Membranes and mammalian glycolipid transferring ... - Åbo Akademi

Accepted Manuscript
Title: Membranes and mammalian glycolipid transferring
proteins
Author: Jessica Tuuf Peter Mattjus
PII:
S0009-3084(13)00151-5
DOI:
http://dx.doi.org/doi:10.1016/j.chemphyslip.2013.10.013
Reference: CPL 4241
To appear in:
Chemistry and Physics of Lipids
Received date: 28-8-2013
Revised date: 29-10-2013
Accepted date: 30-10-2013
Please cite this article as: Tuuf, J., Mattjus, P.,Membranes and mammalian
glycolipid transferring proteins, Chemistry and Physics of Lipids (2013),
http://dx.doi.org/10.1016/j.chemphyslip.2013.10.013
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Membranes and mammalian glycolipid transferring proteins
Jessica Tuuf and Peter Mattjus
Biochemistry, Department of Biosciences, Åbo Akademi University, Turku, Finland.
Keywords: GLTP, FAPP2, glycosphingolipid, membrane binding
Abbreviations:
ARF, ADP-ribosylation factor; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; ER,
endoplasmic reticulum; FAPP1/2, phosphoinositol 4-phosphate adaptor protein-1/2; FFAT,
two phenylalanines (FF) in an acidic tract; GalCer, galactosylceramide; GCS,
glucosylceramide synthase; GlcCer, glucosylceramide; GLTP, glycolipid transfer protein;
GSL, glycosphingolipid; LacCer, lactosylceramide; OSBP, oxysterol-binding protein; PC,
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phosphatidylcholine; PE, phosphatidylethanolamine; PH, pleckstrin homology;
phosphatidylinositol; PI3P, phosphatidylinositol-3-phosphate; PI4P, phosphatidylinositol-4-
phosphate; PM, plasma membrane; POPC, 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine;
PS, phosphatidylserine; SM, sphingomyelin; START, steroidogenic acute regulatory protein
(StAR)-related lipid transfer; TGN, trans-Golgi-network; VAP, vesicle-associated membrane
protein-associated protein
PI,
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Abstract
Glycolipids are synthesized in and on various organelles throughout the cell. Their
trafficking inside the cell is complex and involves both vesicular and protein-mediated
machineries. Most important for the bulk lipid transport is the vesicular system, however,
lipids moved by transfer proteins is also becoming more characterized. Here we review
the latest advances in the glycolipid transfer protein (GLTP) and the phosphoinositol 4-
phosphate adaptor protein-2 (FAPP2) field, from a membrane point of view.
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1. Introduction
The hydrophobic nature of the lipids requires different transport and trafficking mechanisms
compared to water soluble biomolecules. Lipids are synthesized on and in different
organelles, and cells constantly need to adjust and respond to changes in the lipid
requirements. To do this efficiently they need different transport machineries, connected to
the synthesis and degradation pathways. Several mechanisms in the cells are used for lipid
distribution. Most important for the bulk lipid transport is the vesicular system, however, lipid
movement mediated by transfer proteins is also widely characterized. In addition, lipid
transfer proteins have also often been given the role as sensors, responsible for coordinating
the transport routes within the synthesis and degradation machineries. There are several
glycolipid-binding proteins in mammals, proteins that specifically recognize glycolipids. The
sphingolipid activator proteins (saposins) bind to different glycosphingolipids (GSL) and help
in the degradation process of GSLs, with short oligosaccharide chains, in the lysosomes
(Schulze et al., 2009). Mutations in the saposins can cause severe lysosomal storage disorders,
like Gaucher disease (Schnabel et al., 1991). There is also evidence that non-specific lipid
transfer proteins, isolated from beef liver, can recognize GSLs and transfer them between
membranes (Bloj and Zilversmit, 1981). Another emerging group are the glycolipid transfer
proteins. In this review we will focus mostly on glycolipid transfer protein (GLTP), but also
on phosphoinositol 4-phosphate adaptor protein-2 (FAPP2), two proteins that belong to the
GLTP superfamily.
2. Glycolipid transfer protein (GLTP)
GLTP is a small soluble, 24 kDa, protein that has been identified in many organisms. GLTP
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was first discovered in the cytosolic fraction from bovine spleen (Metz and Radin, 1980;Metz
and Radin, 1982) and has since then been found in e.g. liver and brain from various
mammalian sources (Abe et al., 1982;Yamada and Sasaki, 1982a;Yamada and Sasaki,
1982b;Wong et al., 1984). In these studies GLTP was always purified from the cytosolic
fraction and, consequently, the protein was postulated to be cytosolic. It has later been
confirmed that GLTP indeed remains in the cytoplasm and not within other major cellular
organelles (Tuuf and Mattjus, 2007). Homologs to mammalian GLTPs have furthermore been
discovered in both plants (West et al., 2008) and yeast (Saupe et al., 1994). The expression
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levels of GLTP in bovine tissue have been analyzed (Lin et al., 2000). The results show that
the cerebrum and kidney display the highest GLTP mRNA levels, which coincides with
normally quite high amount of glycosphingolipids. GLTP can accelerate the transfer of
glycolipids, both diacylglycerol- and sphingoid-based, between two lipid membranes.
Substrates that can be used by GLTPs include glucosylceramide (GlcCer), lactosylceramide
(LacCer), galactosylceramide (GalCer), sulfatide, GM 1 , and GM 3 (Yamada et al., 1985;Brown
et al., 1985). Furthermore, the initial sugar residue, linked to the ceramide or diacylglycerol
backbone, needs to be in β-configuration for GLTP to recognize it as a substrate (Yamada et
al., 1986). On the other hand, GLTP cannot transfer phosphatidylcholine (PC),
phosphatidylethanolamine (PE), sphingomyelin (SM), phosphatidylinositol (PI), cholesterol
and cholesterol oleate (Yamada et al., 1985). The activity of GLTP is sensitive to changes in
the surrounding pH (West et al., 2006). Results show that GLTP has the highest transfer
activity at pH 7.0, 66% transfer activity at pH 10 and only 11% activity at pH 4.0. This could
give an important clue on GLTP subcellular localization and cellular activity, since GLTP
will encounter different pH conditions within the cell.
