Master’s Programme in Medical
Biosciences
LiU-HU/MEDBIO/SP-A-2011/ 9-SE
Changed Isoform Distribution of
Apolipoprotein C-III in Individuals
with a Mutation in the Enzyme ppGA1NAcT2
Stefan Ljunggren
Scientific project 30 hp/ECTS
Title of project: Changed Isoform Distribution of Apolipoprotein CIII in Individuals with a Mutation in the Enzyme pp-GA1NAcT2
Name of student: Stefan Ljunggren
Main supervisors: Mats Lindahl
Additional supervisors: Helen Karlsson
This project was performed at Division of Occupational and Environmental Medicine,
Department of Clinical and Experimental Medicine, as part of the first year of the Master’s
Programme in Medical Biosciences.
The Scientific project report was orally presented and defended in June, 2011, and approved
in its final form on the 16th of June, 2011.
Changed isoform distribution of apolipoprotein
C-III in individuals with a mutation in the enzyme
pp-GalNAcT2
Stefan Ljunggren
Scientific Project B
30 ECTS
Supervisors: Mats Lindahl and Helen Karlssson
Occupational and Environmental Medicine
Department of Clinical and Experimental Medicine
Popular Summary
Fat molecules are transported through the body in particles known as lipoproteins. These contain many
different proteins which affect the function and effectiveness of the transport. There are different kinds
of lipoproteins in which some transport fat from the dietary uptake out into the body while others have
functions for transporting fat back to the liver for excretion. The system of lipoproteins is important in
cardiovascular disease since high levels of the group that transports fat out to the body are a risk factor
for the disease. At the same time, the group that transports fat back to the liver is protective and high
levels are thought to prevent cardiovascular disease.
In the body, fat is spliced by a protein called lipoprotein lipase. There are proteins which may activate
this process and others that can prevent the activation. Among these that prevent activation is one
called apolipoprotein C-III (apoC-III). At least three types of apoC-III exist depending on which sugar
molecules are attached to the protein. One protein responsible for the attachment of sugar molecules to
apoC-III is called GALNT2 and it has recently been associated to cardiovascular disease. It is possible
that a defect in GALNT2, a so called mutation, can reduce the proteins ability to attach sugars to
apoC-III and therefore affect the ability of apoC-III to prevent activation of lipoprotein lipase. This
may then give more splicing of fats, which protects against cardiovascular disease.
We investigated two mutations in GALNT2 and whether they produced changes in the distribution of
the different forms of apoC-III. One of the two mutations caused that less sugar was attached to apoCIII. People with this mutation have lower levels of fat in their blood as well as high levels of the
protective type of lipoprotein. We therefore believe that this mutation in pp-GalNAcT2 may be the
explanation for why the persons with the mutation have lower levels of fat and higher levels of
protective lipoproteins. In the future, this may be a novel way to decrease the levels of fat in the blood
and so decrease the risk of cardiovascular disease.
i
Abstract
The gene for the sugar transferring enzyme pp-GalNAcT2 has been associated to cardiovascular lipid
metabolism and cardiovascular disease. Recently, a D394A mutation of pp-GalNAcT2 has been
reported to lower the levels of triglycerides and raise HDL-cholesterol levels. Apolipoprotein C-III
(apoC-III) consists of three isoforms due to different levels of glycosylation. Different isoforms may
have different inhibition of lipoprotein lipase which lowers triglyceride levels. The aim of this project
was to investigate whether mutations of pp-GalNAcT2 causes changes in isoform distribution of
apoC-III.
The isoform distribution of apoC-III in plasma of heterozygotes for D314A and wild-type controls was
investigated by two-dimensional gel electrophoresis and western blot. Isoform distribution in VLDL
and HDL of heterozygotes and controls, before and after a fat challenge, was also investigated. The
monosaccharide content in the different isoforms was investigated by tandem mass spectrometry. A
second mutation in pp-GalNAcT2, Q216H, was also investigated with two-dimensional gel
electrophoresis and western blot for its effect upon isoform distribution.
Heterozygotes for the D314A mutation showed an increased proportion of the non-sialylated isoform
C-III0 and decreased proportion of the monosialylated isoform C-III1, compared to their age and
gender matched controls. Monosaccharide content was confirmed for C-III1 but not for C-III0. In
addition, isoform distribution after fat challenge appeared altered in VLDL from heterozygotes. The
second mutation, Q216H, did not display a similar change in isoform distribution as D314A.
The D314A mutation in pp-GalNAcT2, but not the Q216H mutation, caused a change in isoform
distribution in apoC-III. ApoC-III usually inhibits lipoprotein lipase (LPL) which raises systemic
triglycerides. If C-III0 is less able to inhibit LPL, this could explain the found decrease of triglycerides
and increase of HDL-c in heterozygotes for the D314A mutation.
ii
Table of contents
Introduction ........................................................................................................................................... 1
Lipoproteins......................................................................................................................................... 1
Classification ................................................................................................................................... 1
Function ........................................................................................................................................... 1
Atherosclerosis and Lipoproteins .................................................................................................... 2
Lipoprotein lipase (LPL) ................................................................................................................. 2
Glycosylation....................................................................................................................................... 3
Mucin-type O-linked Glycosylation ................................................................................................ 3
ApoC-III .............................................................................................................................................. 3
Background to project ......................................................................................................................... 4
Aim ...................................................................................................................................................... 4
Methods .................................................................................................................................................. 5
Sample collection ................................................................................................................................ 5
Desalting and protein concentration measurement .............................................................................. 5
Two-dimensional gel electrophoresis (2DE) ....................................................................................... 5
Isoelectric focusing (IEF) ................................................................................................................ 5
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) .................................... 6
D314A Mutation.................................................................................................................................. 6
Isoform distribution ......................................................................................................................... 6
Fat challenge.................................................................................................................................... 6
Oligosaccharide analysis ................................................................................................................. 7
Q216H Mutation.................................................................................................................................. 7
Isoform distribution ......................................................................................................................... 7
Results .................................................................................................................................................... 8
D314A ................................................................................................................................................. 8
Isoform distribution ......................................................................................................................... 8
Fat challenge.................................................................................................................................... 8
Oligosaccharide analysis ................................................................................................................. 9
Q216H ............................................................................................................................................... 10
Isoform distribution ....................................................................................................................... 10
Discussion ............................................................................................................................................. 11
References ............................................................................................................................................ 14
iii
Abbreviations
ABCA1
ABCG1
ACN
apo
Asn
CETP
CID
C-III0
C-III1
C-III2
C-III3
DTT
EDTA
ER
ESI-MS/MS
FA
FPLC
Fuc
Gal
GalNAc
GlcNAc
GWAS
HDL
HDL-c
HL
HRP
IDL
IEF
IL-1
KBr
LCAT
LDL
LPL
Lpl
LPS
NH4HCO3
PAGE
PBS
PLTP
pp-GalNAcT
sialic acid/
NeuAc
SDS
SNP
SRB1
TFA
UDP
VLDL
- ATP-binding cassette transporter A1
- ATP-binding cassette transporter G1
- acetonitrile
- apolipoprotein
- asparagine
- cholesterol ester transfer protein
- collision induced disassociation
- non-sialylated apoC-III
- monosialylated apoC-III
- disialylated apoC-III
- hypersialylated apoC-III
- dithiothreitol
- ethylenediaminetetraacetic acid
- endoplasmatic reticulum
- electrospray induced tandem mass spectrometry
- formic acid
- fast protein liquid chromatography
- fucose
- galactose
- N-acetylgalactosamine
- N-acetylglucosamine
- genome wide association study
- high density lipoproteins
- HDL cholesterol
- hepatic lipase
- horseradish peroxidase
- intermediary density lipoprotein
- isoelectric focusing
- interleukin-1
- potassium bromide
- lecithin-cholesterol acyltransferase
- low density lipoproteins
- lipoprotein lipase
- murine lipoprotein lipase
- lipopolysaccharide
- ammonium bicarbonate
- polyacrylamide gel electrophoresis
- phosphate buffered saline
- phospholipid transfer protein
- UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferase
- N-acetylneuraminic acid
- sodium dodecyl sulfate
- single nucleotide polymorphism
- scavenger receptor B1
- trifluoracetic acid
- uridine diphosphate
- very low density lipoprotein
iv
Introduction
Lipoproteins
Lipoproteins are water soluble micelles with an inner core of lipids. The outer hydrophilic layer of the
lipoproteins is made up of phospholipids, free cholesterol and various proteins. These proteins are
called apolipoproteins and they are critical to the function of the lipoproteins. The main function of
lipoproteins is the transport of lipids in the blood stream [1,2]. Apolipoproteins also have important
functions for the distribution of lipids, as cofactors for enzymes in the lipid metabolism and for
maintaining the structure of the lipoproteins [1].
