Structural features in lipoprotein lipase necessary for the mediation of lipoprotein uptake into cells
Lipoprotein lipase (LpL) has been shown to mediate the uptake of lipoproteins into cells. The uptake is initiated by binding of LpL to cell surface proteoglycans and to the low density lipoprotein (LDL) receptor-related protein. This ability of LpL is independent of catalytic activity and depends on...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
1995-11-01
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| Series: | Journal of Lipid Research |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S0022227520397170 |
| _version_ | 1818386308557438976 |
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| author | A Krapp H Zhang D Ginzinger M S Liu A Lindberg G Olivecrona M R Hayden U Beisiegel |
| author_facet | A Krapp H Zhang D Ginzinger M S Liu A Lindberg G Olivecrona M R Hayden U Beisiegel |
| author_sort | A Krapp |
| collection | DOAJ |
| description | Lipoprotein lipase (LpL) has been shown to mediate the uptake of lipoproteins into cells. The uptake is initiated by binding of LpL to cell surface proteoglycans and to the low density lipoprotein (LDL) receptor-related protein. This ability of LpL is independent of catalytic activity and depends on the intact dimeric structure of the lipase and functional residues in the C-terminal domain. The goal of this study was to identify structural features in LpL that are essential in the mediation of lipoprotein uptake. Naturally occurring variants and LpL mutants produced by site-directed mutagenesis were cloned and expressed in COS-cells. A combination of immunoassays and separation on heparin-Sepharose columns was used to determine the molar ratio of monomeric to dimeric LpL in the expression media. The mutants were tested for their ability to mediate the uptake of 125I-labeled beta-VLDL in cultured Hep3b cells in direct comparison with wild type LpL. We found that the concentration of monomer in the media correlated negatively with the effect on the uptake mediated by the dimeric form of LpL. A mutation affecting the catalytic activity (Asp 156Gly) resulted in no significant reduction in the lipase-mediated beta-VLDL uptake. Point mutations in the proposed lipid binding region Trp390Ala or Trp393Ala and the substitution of 390-393 with the homologous hepatic lipase (HL) sequence were also normal, while the deletion of 390-393 reduced the ability to mediate the uptake by about 60% in comparison to wild type. A mutation known to impair heparin binding (Arg294Ala) was also less efficient than the wild type in mediating uptake. In conclusion, it is important to determine the monomer/dimer ratio in mutant preparations as the presence of monomers inhibits the uptake mediated by the dimeric LpL. Moreover, sites involved in heparin and lipid binding between residues 390-421 are important for LpL-mediated lipoprotein uptake. |
| first_indexed | 2024-12-14T03:51:59Z |
| format | Article |
| id | doaj.art-44391a0d80e8429da6745384bd7f94d0 |
| institution | Directory Open Access Journal |
| issn | 0022-2275 |
| language | English |
| last_indexed | 2024-12-14T03:51:59Z |
| publishDate | 1995-11-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Lipid Research |
| spelling | doaj.art-44391a0d80e8429da6745384bd7f94d02022-12-21T23:18:11ZengElsevierJournal of Lipid Research0022-22751995-11-01361123622373Structural features in lipoprotein lipase necessary for the mediation of lipoprotein uptake into cellsA Krapp0H Zhang1D Ginzinger2M S Liu3A Lindberg4G Olivecrona5M R Hayden6U Beisiegel7Medizinische Klinik, Universitätskrankenhaus Eppendorf, Hamburg, Germany.Medizinische Klinik, Universitätskrankenhaus Eppendorf, Hamburg, Germany.Medizinische Klinik, Universitätskrankenhaus Eppendorf, Hamburg, Germany.Medizinische Klinik, Universitätskrankenhaus Eppendorf, Hamburg, Germany.Medizinische Klinik, Universitätskrankenhaus Eppendorf, Hamburg, Germany.Medizinische Klinik, Universitätskrankenhaus Eppendorf, Hamburg, Germany.Medizinische Klinik, Universitätskrankenhaus Eppendorf, Hamburg, Germany.Medizinische Klinik, Universitätskrankenhaus Eppendorf, Hamburg, Germany.Lipoprotein lipase (LpL) has been shown to mediate the uptake of lipoproteins into cells. The uptake is initiated by binding of LpL to cell surface proteoglycans and to the low density lipoprotein (LDL) receptor-related protein. This ability of LpL is independent of catalytic activity and depends on the intact dimeric structure of the lipase and functional residues in the C-terminal domain. The goal of this study was to identify structural features in LpL that are essential in the mediation of lipoprotein uptake. Naturally occurring variants and LpL mutants produced by site-directed mutagenesis were cloned and expressed in COS-cells. A combination of immunoassays and separation on heparin-Sepharose columns was used to determine the molar ratio of monomeric to dimeric LpL in the expression media. The mutants were tested for their ability to mediate the uptake of 125I-labeled beta-VLDL in cultured Hep3b cells in direct comparison with wild type LpL. We found that the concentration of monomer in the media correlated negatively with the effect on the uptake mediated by the dimeric form of LpL. A mutation affecting the catalytic activity (Asp 156Gly) resulted in no significant reduction in the lipase-mediated beta-VLDL uptake. Point mutations in the proposed lipid binding region Trp390Ala or Trp393Ala and the substitution of 390-393 with the homologous hepatic lipase (HL) sequence were also normal, while the deletion of 390-393 reduced the ability to mediate the uptake by about 60% in comparison to wild type. A mutation known to impair heparin binding (Arg294Ala) was also less efficient than the wild type in mediating uptake. In conclusion, it is important to determine the monomer/dimer ratio in mutant preparations as the presence of monomers inhibits the uptake mediated by the dimeric LpL. Moreover, sites involved in heparin and lipid binding between residues 390-421 are important for LpL-mediated lipoprotein uptake.http://www.sciencedirect.com/science/article/pii/S0022227520397170 |
| spellingShingle | A Krapp H Zhang D Ginzinger M S Liu A Lindberg G Olivecrona M R Hayden U Beisiegel Structural features in lipoprotein lipase necessary for the mediation of lipoprotein uptake into cells Journal of Lipid Research |
| title | Structural features in lipoprotein lipase necessary for the mediation of lipoprotein uptake into cells |
| title_full | Structural features in lipoprotein lipase necessary for the mediation of lipoprotein uptake into cells |
| title_fullStr | Structural features in lipoprotein lipase necessary for the mediation of lipoprotein uptake into cells |
| title_full_unstemmed | Structural features in lipoprotein lipase necessary for the mediation of lipoprotein uptake into cells |
| title_short | Structural features in lipoprotein lipase necessary for the mediation of lipoprotein uptake into cells |
| title_sort | structural features in lipoprotein lipase necessary for the mediation of lipoprotein uptake into cells |
| url | http://www.sciencedirect.com/science/article/pii/S0022227520397170 |
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