Download The advantages of being small Stockholm University

The advantages of being small
Glycosyltransferases in many dimensions and glycolipid synthesis in
Mycoplasma pneumoniae
Maria Rosén Klement
Stockholm University
Doctoral thesis
©Maria Rosén Klement, Stockholm 2007
ISBN 978-91-7155-503-8 Pages 1-56
Printed in Sweden by US-AB, Stockholm 2007
Distributor: Stockholm University Libary
Till min familj,
Göran, Emmy
och Thyra
List of Publications
This thesis is based on the following publications:
I. Rosén M.L., Edman M., Sjöström M., and Wieslander Å. Recognition
of fold and sugar linkage for glycosyltransferases by multivariate
sequence analysis. J. Biol. Chem., 2004, 279(37):38683-92.
II. Lind J1., Rämö T1., Rosén Klement M.L., Bárány-Wallje E., Epand
R.M., Epand R.F., Mäler L., and Wieslander Å. High cationic charge
and bilayer interface-binding helices in a regulatory lipid
glycosyltransferase. Biochemistry, 2007, 46(19):5664-77
III. Rosén Klement M.L, Öjemyr L., Tagscherer K., Widmalm G., and
Wieslander Å. A processive lipid glycosyltransferase in the small human
pathogen Mycoplasma pneumoniae: Involvement in host immune
response. Mol. Microbiol., 2007, 65(6):1444-57
IV. Wikström M., Kelly A., Eriksson H., Georgiev, A., Rosén Klement
M.L., Dowhan W., and Wieslander Å. Curvature-engineered
Escherichia coli bilayers reveal critical lipid head-group size for
membrane protein function in vivo. Manuscript
1
These authors have contributed equally
Reprints were made with permission from the publisher
Additional publication
Wieslander, Å. Rosén, M. (2001) Cell membranes and transport. In
“Molecular Biology and Pathogenicity of Mycoplasmas” (S. Razin & R.
Herrmann, eds), Kluwer Academic/Plenum Press
Rajalahti T, Huang F, Rosén Klement M.L., Pisareva T, Edman M,
Sjöström M, Wieslander A, Norling B. Proteins in different Synechocystis
compartments have distinguishing N-terminal features: A combined
proteomics and multivariate sequence analysis. J. Proteome. Res. 2007
6(7):2420-34
Contents
Introduction .....................................................................................................1
The biological membrane................................................................................3
Lipid properties ................................................................................................................ 4
Lipid shape................................................................................................................. 4
Glycolipids ....................................................................................................................... 7
Mollicutes and Mycoplasmas ..........................................................................9
Lipid content and synthesis ........................................................................................... 10
Cholesterol ....................................................................................................................11
Immune response in a Mycoplasma infection ............................................................... 12
Autoimmunity................................................................................................................. 13
Glycosyltransferases.....................................................................................14
Glycosyltransferase classification ................................................................................. 14
Glycosyltransferase fold types ...................................................................................... 16
GT-A .............................................................................................................................. 17
Inverting reaction mechanism .................................................................................. 18
Retaining reaction mechanism................................................................................. 20
GT-B .............................................................................................................................. 21
Inverting mechanism................................................................................................ 22
Retaining mechanism .............................................................................................. 24
Membrane binding ........................................................................................25
Sequence analysis and protein classification ...............................................27
Multivariate data analysis .............................................................................................. 27
Previous studies....................................................................................................... 28
Multivariate projection methods ....................................................................29
Principal component analysis .................................................................................. 29
Partial least squares projection to latent structures ...................................................... 30
Summary of papers.......................................................................................32
Paper I ........................................................................................................................... 32
Paper II .......................................................................................................................... 34
Paper III ......................................................................................................................... 36
Paper IV......................................................................................................................... 37
Concluding remarks ......................................................................................40
Acknowledgment...........................................................................................42
References....................................................................................................42
Abbreviations
Gal
Glc
GalGalDAG
GlcGlcDAG
GalDAG
GlcDAG
GT
LPS
PA
PC
PE
PG
CL
GM1
GA-1
TM
APC
GBS
ACC
PCA
PLS
PLS-DA
Galactose
Glucose
1,2-diacyl-3-O-[α-D-galactopyranosyl-(1→6)-O-βD-galactopyranosyl]-sn-glycerol
1,2-diacyl-3-O-[α-D-glucopyranosyl-(1→2)-O-αD-glucopyranosyl]-sn-glycerol
1,2-diacyl-3-O-( β -D-galactopyranosyl)-sn-glycerol
1,2-diacyl-3-O-(α-D-glucopyranosyl)-sn-glycerol
Glycosyltransferase
Lipopolysaccharide
Phosphatidic acid
Phosphatidylcholine
Phosphatidylethanolamine
Phosphatidylglycerol
Cardiolipin
monosialotetrahexosylganglioside
asialo GM1
Trans membrane
Antigen presenting cell
Guillian Barré syndrome
Auto cross covariances
Principal component analysis
Partial least squares projections to latent structures
PLS discriminant analysis
Introduction
Since the 1950´s the minimal genome concept has been a challenging idea
for many researchers, that is the genome believed to describe life. What is
life? As stated in many textbooks, living organisms should be able to grow
by themselves, react to stimuli, and self replicate, but what genes and
proteins are needed for this? Mollicutes are the smallest self replicating
bacteria known today. After the publication of the genome sequence of
Mycoplasma genitalium (Fraser et al., 1995) the scientific world has
questioned if this bacterium contains the minimal self sufficient gene set or
if even a smaller genome is possible? By comparing M. genitalium, with 468
ORFs with Haemophilus influenzae containing 1703 ORFs 240 genes were
found to be homologous in the two species (Mushegian and Koonin, 1996).
These genes were believed to be the minimal genome set, since the two
bacterial species belong to two ancient bacterial lineages, Gram-positive and
Gram-negative, respectively. However, when analysing the 240 orthologous
genes it became evident that some essential pathways were missing
(Mushegian and Koonin, 1996). This is explained by the presence of nonorthologous genes coding for enzymes performing the same reaction. Hence,
estimating the minimal genome becomes difficult and the metabolome is
also of importance and will vary in different growth conditions. When taking
non-orthologous genes into account a minimal genome set was predicted to
be 256 genes (Mushegian and Koonin, 1996).
In 2006 Glass and colleagues randomly inactivated all genes in M.
genitalium to investigate the minimal number of essential genes in this
minimal bacterium. It was found that 382 out of the 482 were essential
(Glass et al., 2006). This is quite close to the 256 predicted in 1996, however
one should take into account that the growth conditions are of importance.
Surprisingly one of the largest groups of essential genes was found to be
genes with unknown function. M. genitalium was a good candidate for these
experiments due to low genomic redundancy, bacteria with larger genomes
contains higher degree of genomic redundancy. However, using M.
genitalium, a parasite and human pathogen as the base for the minimal cell
can be doubtful, since it has genes that are of importance for the
pathogenicity and interaction with the host cell, e.g. the bacterium needs to
send signals to the host cell to obtain nutrients. These functions are essential
1
to M. genitalium, but would not be of importance to other bacteria. The
largest groups of essential genes all consists of genes encoding proteins
involved in replication, transcription, translation, and repair, the latter is
however only a minor group of genes in Mollicutes sequenced until today.
Mycoplasmas are known to have the ability to fuse with, invade the cell or
live in close contact with the host cell, being obligate parasites (Baseman et
al., 1995; Dimitrov et al., 1993). They are able to incorporate essential
components such as fatty acids and amino acids from the host cell to save
energy (Baseman and Tully, 1997). It is also known that some mycoplasmas
incorporate host lipids into the membrane (Uemura et al., 1988), which
would mimic the host cell and prevent them being discovered by the host
immune system. These bacteria thus have a large fraction of specific
transporters essential for a parasitic life, which would not be essential for
other organisms. Even if the bacterium is small, it has functions that a larger
bacterium does not need, which makes estimations of the minimal genome
difficult.
Mutagenesis experiments, making knockout mutants in M. genitalium have
shown that as mentioned, 382 out of 482 genes are needed for survival.
