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Apolipoprotein M – Studies of Structure and Function

Ahnström, Josefin LU (2009) In Lund University, Faculty of Medicine Doctoral Dissertation Series 2009:76.
Abstract
Apolipoprotein M (apoM), a 25 kDa plasma protein, is found in all major lipoprotein classes, although the majority is found in high density lipoproteins (HDL). ApoM has been suggested to be involved in the formation of and adding to the antiatherogenic functions of HDL, but its function is still not completely known. ApoM has been suggested to be a lipokalin. By studies in vitro using recombinant apoM we were able to show that apoM shares the ligand-binding abilities of lipokalins by being able to bind retinol and its two metabolites, all-trans retinoic acid and 9-cis retinoic acid. After successful crystallization trials we managed to determine the 3D structure of recombinant apoM expressed in E.coli. ApoM displays the typical lipocalin... (More)
Apolipoprotein M (apoM), a 25 kDa plasma protein, is found in all major lipoprotein classes, although the majority is found in high density lipoproteins (HDL). ApoM has been suggested to be involved in the formation of and adding to the antiatherogenic functions of HDL, but its function is still not completely known. ApoM has been suggested to be a lipokalin. By studies in vitro using recombinant apoM we were able to show that apoM shares the ligand-binding abilities of lipokalins by being able to bind retinol and its two metabolites, all-trans retinoic acid and 9-cis retinoic acid. After successful crystallization trials we managed to determine the 3D structure of recombinant apoM expressed in E.coli. ApoM displays the typical lipocalin fold that was predicted, i.e. characterized by an 8-stranded antiparallel β-barrel enclosing an internal ligand-binding pocket, flanked by a α-helix. The ligand-binding pocket in apoM can be subdivided into a hydrophilic upper part, surrounded by several positively and negatively charged residues, and a hydrophobic lower part lined by numerous hydrophobic residues. ApoM was crystallized as a complex with either myristic acid or glycerol-1-myristate in the hydrophobic pocket showing that apoM can work as a fatty acid binding protein. We were also able to detect a binding of D-sphingosine and sphingosine-1-phosphate in ligand-binding studies. The physiological importance of the binding is not known. Mature apoM is circulating with a retained signal peptide. By constructing apoM with a cleavable signal peptide we were able to show that the signal peptide is necessary for the protein’s ability to associate to lipoproteins. We were also able to show that, in contrast to apoM with a cleavable signal peptide, full-length apoM was accumulated in stably transfected HEK293 cells, expressing apoM, unless serum was present. The addition of extra cellular HDL or co-expression of HDL was enough to completely restore the apoM expression. We have in a previous study found a marked positive correlation between plasma apoM and total cholesterol levels in healthy individuals. To investigate whether plasma apoM levels predict the risk of coronary heart disease, apoM was measured in plasma from subject later developing CHD and healthy controls from two prospective case-control studies, FINRISK ´92 and Copenhagen City Heart Study. In conditional logistic regression analyses, apoM was not a predictor of CHD events. We found positive correlations for apoM with both apoA-I and apoB. (Less)
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author
supervisor
opponent
  • Professor Kostner, Gert M., Medical University of Graz, Graz, Austria
organization
publishing date
type
Thesis
publication status
published
subject
keywords
signal peptidase, signal peptide, crystallization, Apolipoprotein, lipocalin, HDL, coronary heart disease
in
Lund University, Faculty of Medicine Doctoral Dissertation Series
volume
2009:76
pages
230 pages
publisher
Department of Clinical Chemistry, Lund University
defense location
Lecture hall, entrance 78, 2nd floor, Malmö University Hospital, Malmö
defense date
2009-09-25 09:00
ISSN
1652-8220
ISBN
978-91-86253-64-6
language
English
LU publication?
yes
id
57660883-82f2-42b5-9969-c4812cb1ee6e (old id 1470400)
date added to LUP
2009-09-03 09:09:41
date last changed
2016-09-19 08:44:51
@phdthesis{57660883-82f2-42b5-9969-c4812cb1ee6e,
  abstract     = {Apolipoprotein M (apoM), a 25 kDa plasma protein, is found in all major lipoprotein classes, although the majority is found in high density lipoproteins (HDL). ApoM has been suggested to be involved in the formation of and adding to the antiatherogenic functions of HDL, but its function is still not completely known. ApoM has been suggested to be a lipokalin. By studies in vitro using recombinant apoM we were able to show that apoM shares the ligand-binding abilities of lipokalins by being able to bind retinol and its two metabolites, all-trans retinoic acid and 9-cis retinoic acid. After successful crystallization trials we managed to determine the 3D structure of recombinant apoM expressed in E.coli. ApoM displays the typical lipocalin fold that was predicted, i.e. characterized by an 8-stranded antiparallel β-barrel enclosing an internal ligand-binding pocket, flanked by a α-helix. The ligand-binding pocket in apoM can be subdivided into a hydrophilic upper part, surrounded by several positively and negatively charged residues, and a hydrophobic lower part lined by numerous hydrophobic residues. ApoM was crystallized as a complex with either myristic acid or glycerol-1-myristate in the hydrophobic pocket showing that apoM can work as a fatty acid binding protein. We were also able to detect a binding of D-sphingosine and sphingosine-1-phosphate in ligand-binding studies. The physiological importance of the binding is not known. Mature apoM is circulating with a retained signal peptide. By constructing apoM with a cleavable signal peptide we were able to show that the signal peptide is necessary for the protein’s ability to associate to lipoproteins. We were also able to show that, in contrast to apoM with a cleavable signal peptide, full-length apoM was accumulated in stably transfected HEK293 cells, expressing apoM, unless serum was present. The addition of extra cellular HDL or co-expression of HDL was enough to completely restore the apoM expression. We have in a previous study found a marked positive correlation between plasma apoM and total cholesterol levels in healthy individuals. To investigate whether plasma apoM levels predict the risk of coronary heart disease, apoM was measured in plasma from subject later developing CHD and healthy controls from two prospective case-control studies, FINRISK ´92 and Copenhagen City Heart Study. In conditional logistic regression analyses, apoM was not a predictor of CHD events. We found positive correlations for apoM with both apoA-I and apoB.},
  author       = {Ahnström, Josefin},
  isbn         = {978-91-86253-64-6},
  issn         = {1652-8220},
  keyword      = {signal peptidase,signal peptide,crystallization,Apolipoprotein,lipocalin,HDL,coronary heart disease},
  language     = {eng},
  pages        = {230},
  publisher    = {Department of Clinical Chemistry, Lund University},
  school       = {Lund University},
  series       = {Lund University, Faculty of Medicine Doctoral Dissertation Series},
  title        = {Apolipoprotein M – Studies of Structure and Function},
  volume       = {2009:76},
  year         = {2009},
}