Classification
Lipoproteins are usually classified according to their density obtained by ultracentrifugation. This
density mostly depends on their triglyceride content and size. There are six major classes of
lipoproteins; chylomicrons, chylomicron remnants, very low density lipoprotein (VLDL), intermediate
density lipoprotein (IDL), low density lipoprotein (LDL) and high density lipoprotein (HDL).
Chylomicrons are the largest lipoproteins with the lowest density of less than 0.93 g/mL. These are
produced in the epithelium and contain apolipoprotein (apo) E, apoC-II and apoB-48. Chylomicron
remnants arise from hydrolysis of triglycerides in chylomicrons.
The next group of lipoproteins includes VLDL, IDL and LDL. These have a density of 0.93-1.006
g/mL, 1.006-1.019 g/mL and 1.019-1.063 g/mL respectively. Just like chylomicrons, these are
triglyceride-rich and contain both apoE and apoC-II (there are however a lot more proteins present). In
contrast to chylomicrons, VLDL, IDL and LDL contain apoB-100 instead of apoB-48. This difference
is important since apoB-100 is a natural ligand for the LDL receptor. VLDL is synthesized in the liver
while IDL and LDL arise from hydrolysis of VLDL and IDL respectively. LDL is cholesterolenriched particles which main functions is the transport of cholesterol to extrahepatic tissues as well to
the liver.
HDL has a density of 1.063-1.210 g/mL. These are produced in the intestine and liver in the form of
disc-shaped precursor forms called pre-ß-HDL. These precursors contains a small amount of
phospholipids and apoA-I. Pre-ß-HDL matures when obtaining cholesterol and triglycerides from
peripheral tissues [3].
Function
Chylomicrons function as transporters of dietary lipids such as triglycerides and cholesterol from the
intestine to peripheral cells. At extrahepatic tissues, triglycerides are hydrolyzed by lipoprotein lipase
(LPL) which reduces their size and density which finally produces the chylomicron remnants. The free
fatty acids generated in the lipolysis acts as a source of energy for the cells [1-3]. ApoC-II in the
chylomicrons are activators of LPL while apoE functions as a ligand to the LDL receptor which is
important in the hepatic uptake of chylomicron remnants. In the hepatocytes, the remnants are used for
synthesis of VLDL.
VLDL functions as transporters of triglycerides and cholesterol from the liver to extrahepatic tissues.
There, LPL hydrolyzes the internal triglycerides which transform the VLDL to IDL. IDL can then be
further hydrolyzed by hepatic lipase (HL) to LDL. Both VLDL and IDL interact with HDL and
transfers apoE and apoC-II to them. VLDL, IDL and LDL are taken up into hepatocytes by the means
of apoB-100 and apoE binding to LDL receptors on the hepatocytes [3].
HDL are the smallest of the lipoproteins and function in the reverse cholesterol transport, which is the
process by which cholesterol is transported from peripheral tissues to the liver for excretion. HDL
obtains cholesterol by absorption of free cholesterol from peripheral cells. This is mainly facilitated by
the binding of apoA-I on HDL to ATP-binding cassette transporter A1 (ABCA1) on the donor cells
[3,4]. ABCA1 also resides in the internal endocytotic machinery and functions in the efflux of
cholesterol from late endosomes to the plasma membrane. Besides the ABCA1 already located in the
endocytotic parts, it can also be internalized and transported from the plasma membrane to help with
the cholesterol efflux from late endosomes. Another important transporter is ATP-binding cassette
transporter G1 (ABCG1) which mediates efflux of phosphatidylcholine and sphingomyelin from the
cells. These may have a regulatory function on the cholesterol efflux from the cells [4]. The
1
amphipathic cholesterol obtained by HDL is then esterified by the enzyme lecithin-cholesterol
acyltransferase (LCAT) which is activated by apoA-I on HDL. Esterified cholesterol, cholesteryl ester,
is more hydrophobic and moves into the core of HDL. When cholesteryl ester is moved into the
particle, this allows more free cholesterol to adsorb to the HDL [3-5]. LCAT is most present and
active on pre-ß-HDL while the number of LCAT decreases when the particles grow with the uptake of
cholesterol [5]. By the activity of LCAT, pre-ß-HDL matures into the spherical α-HDL. There is
however two types of mature α-HDL. The first, which arises during the maturation process, is called
HDL3 and this form is smaller and denser than HDL2 which arises after uptake of cholesterol from
peripheral cells [3,5]. The state of HDL is however not a fixed state. Action of HL causes spherical αHDL to be remodeled to pre-ß-HDL while HDL2 is remodeled to HDL3 by the same enzyme [5].
For removal of cholesteryl ester from the mature spherical α-HDL, there are two pathways. In the
minor or direct pathway, HDL binds directly to scavenger receptor B1 (SRB1) or apoE on hepatocytes
which cause efflux of cholesterol to the hepatocytes [3]. The majority of cholesterol in HDL is
transferred to apoB-containing lipoproteins, such as VLDL, IDL or LDL, by cholesterol ester transfer
protein (CETP). These are subsequently taken up by hepatocytes by the binding of apoE and apoB-100
to LDL receptors on the hepatocytes [3,4]. CETP also promotes the efflux of triglycerides from
triglyceride rich lipoproteins, such as LDL, to HDL [4]. The activity of CETP is inhibited by apoC-I
and apoF which mainly reside on VLDL and LDL respectively [5]. Phospholipid transfer protein
(PLTP), affects the levels of HDL by its transfer of phospholipids from lipoprotein remnants to HDL
precursors. Both PLTP and CETP belongs to the family lipid transfer/lipopolysaccharide (LPS)binding proteins which protects against systemic inflammation [4].
Atherosclerosis and Lipoproteins
Atherosclerosis is generally considered an inflammatory disease in which arterial walls gets thickened.
This thickening is due to atherosclerotic plaques which cause reduced blood flow. Serious
complications of the plaques are ruptures which can lead to thrombosis and subsequent coronary heart
attack.
There are several risk factors for atherosclerosis including age, gender, smoking, hypertension,
hypertriglyceridemia and diabetes. Another risk factor is hyperlipoproteinemia, and especially higher
concentrations of LDL. LDL is a key player in the pathophysiology of atherosclerosis since its
infiltration of an arterial wall with subsequent oxidation of LDL is the start of plaque formation. These
oxidized LDL particles are then phagocytized by macrophages through scavenger receptors. A
continued process may transform the macrophages into a foam cell which are the major constituents of
plaques [6,7]. Oxidized LDL causes the foam cells to produce more inflammatory cytokines such as
interleukin-1 (IL-1). These inflammatory cytokines increases the adhesion of LDL to the endothelium
which subsequently raises oxidized LDL in the arterial wall [7].
Hypercholesterolemia is a risk factor for atherosclerosis. The reverse cholesterol transport is therefore
an important function for preventing atherosclerosis since it lowers the systemic cholesterol levels and
prevents formation of foam cells [3,4]. The levels of HDL and HDL cholesterol (HDL-c) are much
intertwined with the levels of triglycerides in the serum. An increase of triglyceride levels affects the
HDL so that the HDL-c concentrations are lowered and the sizes of both LDL and HDL particles are
reduced. The larger and less dense HDL2 is believed to be more protective against atherosclerosis than
the smaller, earlier HDL3. Raised triglyceride levels therefore leads to reduced protection from
atherosclerosis. By lowering triglyceride levels, with for example fibric acid derivates, the HDL-c
levels as well as the size of HDL and LDL increases. This provides a less atherogenic lipid profile [8].