Within these, the genes encoding three glycosyltransferases (GT) could not
be disrupted by the transposones (Glass et al., 2006), and two of these are
included in the minimal genome set estimated in 1996 (Mushegian and
Koonin, 1996). M. pneumoniae is almost an exact copy of M. genitalium
with 207 additional ORFs that are unique (Himmelreich et al., 1997). When
considering the high amino acid identity between M. genitalium and M.
pneumoniae orthologous, the protein functions are believed to be similar in
the two species and thus be essential in both species.
Why would a glycolipid synthase be essential to the obligate human
parasite, M. pneumoniae and why can’t some lipids be replaced by others?
The goal in this thesis was to identify the glycosyltransferases used in the
glycolipid synthesis in M. pneumoniae. Only three GTs had been proposed
after sequencing the genome, however the function of these could not be
predicted. The closest homologues were all LPS enzymes from Grampositive bacteria, a structure not found in Mollicutes, however
lipoligosaccharides have been found (Smith, 1992). Glycolipids are only a
minor component of the M. pneumoniae membrane, but the variation is
large. How are glycolipids synthesised and how do the enzymes producing
them work? These are the questions that will be addressed in this thesis.
2
The biological membrane
Life would not exist without the biological membrane, the shield that
separates the cell from its surrounding making it possible to have a different
environment within. The membrane consists of both a large variety of
different lipids forming a bilayer and membrane proteins embedded in the
lipid layers. The lipid bilayer is formed due to hydrophobic and hydrophilic
properties, with the hydrophilic head groups shielding the hydrophobic
hydrocarbon chains from the water surrounding it. The physical properties
and the shape of the lipids will influence the function of the membrane (van
den Brink-van der Laan et al., 2004). The biological membrane will also
work as a selective filter, determining all communication with the world
outside. Only a very limited number of molecule species can pass the bilayer
without the assistance of a transporter or channel.
The cellular lipidome consists of thousands of different lipid molecules and
the lipid content and amounts vary greatly between different bacterial and
eukaryotic species (van Meer, 2005). The total lipid content of the cell is
only about 5 % of the dry weight and 40-50% of the membrane with the rest
being proteins (Smith, 1992). The major functions of lipids are the same, but
the way to perform them are different. The major lipids in for example
Echerichia
coli
are
phosphatidylethanolamine
(PE)
75%
phosphatidlyglycerol (PG) 20% and cardiolipin (CL) 5% (all mol %)
(Cronan, 2003). Acholeplasma laidlawii, a well studied Mollicute, contains
15-25% PG and large amounts of glycolipids, 15-50% GlcDAG and
GlcGlcDAG (Rilfors et al., 1993). The thylakoid membrane, the most
common bio-membrane on Earth, is unusual with its low phospholipid
content, PG 10% but large amounts of glycolipids, 50% GalDAG and 30%
GalGalDAG (all mole %) (Lee, 2000). The large differences in lipid
composition of different organisms reflect the different properties of lipids
and the differences in growth. In the next section, the properties of lipids will
be discussed.
3
Lipid properties
Why are there so many kinds of lipids? Lipids differ in head group, back
bone and acyl chain length and chain unsaturation (Dowhan and Bogdanov,
2002). These differences will have effects on the property and function of
the lipid. The physical state of membrane lipids has been found to strongly
affect membrane associated processes (Dowhan, 1997), such as insertion of
membrane proteins, membrane protein function, and binding of membrane
associated proteins. The membrane flexibility and ability to adapt to
different environmental changes is only possible with a large variety of
lipids, since the membrane needs to be fluid and flexible at all times for the
cell to function.
There are many different forces that control the behaviour of the lipids, for
example the attraction or repulsion of the head groups, the molecular
structure and ratio between the volumes of the head groups, the chain length
and saturation, and the hydration forces (Boggs, 1987). Within these, the
attraction or repulsion of charged head groups is one of the most important
properties, which causes the lipids to become more or less densely packed.
This also affects their tendency to form hydrogen bonds. The intramolecular
bonds are important for the stability of the membrane, charged or neutral
lipids with hydrogen-donating and accepting groups will form these
interactions. It is important to remember that the membrane is in a fluid state
and the interhydrogen bonds will constantly break and reform between
different lipid molecules (Boggs, 1987).
Lipid shape
The lipid shape is another important property that will affect the bilayer.
There are three major classes of lipid structures, inverted cone, cylindrical,
and cone shaped (Fig. 1). The first type is most common for
lysophospholipids, having only one acyl chain, phospholipids with short akyl
chains, and detergents. This will cause the lipids to form micells due to a
large head group and small hydrophobic domain, with the alkyl chains
facing inwards (Reviewed in) (Dowhan and Bogdanov, 2002).
Cylindrically shaped lipids such as phosphatidylcholine (PC),
sphingomyelin,
cardiolipin,
phospahtidic
acid
(PA),
and
digalactosyldiacylglycerol, (GalGalDAG) will pack into bilayers, either as
4
ordered gel phase (Lβ) or liquid crystalline phase (Lα). This allows for tight
packing of acyl chains and will efficiently exclude water. These lipids have
head groups and hydrophobic domains of similar size (Dowhan and
Bogdanov, 2002) and will thus have a cylindrical shape (se Fig. 1).
The last group of lipids have smaller head and larger hydrophobic domains,
causing them to become cone shaped. PE and monogalactosyldiacylglycerol
(GalDAG) together with, PA, and CL are examples of lipids with this shape,
the latter, however only in the presence of divalent cations. The ion reduces
the repulsion of the negatively charged head groups, making CL cone shaped
instead of cylindrical. This shape will tend to form a hexagonal (HII) phase
or cubic phase, with the acyl chains facing outwards and the head groups
inwards towards the aqueous core in long tubes (see Fig. 1) (Dowhan and
Bogdanov, 2002).
The inverted cone and cone shaped lipids are considered to be nonbilayerprone and when mixed with bilayer forming lipids, the cone shape will
change the physical properties of the bilayer and cause stress or strain to the
bilayer structure. The chain saturation can also affect the shape of the lipid,
when increasing the unsaturation the acyl chains will become larger the
shape will change from cylindrical to conical, this will also happen with
increased temperature (Dowhan and Bogdanov, 2002).
Figure 1. Phases and molecular shapes of different lipids. The physical shape and
the phase they adopt is shown for the three different shapes, inverted cone,
cylindrical and cone types (Dowhan and Bogdanov, 2002).
5
The liquid crystalline phase, formed by cylindrical lipids has been found to
be essential for the viability of the cell and the lipid content is therefore
adjusted to maintain this property. The strain caused by the insertion of nonbilayer forming lipids, results in the curvature stress that exist in the
membrane. Forcing a non-bilayer prone lipid into a cone shape in a
membrane will result in the exposure of the hydrophobic core to the aqueous
surrounding (Dowhan and Bogdanov, 2002). This is probably driving the
formation of micro domains, separating the different types of lipids that will
decrease the tension in the membrane. The existence of rafts, specific lipid
domains in eukaryotes, which constitutes micro environments has been
under debate for decades. Rafts are believed to be important in many cellular
functions, such as cellular signal transduction and trafficking (Hanzal-Bayer
and Hancock, 2007). Lipid domains in bacteria would produce a unique
milieu for different enzymes and could potentially regulate their functions.
Different lipids could therefore regulate different enzymes in both
eukaryotes and prokaryotes (Matsumoto et al., 2006).
The importance of the existence of different types of lipids can be
illustrated with the mutant AD93 (Cronan, 2003; DeChavigny et al., 1991).
This mutant lacks the non-bilayer prone PE and grows very poorly. Several
membrane proteins have been found to have an altered topology in this
mutant e.g. lactose permease and gamma-aminobutyric acid permease (Xie
et al., 2006; Zhang et al., 2005). The properties of PE have been found to be
important for the insertion of these proteins in the correct transmembrane
orientation. The non-bilayer prone lipid GlcDAG was found to be able to
replace PE and restore both the orientation of LacY and the growth rate of
the mutant (Xie et al., 2006). This shows how important the structure of the
lipids is, and how different enzymes use the different lipids and probably the
bilayer stress that non-bilayer forming lipids causes to insert correctly into
the membrane. Charged lipids are of great functional importance to the cell,
such as the anionic lipid PG, but is only a minor component (20- 30%) in
E.coli and most other membrane. The charged head group is utilised in the
membrane association of many cytoplasmic proteins (Dowhan, 1997). It is
believed for example that the negative charges of PG are not equally
distributed in the membrane. Charge islands would be more attractive
binding sites than a random distribution (Dowhan, 1997). Other lipids in
bacteria are also thought to exist in micro domains such as CL and PE
(reviewed in (Matsumoto et al., 2006)) offering specific environments for
certain proteins and enzymes. Micro domains in the bacterial membrane,
were also believed to be formed by the differences between PE, nonbilayerprone and CL, bilayer-prone. The preference for certain lipids by enzymes is
known, for exampel MurG a membrane bound glycosyltransferase involved
6
in the cell wall synthesis in E. coli binds preferentially to CL (van den Brinkvan der Laan et al., 2003).