The amino acid sequence of GLTPs from different mammals is highly conserved. The protein
sequence of GLTP from pig brain was the first to be determined by Edman degradation (Abe,
1990). Later, it was demonstrated that GLTP contains 209 amino acids and that the sequence
between porcine and bovine is 100% identical (Lin et al., 2000). Human GLTP has almost as
high identity and shows 98% identity compared to porcine and bovine amino acid sequences
(Li et al., 2004). Furthermore, the human GLTP amino acid sequence is 100% identical to the
amino acid sequences of chimpanzee and macaque, 99% to the nonprimate mammal Canis
familaris and 98% identical to the Bos taurus sequence (Zou et al., 2008). Two single-copy
GLTP genes have been found in human cells, on chromosome 11 and 12. The
transcriptionally active GLTP is on chromosome 12 and contains five exons and four introns,
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while the inactive pseudogene is located on chromosome 11 (Zou et al., 2008). The
expression of GLTP mRNA is regulated by two transcription factors, Sp1 and Sp3. These
factors bind to multiple sites in the GC-boxes localized to the promoter of GLTP (Zou et al.,
2011). In the same study, the ability of different sphingolipids to enhance the GLTP
transcription of GLTP was analyzed. Interestingly, only ceramide (but not for example
GlcCer, GM 1 or sulfatide) could enhance the transcription levels of GLTP via the Sp1 and
Sp3 transcription factors. The structure of both bovine and human GLTP has been
determined, both in its apo-form and in complex with different glycolipids (Malinina et al.,
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2004;West et al., 2004;Airenne et al., 2006;Malinina et al., 2006). The GLTP structure
contains eight α-helices, that are arranged in a two-layer fold, a structure that is completely
novel for lipid transferring proteins. The carbohydrate group will bind to a recognition center
on the surface of GLTP, through hydrophobic contacts and hydrogen bonding, and the
hydrocarbon acyl chains will be accommodated into a hydrophobic pocket within the protein
(Malinina et al., 2004). The binding of glycolipids to GLTP will be discussed extensively in
chapter 4.
The formation of GLTP dimers has been discussed in early publications. There are three
cysteine residues in the GLTP sequence, two of them reside inside the protein and the third is
on the surface of GLTP. Abe and coworker suggested that the two internal cysteines might
form a intramolecular disulfide bond, while the cysteine on the outside could form a disulfide
bond with another GLTP molecule (Abe and Sasaki, 1989a;Abe and Sasaki, 1989b). The
disulfide bridge forming ability of GLTP was suggested to function as a means for regulating
GLTP transfer activity, since it was demonstrated that the dimer had almost no transfer
activity in contrast to GLTP with an intermolecular disulfide bridge, which had a high transfer
activity. However, results from crystallization studies show no intra- or intermolecular
disulfide bonds in the GLTP structure (Malinina et al., 2004;Airenne et al., 2006), but based
on structural requirements it was suggested that it might be possible that an internal disulfide
bridge would help in regulating GLTP activity (Airenne et al., 2006). It is still unknown
whether disulfide bond-formation actually occurs in vivo as a regulatory mechanism for
GLTP function. Recently a new transfer protein was discovered that moves ceramide-1-
phosphate, and was named CPTP, ceramide-1-phosphate transfer protein (Simanshu et al.,
2013). Structurally, CPTP has a very similar fold compared to the GLTP fold, however, CPTP
associates with the trans-Golgi network (TGN), nucleus and the plasma membrane (PM). A
CPTP-mediated ceramide-1-phosphate decrease in plasma membranes and increase in the
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Golgi complex stimulates cPLA 2 α release of arachidonic acid, triggering pro-inflammatory
eicosanoid generation.
3. Phosphoinositol 4–phosphate adaptor protein-2 (FAPP2)
There are two closely related homologs of four-phosphate adaptor proteins, type 1 and 2
(FAPP1 and FAPP2). FAPP1 was originally discovered in a study, when trying to identify
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novel proteins that could interact with various phosphatidylinositol-phosphates (Dowler et al.,
2000). The FAPP1 gene is located on chromosome 2 and encodes a protein with 300 amino
acids. FAPP1 has in its amino-terminal a pleckstrin homology (PH) domain and in the C-
terminus resides a proline rich-motif (Godi et al., 2004). FAPP2, on the other hand, is a
protein with 519 amino acids and is encoded by a single-copy gene on chromosome 7. In
addition to a PH domain in the N-terminus, FAPP2 has a GLTP domain in the carboxyterminal,
which makes it a member of the GLTP superfamily (Godi et al., 2004;Kamlekar et
al., 2013) (Fig. 1A). The PH domain of FAPP1 and FAPP2 is 80% identical (Godi et al.,
2004). FAPP2 is able to transfer GlcCer, both fluorescently labeled and natural, but not SM,
PC or ceramide (D'Angelo et al., 2007). Recently it was demonstrated that in addition to
GlcCer, the GLTP domain of FAPP2 can specifically transfer GalCer and LacCer between
membranes, but not the ganglioside GM 1 or the negatively charged sulfatide (Kamlekar et al.,
2013). This substrate specificity makes the GLTP domain of FAPP2 more similar to HET-C2,
a fungal homolog of GLTP, than to human GLTP itself.
The FAPPs appear to be ubiquitously expressed and have a cytoplasmic distribution, though,
with a strong preference towards the TGN via their targeting signals (Godi et al., 2004). For
example, in mouse tissue FAPP2 is highly expressed in kidney, but is also found in testis, the
small intestine, spleen and cerebrum (D'Angelo et al., 2013). Homologs of FAPP2 have been
found in vertebrates and echinoderms (D'Angelo et al., 2008) and sequence analysis of
various FAPP2 homologs reveals that both the PH domain as well as the GLTP domain are
highly conserved (D'Angelo et al., 2012).
By analyzing FAPP2 by small-angle x-ray scattering and analytical ultracentrifugation, it
appears that FAPP2 is dimeric in solution (Cao et al., 2009). Whether this is the case in
biological systems remains to be demonstrated. To date, it has been proven to be difficult to
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crystallize FAPP2, probably because of its large size and flexibility. However, a lowresolution
structure of dimeric FAPP2 by small-angle x-ray scattering has been obtained,
showing a molecular shape that is well extended and curved, with a length of 30 nm (Cao et
al., 2009). The GLTP domain has been analyzed using homology modeling. These models
show a conserved folding structure against human GLTP, with the key residues, important for
the glycolipid transferring properties retained (D'Angelo et al., 2007). A truncated FAPP2
form, with a 212 amino acids C-terminus (the GLTP domain), was used to study the
resemblance to GLTP (Kamlekar et al., 2013). This protein showed a structure with high
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content of alpha-helices and results from experiments using fluorescence emission of the
intrinsic tryptophan residues suggested that the GLTP domain actually adopts a GLTP fold.