Besides the removal of cholesterol from peripheral cells in the arterial wall, HDL also display
antioxidative properties. This is done by enzymes associated in the HDL, mainly paraoxonase [9].
Lipoprotein lipase (LPL)
Lipoprotein (LPL) is synthesized in adipocytes as well as in cardiac and skeletal myocytes. It is
transported to the capillary endothelium where it is bound to heparin sulfate proteoglycans. This
enzyme´s function is to hydrolyze triglycerides in chylomicrons and VLDL. The free fatty acids which
are liberated during the hydrolysis is used for energy in myocytes and stored as energy in adipocytes.
The remnants of the lipoproteins, including surface phospholipids and apolipoproteins, are thereafter
acquired by HDL.
2
In transgenic mice in which murine lipoprotein lipase (Lpl) is overexpressed, the levels of HDL-c in
the blood are significantly increased. Analogously, Lpl knockout mice have lower concentrations of
HDL-c and hypertriglyceridemia. In humans with LPL deficiency, the level of triglycerides is normal
but there is a lower concentration of HDL-c [10].
Glycosylation
Glycosylation of proteins is one of largest classes of post-translational modifications [11,12] since
about half of the human proteins have some kind of glycosylation [11]. The two major types of
glycosylation in humans are called N-linked and O-linked glycosylation. The most common type of Nlinked glycosylation is when N-acetylglucosamine (GlcNAc) is linked to asparagine (Asn) [11,12].
This happens at sequences of Asn-X-serine/threonine where X is any amino acid except proline [12].
There exist several different enzymes, such as N-glycosidase F and N-glycosidase A, which can cleave
the linked oligosaccharide from proteins. These can be used for investigating changes in glycosylation
since they allow complete detachment of oligosaccharides from the protein backbone and subsequent
analysis with mass spectrometry [11,12].
O-linked glycosylation is a diverse class of modifications which occurs on serine, threonine or lysine.
Based upon which monosaccharide that is linked to the protein backbone, seven forms of O-linked
glycosylation can be distinguished. Since each of these monosaccharides have two to three additional
linkage sites to additional monosaccharides and the binding can be in both α and ß configuration, the
complexity of O-linked oligosaccharides are great [11]. Compared to N-linked glycosylation, there is
no general sequence in which glycosylation occurs. These two factors, together with the fact that there
are no enzymes which can cleave the linked oligosaccharides from the protein backbone, makes the Olinked glycosylation more difficult to investigate compared to N-linked. The most common O-linkage
molecule is N-acetylgalactosamine (GalNAc) which constitutes what is called mucin-type O-linked
glycosylation of proteins [11,12].
Mucin-type O-linked Glycosylation
In mucin-type O-linked glycosylation, α-GalNAc from the donor uridine diphosphate-GalNAc
(UDP-GalNAc) is attached to the hydroxyl group of serine and threonine residues on proteins. This
process is initiated by members of the enzyme family UDP-GalNAc:polypeptide α-Nacetylgalactosaminyltransferases (pp-GalNAcTs) in the Golgi [13,14]. There are about 20 members in
the family [14] and they share a common structure in that they contain a short N-terminal cytoplasmic
tail, a transmembrane anchor and finally a large C-terminal luminal segment which resides within the
lumen of the Golgi [15]. C-terminally, there is a ricin-type lectin domain which contains three binding
sites for carbohydrates which are believed to be important for the catalytic activity of the enzymes
[16].
The initial linkage of α-GalNAc to the protein creates what is called a Tn antigen. The Tn antigen can
be elongated by other glycosyltransferases into a set of different core structures. There are at least
seven different core structures. The most common is Core 1 which has a GalNAc acting as the link
between serine/threonine and the monosaccharide Gal. The core structures can also be further
modified by addition of other moieties such as N-acetylneuraminic acid (sialic acid, NeuAc), fucose
(Fuc), GlcNAc and GalNAc [11,13]. To further complicate things, Gal and GlcNAc can be sulphated
at positions 3 and 6 while NeuAc can be modified on positions 4, 7, 8 and 9 by O-acetyl ester groups
[11].
ApoC-III
ApoC-III is a protein produced in the liver and intestines which can be found in chylomicrons, VLDL,
LDL, IDL and HDL. In a fasting state of normolipidic (normal level of lipids) individuals, it is mainly
located on HDL while in a fed state it transfers to chylomicrons and VLDL [17,18]. It has been shown
that apoC-III is mainly located to VLDL in hypertriglyceridemic individuals who also display a larger
total amount of total apoC-III in the plasma [18]. The protein is synthesized as a 99 amino acid long
3
precursor with a 20 amino acid signal. This signal is spliced in the endoplasmatic reticulum (ER)
which gives the final protein length [17]. ApoC-III main function is inhibition of LPL which leads to
increased amounts of triglycerides in plasma [18-20] Besides the LPL inhibition, apoC-III also
functions as an inhibitor of apoE-containing VLDL binding to hepatic receptors [21,22] and apoB
binding to the LDL receptor with subsequent uptakes [23].
ApoC-III contains one single O-glycosylation site at T94 (threonine at position 94 including signal).
Attached to this site is a Core 1-structure on which none, one or two molecules of sialic acid may be
attached. The different numbers of sialic acids give rise to the three main isoforms of apoC-III, called
non-sialylated (C-III0), monosialylated (C-III1) and disialylated (C-III2) according to number of sialic
acids [24-26]. However, it has also been reported that C-III0 not just lacks sialic acids, but the whole
oligosaccharide structure [27]. In a more recent communication, it was showed that C-III0 consists of
four different isoforms (hereby called sub-isoforms to distinguish from C-III2 and C-III1). They
proposed that the most common sub-isoform is a variant in which only GalNAc is linked to threonine
[28]. There are also a fourth isoform, called hypersialylated apoC-III (C-III3), which has three sialic
acids due to hypersialylation [29].
C-III1 and C-III2 have similar production and clearance rates while C-III0 has lower production and
clearance rate. The production rate has been shown to be positively correlated with VLDL
triglycerides and cholesterol. C-III0 is therefore least associated with hyper-triglyceridemia, probably
due to lower inhibition of LPL [30]. The relative levels of the isoforms changes during life. The
proportion of C-III0 and C-III1 increases with age while the proportion of C-III2 decreases with age. By
isoelectric focusing it has been shown that chronic alcohol abuse does not affect the isoform
distribution [31].
Background to project
This project is part of a larger project in which the novel loss-of-function mutation D314A in ppGalNAcT2 was investigated due to increased HDL-c and decreased triglyceride levels in
heterozygotes compared to age and gender matched family controls. An important finding was that an
apoC-III based peptide, including the glycosylation site at position 94, is a substrate for ppGalNAcT2. The mutant form of pp-GalNAcT2 were however less able to glycosylate the peptide due
to lower activity. Neuroaminidase treatment of apoC-III, which removes the sialic acids, reduced the
ability of apoC-III to inhibit LPL.
Aim
The aim of the project was to investigate how the mutations D314A and Q216H in the enzyme ppGalNAcT2 affect the isoform distribution of apoC-III. Furthermore, to investigate if intake of fat
changes the isoform distribution in VLDL and HDL of heterozygotes for D314A. Finally, to determine
the sugar content of apoC-III with mass spectrometry.
The working hypothesis of the project was that the mutations will indeed change the isoform
distribution in a way that could explain the altered levels of triglycerides and HDL-c found in the
heterozygotes for the mutations.
4
Methods
Sample collection
Isolated samples were kindly provided by Amsterdam Academic Medical Center. Whole blood had
been collected, with EDTA, from voluntary heterozygotes for a D314A or Q216H mutation in the
enzyme pp-GalNAcT2 and from voluntary age and gender matched family members lacking the
described mutations. Plasma had been isolated by routine centrifugation and kept in -70˚C.