Glycolipids
As described above, the glycolipid content of various membranes varies
greatly. One of the organisms that has been very well studied and from
which most information of the importance of glycolipids originates is A.
laidlawii. Studies have shown that unsaturated GlcDAG is more nonbilayerprone then saturated GlcDAG, with the unsaturated being more common in
this bacterium. To overcome the tension created by unsaturated GlcDAG,
the ratio of GlcDAG to GlcGlcDAG (bilayer-prone) decreases when the
bacterium changes the properties of the membrane by altering the glycolipid
content, instead of the unsaturation of the fatty acid chains (Wieslander et
al., 1978). The latter is more common in other bacteria e.g. E. coli. Why
would a bacterium use this system to make new glycolipids instead of just
changing the saturation of e.g. PG or PE? The glycolipid content in bacteria
lacking a cell wall, e.g. Mycoplasmas is found to be higher than in bacteria
having a this structure. Glycolipids have the ability to form hydrogen bonds
between the sugar molecules and would therefore stabilise membrane
without the need of a cell wall, (Boggs, 1987). However, the glycolipid
content has been found to vary greatly in this bacterial group. This could
however be explained by the presence of a lipoglycan layer in some spieces,
replacing the glycolipids (Smith, 1992).
Glycolipids have also been found to be of major importance for
photosynthesis. The transition temperature is higher for glycolipids than for
phospholipids, increasing the potential to form clusters in the membrane
(Curatolo, 1987b). The glycolipid GalDAG is also found to drive the
stacking of the membrane to form grana. This stacking allows for efficient
packing of the thylakoid membrane within the chloroplast and leads to the
separation of photosystem I and II (Lee, 2000). This glycolipid is a
nonbilayer-prone lipid, due to its smaller head group and bulky highly
unsaturated chains. GalDAG has been found to be highly associated with the
light harvesting complex II, with 80 molecules per protein complex
(Simidjiev et al., 2000). The binding to the membrane proteins forces the
lipids to become more bilayer prone in structure. The strained nonbilayer
properties of GalDAG and the energy stored in this tension is believed to
change or modulate membrane protein packing properties and also the
function (Lee, 2000). This phenomenon has also been seen in cytochrome c
oxidase that forces CL into a bilayer prone state (Rietveld et al., 1987). The
7
ratio between GalDAG and GalGalDAG in the thylakoid membrane has
been found be similar to the PE/PC ratio found in most plasma membranes,
hence the proportion of bilayer prone and nonbilayer-prone is the same in
the two membranes. It is believed that the reason for using glycolipids in the
thylakoid is to make it more stable due to the intra hydrogen bonds
(Curatolo, 1987a). The membrane stability is of major importance due to the
transfer of charge and where the trans-bilayer ionic environment must be
keept constant.
8
Mollicutes and Mycoplasmas
Most of the work in this thesis has been done on or with enzymes from M.
pneumoniae a bacterium classified as a Mollicute. This group of bacteria is
the smallest capable of autonomous growth and is characterised by the small
genomes (0.58 kb to 2.2 Mb), their low G+C % content (23-40%)
(Johansson and Pettersson, 2002), and their lack of cell wall. Until today 200
different species have been identified. Mollicutes belong to the phylum
Firmicutes to which also other Gram-positive bacteria with low G+C %
content belong, such as Bacilli and Clostridia (see Fig 2). Studies of the 16S
rRNA show that the Mollicutes diverged from the Streptococcus branch of
the Gram positive bacteria with low G+C content about 605 million years
ago. By genome reduction different branches of the Mollicutes were
developed during 400 million years, giving the different families of
Mollicutes found today (Manillof, 2002).
Figure 2 Schematic representation of relationships within the Firmicutes (Wolf et
al., 2004)
In Mollicutes, the majority of the lipids are located in the cytoplasmic
membrane and constitute between 25-35% of the dry weight (Hayashi and
Wu, 1990). Lipid synthesis is diverse and sequence analysis has revealed
that the identified phospholipid synthesis (see Fig. 3), must differ from other
bacteria with known metabolic pathways. Analogous enzymes/genes from E.
coli and other bacteria with known lipid synthesis have not been found in the
9
sequenced Mycoplasma genomes. One of the two acyltransferases, used in
the phosphatidic acid synthesis is for example missing, in M. pneumoniae,
M. genitalium, and Ureaplasma urealyticum and phosphatidylglycerol
phosphate phosphatase in M. pulmonis (Chambaud et al., 2001; Fraser et al.,
1995; Glass et al., 2006; Himmelreich et al., 1996). However, the one found
might be adding both acyl chains on the glycerol in these species.
Most of the studies on the lipid content and lipid synthesis in Mollicutes
were performed in the late sixties and not much work has been done since
then. The lipid content in approximately 20 different Mycoplasma species
have been characterised (Smith, 1992). Later studies have mainly been
focusing on A. laidlawii, M. pneumoniae, M. fermentans. Mollicutes have no
ability to synthesise fatty acids, nor change the fatty acid chain length or
saturation with a few exceptions, e.g. Acholeplasmas are capable of
synthesising saturated fatty acids (Smith, 1992) and also elongate exogenous
chains (McElhaney, 1992). Mollicutes depend on the environment for the
supply of cholesterol, an essential component for the structure and properties
of the membrane. Acholeplasma is an exception, where cholesterol is not
essential. These components together with some phospholipids, e.g. PC and
PE are known to be incorporated into the membrane from the surroundings.
These lipids can also be further metabolised or the saturation of the fatty
acids in PC changed (Rottem et al., 1986).
The lack of cell wall peptidoglycan layer in mollicutes makes them unique
in the bacterial kingdom. This property places them in a direct contact with
the surrounding, making it easier to take up essential components. A
lipoglycan layer has been found in some species, for example in
Acholeplasma, Anaeroplasma, Ureaplasma and a few Mycoplasma. This
layer is firmly attached to the membrane and constitutes about 10% of the
dry weight (Smith, 1992). The absence of a cell wall has made Mollicutes an
ideal organism for studying the biomembrane, making it easy to remove and
add membrane components that are otherwise difficult.
Lipid content and synthesis
The membrane of Mollicutes contains mainly three classes of lipids,
phospholipids, glycolipids, and neutral lipids (Smith, 1992). The major lipids
in the membrane are phospholipids and especially PG. The lipid synthesis
pathways are believed to be very similar in most Mollicutes, however the
pathways have been mostly studied in A. laidlawii. PA is the key lipid in the
biosynthesis of both glycolipids and phospholipids in A. laidlawii
(McElhaney, 1992) and believed to be in most Mollicutes (Fig. 3). PA is
synthesised by acylation of sn-glycerol-3-phosphate with two fatty acids. PA
10
can be dephosphorylated to a diacylglycerol, DAG by a PA phosphatase
(Berg and Wieslander, 1997). DAG can be glucosylated in two steps to form
GlcDAG and GlcGlcDAG (Dahlqvist et al., 1992). The glycolipids can be
further acylated by a third acyl chain to form MAGlcDAG and
MAGlcGlcDAG (Hauksson et al., 1994a, b). Phosphoglycolipids,
GPGlcGlcDAG and MABGPGlcGlcDAG from GlcGlcDAG are also
synthesised by A. laidlawii however by an unknown process (Hauksson et
al., 1994b). PA is also a precursor in the phospholipid synthesis, where
cytidine diphosphate diglyceride is formed from CTP and PA.
Phosphatidylglycerol-3-phosphate is formed by CDP-diacylglycerol and
glycerol phosphate and further metabolised of a phosphate group to form
PG. Two PG molecules can form a diphosphatidylglycerol or cardiolipin
(Smith, 1992) (Fig. 3).
