for 90 min, followed by lipid extraction and scintillation counting (32Bengtsson-Olivecrona G. Olivecrona T. Assay of lipoprotein lipase and hepatic lipase.in: Skinner R.E. Converse C.A. In Lipoprotein Analysis: A Practical Approach. Oxford University Press, Oxford, UK1992: 169-185Google Scholar). We tested the movement of LPL from V5-LPL–loaded HepG2 cells to agarose beads harboring recombinant human GPIHBP1. To prepare GPIHBP1-agarose beads, we added 8.5 μg GPIHBP1 proteins [wild-type GPIHBP1, GPIHBP1-W109S, or GPIHBP1Δ(25–50)] or buffer alone to 100 μl antibody 11A12–coated agarose beads (33Beigneux A.P. Gin P. Davies B.S.J. Weinstein M.M. Bensadoun A. Fong L.G. Young S.G. Highly conserved cysteines within the Ly6 domain of GPIHBP1 are crucial for the binding of lipoprotein lipase.J. Biol. Chem. 2009; 284: 30240-30247Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The agarose beads were then placed adjacent to the coverslips containing V5-LPL–loaded HepG2 cells in 6-well plates and incubated for 2 h at 4°C. The beads (spatially separated from the coverslips) were then collected and washed, and any LPL that had moved to GPIHBP1 on the surface of the beads was eluted with SDS sample buffer (10 min at 90°C). The amounts of GPIHBP1 and LPL were analyzed by Western blotting. Antibody 11A12–coated agarose beads that had been incubated with wild-type GPIHBP1 (or buffer alone) were injected into the interscapular brown adipose tissue of wild-type or Gpihbp1−/− mice (50 μl of agarose beads containing 6.5 μg GPIHBP1 injected into each pad). After 45 min, the mice were euthanized and perfused with PBS followed by 3% paraformaldehyde. The brown adipose tissue pad was then harvested and embedded in OCT medium on dry ice. Tissue sections (10 μm) were fixed with methanol at −20°C for 10 min, permeabilized with 0.2% Triton X-100 for 5 min, and blocked at room temperature with 5% donkey serum, 10% fetal bovine serum, and 0.2% BSA in PBS/Mg/Ca. Tissues were incubated overnight at 4°C with a goat polyclonal antibody against mouse LPL (7 μg/ml) (26Page S. Judson A. Melford K. Bensadoun A. Interaction of lipoprotein lipase and receptor-associated protein.J. Biol. Chem. 2006; 281: 13931-13938Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar) and a rabbit polyclonal antibody against mouse CD31 (Abcam; 1:50), followed by a 45 min incubation at room temperature with Alexa647-conjugated antibody 11A12 (3 μg/ml), Alexa568-conjugated donkey anti-goat IgG (Thermo Fisher Scientific; 1:200), and Alexa488-conjugated donkey anti-rabbit IgG (Thermo Fisher Scientific; 1:200). After washing, the cells were fixed with 3% paraformaldehyde for 5 min and stained with DAPI to visualize DNA. Microscopy was performed as described earlier. The mice were fed a chow diet and housed in a barrier facility with a 12 h light-dark cycle. All studies were approved by the University of California Los Angeles Animal Research Committee. Davies et al. (10Davies B.S.J. Beigneux A.P. Barnes II, R.H. Tu Y. Gin P. Weinstein M.M. Nobumori C. Nyrén R. Goldberg I.J. Olivecrona G. et al.GPIHBP1 is responsible for the entry of lipoprotein lipase into capillaries.Cell Metab. 2010; 12: 42-52Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar) reported that LPL in the hearts of Gpihbp1−/− mice is mislocalized, with most of it located on or near the surface of cells (both cardiomyocytes and endothelial cells). We wanted to determine whether the LPL in Gpihbp1−/− mice was also mislocalized in the lactating mammary gland, a site with high levels of LPL expression (34Wang Y. Tong J. Li S. Zhang R. Chen L. Wang Y. Zheng M. Wang M. Liu G. Dai Y. et al.Over-expression of human lipoprotein lipase in mouse mammary glands leads to reduction of milk triglyceride and delayed growth of suckling pups.PLoS One. 2011; 6: e20895Crossref PubMed Scopus (11) Google Scholar). In wild-type mice, most of the LPL in the mammary gland was bound to capillaries, colocalizing with the endothelial cell marker, CD31 (Fig. 1A, B). In Gpihbp1−/− mice, LPL did not colocalize with CD31 and, instead, was mislocalized in the interstitial spaces on or near mammary epithelial cells (Fig. 1C). We have assumed that the LPL within the interstitial spaces of Gpihbp1−/− mice was bound to HSPGs because the LPL in those mice was readily released into the plasma with an injection of heparin (25Weinstein M.M. Yin L. Beigneux A.P. Davies B.S. Gin P. Estrada K. Melford K. Bishop J.R. Esko J.D. Dallinga-Thie G.M. et al.Abnormal patterns of lipoprotein lipase release into the plasma in GPIHBP1-deficient mice.J. Biol. Chem. 2008; 283: 34511-34518Abstract Full Text Full Text PDF PubMed Scopus (59) Google Sch
Referência(s)