For analysis of glycosylation in the D314A group, HDL was isolated using two-step density
ultracentrifugation. The first step of ultracentrifugation was done by mixing 4.5 mL plasma with 0.5
mL 5% sucrose solution containing ethylenediaminetetraacetic acid (EDTA, 10 mg/mL) and 1.632g
potassium bromide (KBr) in a Beckman ultracentrifuge tube (Beckman, ultraclear™ 16 x 76 mm2,
total volume 10.4 mL). This gave the solution a density of 1.22 g/mL. Gently a layer of ice-cool
phosphate buffered saline (PBS) was added on top in the tube. Ultracentrifugation was done in a
Beckman XL-90 equipped with a pre-cooled Ti 70 rotor (fixed angle, Beckman Instruments, US, CA)
at 290 000 x g in 4˚C for 2 h for LDL and 4 h for HDL. The phase containing LDL or HDL was
extracted using a syringe and needle and directly put in a new Beckman ultracentrifuge tube. Tube was
filled with KBr solution (0.133 g/mL) containing EDTA (1 mg/mL) which has a density of 1.10 g/ml.
The second round of ultracentrifugation was performed at 290 000 x g in 4˚C for 2 h for LDL and 4 h
for HDL. Isolated LDL and HDL fractions were extracted with syringes and transferred to test tubes.
Desalting and protein concentration measurement
Plasma and HDL fractions were desalted using ammonium bicarbonate (NH4HCO3, 12mM, pH 7.1)
and PD-10 desalting columns (Sephadex™ G-25 Medium, GE Healthcare, UK) according to
manufacturer´s instructions. Protein concentration in desalted samples was determined by mixing 15
µL desalted sample with 250 µL BioRad protein assay solution (BioRad, US, CA) in a 96 well plate.
The change of absorbance, which happens when the dye bind to proteins, was detected with a FluoStar
spectrophotometer (BMG LabTech GmbH, Germany).
Lipoprotein fractions were freeze dried by keeping the samples at -60˚C in vacuum over night.
Two-dimensional gel electrophoresis (2DE)
Due to glycosylation of apoC-III, three well describes isoforms can be distinguished based upon their
number of sialic acids. Sialic acids are charged molecules which will affect the net charge of the
isoforms. However, isolation by one-dimensional gel electrophoresis is insufficient to isolate the
described sub-isoforms of C-III0 due to similar masses [28]. Instead, two-dimensional gel
electrophoresis (2DE) can be used since it separates the proteins both on their molecular weight and
charge. This allows distinction between the three isoforms of apoC-III.
Isoelectric focusing (IEF)
Freeze dried HDL samples were dissolved in 150-200 µL sample solution containing 0.54 g/mL urea
(Sigma-Aldrich, US, MO), 10 mg/mL dithiothreitol (DTT, Sigma-Aldrich), 2% Pharmalyte™ 4-7 (GE
Healthcare), 0.04 g/mL CHAPS (Sigma-Aldrich) and 10 µL/mL bromphenolblue. When using plasma,
5 µL of desalted plasma were mixed with 95 µL sample solution. For HDL, a volume corresponding to
500-800 µg of protein was mixed with rehydration solution containing 8M urea, 4% CHAPS and a
small amount of Orange G (Sigma-Aldrich) to the final volume 350 µL. Plasma mixed with sample
solution (100 µL) was mixed with 250 µL rehydration solution.
The solution was placed in the bottom of a ceramic IEF holder and an Immobiline IPG DryStrip pH 47 (18 cm, GE Healthcare) was placed on top. The strip and protein solution was covered with
Immobiline DryStrip Cover Fluid (GE Healthcare) and IEF was performed for approximately 48000
Vhrs and 54000 Vhrs for plasma and HDL respectively.
5
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
A homogenous gel containing a 5% acrylamide stacking gel (crosslinker concentration 5%) and a 14%
acrylamide resolution gel (crosslinker concentration 1.5%) was cast on a gel-bond (GE Healthcare).
The Immobiline DryStrips from the IEF were two-step equilibrated using an equilibration buffer
containing 6M urea, 30.5% glycerol, 4% SDS and 50 mM Tris-HCl set to pH 8.8. The first step also
contained 20 mg/mL DTT while the second contained 0.09 g/mL iodacetamide and 93‰
bromphenolblue.
Homecast gel and Immobiline DryStrip was assembled on a Multiphor II electrophoresis unit (Ge
Healthcare). For HDL investigation, 7 µL of precision plus molecular weight ladder (BioRad) was
added. In western blot, DryStrips were cut at the middle to fit two strips on the same 2D gel. The
acidic end of a DryStrip (which holds apoC-III) from a heterozygote were put besides the DryStrip
from its gender matched control on one 2D gel. 2DE was performed at 60V and 30A over night.
D314A Mutation
Isoform distribution
In the plasma investigation of D314A, a polyvinylidene fluoride (PVDF) membrane (BioRad) was
pre-wet in methanol for 15 s before washing in dH2O for 2 min. Then the membrane was placed in
transfer buffer, containing 25 mM Tris, 192 mM glycine and 10% methanol in dH2O, for 30 min.
Fiberpads and filterpads were also placed in transfer buffer before placed in a transfer cassette. The gel
was shaved from the bond and immediately placed on one of the filterpads. The equilibrated PVDF
membrane was placed on top of the gel and the rest of the transfer cassette assembled. The cassette
was placed in a transfer vessel which was filled with transfer buffer. Western blot was performed at
100V and 0.4 A for 1h. Directly after the blot, the PVDF membrane was placed in blocking solution
containing 5% non-fat dry milk (BioRad) in Tris-HCl buffered saline (TBS) pH 7.5, containing 20
mM trizma base (Sigma Aldrich) and 500 mM NaCl (Sigma Aldrich) for 1 h.
After blocking, the membrane was washed 2x5 min in TBS containing 0.05% Tween-20 (TTBS,
BioRad). Primary antibodies, polyclonal rabbit anti-apoC-III (Abcam, 21032, 1:5000) diluted in 2%
non-fat dry milk in TTBS, was applied to the membrane and incubated in room temperature on a
shaker over night. After incubation the membrane was washed with TTBS before secondary
antibodies, goat anti-mouse with conjugated horseradish peroxidase (HRP, 1:40000, BioRad), was
applied and the membrane incubated on shaker for 1h. Membrane was washed with TTBS before 5
mL mixed Amersham enhanced chemiluminescence (ECL) plus solution (GE Healthcare) was added
and incubated for 1 minute. The chemiluminescence was detected using X-ray films. Exposure time of
membranes was adjusted to achieve sufficient staining. The X-ray films were digitalized using FluorS
camera system (BioRad) and analyzed with PDQuest Software (BioRad).
Staining of apoC-III isoforms was calculated as percent of total apoC-III staining. Images were
normalized to total quantity of apoC-III spots in each sample. Statistical analysis of changes in
isoforms distribution was done by using Mann-Whitney U-test in Graphpad Prism 5 (Graphpad
Software Inc., US, CA).
Fat challenge
Isolated VLDL and HDL fractions, before and after fat challenge, were kindly provided by the
Amsterdam Academic Medical Center. Heterozygotes for the D314A mutation (n=3) in pp-GalNAcT2
and a control (n=1) were subjected to a fat challenge in the form of cream corresponding to 40 g of fat
for every square meter of body surface. Blood was sampled before fat ingestion and 4 hours after
ingestion. Plasma was first isolated by centrifugation and lipoprotein fractions were subsequently
isolated by using fast protein liquid chromotography (FPLC).
VLDL and HDL fractions were then subjected to desalting with PD-10 columns and subsequently
2DE as previously described. The isoform distribution was investigated by using Western blot with
anti-apoC-III (Abcam, 21032, 1:5000) as described above for D314A isoform distribution. The total
volume from desalting and subsequent freeze drying (corresponding to approximately 20 µg) was used
for VLDL analyses. For HDL, a volume corresponding to 200 µg protein was used in IEF and 2DE.