Figure 3. Metabolic pathways for the synthesis of glycolipids and phospholipids in
Mollicutes. GP, glycerol-phosphate; PA, phosphatidic acid; CPD-DAG, Cytidine
diphosphate diacylglycerol; DAG, diacylglycerol; MH, monohexose; DH, dihexose;
TH, trihexose; PG, phosphatidylglycerol; APG, acylphosphatidylglycerol; CL,
cardiolipin; and Cer, ceramide. All steps between PA and PG are not shown.
Cholesterol
Cholesterol is as stated previously, an essential component in Mollicutes
and is inserted in between the long hydrophobic fatty acid chains in the
membrane and account for between 4 to 20% of the total lipid content
(Smith, 1992). Cholesterol enhances the formation of a liquid ordered phase
(Simons and Vaz, 2004) and decreases the surface charge caused by PG and
Cardiolipin in the membrane, by modifying the intermolecular interaction
between these polar head groups (Tarshis et al., 1993). However, cholesterol
11
has been found to have a preference for sphingolipids, a component found in
many Mycoplasma species, due to the hydrogen bonding ability and the
saturation of the chains that is naturally occurring in sphingolipids (Li et al.,
2001). All sphingolipids have both hydrogen bond acceptors and donors (the
carbonyl group and the amide group), whereas DAG based lipids such as PC
only have the hydrogen bond acceptor, the carbonyl group (Li et al., 2001).
By incorporating cholesterol into the membrane, the bilayer can consist of
lipids in a physical intermediate state between gel and liquid–crystalline
states (Ladbrooke et al., 1968; Shah and Schulman, 1967), forming
membrane domains. The cholesterol content has also been found to be very
important in the fusion of Mycoplasma cells, e.g. M. capricolium with the
eukaryotic host cell (Franzoso et al., 1992; O'Toole and Lowdell 1990), but
is believed to be important for other cholesterol containing Mollicutes such
as M. pneumoniae. This is probably due to the Mycoplasma membrane
becoming more like the host cell making it easier to fuse the membranes.
Immune response in a Mycoplasma infection
M. pneumoniae is a common human pathogen, and is the bacterium behind
pneumonia in about 20% of all the cases, and up to 50% of all pneumonia in
the age group of 20-30 years. It is often referred to as walking pneumonia,
due to rather week symptoms and long incubation time. As most other
Mycoplasmas it is a mucosal pathogen, living in close association with
epithelial cells (Waites and Talkington, 2004). The progression of an M.
pneumoniae infection is slow and when the bacterium reaches the lower
respiratory tract, it meets the host defence. Macrophages become activated
and start to phagocytose the bacteria. The macrophages, also called antigen
presenting cell, (APC) present mainly peptides from digested surface
associated proteins and LPS on the surface of the cell through MHC I and
MHC II molecules. One of the most important peptide antigens for the
immune system in mycoplasma infections are lipoproteins, due to their
surface exposure in the bacterium (Chambaud et al., 1999).
Recently it was discovered that a new class of molecules, named CD1,
present lipids instead of peptides on the surface of the antigen presenting
cells (Porcelli and Modlin, 1999). The activation of invariant nature killer
cells (iNKT) cells through these molecules has been found to be an
important factor in the defense of bacteria that lack a cell wall (Kinjo et al.,
2006). Kinjo and co-workers showed that the glycolipids, α-GalDAG from
the human pathogen Borrelia burgdorferi is presented by the CD1d
molecule on APC (Kinjo et al., 2006). Activation of iNKT cells by CD1d
and through the T cell receptor will also activate B-cells to proliferate and
12
produce immunoglobulins (Galli et al., 2003). The sugar preference and
orientation for CD1d binding is still not clearly understood. Both α-GalCer
and β- GalCer (Parekh et al., 2004) as well α-GlcCer (Brigl et al., 2006) can
bind to CD1d and activate iNTK cells. When analysing the structure of a
CD1d molecule with the bound α-GalCer substrate, it was however
discovered that the molecule have structural preferences for the alpha
configuration. The structure shows that a beta linkage between the ceramide
and the galactose would disrupt the interaction between the CD1d molecule
and the T-cell receptor (Borg et al., 2007). The activation of the immune
system through a MHC unrestricted pathway has previously been reported
after a Mycoplasma infection (Ruuth and Praz, 1989). These results indicate
that glycolipids from M. pneumoniae and other cell wall lacking bacteria
could be processed in similar fashion and be presented by CD1d or by a
similar molecule. The activation of iNKT stimulates the immune system and
activates the production of antibodies.
Autoimmunity
M. pneumoniae is known to rarely, in 0.1% of the cases cause GuillainBarré syndrome (GBS) (Tsiodras et al., 2005). This autoimmune disease
breaks down the myelin sheets surrounding nerve cells, which causes limb
weakness and loss of nerve reflexes. The auto antibodies recognise
glycolipids, the major component in the myelin sheets, indicating that the
bacteria infecting the patient prior to GBS have these epitopes (Kumada et
al., 1997). The GM1 lipid sugar structure, the most common epitope for
antibodies in GBS patients, has been found in some strains of
Campylobacter jejuni, indicating molecular mimicry as the cause of GBS
(Gilbert et al., 2000). In GBS patients where the disease is preceded by an
M. pneumoniae infection, anti-GalCer antibodies are most common, and
anti-GA1 (Kusunoki et al., 1995; Kusunoki et al., 2001) and GM1 have also
been found (Susuki et al., 2004). Susuki and co-workers showed that the
GM1 sugar structure is probably a part of the membrane of M. pneumoniae
(Susuki et al., 2004). Antibodies directed against this lipid react to total lipid
extracts from M. pneumoniae. Cross reactivity between anti-GM1 and antiGalCer has however been seen. The bacterial GTs, found to be essential by
knock out studies, must have the ability to use the eukaryotic ceramide as
base in the sugar lipid synthesis. Molecular mimicry described above is a
way to hide from the immune system, however once the bacterium has been
discovered, the production of autoantibodies can become lethal to the
patient.
13
Glycosyltransferases
The turn-over of sugars is one of the most widespread enzymatic functions
on this planet. Approximately 1-2% of the coding regions of all genomes are
glycosyltransferase genes (Davies and Henrissat, 1995). However, a massive
gene loss has been observed in bacteria with a parasitic or symbiotic life
style (Davies and Henrissat, 1995). For example, M. pneumoniae has only
0.4% predicted GTs, or three genes encoded in its genome, instead of the
expected 10. However, these GTs are all essential indicating their
importance for supporting life, probably being the minimal number of GTs
bacteria need.
The energy stored in glycogen and starch is one of the most important
metabolic energy sources. Sugar structures are not only used as energy
storage, but also e.g. building blocks. The production of lipopolysaccharide
(LPS) is of major importance for the survival of free living bacteria,
protecting the cell from osmotic lysis and making a first barrier for
aggressive macromolecules and the host defence. Glycolipids, discussed
above, are other important products made by GTs and are essential to
photosynthesis and for membrane stability in many bacteria. The
galactoceramide molecule is another example, being an essential component
of nerve cells, isolating the cell from its surrounding, making it possible to
rapidly transfer electric impulses along the axon (Ruvolo, 2003). However,
only the function of glycosyltransferases, transferring an activated sugar to
an acceptor will be discussed here.
Glycosyltransferase classification
Glycosyltransferases have been classified by Henrissat and colleagues in a
system called CAZy (Coutinho and Henrissat, 1999). The list of GT families
is growing monthly and the data base consists of over 20 000 amino acid
sequences, with an established or predicted function. The GTs are divided
into 90 families according to function, with 21 of these having at least one
GT with an established structure (CAZy GT family 1, 2, 5–9, 13, 15, 20, 27,
28, 35, 42–44, 63, 64, 72, 78, and 80) (Martinez-Fleites et al., 2006). The
classification is amino acid sequence-based, a GT is grouped to a specific
14
CAZy family if it has a BLAST probability value lower than 10-3 over a
stretch of 100 amino acids and a hydrophobic cluster pattern that is similar to
other members in that group (Campbell et al., 1997). The origin of the
sequences varies greatly, with some families containing enzymes from all
kingdoms, e.g. CAZy GT 2 and GT 4 whereas others contain only one
taxonomic group. These two families, CAZy GT 2 being inverting and
CAZY GT 4 retaining are probably the two ancestor families to all others
(Martinez-Fleites et al., 2006). The fact that they also contain all kingdoms
and that Archea GTs mainly are classified into these two families make this
a good assumption (Martinez-Fleites et al., 2006).