However, there are some differences in FAPP2 compared to GLTP, e.g. FAPP2 has a more
selective substrate specificity which is due to a restricted glycolipid head group recognition
center (Kamlekar et al., 2013). The FAPP1 PH domain has been analyzed using nuclear
magnetic resonance-based solution structures in a free state, together with micelles or
phosphatidylinositol-4-phosphate (PI4P) (Lenoir et al., 2010). Shortly after, the crystal
structure of FAPP1 PH domain was determined (He et al., 2011). The PH domain was
reported to consist of a β-barrel capped by an α-helix at one edge. Three additional loops
connect the strands; a long β1- β2 loop, a short β6- β7 loop and a β3-β4 loop that is partially
hidden in the structure. Between the β4 and β5 strand one can also find a one-turn α-helix.
Hydrogen-deuterium exchange mass spectrometry has been used on FAPP2 to look at
conformational changes upon GlcCer binding (D'Angelo et al., 2013). The GLTP and the PH
domain appear to be separated by a very flexible linker region and results show that when
FAPP2 binds GlcCer a conformational change will occur in the whole structure, not only in
the glycolipid-binding domain, but also the linker region and the PH domain will be affected.
4. Glycolipid binding
The broad specificity of GLTP for various glycosphingolipids, for example glycolipids with
large variations in their head group structure, is due to the hydrogen bonding adaptable head
group recognition site localized at the entrance to the hydrophobic tunnel of the protein. This
enables GLTP to selectively bind the GSL head and the proximal ceramide amide group with
multiple hydrogen bonds.
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GLTP from different species all have the characteristic feature of an adaptive hydrophobic
cavity. The physical function of GLTP is to protect the lipid substrate hydrocarbon chains
from the aqueous environment once the glycolipid is extracted from the membrane. Due to the
large variation in the chain lengths and saturation of natural GSLs, an adaptable cavity seems
to be the evolutionary structural optimized form. This is in contrast to the precursor synthases
responsible for ceramide syntheses, that seem to have developed into six different ceramide
synthases for various chain lengths of ceramides (Tidhar and Futerman, 2013). Part of the
hydrophobic tunnel of GLTP is partly collapsed in the apo-state. The 3D structure of GLTP,
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as determined by crystallization (West et al., 2004), reveals a sandwiched all-alpha-helical
conformation (Malinina et al., 2004;Airenne et al., 2006) (Fig. 2A). Several amino acid
residues have been identified based on the crystal structure studies, that are required for
glycolipid binding (Malinina et al., 2004;Malakhova et al., 2005;Airenne et al., 2006;Malinina
et al., 2006). Tryptophan at position 96 (helix 4) is in a key role in the forming of a platform
of hydrogen bonds between the hydroxyl group of the first glucose or galactose. For FAPP2
the corresponding tryptophan required for glycolipid transfer activity is at position 407.
Further hydrogen bonds are formed between His-140 and the ceramide amide region of the
glycolipid backbone (Fig. 2B). By comparison of the apo-GLTP and GLTP with a lipid
bound, it is evident that there is a shifting of the alpha-2 and alpha-6 helices and by changing
the side chain conformations of residues Phe-42, Ile-45, Phe-148 and Leu-152 when a lipid is
bound. Presumable, when the glycolipid acyl chains come in contact with these hydrophobic
amino acid side chains inside the hydrophobic tunnel, they change their conformation, and
consequently, a shift in helix-2 and helix-6 occurs. We believe that this acts as the process by
which GLTP desorbs from the membrane into the surrounding aqueous solution. Neither
GLTP nor FAPP2 contain the “KRTIQK“ amino acid motif described by Fantini and
coworkers as a glycolipid-binding algorithm (Fantini et al., 2006). They identified this domain
in several amino acid sequences from Helicobacter pylori bacterial adhesion protein adhesin
A (HpaA). HpsA is known to interact with membranes containing glycolipids. HpaA, to our
knowledge, appears not to be able to transfer glycolipids.
4.1 Sphingosine in versus sphingosine out
The cavity that accommodates the hydrophobic parts of the GSL is adaptable and aligned with
nonpolar side chains effectively excluding water (Malinina et al., 2004;Airenne et al., 2006).
Thorough examination of different crystal structures of GLTP with different bound GSLs
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with varying acyl chain composition reveals that either one or both acyl chains are
accommodated inside the hydrophobic tunnel. In the sphingosine-in form, both chains are
inside, whereas in the sphingosine-out conformation, the amide-linked chain occupies more of
the hydrophobic cavity preventing the sphingosine chain to enter (Fig. 3A). Most of the
crystallized forms of GLTP are in the sphingosine-out conformation. However, it is still not
clear if GLTP has its substrate bound in the sphingosine-out mode in biological systems. It is
neither known how different diacylglycerol-based glycolipids are bound by GLTP, if they
also adopt both a sphingosine-in and sphingosine-out binding mode. Encapsulation of both
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chains has only been observed for wildtype GLTP monomers and with either GlcCer or
LacCer containing an amide-linked oleoyl (18:1 Δ 9 ) acyl chain (Malinina et al.,
2004;Samygina et al., 2011). A proposed mechanism for the accommodation of the
sphingosine chain inside GLTP involves His-140 hydrogen bonding to the ceramide amide
group and movement of two phenylalanines (Phe-42 & Phe-148), creating additional space for
chain encapsulation. A mutation of the residue His-140 leads to a complete inactivation of
GLTP (Malinina et al., 2004). Another mutant GLTP (D48V) crystallizes with the 24:1 Δ 9
sulfatide in a sphingosine-in form. The mutant has a structural resemblance very close to that
of the wildtype GLTP with sphingosine-in with bound 18:1 GlcCer. The D48V mutation as
well as another double mutant A47D/D48D are interesting proteins in that they do not bind
neutral GSL, but can be engineered to become transfer proteins for 24:1 Δ 9 sulfatide
(Samygina et al., 2011).
What determines the sphingosine-in conformation, and is this simply a crystallization
phenomenon? There are very small conformational differences between the sphingosine-in
and sphingosine-out GLTP-lipid complexes. At a first glance, it appears that increasing the N-
linked acyl chain length, and due to the adaptable hydrophobic cavity of GLTP, sphingosine
appears to be simply forced out by a long chain. However, co-crystallization with short chains
N-linked chains, such as C8 & C10, did not allow for a sphingosine-in alignment. Most likely
the shorter acyl chain never reaches all the way to the bottom of the hydrophobic tunnel and,
consequently, the tunnel remains narrow preventing the sphingosine from entering. Another
peculiarity that has been complicating the GLTP crystal structure studies has been the
presence of short extraneous hydrocarbon chains not originating from the bound GSL, found
inside the hydrophobic cavity (Malinina et al., 2004;West et al., 2004;Airenne et al., 2006).