6
Statistical analysis of changes in isoforms distribution after fat challenge in heterozygotes (comparison
of timepoint 0 and timepoint 4) was done by using Students t-test in Graphpad Prism 5 (Graphpad
Software Inc.). Controls were not statistically analyzed.
Oligosaccharide analysis
Silver staining of HDL
For sugar analysis, the gel was directly placed in fix containing 50% methanol and 5% acetic acid after
2DE. The gel was stained by a modified protocol from Shevchenko [32]. First, the gel was washed
with 50% methanol and then directly put in water to remove any residual methanol and acetic acid.
The gel was desensitized using 0.02% sodium thiosulphate and washed twice with dH20. The next step
is the silver staining step in which the gel is subjected to 0.1% silver nitrate. Then the gel was washed
twice in dH20 before developed using 0.04% formaldehyde in 2% sodium bicarbonate decahydrate. To
terminate the development, the gel was washed in 0.5% glycine and thereafter kept in dH20.
The protein gels were digitized with FluorS camera system (BioRad) and analyzed using PDQuest
Software (BioRad).
Trypsination
ApoC-III spots in HDL were picked with a spot-picking tool. These were destained using a solution
with 30 mM potassium ferrycyanide and 100 mM sodium thiosulfate. After 3 min, the gel was washed
in dH2O before 200 mM NH4HCO3 was added. Gel pieces were washed in dH2O before dehydrated for
5 min in 100% acetonitrile (ACN) and subsequently completely dried using vacuum centrifugation.
The gel pieces were trypsinated with 10 µg/mL trypsin (Promega, US, WI) in 25 mM NH4HCO3 for
16 hours at 37˚C. After incubation, the supernatant was transferred to a new test tube and completely
dried by vacuum centrifugation. Samples were reconstituted in 0.1% trifluoretic acid (TFA). The
solution was desalted using ZipTipC18 (Millipore, US, MA). In short, the Ziptips were washed with
50% ACN, equilibrated with 0.1% TFA before the sample with peptides was loaded. After wash with
0.1% TFA, sample were eluted in 6-10 µL 50% ACN.
Electrospray induced Tandem Masspectrometry (ESI-MS/MS)
Desalted samples were mixed with 2 µL formic acid (FA) to increase ionization of peptides. Sample (1
µL) was transferred to silver-coated glass capillary and mounted in an electrospray induced tandem
mass spectrometer (ESI-MS/MS, API Q-STAR Pulzer, Applied Biosystems, US, CA).
Voltage was set to 900V and a full scan was performed. Peaks corresponding to trypsinated apoC-III
peptides containing position 94, were subjected to collision-induced disassociation (CID) and
fragments were detected by tandem mass spectrometry. Spectra from MS/MS were manually
interpreted using Analyst software (Applied Biosystems). Ions representing different fragments of the
peptide (a-, b- and y-ions) were matched against a theoretical fragmentation including possible
oligosaccharides. Peaks with a mass accuracies < 200 ppm and intensities ≥ 2.0 was considered
significant.
Q216H Mutation
Isoform distribution
Western blot of Q216H was performed as described above for the D314A mutation, with a few
exceptions. Firstly, 5 mL mixed Amersham ECL Prime solution (GE healthcare) was used instead of
ECL Plus as chemiluminescence for X-ray film staining. The membranes were kept in ECL Prime for
5 min, according to the manufacturer´s instructions. The films were digitized using Versadoc MP4000 camera system (BioRad).
Statistical interpretation of clinical data was done by Students t-test in Graphpad Prism 5 (Graphpad
Software Inc.).
7
Results
D314A
Isoform distribution
The western blot of D314A 2DE gels showed a significant larger proportion of C-III0 in the
heterozygotes compared to age and gender matched family controls. At the same time, the
heterozygotes displayed a smaller proportion of C-III1 (figure 1 and 2).
C-III2
31%
C-III1
69%
C-III0
0%
C-III2
35%
Control
C-III1
52%
C-III0
13%
Heterozygote
Figure 1. Representative Western blot image of the isoform distribution in a heterozygote for a D314A mutation in ppGalNAcT2 and an age and gender matched control. Numbers represent proportion of each isoform.
D314A mutation
*
% of total ApoCIII
70
Control
Heterozygote
60
50
40
**
30
20
10
0
ApoC-III2
ApoC-III1
ApoC-III0
Figure 2. Isoform distribution of apoC-III in plasma from heterozygotes for a D314A mutation in pp-GalNAcT2 and their age
and gender matched controls obtained by Western blot. Bars represent means and whiskers SD of controls (n=7) and
heterozygotes (n=7). * = p < 0.05, ** = p < 0.01.
Fat challenge
VLDL fractions from heterozygotes for the D314A mutation displayed a relative low proportion of CIII0 in the fasting state (T0). After fat consumption (T=4), the proportion of C-III0 increased
significantly in the heterozygotes but not in the control (figure 3 and 4). In the HDL fractions, no
significant difference was found in the heterozygotes (data not shown).
8
C-III3 C-III2 C-III1 C-II
2% 15% 78%
C-III0
5%
Control T0
C-III3 C-III2 C-III1 C-II
7% 48% 44%
C-III0
1%
Control T4
C-III3 C-III2 C-III1 C-II
3% 47% 49%
C-III0
1%
C-III3 C-III2 C-III1 C-II
12% 44% 33%
C-III0
12%
Heterozygote T4
Heterozygote T0
Figure 3. Representative Western blot images of apoC-III isoform distribution in the VLDL fraction of a D314A
heterozygote in pp-GalNAcT2 and a gender matched control before (T=0) and 4h after (T=4) fat challenge. C-II represents
unspecific binding of antibody to apolipoprotein C-II. Numbers represent proportion of each isoform.
VLDL Heterozygote
% of total ApoC-III
70
T0
T4
60
50
40
30
20
*
10
0
ApoC-III3
ApoC-III2
ApoC-III1
ApoC-III0
Figure 4. Isoform distribution of apoC-III in VLDL from heterozygotes with a D314A mutation in pp-GalNAcT2 obtained by
Western blot. Bars represent means and whiskers SD at the fasting state (T0) and 4h after fat consumption (T4) in
heterozygotes (n=3). * = p < 0.05.
Oligosaccharide analysis
The oligosaccharide content of C-III0 and C-III1 was investigated using ESI-MS/MS. In the apoC-III1
isoform, the mass 1013.5 Da corresponding to a triple charged peptide, position 79-99 with GalNAcGal-NeuAc attached was selected. Selected peptide was fragmented with CID which produced a-, band y-ions. By manual interpretation, the ions were matched with peptide fragments which allowed a
large amount of sequence coverage (figure 5). Typical peaks with the masses 204, 292 and 366 Da,
corresponding to single charged oxonium ions of GalNAc, NeuAc and GalNAc-Gal respectively, was
found. Several different masses corresponding to peptides in C-III0 were investigated. No peptide was
however sequenced due to insufficient amount of protein for ESI-MS/MS.
9
Figure 5. Sequence information of apoC-III1 obtained by ESI-MS/MS. Peak corresponding to +3 ion of peptide with position
79-99 including potential oligosaccharide (at threonine, position y6/a16/b16). Ions considered include a and b ions in Nterminal to C-terminal direction and y ions in C-terminal to N-terminal direction. Summary of the obtained sequence is
located in top of the image. All ions corresponding to y>5 or b>15 includes N-acetylgalactosamine (GalNAc), Galactose
(Gal) and Sialic acid (NeuAc) if not otherwise stated.
Q216H
Isoform distribution
Investigated carriers of Q216H mutation and gender matched controls showed no significant
differences according to age, HDL-c and triglyceride levels (table I).
There were no significant difference in isoform distribution between heterozygotes and gender
matched controls for the Q216H mutation (figure 6).
Table I. Lipid data for Q216H mutation. Numbers within parenthesis represents SD.