Glycosyltransferases use an activated sugar donor, where an α
configuration between the sugar and the nucleoside diphosphate, nucleoside
monophosphate or lipid phosphosugar is the most common (Tarbouriech et
al., 2001) phosphate, UDP and TDP, is believed to be used by around 60%
of all predicted GTs (Hu and Walker, 2002). The linkage of the sugar
nucleotide is either retained or inverted between the sugar and the acceptor,
hence giving the naming retaining or inverting GTs (Fig. 5).
Figure 5. The stereochemical outcome. An α-D-linked activated sugar donor can
give rise to a product with the anomeric configuration either retained or inverted.
The sequence similarity within each CAZy family can be very low, and
where not all members share sequence identity or similarity, eg CAZy GT 2
and GT 4. This is due to the large variation of the origin of the GTs. The fold
is however believed to be conserved within each family, and there is mostly
no or very low sequence similarity between different families (Campbell et
al., 1997; Davies and Henrissat, 1995; Henrissat and Davies, 1997).
However, only one GT in family 2 (Charnock and Davies, 1999) and three in
CAZy GT 4 (Guerin et al., 2005; Martinez-Fleites et al., 2006), have been
crystallised, making this generalisation uncertain for these two large
families. The substrate diversity within some large families varies greatly,
for example in the large families 1, 2, 4, although they still share substantial
sequence similarities (Henrissat and Davies, 2000). Other families consist of
GTs utilising the same substrate, e.g. CAZy GT 3. There are also examples
of almost identical enzymes using different substrates. The blood group A
15
and B transferases from CAZy GT 6 differ in only four amino acids, but still
use different sugar donors (Yamamoto et al., 1990). Only two of these four
amino acids are found in the active site in the crystal structure, involved in
the binding of the different sugars, UDP-Galactose and UDPAcetylgalactoseamine (Patenaude et al., 2002). The opposite has also been
recorded, enzymes having very low or no sequence similarity can utilise the
same substrates (Breton et al., 2001), indicating convergent evolution. The
CAZy GT families are believed to be classified according to both structure
and mechanism, however this may not be completely correct for all families.
Distant similarities have been found between families with different
mechanism, retaining and inverting, and some families have been spliced
into new families, when the mechanism has been characterised for some
family members (Breton et al., 2006). The prediction of substrate specificity
and mechanism of a newly discovered GT are therefore almost impossible.
Glycosyltransferase fold types
Within the 21 families with an establish fold, only two fold types have
been characterised until today, named GT-A and GT-B (Ang et al., 2000;
Bourne and Henrissat, 2001; Coutinho et al., 2003; Davies et al., 2005) (Fig.
4). Both fold types are globular proteins and with partial Rossman folds
(Bourne and Henrissat, 2001), with GT-A having anti parallel beta sheets
and GT-B parallel. The Rossman fold was first identified in proteins binding
diphosphate containing co-factors such as NADH (Buehner et al., 1973).
Even though GT-B consists of two Rossmann fold domains and GT-A of
one, no sequence similarities have been found between the two different fold
families, indicating that they have evolved separately (Liu and Mushegian,
2003). The idea of a gene duplication of a GT-A fold enzyme is otherwise
easy to assume. The topology with parallel beta-sheets found in the GT-B
family is quite common in the SCOP database. However, when analysing the
GT-A family, it is evident that this family have anti-parallel beta-sheets that
seem to be unique in the fold database (Liu and Mushegian, 2003). The fold
is otherwise very similar between the two families.
Most glycosyltransferases use activated sugar donors where UDP and other
sugar nucleotides are the most common. A third glycosyltransferase group
(GT-C), (see Fig. 4) has been discovered by iterative BLAST searches
followed by structural comparisons (Liu and Mushegian, 2003). However,
the latter families are only predicted and no crystal structures have yet been
solved. Proteins with this fold are all integral membrane proteins with the
active site in the loops, and with the transmembrane helix number varying
between 8 and 13. This family can also be found with Hidden Markov
16
method searches within the GT families (Liu and Mushegian, 2003). This
method have also identified a fourth family, unique to eukaryotes, named
GT-D, consisting of only 3 CAZy GT families (Kikuchi et al., 2003). There
are however, no fold models for this enzyme group. The structural diversity
is believed to be very small for GTs, however the opposite is seen for the
glycosylhydrolases, breaking sugar linkages, where the structural diversity is
large (Davies et al., 2005).
Figure 4. The different fold families discovered until today within the GT
enzyme groups (Charnock and Davies, 1999; Ha et al., 2000).
GT-A
The GT-A fold family is the largest with 25 CAZy families classified into
this group, compared to 12 for the GT-B fold family. The fold consists of a
Rossman fold, with two dissimilar domains, with the nucleotide sugar
binding domain N-terminally and the acceptor binding domain in the Cterminal. Almost all enzymes with a GT-A fold type have a DxD motif in the
N-domain. This motif is found to interact with the phosphate groups of the
nucleotide donor through a divalent cation, most commonly Mn2+. There are
differences in the function of the DxD motif when comparing inverting and
retaining GTs of the GT-A fold. The two aspartate residues, interacting with
the ion in the retaining GTs, but only the last aspartate interacting with the
ion in inverting GTs (Breton et al., 2006). The variable amino acid, most
often a polar or small and non cyclic is usually involved in binding the
ribose. For example, in the inverting enzyme SpsA (CAZy GT 2) the motif is
xDD where the first aspartate is known to interact with the ribose group of
the nucleotide and the second aspartate interacts with the ion (Tarbouriech et
al., 2001). The DxD motif is not fully conserved in the GT-A family with for
example SpsA having, xDD and DxH has been found in the retaining family
27 (Fritz et al., 2004). The inverting C2GnT-L (CAZy GT 14) completely
17
lacks this motif. The function of the metal ion is replaced by the two basic
amino acids arginine and lysine (Pak et al., 2006). This active site
arrangement has also been seen in the GT-B fold family discussed below,
indicating that the reaction mechanism is a junction between the two fold
families GT-A and GT-B.
Cst II from Campylobacter jejeni believed by some to have a GT-A like
fold, also lacks the DxD motif. It is known to be further activated by Mg2+
and Mn2+, even though the ions are not known to be essential (Chiu et al.,
2004). Due to the absence of the DxD motif, an ion in the active site, as well
as a catalytic base, the reaction mechanism is believed to be similar to the
retaining GTs discussed below, the product is although inverted (Chiu et al.,
2004). This structure is however not a clear GT-A fold. The flexible loops
found in most GT-A structures are here containing short helixes that will
fold over the active site (Chiu et al., 2004). The secondary structure
arrangement in the cst II enzyme is also different from the GT-A family and
has been designate to a fold family of its own by SCOP (Murzin et al., 1995;
Pak et al., 2006).
Inverting reaction mechanism
The inverting mechanism is the mostly widely studied, and it has been
easier to “capture” intermediates during structure analysis. The mechanism
is believed to be a SN2 type, with the bound divalent cation being the acid
catalyst and involved in the coordination of the substrate and the donor
(Breton et al., 2006). The inverting mechanism uses a base to deprotonate
the reactive hydroxyl of the sugar acceptor to further activate it (Charnock et
al., 2001). This base is aspartate-191 in SpsA, (CAZy GT 2) (Charnock and
Davies, 1999), conserved in CAZy GT families 2, 7 (Gastinel et al., 1999)
and 13 (Gordon et al., 2006; Unligil et al., 2000) and replaced by a
glutamate in CAZy GT families 14 (Pak et al., 2006), and 43 (Pedersen et
al., 2000). In the later families the sugar acceptor is known to be hydrogen
bonded to the glutamate in the position for a nucleophilic attack on the UDPsugar donor (Pak et al., 2006; Pedersen et al., 2000). The function of the
conserved aspartate has also been verified by mutational studies (GarinotSchneider et al., 2000; Keenleyside et al., 2001). These enzymes use three
aspartates, (Asp 39, 98, 99 in SpsA) to coordinate the nucleotide in the donor
sugar and a fourth aspartate, Asp 191 or glutamate acts as the catalytic base
(Charnock et al., 2001). Aspartate 99 is also known to coordinate the
divalent cation (Tarbouriech et al., 2001).
