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Novel factors affecting clearance of triacylglycerol-rich lipoproteins from blood
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Physiological chemistry. (Olivecrona)
2010 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Nya faktorer som påverkar upptaget av triglycerider från blodet (Swedish)
Abstract [en]

Apolipoprotein (apo) A-V is the most recently discovered member of a protein family responsible for the structure and metabolic fate of plasma lipoproteins. While most of the apolipoproteins are well characterized with regard to structure, interactions and function, the role of apoA-V is not well understood. ApoA-V is synthesized only in liver and is present in blood at much lower concentration than the other apolipoproteins. Although apoA-V is firmly established as an important determinant for plasma triacylglycerol (TG) metabolism, the mechanism is unclear. ApoA-V has been suggested to act through 1) an intracellular mechanism affecting lipoprotein assembly and secretion, 2) direct or indirect activation of lipoprotein lipase (LPL), or 3) interaction with endocytotic lipoprotein receptors.

Two other novel players involved in the clearance of lipoproteins are angiopoietin-like protein (ANGPTL) 3 and 4. Previous studies have shown that the coiled-coil domain (ccd) of ANGPTL3 and -4 can inactivate lipoprotein lipase (LPL). The functional site of action of LPL is at the capillary endothelium, but the enzyme is synthesized mostly in adipocytes and myocytes and has to be transported by trancytosis to the luminal side of endothelial cells. Both ANGPTLs are present in tissues and in the circulating blood, but it is not known were the inactivation of LPL normally takes place.

The aim of this thesis was to investigate the mechanism by which apoA-V exerts its effect on TG metabolism and to investigate in further detail how ANGPTLs act on the LPL system.

Binding of apoA-V to receptors involved in lipoprotein metabolism was investigated by surface plasmon resonance technique (SPR). ApoA-V was found to bind to the LDL receptor related protein 1 (LRP1) and to the mosaic type 1 receptor sorLA. Binding could be competed by receptor associated protein (RAP) or by heparin, and was calcium dependent. We concluded that apoA-V binds to the LA-repeats of these receptors. In further experiments apoA-V was shown to increase binding of TG-rich chylomicrons to the receptors. This demonstrated a possible mechanism for the TG-lowering effect of apoA-V in vivo.

A putative binding region in apoA-V for heparin and receptors was investigated by site-directed mutagenesis. Two positively charged amino acid residues were changed (Arg210Glu/Lys211Gln), resulting in decreased binding to heparin and to LRP1 and thus the localization of one important functional region in apoA-V.

Since the receptor sorLA also contains a Vsp10p domain, another Vsp10p domain family member, sortilin, was investigated. ApoA-V was found to interact also with this receptor. In experiments with human embryonic kidney cells transfected with sorLA or sortilin, apoA-V was found to bind to cell surfaces and to be rapidly internalized while co-localized with the receptors on the way to lysosomes for degradation.

Additional apoA-V mutants, identified in patients with severe hypertriglyceridemia, were investigated with regard to effects in vitro on LPL activity and receptor binding. The most severe mutants displayed null binding to LRP1, whereas the effect on LPL activity was retained. These results suggest that lack of receptor interaction mirrors the loss of biological function in a better way than the in vitro effect on LPL activity.

We noted that ccd-ANGPTL3 and -4 did not prevent the LPL-mediated uptake of chylomicron-like lipoproteins in primary murine hepatocytes. Therefore LPL activity was measured after pre-incubation with ccd-ANGPTL3 or 4 in the presence or absence of TG-rich lipoproteins. Physiological concentrations of lipoproteins were found to protect LPL from inactivation by ccd-ANGPTLs. Investigation by SPR demonstrated that the ccd-ANGPTLs did not bind to the lipoproteins. Other experiments showed that less than 1% of ANGPTL4 in human serum was bound to TG-rich lipoproteins. This implies that the known binding of LPL to TG-rich lipoproteins stabilizes the enzyme and protects it from inactivation by ANGPTLs. We conclude that the normal levels of ANGPTLs in plasma are too low to affect the LPL-system and that inactivation of the enzyme by ANGPTLs is more likely to occur locally in the extracellular interstitium of tissues where LPL is en route to its endothelial binding sites and where the concentrations of the TG-rich lipoproteins are low.

Place, publisher, year, edition, pages
Umeå: Umeå university , 2010. , 94 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1352
Keyword [en]
lipoprotein, triacylglycerol, apolipoprotein A-V, ANGPTL
National Category
Cardiac and Cardiovascular Systems
Research subject
Medical Biochemistry
Identifiers
URN: urn:nbn:se:umu:diva-33792ISBN: 978-91-7459-006-7 (print)OAI: oai:DiVA.org:umu-33792DiVA: diva2:318166
Public defence
2010-05-28, Betula, Byggnad 6M, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2010-05-21 Created: 2010-05-06 Last updated: 2010-05-21Bibliographically approved
List of papers
1. Apolipoprotein A-V interaction with members of the low density lipoprotein receptor gene family
Open this publication in new window or tab >>Apolipoprotein A-V interaction with members of the low density lipoprotein receptor gene family
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2007 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 12, 3896-3904 p.Article in journal (Refereed) Published
Keyword
Amino Acid Substitution, Apolipoproteins A/*chemistry/genetics/metabolism, Calcium/*chemistry/metabolism, Endocytosis/physiology, Heparin/*chemistry/metabolism, Homeostasis/genetics, Humans, LDL-Receptor Related Proteins/*chemistry/metabolism, Lipid Metabolism/physiology, Membrane Transport Proteins/*chemistry/metabolism, Plasma/chemistry/metabolism, Protein Binding/genetics, Surface Plasmon Resonance
National Category
Other Clinical Medicine
Research subject
Clinical Chemistry
Identifiers
urn:nbn:se:umu:diva-6832 (URN)17326667 (PubMedID)
Available from: 2008-01-11 Created: 2008-01-11 Last updated: 2017-12-14Bibliographically approved
2. Endocytosis of apolipoprotein A-V by members of the low density lipoprotein receptor and the VPS10p domain receptor families.
Open this publication in new window or tab >>Endocytosis of apolipoprotein A-V by members of the low density lipoprotein receptor and the VPS10p domain receptor families.
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2008 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 38, 25920-25927 p.Article in journal (Refereed) Published
Abstract [en]