Wild type family controls (n=7)
Q216H mutation carriers (n=7)
Gender
5 Female, 2 Male
5 Female, 2 Male
Age
36.7 (21.9)
48.4 (18.8)
HDL-c
1.6 (0.3)
1.9 (0.5)
Triglycerides
1.1 (0.8)
1.2 (0.5)
Q216H mutation
Control
Heterozygote
% of total ApoCIII
60
40
20
0
ApoC-III3
ApoC-III2
ApoC-III1
ApoC-III0
Figure 6. Isoform distribution of apoC-III in heterozygotes with Q216H mutation in pp-GalNAcT2 and their gender matched
controls obtained by Western blot. Bars represent means and whiskers SD.
10
Discussion
In a meta-analysis of four genome-wide association studies (GWAS), including more than 100 000
humans, a single nucleotide polymorphism (SNP) in the gene for GalNAcT2 was found to be
associated with increased levels of HDL-c and decreased levels of triglycerides. In mice, overexpression of the gene caused decreased levels while knock-down caused increased levels of HDL-c
[33].
In this study, we found that for the D314A mutation, the proportion of C-III0 increased while C-III1
decreased in heterozygotes compared to controls. As previously stated, C-III0 is the least associated, of
the three isoforms, with levels of triglycerides in humans [30]. It has also been shown that by
removing the sialic acids from apoC-III in VLDL by the use of neuroaminidase, the LPL inhibiting
activity is reduced [34]. In contrast, it has been reported that there is no difference in LPL inhibiting
activity between C-III0 and C-III1 [35] and that several patients suffering from familial
hypertriglyceridemia have increased proportion of C-III0 [36]. However, the results of this project
together with the findings in the larger study showing decreased activity of D314A pp-GalNAcT2
against apoC-III peptide substrate, reduced ability to inhibit LPL in carriers and that neuroaminidase
treatment of apoC-III decreases the LPL inhibition, implicates that heterozygotes would be less able to
inhibit LPL via less sialylated apoC-III. This would explain the decrease in triglyceride levels in the
heterozygotes as well as the increase in HDL-c. Interestingly, the Q216H mutation did not show a
similar change in apoC-III isoform distribution as D314A. At the same time, the Q216H
heterozygotes, in contrast to D314A heterozygotes, did not have altered HDL-c and triglyceride levels.
Both the D314A and Q216H mutation reside within catalytical domains of pp-GalNAcT2 (according
to Uniprot). However, they reside within two different domains and D314A is the only one which
produces a charge alteration. This may explain the difference in pp-GalNAcT2 function and
subsequent alteration of isoform distribution in apoC-III between the two mutations.
In the fat challenge tests, only a small number of heterozygotes were investigated. The results are
therefore preliminary. However, the results indicate that the proportion of C-III0 increased in VLDL
from the heterozygotes, while this was not found in the single control. The significance of an increased
proportion of C-III0 in VLDL in the heterozygotes with the D314A mutation is unclear. It may reflect
that upon fat ingestion, new VLDL is synthesized and that in the heterozygotes there is a larger
proportion which is non-sialylated compared to the controls. In normolipidic subjects, total apoC-III is
translocated from HDL to VLDL after ingestion of fat while the carriers of the D314A mutation have a
translocation of total apoC-III from VLDL to HDL. A second explanation for the apparent increase in
VLDL C-III0 may then be that C-III2 and C-III1 are more readily translocated compared to C-III0. We
were however unable to detect an increased distribution of sialylated isoforms in HDL. A third
explanation could be that both C-III2 and C-III1 have a faster clearing rate in VLDL, IDL and some
LDL [30]. This clearing is still not understood and may therefore depend upon translocation between
lipoproteins or by removal from the lipoproteins (maybe by binding to functional apoC-III to
receptors). More studies are therefore needed to be able to draw any conclusion about differences in
apoC-III isoform distribution after fat challenge.
By using ESI-MS/MS, we were able to sequence C-III1 and also detect peaks with the masses 366, 204
and 292 Da which are previously described as the oxonium ions (single charge) of HexNAc-Hex,
HexNAc and NeuAc respectively [37]. However, HexNAc can either be GalNAc or GlcNAc, since
they have the same mass. The same goes for Hex which can be either Gal or Glucose (Glc). However,
based on previous studies about apoC-III [25,26], and that O-linked GlcNAc is not further elongated
[11], it is safe to assume that the ions found represent GalNAc-Gal, GalNAc and NeuAc. As illustrated
in figure 3, C-III0 probably consists of at least three sub-isoforms. There is however diversity in the
distribution and sometimes even a fourth sub-isoform can be seen. This is in line with a previous
report that stated that C-III0 consists of four different sub-isoforms of which none was fully
characterized [28]. We were unable to sequence apoC-III0 and determine which eventual
monosaccharides contribute to the sub-isoforms due to low amount of protein. As a future direction of
the project, apoC-III should be enriched with for example immuno-affinity purification. Hopefully this
will provide sufficient amounts of the sub-isoforms to fully characterize them.
Overexpression of apoC-III in transgenic mice has been shown to cause hypertriglyceridemia in the
animals [38] while knockout mice have reduced levels of triglycerides [39]. Mice expressing human
11
apoC-III and a defect LDL receptor have been bred and these showed increased levels of VLDL, IDL
and LDL at the same time as they were more prone to develop atherosclerosis. The effect of human
apoC-III expression and LDL receptor deletion appeared to be synergistic which points toward a
connection in vivo, at least in mice [38]. In humans, it has been shown that a nonsense-mutation in
apoC-III reduced the levels of apoC-III in the blood and provides higher HDL-c and lower
triglycerides [40]. Despite much research into the function of apoC-III, the clinical significance of
different isoforms of apoC-III is still not clear. As previously stated, some have found that there are no
significant differences while others propose that C-III0 is less efficient as an inhibitor of LPL and
therefore provides a favourable lipid profile. It is therefore too early to draw any final conclusions
regarding whether our found differences in D314A heterozygotes (and lack of difference for Q216H)
really is clinical relevant. However, the isoform change in D314A is still a probable candidate for the
found differences in triglyceride levels and HDL-c in the heterozygotes. If this in the future will prove
to be a significant finding with clinical relevance, the disruption of pp-GalNAcT2 may be a novel way
to reduce the function of apoC-III and therefore lower triglyceride levels and raise HDL-c levels. More
research is however needed to fully characterize apoC-III isoforms and further investigate whether
disruptions of the enzyme pp-GalNAcT2 with subsequent change in apoC-III may be used as a clinical
tool to prevent cardiovascular disease.
In summary, we have found that heterozygotes for the mutation D314A in pp-GalNAcT2 have an
increase of C-III0 proportion in plasma and that the proportion of C-III0 seems to increase in VLDL
after ingestion of a large amount of fat. The monosaccharide content of the C-III1 isoform was
confirmed while C-III0 still needs to be investigated further. Heterozygotes for the Q216H mutation in
pp-GalNAcT2 did not display a similar change in C-III isoform proportions as D314A.
12
Acknowledgements
First, I would like to thank both my supervisors Mats and Helen. Thank you Mats for accepting me to
your group, all help in writing and for making me improve as a scientist. Thank you Helen for all the
practical help, interesting theoretical discussions as well as all the laughs in the lab.
Secondly, thanks to Louise, Bijar and Patrik. You all helped me with different aspects of the project as
well as always keeping my spirits up.
Thanks to Inger and Jan for always helping me with the practical stuff whenever I needed someone
who knew better.
Thanks to Sara, Anders, Liselott and Sasha for being at the lab to share the fun and work.
Thanks to all other students and personnel at Occupational and Environmental Medicine.
Last, but never least, thank you my love, my fiancée, my everything Therese. Without you at my side,
I would have lost so much. Olive Juice. Always.
13
References
1. Mahley, R.W., Innerarity, T.L., Rall, S.C.,Jr & Weisgraber, K.H. 1984, "Plasma
lipoproteins: apolipoprotein structure and function", Journal of lipid research, vol. 25, no.
12, pp. 1277-1294.