18
Figure 6. Coordination of the UDP-sugar in the Gal-T1 in CAZy GT 7 and
the conserved amino acids in other GT-A enzymes with an established fold
(Tarbouriech et al., 2001).
All GT-A structures solved have a flexible loop that is believed to be
involved in the active site and the binding of the substrate (Boix et al., 2001;
Ramakrishnan et al., 2002b; Ramakrishnan et al., 2006), but this has only
been functionally characterised in the inverting Gal-T1 (CAZy GT 7)
enzyme (Ramakrishnan et al., 2006) and partially in LgtC (CAZy GT 8)
(Persson et al., 2001). The Gal-T1 enzyme has been very extensively studied
and was first crystallised in 1999, and has since then been crystallised with
various substrates and at different resolutions (Persson et al., 2001;
Ramakrishnan and Qasba, 2001; Ramakrishnan et al., 2002a; Ramakrishnan
et al., 2002b; Ramakrishnan et al., 2006). The mechanism established until
today involves first the binding of Mn2+ followed by UDP-Gal and the
acceptor substrate. The binding of the ion has been found to be essential for
the binding of the donor (Ramakrishnan et al., 2006). This is probably true
for most inverting GT-A enzymes. The formation of the acceptor binding
site has also been seen in the GnT-I enzyme (CAZy GT 13) (Gordon et al.,
2006). The metal ion is bound in an octahedral coordination, where it
interacts with one or two residues in the DxD motif, two oxygen atoms in the
UDP molecule and finally with three water molecules (Qasba et al., 2005).
The binding of the ion and the donor induces a conformational change in the
flexible loops described above, leading to a change in the coordination of the
water molecules around the ion. This change creates an active ground state
in the enzyme that can assist catalysis and makes the acceptor binding site
accessible to the solvent (Fig. 7) (Qasba et al., 2005). This conformational
change and the change in the coordination of different water molecules also
assist the rotation of the phosphate group in the donor sugar nucleotide,
preventing the reaction to revert (Ramakrishnan et al., 2006). The water
molecules found to be coordinated with the ion have been found to be
conserved in the structures solved where the reaction is dependent on a
19
divalent ion (Ramakrishnan et al., 2006). The ion is also believed to
neutralise the nucleotide once the sugar has been cleaved off, helping it to
leave the enzyme (Qasba et al., 2005). This function has been replaced by
two basic residues in the CAZy GT 14 family and in the CstII enzyme that
has a novel fold. These enzymes are also believed to be able to bind the
acceptor in the absence of the donor (Pak et al., 2006).
Figure 7. Schematic drawing of the conformational changes during catalysis
observed in GNT1. i) The binding of the ion makes it possible for the ii) sugar donor
to bind. iii) The loop folds in to the structure, iv) this conformational change creates
a binding site for the acceptor and the reaction can take place. The donor and the
product leaves the active site and the cycle start once again (Qasba et al., 2005).
Retaining reaction mechanism
The retaining reaction mechanism is not as well understood, but is
believed by many to involve a double displacement reaction where the donor
sugar is first transferred to an amino acid in the protein and then transferred
to the acceptor, as seen in glycosylhydrolases (Qasba et al., 2005). To
support the double displacement mechanism, a base is needed in the active
site (Tvaroska, 2004). However, this has not been identified in the only
retaining enzyme crystallised in the presence of donor or substrate, the LgtC
enzyme (CAZy GT 8) (Persson et al., 2001). Molecular modelling has
shown that the most likely reaction mechanism occurs in one step, where an
oxygen in the acceptor attacks a carbon in the donor, breaking the bond
between the sugar and the nucleotide (Tvaroska, 2004). This is in agreement
with the proposed mechanism from crystal structure data from LgtC
(Persson et al., 2001). In this suggested mechanism, there is no need for a
catalytic acid or base in the protein. No intermediates, expected to be present
in a double displacement mechanism, have been captured in the structures
20
solved until today, further indicating the mechanism described above. The
active site is only providing the right environment for the reaction to take
place and is not actively taking part in the reaction. This would also explain
why there are no conserved conformational signatures in this enzyme family,
except for the binding of the sugar donor and the acceptor (Flint et al.,
2005).
In the structure of LgtC, the metal ion is coordinated with the nucleotide
and three amino acids in the protein, histidine 244, aspartate 103 and
aspartate 105, the DxD motif as in the inverting Gal-T1. The three amino
acids are conserved within the CAZy GT families (Persson et al., 2001). As
in the inverting mechanism the sugar donor binds first to the enzyme,
followed by movement of the flexible loops, forming the active site, making
it possible for the acceptor to bind (Persson et al., 2001). The metal ion is
believed to bind early in the reaction, and as in the inverting mechanism
acting as an acid catalyst (Persson et al., 2001). In the structures of the
inverting Gal-T1 enzyme, water has been found to be important in the
reaction mechanism, but is excluded once the acceptor binds (Ramakrishnan
et al., 2006; Snajdrova et al., 2004). The retaining enzyme group seems
more diverse than the inverting. In the latter, flexible loops are of importance
in the donor and acceptor binding, however in the retaining Kre2p/Mnt1p
enzyme (CAZy GT 15), this has not been observed. The movement of the
protein domains or loops during binding of donor and acceptor is minor in
this protein. On the other hand, a few side chains rotate upon binding of the
sugar donor. This small movement makes it possible once again for the
acceptor to bind (Lobsanov et al., 2004), but no large conformational
changes have been seen as for inverting GT-A.
GT-B
The GT-B fold family group of enzymes are the most diverse in sugar
acceptor usage when compared to GT-A. Vancomycin, LPS, glycolipids, and
proteins, and DNA are some of the products that this group of enzymes is
involved in producing (Hu and Walker, 2002). The GT-B structure is better
suited for large acceptor molecules compared to GT-A, due to the large cleft
in between the two domains (Fig. 4) (Breton et al., 2006). The GT-A fold
with its compact structure would have difficulties in binding large acceptor
molecules. The GT-B fold consists of two separate Rossmann fold domains
with a linker region and the catalytic site in between the two domains. Most
GT-B structures have a kink in the last C- terminal helix making it cross
over into the N-domain (Gibson et al., 2004; Hu and Walker, 2002). The Cterminal of proteins with this structure is well conserved and the N-terminal
21
domain is diverse, reflecting the diversity in used sugar acceptors used and
the conservation of used sugar nucleotides (Breton et al., 2006).
A sequence motif encoding a α/β/α subdomain has been identified in the
GT-B family (Hu and Walker, 2002), (Fig. 8) that consists of Cα4, Cβ5, and
Cα5 but with few other invariant residues. It has been found to be involved
in binding of the sugar donor (Hu and Walker, 2002), with the first helix
being involved in the sugar binding and the second in binding the pyranose.
The first helix that is enriched in positively charged amino acids is believed
to stabilise the binding of the negatively charged oxygen in the nucleotide
(Hu and Walker, 2002). The GT-B family lacks the otherwise common DxD
motif, but it is known that most GT-B enzymes are further activated by
divalent cations. No ions have however been found in the active site in the
crystal structures solved (Breton et al., 2006; Gibson et al., 2004; Hu et al.,
2003; Mulichak et al., 2001). The α/β/α subdomain described above, is
believed to have the same function as the cation in GT-A (Hu and Walker,
2002). In for exampel OtsA (CAZy GT 20), the phosphate groups of UDP
are in direct contact with arginine 262 and leucin 267 in the C-domain and a
conserved Gly,Gly,Leu motif in the N-domain instead of through an ion
(Gibson et al., 2004).
Inverting mechanism
The inverting mechanism is the most studied in the GT-B fold family as
in GT-A, but the exact reaction mechanism and conformational changes are
not as well established as for the GT-A group. The inverting reaction
mechanism is believed to go through an SN2 mechanism (Breton et al.,
2006). As in the GT-A fold family the enzymes undergo conformational
changes after the sugar donor has bound, however the GT-B fold type has
shorter loops involved in the donor and acceptor binding than the GT-A.
