Structural features in lipoprotein lipase necessary for the mediation of lipoprotein uptake into cells

A. Krapp, H. Zhang, D. Ginzinger, M. S. Liu, A. Lindberg, G. Olivecrona, M. R. Hayden, U. Beisiegel

Research output: Contribution to journalArticle

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Abstract

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 immunosorbent 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 β-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 (Asp156Gly) resulted in no significant reduction in the lipase-mediated β-VLDL uptake. Point mutations in the proposed lipid binding region Trp390Ala or Trp393Ala and the substitution of 391-393 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 391-421 are important for LpL- mediated lipoprotein uptake.

Original languageEnglish (US)
Pages (from-to)2362-2373
Number of pages12
JournalJournal of Lipid Research
Volume36
Issue number11
StatePublished - Jan 1 1995

Fingerprint

Lipoprotein Lipase
Lipoproteins
Lipase
Monomers
Heparin
Catalyst activity
LDL-Receptor Related Proteins
Lipids
Immunosorbents
Mutagenesis
Mutation
LDL Receptors
COS Cells
Proteoglycans
Site-Directed Mutagenesis
Point Mutation
Dimers
Cultured Cells
Substitution reactions

Keywords

  • hepatoma cells
  • LDL receptor-related protein
  • LpL mutants

ASJC Scopus subject areas

  • Endocrinology

Cite this

Krapp, A., Zhang, H., Ginzinger, D., Liu, M. S., Lindberg, A., Olivecrona, G., ... Beisiegel, U. (1995). Structural features in lipoprotein lipase necessary for the mediation of lipoprotein uptake into cells. Journal of Lipid Research, 36(11), 2362-2373.

Structural features in lipoprotein lipase necessary for the mediation of lipoprotein uptake into cells. / Krapp, A.; Zhang, H.; Ginzinger, D.; Liu, M. S.; Lindberg, A.; Olivecrona, G.; Hayden, M. R.; Beisiegel, U.

In: Journal of Lipid Research, Vol. 36, No. 11, 01.01.1995, p. 2362-2373.

Research output: Contribution to journalArticle

Krapp, A, Zhang, H, Ginzinger, D, Liu, MS, Lindberg, A, Olivecrona, G, Hayden, MR & Beisiegel, U 1995, 'Structural features in lipoprotein lipase necessary for the mediation of lipoprotein uptake into cells', Journal of Lipid Research, vol. 36, no. 11, pp. 2362-2373.
Krapp A, Zhang H, Ginzinger D, Liu MS, Lindberg A, Olivecrona G et al. Structural features in lipoprotein lipase necessary for the mediation of lipoprotein uptake into cells. Journal of Lipid Research. 1995 Jan 1;36(11):2362-2373.
Krapp, A. ; Zhang, H. ; Ginzinger, D. ; Liu, M. S. ; Lindberg, A. ; Olivecrona, G. ; Hayden, M. R. ; Beisiegel, U. / Structural features in lipoprotein lipase necessary for the mediation of lipoprotein uptake into cells. In: Journal of Lipid Research. 1995 ; Vol. 36, No. 11. pp. 2362-2373.
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abstract = "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 immunosorbent 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 β-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 (Asp156Gly) resulted in no significant reduction in the lipase-mediated β-VLDL uptake. Point mutations in the proposed lipid binding region Trp390Ala or Trp393Ala and the substitution of 391-393 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 391-421 are important for LpL- mediated lipoprotein uptake.",
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AU - Zhang, H.

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AU - Liu, M. S.

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AU - Olivecrona, G.

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N2 - 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 immunosorbent 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 β-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 (Asp156Gly) resulted in no significant reduction in the lipase-mediated β-VLDL uptake. Point mutations in the proposed lipid binding region Trp390Ala or Trp393Ala and the substitution of 391-393 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 391-421 are important for LpL- mediated lipoprotein uptake.

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