These most likely originate from the bacterial expression of the protein used for
crystallization. Finding hydrocarbons and detergents inside the crystal structure of
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hydrophobic proteins is seen frequently, and is sometimes crucial for obtaining protein
crystals (Haapalainen et al., 2001;Guan et al., 2001;Wright et al., 2003). The extraneous
hydrocarbon seen in the apo-GLTP crystal structures is displaced from the hydrophobic
tunnel when GLTP forms a complex with the glycolipid (Malinina et al., 2004;Airenne et al.,
2006). It could be possible that in vivo, GLTP could also carry a hydrophobic molecule when
no glycolipid is bound. It is not clear if the molecule enters from the glycolipid binding site or
enters into the hydrophobic cavity from the opposite end, through the openings at the end of
the tunnel, formed by helix-1, helix-7 and helix-8. The opening is structurally wide enough to
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let a hydrocarbon chain pass. The far end opening of the hydrophobic cavity can be seen in
Fig. 3A, black arrow.
4.2 Crystal-Related Dimerization in GSL-GLTP Complexes
In a recent paper, the dimerization states of GLTP were analyzed (Samygina et al., 2013).
Previously, homodimers of GLTP has been found in various crystal structures when GLTP
has been complexed with different glycosphingolipids. It appears that when GLTP binds the
glycolipid in the sphingosine–out mode, a dimer will form (Malinina et al., 2006). This event
is suggested to be due to interaction of two different GLTP molecules as well as the adjacent
sphingosine hydrocarbon chains (Samygina et al., 2013). The authors proposed that the
function of GLTP might be regulated by forming reversible homodimers. This is further
supported by the finding that when wildtype GLTP and a mutant GLTP (D48V) bind 24:1
sulfatide in the absence of lipid membranes, a homodimer of GLTP will form in solution
(Samygina et al., 2011). In contrast to these results it was shown, based on analytical
ultracentrifugation experiments, size-exclusion chromatography and affinity-tag
immunoadsorbtion experiments, that both apo-GLTP and GLTP complexed with GalCer
(24:1) are monomeric in solution (Malinina et al., 2006). However, the authors still proposed,
based on optimal docking area (ODA) energy contact calculations, that upon binding of
GalCer in the sphingosine-out mode, the sphingosine chain would interact with a sphingosine
molecule on a partner GLTP molecule, thereby forming crystal-related cross dimers.
The crystal-related dimerization of GLTP allows the two sphingosine chains to interact with
each other presumably in a more energetically favorable conformation than being inside the
tunnel. Is the sphingosine-out form, and the subsequent dimerization of GLTP the biologically
active carrier of GSLs in vivo? More than a dozen crystal structures of GLTP have the bound
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glycolipid in the sphingosine-out conformation. Could this simple be a form generated during
high protein concentrations and under limited hydration? The sphingosine chain might be
jutted outwards into a low hydration environment, hydrophobic amino acid residues in close
proximity to the tunnel entrance (Fig. 3B, red patch indicated with a black arrow) and
extraneous hydrocarbons occupying the far end of the tunnel might drive a sphingosine-out
conformation. It is of vital importance to keep in mind that all dimers form when no
membrane surfaces are present. Analysis of GLTP in solution over a wide range of
concentrations using light scattering and gel-filtration all indicate that in the presence of
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membranes GLTP is a monomer (Zhai et al., 2009).
4.3 GLTP-like proteins
The Arabidopsis thaliana GLTP-like protein (Brodersen et al., 2002), ACD11 has also
conserved residues, the GLTP amino acids Asp-48 and His-140 participate in the hydrogen
binding to the sphingosine base and are conserved in ACD11 as Asp-60 and His-143
respectively (Petersen et al., 2008). ACD11 appears to be able to adopt a GLTP-like structure
(Petersen et al., 2008). ACD11 appears not to be capable of transferring and (binding?)
glycosphingolipids in vitro, only the single chain sphingosine (Brodersen et al., 2002).
Replacing the sphingosine hydrogen bond forming amino acids in ACD11 (Asp-60 & His-
143) causes a complete loss of transfer activity. However, if tryptophan is introduced in
position 103, to make ACD11 resemble GLTP, it does not improve its glycolipid transfer
activity.
5. Membrane composition consequences on protein activity
The composition of the membrane has a great impact on protein function and activity.
Processes like binding to membranes and glycolipid extraction by the GLTPs have been
studied quite intensively. We will here give the latest insights in such events.
5.1 Membrane binding
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The rate-limiting step in the glycolipid transfer process is thought to be the GLTP-glycolipid
complex formation, or the dissociation of the protein-lipid complex from the membrane into
the surrounding aqueous environment (Rao et al., 2005). The initial GLTP binding to the
membrane, however, is not the limiting process and appears to have a minimal contribution to
the overall changes to the enthalpy and entropy for the overall transfer process. Once docked
to the membrane surface, GLTP is searching for the glycolipid substrate in the membrane.
This process is probably a combination of both a lateral flow of both GLTP and lipids. GLTP
transfer activity is different depending on the membrane composition and the lateral
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miscibility of the lipids in the membrane. If the glycolipid is in a membrane in a fluid phase
the glycolipid removal is favored. GLTP binds more strongly to sphingomyelin containing
membranes than to phosphatidylcholine membranes. This is true regardless of acyl chain
composition, i.e. saturated or unsaturated (Ohvo-Rekilä and Mattjus, 2011).
5.2 Lipid extraction
Membrane composition. It has been shown that the rate of transfer by GLTP is sensitive to the
membrane composition surrounding the glycolipid. The positively charged areas of GLTP
affect how well GSLs are moved from donor to acceptor membranes. GalCer in negatively
charged vesicles (5 or 10 mol% negative lipids) is transferred significantly slower than from
the donor vesicles composed of neutral membranes (Mattjus et al., 2000). The same amount
of negative charge in acceptor vesicles does not impede the transfer rate as effectively.
Positively charged lipids in the donor vesicle membrane neither affect the GSL transfer
(Mattjus et al., 2000). GLTP is positively charged at neutral pH (pI=9.0) and will be attracted
to the negatively charged donor membrane through electrostatic interactions between the
protein and the membrane surface, resulting in a slow transfer. It is speculated that the GLTP
off-rate from the donor surface becomes slow and consequently the transfer rate is slow.