Apolipoprotein A-V (apoA-V) is present in low amounts in plasma and has been found to modulate triacylglycerol levels in humans and in animal models. ApoA-V displays affinity for members of the low density lipoprotein receptor (LDL-R) gene family, known as the classical lipoprotein receptors, including LRP1 and SorLA/LR11. In addition to LDL-A binding repeats, the mosaic receptor SorLA/LR11 also possesses a Vps10p domain. Here we show that apoA-V also binds to sortilin, a receptor from the Vsp10p domain gene family that lacks LDL-A repeats. Binding of apoA-V to sortilin was competed by neurotensin, a ligand that binds specifically to the Vps10p domain. To investigate the biological fate of receptor-bound apoA-V, binding experiments were conducted with cultured human embryonic kidney cells transfected with either SorLA/LR11 or sortilin. Compared with nontransfected cells, apoA-V binding to SorLA/LR11- and sortilin-expressing cells was markedly enhanced. Internalization experiments, live imaging studies, and fluorescence resonance energy transfer analyses demonstrated that labeled apoA-V was rapidly internalized, co-localized with receptors in early endosomes, and followed the receptors through endosomes to the trans-Golgi network. The observed decrease of fluorescence signal intensity as a function of time during live imaging experiments suggested ligand uncoupling in endosomes with subsequent delivery to lysosomes for degradation. This interpretation was supported by experiments with (125)I-labeled apoA-V, demonstrating clear differences in degradation between transfected and nontransfected cells. We conclude that apoA-V binds to receptors possessing LDL-A repeats and Vsp10p domains and that apoA-V is internalized into cells via these receptors. This could be a mechanism by which apoA-V modulates lipoprotein metabolism in vivo.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Clinical Chemistry
Identifiers
urn:nbn:se:umu:diva-24657 (URN)10.1074/jbc.M802721200 (DOI)18603531 (PubMedID)
Available from: 2009-07-08 Created: 2009-07-08 Last updated: 2017-12-13Bibliographically approved
3. Effects of six APOA5 variants, identified in patients with severe hypertriglyceridemia, on in vitro lipoprotein lipase activity and receptor binding
Open this publication in new window or tab >>Effects of six APOA5 variants, identified in patients with severe hypertriglyceridemia, on in vitro lipoprotein lipase activity and receptor binding
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2008 (English)In: Arteriosclerosis, Thrombosis and Vascular Biology, ISSN 1079-5642, E-ISSN 1524-4636, Vol. 28, no 10, 1866-1871 p.Article in journal (Refereed) Published
Abstract [en]

OBJECTIVE: The purpose of this study was to identify rare APOA5 variants in 130 severe hypertriglyceridemic patients by sequencing, and to test their functionality, since no patient recall was possible.

METHODS AND RESULTS: We studied the impact in vitro on LPL activity and receptor binding of 3 novel heterozygous variants, apoAV-E255G, -G271C, and -H321L, together with the previously reported -G185C, -Q139X, -Q148X, and a novel construct -Delta139 to 147. Using VLDL as a TG-source, compared to wild type, apoAV-G255, -L321 and -C185 showed reduced LPL activation (-25% [P=0.005], -36% [P<0.0001], and -23% [P=0.02]), respectively). ApoAV-C271, -X139, -X148, and Delta139 to 147 had little affect on LPL activity, but apoAV-X139, -X148, and -C271 showed no binding to LDL-family receptors, LR8 or LRP1. Although the G271C proband carried no LPL and APOC2 mutations, the H321L carrier was heterozygous for LPL P207L. The E255G carrier was homozygous for LPL W86G, yet only experienced severe hypertriglyceridemia when pregnant.

CONCLUSIONS: The in vitro determined function of these apoAV variants only partly explains the high TG levels seen in carriers. Their occurrence in the homozygous state, coinheritance of LPL variants or common APOA5 TG-raising variant in trans, appears to be essential for their phenotypic expression.

National Category
Other Clinical Medicine
Research subject
Clinical Chemistry
Identifiers
urn:nbn:se:umu:diva-24658 (URN)10.1161/ATVBAHA.108.172866 (DOI)18635818 (PubMedID)
Available from: 2009-07-08 Created: 2009-07-08 Last updated: 2017-12-13Bibliographically approved
4. Triacylglycerol-rich lipoproteins protect lipoprotein lipase from inactivation by ANGPTL 3 and ANGPTL 4
Open this publication in new window or tab >>Triacylglycerol-rich lipoproteins protect lipoprotein lipase from inactivation by ANGPTL 3 and ANGPTL 4
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(English)Manuscript (preprint) (Other academic)
Keyword
angptl LPL lipiprotein triglyceride
National Category
Cardiac and Cardiovascular Systems
Research subject
Medical Biochemistry
Identifiers
urn:nbn:se:umu:diva-33790 (URN)
Available from: 2010-05-06 Created: 2010-05-06 Last updated: 2010-05-21Bibliographically approved

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