2. Hoofnagle, A.N. & Heinecke, J.W. 2009, "Lipoproteomics: using mass spectrometrybased proteomics to explore the assembly, structure, and function of lipoproteins",
Journal of lipid research, vol. 50, no. 10, pp. 1967-1975.
3. Tulenko, T.N. & Sumner, A.E. 2002, "The physiology of lipoproteins", Journal of nuclear
cardiology, vol. 9, no. 6, pp. 638-649.
4. Schmitz, G. & Grandl, M. 2009, "The molecular mechanisms of HDL and associated
vesicular trafficking mechanisms to mediate cellular lipid homeostasis", Arteriosclerosis,
thrombosis, and vascular biology, vol. 29, no. 11, pp. 1718-1722.
5. Borggreve, S.E., De Vries, R. & Dullaart, R.P. 2003, "Alterations in high-density
lipoprotein metabolism and reverse cholesterol transport in insulin resistance and type 2
diabetes mellitus: role of lipolytic enzymes, lecithin:cholesterol acyltransferase and lipid
transfer proteins", European journal of clinical investigation, vol. 33, no. 12, pp. 10511069.
6. Choy, P.C., Siow, Y.L., Mymin, D. & O, K. 2004, "Lipids and atherosclerosis",
Biochemistry and cell biology = Biochimie et biologie cellulaire, vol. 82, no. 1, pp. 212224.
7. Ross, R. 1999, "Atherosclerosis--an inflammatory disease", The new England journal of
medicine, vol. 340, no. 2, pp. 115-126.
8. Tsai, M.Y. & Ordovas, J.M. 2009, "APOC3 mutation, serum triglyceride concentrations,
and coronary heart disease", Clinical chemistry, vol. 55, no. 7, pp. 1274-1276.
9. Ragbir, S. & Farmer, J.A. 2010, "Dysfunctional high-density lipoprotein and
atherosclerosis", Current atherosclerosis reports, vol. 12, no. 5, pp. 343-348.
10. Jin, W., Marchadier, D. & Rader, D.J. 2002, "Lipases and HDL metabolism", Trends in
endocrinology and metabolism, vol. 13, no. 4, pp. 174-178.
11. Wopereis, S., Lefeber, D.J., Morava, E. & Wevers, R.A. 2006, "Mechanisms in protein Oglycan biosynthesis and clinical and molecular aspects of protein O-glycan biosynthesis
defects: a review", Clinical chemistry, vol. 52, no. 4, pp. 574-600.
12. Jensen, P.H., Kolarich, D. & Packer, N.H. 2010, "Mucin-type O-glycosylation--putting
the pieces together", The FEBS journal, vol. 277, no. 1, pp. 81-94.
13. Hang, H.C. & Bertozzi, C.R. 2005, "The chemistry and biology of mucin-type O-linked
glycosylation", Bioorganic & medicinal chemistry, vol. 13, no. 17, pp. 5021-5034.
14
14. Gerken, T.A., Jamison, O., Perrine, C.L., Collette, J.C., Moinova, H., Ravi, L.,
Markowitz, S.D., Shen, W., Patel, H. & Tabak, L.A. 2011, "Emerging paradigms for the
initiation of mucin type protein O-glycosylation by the polypeptide GalNAc transferase
(ppGalNAc T) family of glycosyltransferases", The journal of biological chemistry, vol.
286, no. 16, pp. 14493-14507.
15. Ten Hagen, K.G., Fritz, T.A. & Tabak, L.A. 2003, "All in the family: the UDPGalNAc:polypeptide N-acetylgalactosaminyltransferases", Glycobiology, vol. 13, no. 1,
pp. 1R-16R.
16. Fritz, T.A., Raman, J. & Tabak, L.A. 2006, "Dynamic association between the catalytic
and lectin domains of human UDP-GalNAc:polypeptide alpha-Nacetylgalactosaminyltransferase-2", The journal of biological chemistry, vol. 281, no. 13,
pp. 8613-8619.
17. Kawakami, A. & Yoshida, M. 2009, "Apolipoprotein CIII links dyslipidemia with
atherosclerosis", Journal of atherosclerosis and thrombosis, vol. 16, no. 1, pp. 6-11.
18. Batal, R., Tremblay, M., Barrett, P.H., Jacques, H., Fredenrich, A., Mamer, O., Davignon,
J. & Cohn, J.S. 2000, "Plasma kinetics of apoC-III and apoE in normolipidemic and
hypertriglyceridemic subjects", Journal of lipid research, vol. 41, no. 5, pp. 706-718.
19. Ito, Y., Azrolan, N., O'Connell, A., Walsh, A. & Breslow, J.L. 1990,
"Hypertriglyceridemia as a result of human apo CIII gene expression in transgenic mice",
Science, vol. 249, no. 4970, pp. 790-793.
20. Lambert, D.A., Catapano, A.L., Smith, L.C., Sparrow, J.T. & Gotto, A.M.,Jr 1997, "A
new fluorometric method for measuring the action of C apolipoproteins on milk
lipoprotein lipase", Clinica chimica acta; international journal of clinical chemistry, vol.
264, no. 1, pp. 75-90.
21. Sehayek, E. & Eisenberg, S. 1991, "Mechanisms of inhibition by apolipoprotein C of
apolipoprotein E-dependent cellular metabolism of human triglyceride-rich lipoproteins
through the low density lipoprotein receptor pathway", The journal of biological
chemistry, vol. 266, no. 27, pp. 18259-18267.
22. Morita, S.Y., Sakurai, A., Nakano, M., Kitagawa, S. & Handa, T. 2011, "Presence of
apolipoprotein C-III attenuates apolipoprotein E-mediated cellular uptake of cholesterolcontaining lipid particles by HepG2 cells", Lipids, vol. 46, no. 4, pp. 323-332.
23. Clavey, V., Lestavel-Delattre, S., Copin, C., Bard, J.M. & Fruchart, J.C. 1995,
"Modulation of lipoprotein B binding to the LDL receptor by exogenous lipids and
apolipoproteins CI, CII, CIII, and E", Arteriosclerosis, thrombosis, and vascular biology,
vol. 15, no. 7, pp. 963-971.
24. Maeda, H., Hashimoto, R.K., Ogura, T., Hiraga, S. & Uzawa, H. 1987, "Molecular
cloning of a human apoC-III variant: Thr 74----Ala 74 mutation prevents Oglycosylation", Journal of lipid research, vol. 28, no. 12, pp. 1405-1409.
15
25. Vaith, P., Assmann, G. & Uhlenbruck, G. 1978, "Characterization of the oligosaccharide
side chain of apolipoprotein C-III from human plasma very low density lipoproteins",
Biochimica et biophysica acta, vol. 541, no. 2, pp. 234-240.
26. Brewer, H.B.,Jr, Shulman, R., Herbert, P., Ronan, R. & Wehrly, K. 1974, "The complete
amino acid sequence of alanine apolipoprotein (apoC-3), and apolipoprotein from human
plasma very low density lipoproteins", The Journal of biological chemistry, vol. 249, no.
15, pp. 4975-4984.
27. Ito, Y., Breslow, J.L. & Chait, B.T. 1989, "Apolipoprotein C-III0 lacks carbohydrate
residues: use of mass spectrometry to study apolipoprotein structure", Journal of lipid
research, vol. 30, no. 11, pp. 1781-1787.
28. Bruneel, A., Robert, T., Lefeber, D.J., Benard, G., Loncle, E., Djedour, A., Durand, G. &
Seta, N. 2007, "Two-dimensional gel electrophoresis of apolipoprotein C-III and other
serum glycoproteins for the combined screening of human congenital disorders of O- and
N-glycosylation", Proteomics - clinical applications, vol. 1, no. 3, pp. 321-324.
29. Jabs, H.U. & Assmann, G. 1987, "Characterization of an apolipoprotein C-III mutant by
high-performance liquid chromatography and time-of-flight secondary ion mass
spectrometry", Journal of chromatography, vol. 414, no. 2, pp. 323-333.