GLTP cannot transfer GalCer and GlcCer from vesicles made of saturated SMs, like
palmitoyl-SM, regardless of curvature of the donor vesicle membranes (Mattjus et al.,
2002;Nylund and Mattjus, 2005). Addition of cholesterol has only minimal effect on the
transfer rate. If donor vesicles are made of different SM analogues, that more resemble a PC,
transfer is possible. If the 3-hydroxy group of SM is replaced by a hydrogen, and the trans-4,5
double bond of sphingosine is reduced, transfer will occur (Nylund et al., 2006). GLTP is also
able to transfer glycolipids from membranes composed of unsaturated SMs, however much
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slower than from a chain-matched PC (Mattjus et al., 2002;Nylund and Mattjus, 2005).
Membrane curvature. The transfer rates for GLTP are more than 5 times greater for small
highly curved fluid 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) membranes
compared to that for large membranes, that are comparable to a planar bilayer (Rao et al.,
2005;Nylund et al., 2007). The transfer rate of fluorescently labeled GalCer from fluid POPC
vesicles decreases in a linear fashion as the vesicles become larger, to an almost undetectable
rate for vesicles above 800 nm in diameter. From planar monolayers no glycolipid transfer
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occurs (Nylund et al., 2007). If the membrane is composed of 1,2-dipalmitoyl-sn-glycero-3-
phosphocholine (DPPC) there is no difference in the anthrylvinyl-GalCer transfer rates
regardless of the donor vesicle size except for the smallest vesicles (50 nm in diameter) that
are significantly higher. It is noteworthy that the transfer is very small from gel-like
membranes in general. It is speculated that GLTP can bind clustered glycolipids in a fluid
environment such as POPC, but with limited accessibility to glycolipids well dispersed into a
more tightly packed environment such as that in DPPC and palmitoyl-SM (Nylund and
Mattjus, 2005;Nylund et al., 2006;Brown and Mattjus, 2007;Nylund et al., 2007;Mattjus,
2009;Ohvo-Rekilä and Mattjus, 2011). Recently Brown and colleagues found that GLTP was
able to extract BODIPY-GlcCer from a planar monolayer composed of POPC and low
amounts (physiological) of either PE or phospatidic acid (PA) (15 and 5 mol% respectively)
at bilayer comparable surface pressures (30-35 mN/m) (Zhai et al., 2013). It is unclear what is
happening with phosphatidylserine (POPS) in these experiments, it appears not to stimulate
GlcCer transfer to the same extent as PE and PA (Zhai et al., 2013), but rather penetrate the
monolayer. Previously Nylund and co-workers showed that the exclusion pressure for GLTP
i.e. the pressure where GLTP no longer can penetrate the monolayer, was clearly the highest
with DPPS (30.7 mN/m) of all lipids analyzed, even negatively charged sulfatide (Nylund et
al., 2007). GLTP is positively charged at neutral pH and penetrates negatively charged
monolayer lipid films at higher surface pressures than those of neutral lipid films. However,
DPPS is also more easily penetrated than the glycolipid sulfatide. The relatively larger
penetration of GLTP into the lipid monolayers at low surface pressures does not necessarily
indicate a direct interaction of the protein with the lipid molecules, as GLTP alone is surfaceactive
and can spontaneously migrate from the subphase to a lipid free interface. This shows
that the extraction of GLTP can be enhanced by alterations in the matrix lipid, independent of
any additional proteins. The hydration around the GSL is likely to be the driving force for the
effects seen by PE and PA. Saturated-chain PEs (12:0, 14:0 & 16:0) are not miscible with
Accepted Manuscript
chain-mixed GlcCer either above or below T m of the phospholipid. However, the unsaturated
POPE, OPPE and SOPE are all miscible with GlcCer at physiological temperatures (Quinn,
2011;Quinn, 2012). It is likely that GlcCer and GalCer interact more favorable with POPE
than with POPC in monolayers because of the smaller and less hydration of the PE head. The
hydration of GlcCer and GalCer more resembles PE than PC. In the monolayer system one
leaflet is missing, this inevitably allows the lipids to move more freely out from the
membrane plane, less hydrophobic forces keep the lipid stronger into the membrane.
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5.3 GLTP membrane binding, desorption mechanism and lipid release
Trp-142 has been confirmed to be essential in the GLTP membrane binding process. In
FAPP2 and HET-C2 the conserved amino acids are Trp-447 and Phe-149 respectively.
Several other hydrophobic amino acids take part in the docking of GLTP onto the membrane
surface (Malinina et al., 2004;Rao et al., 2005;Airenne et al., 2006;West et al., 2006;Neumann
et al., 2008;Zhai et al., 2009;Kamlekar et al., 2010;Kenoth et al., 2010). A coloring of the
amino acids according to their hydrophobic scale is presented in Fig. 3B. The most
hydrophobic amino acids are shown in red, and amino acids with lower hydrophobicity in
lighter red shades, white being fully hydrophilic in nature (Eisenberg et al., 1984). Examining
the changes to the crystal structure of GLTP after the glycolipid is bound to the hydrophobic
tunnel reveals several movements in two interhelical loops and two different helices. Alphahelix
2 moves along its axis and outwards and alpha-helix 6 moves outwards. The net result of
these movements is an expansion of a hydrophobic tunnel to accommodate the lipid, either
with one or two chains inside. A putative membrane binding domain with high
hydrophobicity is indicated with a black arrow in Fig. 3B. There are only small changes in
this domain between the apo-GLTP and the ligand-bound form. The domain is formed by a
group of tryptophan, tyrosine, isoleucine and nonpolar residues on the protein surface, in
close proximity to the glycolipid-binding site. The small change in the hydrophobic domain
suggests that after GSL binding also other parts of the protein are responsible for the release
of GLTP from the membrane.
The mechanism how the GSL is lifted from the membrane into the GLTP cavity is unclear.
Perhaps the ability of the sphingosine chain to bind to the outer grow of GLTP will allow the
amide-linked chain to enter the hydrophobic tunnel, specifically recognized and bound by the
amino acids of the sugar binding pocket of GLTP. We speculate that the mechanism could be
Accepted Manuscript
that the membrane-binding domain of GLTP generates a disturbance in the hydrogen-bond
network between the water molecules and the membrane lipid head groups, allowing an acyl
chain from the lipids in the membrane to move upward towards the hydrophobic amino acids,
out from the hydrophobic interior, onto the GLTP surface. If the acyl chain belongs to a lipid
that is also recognized by the sugar-binding pocket of GLTP, a lipid binding takes place. If
the chain belongs to a lipid that is not recognized, it simply moves back into the membrane
core, and the next lipid is allowed to be tested by GLTP. Once a glycolipid binding has
occurred and the glycolipid has been captured by GLTP, the second chain would then be
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pulled along and would enter the hydrophobic tunnel. Once GLTP lifts of the interface, as a
consequence of the helical movements and structural changes in the membrane-binding
domain, the sphingosine chain would be forced away form the aqueous environment into a
more energetically favorable milieu inside GLTP. Very little is known about the release of the
bound glycolipid from GLTP, once it encounters new membranes. For instance, the binding
of FAPP2 carrying glucosylceramide is stronger than an empty apo-FAPP2 (D'Angelo et al.,
2013), if this is the case also for GLTP is at present unknown.