When the sugar donor binds to the N-domain in MurG, (CAZy GT 28) (Hu
et al., 2003), and GtfA (Mulichak et al., 2003) and GtfD (CAZy 1)
(Mulichak et al., 2004) a loop in this domain, the GGS loop undergoes
notable changes. It moves towards the α/β/α subdomain located in Cdomain of the protein and mediates contact between residues in both the N
and the C-domain. This conformational change has been found to be
essential for activity (Hu et al., 2003; Mulichak et al., 2003). The movement
of the N- and C-domain has also been observed in WaaG (CAZy GT 4)
(Martinez-Fleites et al., 2006). The movement of the GGS loop is believed
to be important for substrate binding. The conformational change moves an
aromatic amino acid, conserved in many GT-B proteins and works as a lid in
the binding pocket by stacking over the uracil ring of the donor sugar (Hu et
22
al., 2003; Mulichak et al., 2003; Mulichak et al., 2004). Many GT-B
enzymes have a conserved arginine or histidine that is part of the R/H/Ex7E
motif found in the GT-B family. These residues are located on the first helix
of the α/β/α described above and are believed to stabilises the leaving group
through electrostatic interactions or protonation (Ha et al., 2001; Mulichak et
al., 2003). The conserved glutamate in the R/H/EX7E motif is involved in
donor binding and not catalysis like the first residues. Mutational studies in
AceA (CAZy GT 4), a protein not yet crystallised have shown that the
glutamate residue in the EX7E motif is essential for activity (Fig. 8) (Abdian
et al., 2000).
A
B
C
NmMurG
EcMurG
AoGtfD
AoGtfB
EcWaaG
SpMGS
BbMGS
AlDGS
--CVEFITDMVSAYRDADLVICRAGALTIAELTAAGLGALLVPYPHAVDDHQTANHKVTEFIDDMAAAYAWADVVVCRSGALTVSEIAAAGLPALFVPFQHK-DRQQYWNA
-FAIDEVN-FQALFRRVAAVIHHGSAGTEHVATRAGVPQLVIPRNTD---QPYFA-FAIGEVN-HQVLFGRVAAVIHHGGAGTTHVAARAGAPQILLPQMAD---QPYYARNDVSELMAAADLLLHPAYQEAAGIVLLEAITAGLPVLTTAVCGYAHYIADANCGTVI
IAPSETALYYKAADFFISASTSETQGLTYLESLASGTPVIAHGNPYLNNLISDKMFG--IPWEEIYYYYKISDIFASLSKSEVYPMTVIEALTAGIPAILINDYIYKDVIKEGIN---VDGAVIKGAFSGADCVFFPSYEETEGIVVLEGLASKTPVVLRDIPVYYDWLFHK------
Figure 8. A; The structure of MurG, with the α/β/α subdomain in blue. B; The
α/β/α subdomain, with the conserved H/R/EX7E/V motif in green and the donor
substrate in green. C. An alignment of the α/β/α sub-domain. The protein names in
italic are retaining enzymes and the other four are inverting. The conserved R/H/E
residue, marked in red, is believed to stabilise the positive charge in the transition
state and the E/V residue, marked in red, involved in binding the hydroxyls on the
ribose sugar of the UDP group. (The figure A and B were made in PDB viewer and
PovRay)
The exact catalytic residues have not definitely been established by
capturing reaction intermediates for inverting GT-B enzymes. The only
residues at the right position from the donor sugar and the acceptor are serine
10 and aspartate 13 in GtfA that can form hydrogen bonds to the reactive
23
hydroxyl groups of the sugar donor (Mulichak et al., 2003). However,
mutation of the serine and aspartate in GtfA did not abolish activity
(Mulichak et al., 2001). The corresponding residues in WaaC (CAZy GT 9)
are aspartate 13 and 261, histidine 22 and aspartate 121 in UGT71G1 (CAZy
GT 1). The importance of these residues was confirmed by mutagenesis,
after which the activity of the enzymes was almost abolished (Grizot et al.,
2006; Shao et al., 2005). Aspartate 261 (WaaC) is conserved in several GTB inverting enzymes, but not the histidine required in the proposed
mechanism.
Retaining mechanism
The retaining reaction mechanism is believed to be similar to the GT-A
retaining group, being Substitution Nucleophilic internal like (SNi). It is
however not entirely understood due to the lack of both sugar donor and
acceptor in the solved crystal structures. As in the retaining family of GT-A,
no conserved amino acids that could act as a nucleophile have been found
(Martinez-Fleites et al., 2006). A conformational change occurring when the
acceptor and donor are bound has been observed in OtsA. As stated above
most interactions between the enzyme and the donor is in the C-domain of
the protein, the conformational change occurring in a loop is believed to
provide a cross talk between the two domains (Gibson et al., 2004). It is
however not clear if the sugar donor needs to bind to enzyme for the
acceptor to be able to bind, as in the GT-A fold family (Gibson et al., 2004).
In both OtsA and WaaG most interactions between the sugar in the donor
and the enzyme are with main chain amide groups and not side chains,
except for the caroxylate moiety recognising the sugar that is donated by
either a glutamate 281 (WaaG) or an aspartate (OtsA). This carboxylate is
found in many retaining GT-B enzymes and is believed to stabilise the
positive charge in the transition state (Martinez-Fleites et al., 2006). The
extensive use of backbone amides instead of side chains is consistent with
the flexibility reported for the donor (Gibson et al., 2002; Gibson et al.,
2004). OtsA homologues have been reported to be able to use different sugar
nucleotides. The donor sugar binding is expected to vary depending on the
specificity of the enzyme and the usage of different nucleotides.
24
Membrane binding
Many glycosyltransferases act on membrane bound substrates and to be
able to interact with the substrate, the enzyme needs to be membrane
associated. This is solved in mainly three ways; peripheral association with
the membrane by an amphipathic helix, that will snorkel into the membrane
to hide the hydrophobic amino acids; integral membrane proteins with one or
several transmembrane (TM) helices that will anchor the protein to the
membrane with globular domains outside the membrane, and finally
transmembrane proteins with several TM segments and that have the active
site in the loops connecting the TM segments. All three classes are found
among glycosyltransferases, with the first being most common in the GT-B
family (Leipold et al., 2007; van den Brink-van der Laan et al., 2003), and
with a few anchoring TM segments being most common in the GT-A fold
family. There are however exceptions with TM segments in the GT-B family
and amphipathic helices in the GT-A. The last class of membrane bound
enzymes, containing mainly TM segments, has been found only in the third
structural GT, family GT-C. However, only the membrane association
through amphipathic helices will be discussed here.
The binding of a protein to the membrane is dependent on the nature of
binding peptide segments and the lipid environment. Most studies have been
done on peptides, but in nature, the peptide is usually a part of a full length
protein. The binding domains of membrane associated proteins are often
amphipathic helices that are enriched in cationic amino acids (Ladokhin and
White, 2001). This interaction yields more or less strong associations with
the membrane, and the helix is often infiltrated into the membrane for a
more stable interaction (Seelig, 2004). The binding of the protein to the
membrane is dependent on electrostatic forces, van der Waals interactions,
and hydrogen bonds. The depth to which the helix penetrates into the
membrane varies between different proteins, and depends on
hydrophobic/hydrophilic equilibrium between the molecular groups, and
forces involved (Seelig, 2004). However, most amphipathic helices are
inserted to the glycerol backbone level of the membrane lipids. Not all
membrane proteins interact with the membrane in this way. There are also
proteins with specific lipid binding domains usually found in loops, binding
to specific single lipids (Cornell and Taneva, 2006), however the nature of
these will not be discussed here.
25
Enzymes acting on membrane bound substrates often use amphitropism,
to regulate the activity by changing the binding strength to the membrane.