30. Mauger, J.F., Couture, P., Bergeron, N. & Lamarche, B. 2006, "Apolipoprotein C-III
isoforms: kinetics and relative implication in lipid metabolism", Journal of lipid research,
vol. 47, no. 6, pp. 1212-1218.
31. Wopereis, S., Grunewald, S., Morava, E., Penzien, J.M., Briones, P., Garcia-Silva, M.T.,
Demacker, P.N., Huijben, K.M. & Wevers, R.A. 2003, "Apolipoprotein C-III isofocusing
in the diagnosis of genetic defects in O-glycan biosynthesis", Clinical chemistry, vol. 49,
no. 11, pp. 1839-1845.
32. Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. 1996, "Mass spectrometric sequencing
of proteins silver-stained polyacrylamide gels", Analytical Chemistry, vol. 68, no. 5, pp.
850-858.
33. Teslovich, T.M., Musunuru, K., Smith, A.V., Edmondson, A.C., Stylianou, I.M., Koseki,
M., Pirruccello, J.P., Ripatti, S., Chasman, D.I., Willer, C.J., Johansen, C.T., Fouchier,
S.W., Isaacs, A., Peloso, G.M., Barbalic, M., Ricketts, S.L., Bis, J.C., Aulchenko, Y.S.,
Thorleifsson, G., Feitosa, M.F., Chambers, J., Orho-Melander, M., Melander, O.,
Johnson, T., Li, X., Guo, X., Li, M., Shin Cho, Y., Jin Go, M., Jin Kim, Y., Lee, J.Y.,
Park, T., Kim, K., Sim, X., Twee-Hee Ong, R., Croteau-Chonka, D.C., Lange, L.A.,
Smith, J.D., Song, K., Hua Zhao, J., Yuan, X., Luan, J., Lamina, C., Ziegler, A., Zhang,
W., Zee, R.Y., Wright, A.F., Witteman, J.C., Wilson, J.F., Willemsen, G., Wichmann,
H.E., Whitfield, J.B., Waterworth, D.M., Wareham, N.J., Waeber, G., Vollenweider, P.,
Voight, B.F., Vitart, V., Uitterlinden, A.G., Uda, M., Tuomilehto, J., Thompson, J.R.,
Tanaka, T., Surakka, I., Stringham, H.M., Spector, T.D., Soranzo, N., Smit, J.H., Sinisalo,
J., Silander, K., Sijbrands, E.J., Scuteri, A., Scott, J., Schlessinger, D., Sanna, S.,
Salomaa, V., Saharinen, J., Sabatti, C., Ruokonen, A., Rudan, I., Rose, L.M., Roberts, R.,
Rieder, M., Psaty, B.M., Pramstaller, P.P., Pichler, I., Perola, M., Penninx, B.W.,
Pedersen, N.L., Pattaro, C., Parker, A.N., Pare, G., Oostra, B.A., O'Donnell, C.J.,
16
Nieminen, M.S., Nickerson, D.A., Montgomery, G.W., Meitinger, T., McPherson, R.,
McCarthy, M.I., McArdle, W., Masson, D., Martin, N.G., Marroni, F., Mangino, M.,
Magnusson, P.K., Lucas, G., Luben, R., Loos, R.J., Lokki, M.L., Lettre, G., Langenberg,
C., Launer, L.J., Lakatta, E.G., Laaksonen, R., Kyvik, K.O., Kronenberg, F., Konig, I.R.,
Khaw, K.T., Kaprio, J., Kaplan, L.M., Johansson, A., Jarvelin, M.R., Janssens, A.C.,
Ingelsson, E., Igl, W., Kees Hovingh, G., Hottenga, J.J., Hofman, A., Hicks, A.A.,
Hengstenberg, C., Heid, I.M., Hayward, C., Havulinna, A.S., Hastie, N.D., Harris, T.B.,
Haritunians, T., Hall, A.S., Gyllensten, U., Guiducci, C., Groop, L.C., Gonzalez, E.,
Gieger, C., Freimer, N.B., Ferrucci, L., Erdmann, J., Elliott, P., Ejebe, K.G., Doring, A.,
Dominiczak, A.F., Demissie, S., Deloukas, P., de Geus, E.J., de Faire, U., Crawford, G.,
Collins, F.S., Chen, Y.D., Caulfield, M.J., Campbell, H., Burtt, N.P., Bonnycastle, L.L.,
Boomsma, D.I., Boekholdt, S.M., Bergman, R.N., Barroso, I., Bandinelli, S., Ballantyne,
C.M., Assimes, T.L., Quertermous, T., Altshuler, D., Seielstad, M., Wong, T.Y., Tai,
E.S., Feranil, A.B., Kuzawa, C.W., Adair, L.S., Taylor, H.A.,Jr, Borecki, I.B., Gabriel,
S.B., Wilson, J.G., Holm, H., Thorsteinsdottir, U., Gudnason, V., Krauss, R.M., Mohlke,
K.L., Ordovas, J.M., Munroe, P.B., Kooner, J.S., Tall, A.R., Hegele, R.A., Kastelein, J.J.,
Schadt, E.E., Rotter, J.I., Boerwinkle, E., Strachan, D.P., Mooser, V., Stefansson, K.,
Reilly, M.P., Samani, N.J., Schunkert, H., Cupples, L.A., Sandhu, M.S., Ridker, P.M.,
Rader, D.J., van Duijn, C.M., Peltonen, L., Abecasis, G.R., Boehnke, M. & Kathiresan, S.
2010, "Biological, clinical and population relevance of 95 loci for blood lipids", Nature,
vol. 466, no. 7307, pp. 707-713.
34. Holdsworth, G., Stocks, J., Dodson, P. & Galton, D.J. 1982, "An abnormal triglyceriderich lipoprotein containing excess sialylated apolipoprotein C-III", The journal of clinical
investigation, vol. 69, no. 4, pp. 932-939.
35. Lambert, D.A., Catapano, A.L., Smith, L.C., Sparrow, J.T. & Gotto, A.M.,Jr 1996, "Effect
of the apolipoprotein C-II/C-III1 ratio on the capacity of purified milk lipoprotein lipase
to hydrolyse triglycerides in monolayer vesicles", Atherosclerosis, vol. 127, no. 2, pp.
205-212.
36. van Barlingen, H.H., Kock, L.A., de Man, F.H., Erkelens, D.W. & de Bruin, T.W. 1996,
"In vitro lipolysis of human VLDL: effect of different VLDL compositions in
normolipidemia, familial combined hyperlipidemia and familial hypertriglyceridemia",
Atherosclerosis, vol. 121, no. 1, pp. 75-84.
37. Carr, S.A., Huddleston, M.J. & Bean, M.F. 1993, "Selective identification and
differentiation of N- and O-linked oligosaccharides in glycoproteins by liquid
chromatography-mass spectrometry", Protein science : a publication of the protein
society, vol. 2, no. 2, pp. 183-196.
38. Masucci-Magoulas, L., Goldberg, I.J., Bisgaier, C.L., Serajuddin, H., Francone, O.L.,
Breslow, J.L. & Tall, A.R. 1997, "A mouse model with features of familial combined
hyperlipidemia", Science, vol. 275, no. 5298, pp. 391-394.
39. Maeda, N., Li, H., Lee, D., Oliver, P., Quarfordt, S.H. & Osada, J. 1994, "Targeted
disruption of the apolipoprotein C-III gene in mice results in hypotriglyceridemia and
protection from postprandial hypertriglyceridemia", The Journal of biological chemistry,
vol. 269, no. 38, pp. 23610-23616.
17
40. Pollin, T.I., Damcott, C.M., Shen, H., Ott, S.H., Shelton, J., Horenstein, R.B., Post, W.,
McLenithan, J.C., Bielak, L.F., Peyser, P.A., Mitchell, B.D., Miller, M., O'Connell, J.R.
& Shuldiner, A.R. 2008, "A null mutation in human APOC3 confers a favorable plasma
lipid profile and apparent cardioprotection", Science (New York, N.Y.), vol. 322, no. 5908,
pp. 1702-1705.
18