6. Subcellular Targeting of GLTP and FAPP2
There are numerous sequences, motifs and domains within proteins that help them find their
right destinations in the cell. Both protein-protein interactions as well as protein-lipid
interactions are important in guiding cellular components to various subcellular
compartments. The C1 and/or C2 domains of for example protein kinase C, diacylglycerol
kinases and some Ca 2+ -dependent phospholipases, bind most commonly to phospholipids in
cell membranes (Cho, 2001). Another domain, the steroidogenic acute regulatory protein
(StAR)-related lipid transfer (START) domain, is important in lipid trafficking and
metabolism and the different START domains identify several lipids, including cholesterol
and ceramide (Alpy and Tomasetto, 2005). The Zn 2+ -binding FYVE domains (about 70
residues) are, on the other hand, very specific for phosphatidylinositol-3-phosphates (PI3P)
(Gaullier et al., 1998;Patki et al., 1998) and are responsible for endosomal targeting
(Stenmark et al., 1996). Another group of phosphoinositides-binding domains are the PX
domains, found in a diverse set of proteins (Simonsen and Stenmark, 2001). Many of the PX
domain-containing proteins also preferentially recognize PI3P and localize to the endosomes
(Tessier and Woodgett, 2006). The PH domains are very divergent in their sequences, but
Accepted Manuscript
mediate several protein-protein interactions as well as recognizing a variety of phosphorylated
derivatives of phosphatidylinositol (Scheffzek and Welti, 2012). The specific binding of PH
domains to distinct phosphoinositides, located at different sites in the cell, are especially
important amongst lipid-binding proteins, since this recruitment decide the protein subcellular
localization and may regulate protein activity (Levine and Munro, 2002). Although there are a
large variety of domains regulating protein trafficking and activity, more targeting signals will
for sure be found in the future. So this raise the important question, how can the cytoplasmic
proteins GLTP and FAPP2 find their specific sites of action?
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6.1 GLTP
GLTP is a cytoplasmic protein and results obtained from different experimental methods
suggest that GLTP does not reside within the endoplasmic reticulum (ER), the nucleus, the
mitochondria or the Golgi (Tuuf and Mattjus, 2007). However, analysis of the amino acid
sequence of GLTP from different organisms, indicate that there still might exist motifs or
sequences within GLTP that would enable targeting of the protein to different compartments
in the cell. In 2009 a motif in GLTP was characterized, a motif the authors referred to as the
FFAT-like motif (Tuuf et al., 2009). Previously it has been shown that several lipid-binding
proteins possess a conserved motif designated FFAT (two phenylalanines (FF) in an acidic
tract) (Loewen et al., 2003), a targeting signal which directs them to the cytosolic surface of
the ER for association with the vesicle-associated membrane protein-associated proteins
(VAMP-associated proteins or VAPs) (Wyles et al., 2002;Loewen et al., 2003;Kawano et al.,
2006). The VAPs have been imlicated in several important processes in the cell, like vesicle
transport (Skehel et al., 1995), the unfolded protein response (Kanekura et al., 2006) and in
the neurodegenerative disorder Amyothrophic lateral sclerosis, type 8 (Nishimura et al.,
2004). Two of the isoforms of the VAPs, VAP-A and VAP-B, are mainly located in the ER
(Weir et al., 2001;Nishimura et al., 2004), and it is proposed that these integral membrane
proteins localize to specific ER contact sites, which could act as meeting points for several
lipid transfer proteins (Levine and Loewen, 2006). The consensus sequence of the FFAT
motif, almost exclusively found in lipid-binding proteins, is reported to contain the residues
EFFDAxE appearing in an acidic environment (Loewen et al., 2003). In the same study it was
demonstrated that minor substitutions in some of the amino acids are allowed, while other
residues are essential for an interaction to occur with the VAPs. This conclusion is in
agreement with more recent structural and experimental studies, where it is demonstrated that
Accepted Manuscript
many amino acid substitutions in the FFAT motif can indeed be tolerated (Kaiser et al.,
2005;Mikitova and Levine, 2012). The FFAT-like motif identified in GLTP contains the
amino acids 32 PFFDCLG 38 , a region which is also responsible for VAP-A-binding in vitro
(Tuuf et al., 2009) (Fig. 1B). This FFAT-like motif is closely related to EFFDAxE and
contains the two phenylalanines (F) and the aspartate (D) residue, together with proline,
cysteine, leucine and glycine. The fact that the FFAT-like motif is exposed on the surface of
the protein (Airenne et al., 2006) also indicates that it is structurally available for a interaction
with the VAPs (Fig. 2A). However, in a recent analysis on proteins with FFAT-like motifs the
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authors proposed that the FFAT-like motif in GLTP might not be optimal situated for
interaction with VAP (Mikitova and Levine, 2012). This is due to 32 PFFDCLG 38 in GLTP is
residing in a α-helix and not in an β-strand-like conformation, a structure that has previously
been found in FFAT-containing proteins in VAP-FFAT-binding studies (Kaiser et al.,
2005;Furuita et al., 2010). However, and interestingly, another FFAT-like-motif in the GLTP
amino acid sequence has been identified (Mikitova and Levine, 2012). This sequence is weak,
contains the residues 22 PFLEAVS 28 and lies upstream the region 32 PFFDCLG 38 previously
found in GLTP (Tuuf et al., 2009) (Fig. 1B). It was suggested that both the FFAT-like motifs
might act in concert, and consequently, a stronger VAP interaction would be obtained. Other
proteins, with FFAT-like motifs even further from the consensus sequence, have been
reported to interact with the VAPs under physiological conditions (Saita et al.,
2009;Saravanan et al., 2009), which could imply that GLTP also might associate with the
VAPs in vivo and not just in vitro. However, whether GLTP actually interacts with the VAPs
in biological systems still remains unclear.
Although GLTP is suggested to have a cytosolic distribution (Tuuf and Mattjus, 2007), it
should not be excluded that GLTP might interact with various organelles during different
stages in the cell cycle, upon certain cellular signals or that the association of GLTP with
organelle membranes might be weak. In a large phosphoproteomic study on phosphorylation
sites using mass spectrometric methods, there was a site identified in the GLTP sequence that
was phosphorylated, threonine 69 (Olsen et al., 2010). This might raise the possibility that the
subcellular localization of GLTP and/or interactions with other proteins might be regulated by
phosphorylation/dephosphorylation events. The phosphorylation status of ceramide transfer
protein and oxysterol-binding protein (OSBP), which also interact with the VAPs, can affect
the targeting of the proteins to different organelles (Kumagai et al., 2007;Fugmann et al.,
Accepted Manuscript
2007;Saito et al., 2008;Tomishige et al., 2009;Nhek et al., 2010). Other possible posttranslational
modifications of GLTP that could act as regulatory mechanisms could involve
ubiquitylation or perhaps attachment of various lipids. Interestingly, a recent study shows that
GLTP can be ubiquitylated (Wagner et al., 2011).
6.2 FAPP2
The FAPPs possess a PH domain, which is important for targeting of the protein. This domain
specifically recognizes PI4P and the GTPase ADP-ribosylation factor (ARF) on the TGN
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(Godi et al., 2004) (Fig. 1A). The same mechanism is utilized by OSBP in yeast, that uses its
PH domain to associate with both ARF1 and PI4P in the Golgi complex (Levine and Munro,
2002) . In vitro studies on the GlcCer transport ability of FAPP2 showed that if ARF1 and
PI4P were added to acceptor membranes, the transport of GlcCer mediated by FAPP2
between lipid donors to acceptors was enhanced (D'Angelo et al., 2007). It is the PI4P that
dictates the targeting and upon GlcCer binding, FAPP2 translocates to the PI4P binding site
on TGN (D'Angelo et al., 2013). However, the phospatidylinositol-4-OH kinase-β is recruited
to the Golgi by the ARFs, which consequently links PI4P production and ARF localization
together (Godi et al., 1999). The binding of the FAPP1 PH domain to ARF1 and PI4P has
been analyzed (He et al., 2011). It appears that PI4P is absolutely necessary for the PH
domain to be inserted into the membrane, and that the PI4P binding is pH-dependent, showing
a stronger binding at pH 6.5 than 7.4. Furthermore, in the same study it was demonstrated that
although the binding sites of ARF1 and PI4P are in close proximity, they can bind
independently of each other and on the same time. These features of the PH domain
contribute to the specific targeting of the FAPPs to the Golgi complex. FAPP2 with a bound
GlcCer is targeted to the TGN via its PH domain and is capable to bind to PI4P and small
GTPase ARF1 proteins (Godi et al., 2004). The mechanism how the bound GlcCer would
increase the affinity of FAPP2 towards PI4P in the TGN remains unclear. Presumable, when
GlcCer is bound to the GLTP domain of FAPP2, the protein adopts another conformation,
different than the apo-form, allowing for an association by the PH domain to the membranes
in the TGN. Once the GlcCer is released, the apo-FAPP2 leaves the membrane, and the
affinity to bind PI4P by the PH domain again becomes lower. How the release of GlcCer
takes place is not known (D'Angelo et al., 2013). GlcCer needs to be located on the inner
leaflet of the TGN membranes in order to become further glycosylated to LacCer and further
to Gb 3 by the enzymes residing in the lumen of the Golgi. It is tempting to speculate that
when GlcCer exits the hydrophobic cavity of FAPP2, it would directly be pushed, with the
Accepted Manuscript
glucose head group first, directly to the inner leaflet. This mechanism would render a GlcCer
flipping from the outer to the inner leaflet of the TGN unnecessary.
FAPP2 in mammals also contain a weak FFAT-like motif, defined as TFFSTMN (Mikitova
and Levine, 2012) (Fig. 1B). However, results from experiments from the same study showed
that FAPP2 did not target the VAPs on ER as wildtype, but if the serine at position 4 was
pseudophosphorylated, it showed a weak targeting to the VAPs. To our knowledge, it has not
yet been demonstrated that FAPP2 actually interacts with the VAPs in vivo. In addition, there
17
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are several phosphorylation sites in FAPP2 that has been identified, however, whether these
modifications are of importance for the function or subcellular localization of FAPP2 remains
to be determined.
7. Biological functions of GLTP and FAPP2
The substrate specificity of both GLTP and FAPP2 suggests that the members of the GLTP
superfamily are vital for regulating the GSL metabolism. Amongst the lipid transfer proteins,
exist both genuine lipid transporters (Hanada et al., 2003) and/or sensors of various lipids
(Litvak et al., 2005;Vihervaara et al., 2011). What about the proteins that are specific for
glycosphingolipids? The biological function of GLTP is still not clear. However, implications
have been made that GLTP might catalyze the transfer of GlcCer from the Golgi to the PM
(Warnock et al., 1994;Halter et al., 2007). Other reports propose that GLTP might in addition
function as an intracellular sensor of GSLs. Malakhova and coworkers mutated the liganding
site in GLTP and looked at the ability of GLTP to acquire and release its substrate
(Malakhova et al., 2005). The authors speculated that in addition to acting as a glycolipid
carrier, GLTP might function as a GlcCer sensor or a presenter of glycolipids to protein
receptors, since the results showed that wildtype GLTP was not keen to release the ligand to
POPC membranes. Furthermore, GLTP appears to sense changes in the amount of newly
synthesized GlcCer (Kjellberg and Mattjus, 2013). When GLTP is overexpressed in HeLa
cells, de novo synthesis of GlcCer, but not LacCer or GalCer, is enhanced (Tuuf and Mattjus,
2007). Downregulation of GLTP resulted in no changes in de novo syntheses of the
sphingolipids, which made the authors suggest that GLTP is not necessarily involved in
GlcCer transport, bur rather has a sensor function (Tuuf and Mattjus, 2007). Together, these
studies indicate that GlcCer is the relevant lipid substrate for GLTP. This goes well with
Accepted Manuscript
GlcCer being the only glycolipid synthesized at the cytoplasmic side of organelle membranes
(Futerman and Pagano, 1991;Jeckel et al., 1992). Another proposed function of GLTP is as a
regulator of adjusting levels of cellular ceramide and, consequently, also the production of
GSLs (Zou et al., 2011).
FAPP2 has been implicated in several biological processes in the cell. Initially, FAPP2 was
suggested to be involved in the transport of cargo between the TGN and the PM (Godi et al.,
2004;Vieira et al., 2005). FAPP2 has also a tubulation activity and is suggested to be
18
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esponsible for tubulating the membranes in the TGN through its PH domain, which gives
further evidence for a role of FAPP2 in Golgi-PM trafficking (Cao et al., 2009). The PH
domain of FAPP1 is furthermore able to tubulate membranes (Lenoir et al., 2010). Another
major role of FAPP2 was demonstrated by D´Angelo and coworkers, when they discovered
that FAPP2 was an important factor in the glycosphingolipid synthetic pathways (D'Angelo et
al., 2007). FAPP2 was responsible for transporting GlcCer from the cytosolic side of cis-
Golgi to the later Golgi compartments, where the more complex GSLs are synthesized
(Lannert et al., 1998). This transport was shown to be non-vesicular and require both the
presence of ARF and PI4P on the later Golgi (D'Angelo et al., 2007). In a recent work it has
been shown that GlcCer can undergo two fates. After GlcCer is synthesized in can either flip
into the lumen of Golgi and be a precursor molecule for LacCer and GM 3 or alternatively, it
can be transported by FAPP2 through the cytosol to TGN for globoside production, Gb 3
(D'Angelo et al., 2013). This interesting and novel finding shows how the sorting and
branching of GlcCer by two different transport mechanisms, the non-vesicular (FAPP2) and
the vesicular, will decide which GSL end product is produced. However, FAPP2 has also
been shown to mediate a GlcCer-transport in another direction, from the cis-Golgi back to the
ER (Halter et al., 2007). Here it is suggested that after GlcCer reaches the ER, it would flipflop
into the lumen of ER and be transported through Golgi by vesicle transport to the TGN
where it could be processed to more complex GSLs. The flip-flop of GlcCer into the lumen of
ER might be catalyzed by a ATP-independent phospholipid flippase (Chalat et al., 2012). A
possible VAP-interaction, as previously discussed in chapter 6.2, might enable ER targeting
of FAPP2 (Mikitova and Levine, 2012).
8. Conclusion
What is known about the depth of GLTP penetration into the membrane surface (Fig. 4)?
Accepted Manuscript
Experimental evidence indicated that the GLTP binding to the membrane is nonperturbing
and not deep (Rao et al., 2005;Nylund et al., 2007). A theoretical orientation of proteins in
membranes (OPM) analyses indicate a membrane penetration depth for GLTP of 3.4 Å with a
tilt angle of 86° for the apo-GLTP and 3.9 Å and 87° for GLTP with a LacCer bound. It
would be interesting to relate this predicted penetration depth with the membrane curvature,
head group hydration and the depth of the GSLs in the surrounding lipid microenvironment,
to mechanistically understand how the GLTP lipid extraction from the membrane takes place.
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The glycolipid depth is dependent on the acyl chain length and saturation of the ceramide
backbone, and consequently, the lipid species is directly affecting the exposure of the
saccharide moieties into the plane of the bilayer. This aglycone (non-sugar part of the
glycolipid) modulation of glycolipids will strongly affect how they are recognized for
instance by their transfer proteins (Lingwood, 1996;Lingwood, 2011). It is clear that all
membrane GSLs are not distributed equally over the cellular membranes and their amount
only controlled by their respective synthesis and breakdown machineries. It is also
fundamental to know whether the GSLs are transported by vesicular means or by proteins.
The function of the glycolipid transferring proteins is slowly emerging while new members
are found, like the CPTP protein. This further adds complexity to the intracellular lipid
trafficking picture.
Acknowledgements
We thank Anders Backman for help with the images.
References
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Figure legends
Fig. 1. A) Schematic presentation of GLTP, FAPP1 and FAPP2 sequences, showing the
important PH and glycolipid binding domains. The binding sites of PI4P within the PH
domain and the FFAT-like motifs within GLTP are highlighted. B) The consensus sequence
of the FFAT motif is shown in bold (Loewen et al., 2003), together with the FFAT-like
sequences in GLTP and FAPP2 in italic letters (Mikitova and Levine, 2012), and a second
FFAT-like motif in the GLTP sequence written in normal letters (Tuuf et al., 2009).
Figure 2. Structural features of GLTP. (A) A typical all alpha-helical GLTP fold with the 8
helices colored as follows; helix 1 (dark blue), helix 2 (light blue), helix 3 (dark green), helix
4 (light green), helix 5 (yellow), helix 6 (dark yellow), helix 7 (orange) and helix 8 (red). The
location of the FFAT-like motif is in purple on helix 1. (B) The GSL sugar-binding pocket of
GLTP (1BV7) with gangliosides GM 3 bound. Note that only the glucose residue of the
ganglioside GM 3 head group is shown. The GSL molecule has the sphingosine chain directed
outwards and a long amide linked acyl chain inside the tunnel. Amino acid residues Asp-48,
Asn-52 (above) and His-140 (below) are shown in different shads of blue, and Trp-96 in red,
are all responsible for stabilizing the glycolipids inside the structure. Additional hydrophobic
interactions between amino acids aligning the interior of the tunnel and with the acyl chain(s)
of the lipid further locks the lipid in position. The yellow lines represent hydrogen bonds
between the three amino acids and the GSL.
Figure 3. (A) Structural presentation of sphingosine-in and sphingosine-out forms of the
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glycosphingolipid binding of GLTP. The far end opening of the hydrophobic cavity can be
seen in the left picture (black arrow). GalCer with N-linked linoleic acid (18:2 Δ 9,12 ) and with
the sphingosine chain in a sphingosine-out conformation (blue) next to GalCer with N-linked
oleic acid (18:1 Δ 9 ) in a sphingosine-in conformation (yellow). (B) Hydrophobicity scale
coloring of GLTP amino acids, red most hydrophobic and white hydrophilic. A putative
membrane interaction domain with high hydrophobicity is indicated with a black arrow (left
picture. The right picture is an 180 degree rotation, revealing a red area originating from the
amino acids lining the far end of the hydrophobic tunnel (white arrow).
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Figure 4. Schematic illustration of the hypothetical GLTP (in cyan) positioning at a
phosphtidylcholine membrane surface with a glucosylceramide bound to GLTP (only one
membrane leaflet is shown), the right picture is an 180 degree rotation. The yellow colored
amino acids indicate the location of the FFAT-like domain in GLTP. The phosphatidylcholine
molecules are rendered in the van der Waals volume. Lipid coloring: black, carbon; red,
oxygen; orange, phosphorus and white, hydrogen. The approximate length of a membrane
leaflet is indicated.
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Figure 2
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