Most amphitropic enzymes are active when membrane bound and inactive
when soluble. The activity is altered by binding soluble ligands or by an
alteration in the membrane lipid composition that will change the membrane
affinity (Cornell and Northwood, 2000). For example CTP:phosphocholine
cytidylyltransferase that is involved in the synthesis of PC is regulated by the
lipid environment and by phosphorylation of the protein (Cornell and
Northwood, 2000). The amphipathic helix that is involved in the regulation
has mostly acidic residues at the polar face, the interfacial zone consists of
mostly basic residues, and the non-polar face mostly of hydrophobic residues
that drive the membrane binding. The hydrophobic residues are believed to
be buried in the structure when the protein is soluble. The many basic
residues, in the interface region of the helix are believed to mediate the
binding to a negatively charged membrane, followed by the hydrophobic
interactions between the amino acid and the lipid (Cornell and Northwood,
2000). Hence, changes in the lipid content of the membrane will change the
strength of the binding. There are also membrane proteins that sense the
curvature of the membrane. These proteins have amphipathic helices that are
enriched in serine and threonine residues on the polar face of the helix (Drin
et al., 2007). These types of regulation are also believed to be used by GTs,
especially by GT enzymes using lipids as substrates. A. laidlawii MGS,
synthesising GlcDAG is activated by negatively charged lipids such as PG
(Karlsson et al., 1994), however the enzyme activity is not affected by the
curvature (Karlsson et al., 1997). The DGS enzyme from the same species,
synthesising GlcGlcDAG is regulated by both PG and sensing the curvature
of the membrane (Vikström et al., 1999). These two enzymes will work
together to keep the membrane properties constant, MGS is only active at
high PG levels in the membrane, making it less charged, and DGS will
regulate the production of nonbilayer-prone or bilayer-prone lipids, hence
GlcDAG or GlcGlcDAG. MGS is known to bind to the membrane through
an amphipathic helix (Lind et al., 2007).
26
Sequence analysis and protein classification
Traditionally the methods that have been used to compare and classify
proteins are mainly based on sequence alignments, where only amino acid
identity or similarity in the same position is accounted for (e.g. BLAST).
However, a small number of gaps are allowed. To classify and predict the
function and fold of new proteins with low sequence similarity to proteins of
known function other methods are needed. It is believed that protein
sequences behave in manners far from random, and the amino acid sequence
should therefor be organised to reflect the corresponding function and
structure (Rackovsky, 1998). Hence, the property patterns are believed to be
the same in proteins with the same (or similar) structure or function and the
property patterns are thought to be conserved even at low sequence
similarities. These thoughts are especially clear for proteins with repeated
motifs, e.g. TIM barrels (Rackovsky, 1998; Wold and Sjöström, 1998), but
should be valid for other protein families as well. Proteins with the same
function, but with different folds are still believed to have certain local
property patterns in the sequence that determine the function. With a
multivariate sequence analysis method, based on amino acid properties,
proteins with no sequence similarity can be grouped together and conserved
property patterns can be visualised within the proteins.
Multivariate data analysis
To be able to use a multivariate approach, the protein sequence needs to
be translated into numbers, variables. This is done by translating the amino
acid sequence into Z-scores describing different amino acid properties. The
scores used in this study are a combination of 29 different physico-chemical
variables calculated by principal component analysis, PCA (Hellberg et al.,
1987). The scores are experimentally determined e.g. NMR, HPLCretention, pKa, and logP and theoretical values such as Mw and van der
Waals volume. The three Z-scores used, named z1 to z3 describes
hydrophilicity, side chain bulk volume and polarisability/charge
respectively.
27
The periodicity of the amino acid properties in a protein is described by
calculating the auto-covariance and cross-covariance (ACC). The covariance
is analysed with different “window” sizes, i.e. lags where the amino acid
properties at different positions from each other, up to the decided
lag/window are calculated and an average of all positions along the sequence
is obtained. The difference between the two methods is that auto-covariance
is using the same property along the sequence and cross-covariance a
combination of different properties (Fig. 9). This method provides an
analyses of the relationship between close and more distant neighbouring
amino acids and not only amino acid identity and similarity. The obtained
data matrix is then analysed by a multivariate method described below.
Figure 9. The amino acid sequence is translated into Z-values that are used to
calculate the auto covariance and the cross covariance variables up to the decided
lag.
Previous studies
The above method has been used to successfully classify ABC
transporters in M. pneumoniae and other genomes, and signal peptides in E.
coli, Mycoplasma and other Gram-positive bacteria (Edman et al., 1999;
Edman, 2001), predict the localisation of proteins in Synechocystis (Rajalahti
et al., 2007), and to classify and identify sequence difference between
GPCRs (Lapinsh et al., 2002).
28
Multivariate projection methods
Principal component analysis
Multivariate data analysis can be used to analyse the ACC data. The
method has been developed to analyse the numerous amounts of research
data, obtained from many different techniques used today. The multivariate
data analysis programs is based on a so called projection method where a
swarm of points in a K-dimensional space is used, where K can be any
number greater the two. One dimension for each variable and one point for
each observation is projected down in one plane, a two dimensional space.
The coordinates of the points in the new hyperplane contain a compressed
representation of the observation in the original data table. The resulting
vectors, when compressing the original matrix, are scores (t-vectors) that
describe the relationship between the objects, and loadings that describe the
contribution of the variables to the score-vectors. The compression is done to
find underlying structures in the data set using a set of new latent variables,
principal components. The first principal component t1,p1 describes most of
the variation in the data set and the second (t2,p2) the second most and so
on. The two first principal components are orthologous to each other,
meaning that they are independent of each other. When reducing the data set,
it becomes easier to obtain an overview of the variation, and outliers in the
group can be identified. This compression is possible when the variables are
correlated to each other. When using this technique to separate and classify
protein groups it is believed that proteins with similar function or structure
have similar sequence periodicity as described above (Wold and Sjöström,
1998). To analyse the results, the t and p scores are plotted, where the t
scores are plotted to visualise objects with similar properties and the
loadings to search for the important variables. The loadings are later used to
find the relationship between different objects. However, it should be noted
that the new principal components are a combinations of all variables and
can usually not be assigned to a specific one.
To simplify the projection method, one can think about it as a looking at
a shadow of a 3D object. When holding a pair of scissors in front of a light,
29
holding it like in Fig. 10, A the shadow will describe the object well,
however a 45º rotation will make it harder and turning it 45º further will
make it impossible to identify the object. Hence, the direction in space that is
chosen when projecting the 3D object into a 2D plane is of importance. The
direction that has the largest variation should be chosen. In this example, 3
variables (3-dimensions) are projected down to new principal components.
The first two dimensions/variables, describe the object best and the width is
not needed.
Figure 10. Projection of a K dimensional object onto a 2D plane, here a pair of
scissors (a 3D object) is projected on to a sheet of paper by the help of a light source.
The image, the shadow, represents two dimensions of the 3D object. The object is
rotated 45° in two steps to generate different shadows of the same object. The same
method is used with multivariate analysis programs. A projection of a 3 dimensional
problem that is projected down into two dimensions and the resulting t vectors are
plotted as a score plot.
Partial least squares projection to latent structures
Partial least squares projection to latent structures (PLS), is used to find
relationships between X-matrix observations and a response matrix Y. The Y
matrix contains additional descriptions of the samples in X. PLSdiscriminator analysis, PLS-DA is a special case of PLS, used to find class
characteristics within objects in a X matrix. In this special case, the Y-matrix
is composed of dummy variables instead of e.g. biological activity. Each
member of a group has the value 1 and non-members 0. PLS-DA uses the
fact that objects belonging to the same class are expected to have common
features and should behave similarly in the score plot. The method also gives
the opportunity to predict new unclassified sequences. To evaluate the
complexity of a PLS model, cross validation is preformed, objects are
randomly taken out one by one during the calculation, and are predicted into
30
the model and the Y-values calculated (Wold 1978). This is an internal
validation method, where objects that create the model are used to validate
it. The validation is repeated until all objects have been left out once. The
cross validation is done to calculate the Q2 value. Hence, the ability of the
model to predict the object back to the same place as it had in the plot before
it was taken out, is calculated. The Q2 value describes how much of the Y
matrix can be predicted by the model, hence if all objects that are taken out
and predicted back again are predicted to the same position a Q2 score of 1 is
obtained. A Q2 larger than 0.1 corresponds to 95% significance. As in PCA a
hyper plane is calculated from the original X matrix that gives t-scores, and
the Y variables u-scores. These scores can be plotted to analyse the data and
w*c plots are made to evaluate the correlation between the X and the Y
variables. Hence, these plots are used to identify variables that are important
for the separation of the groups. With PLS-DA it is possible to predict
whether a object belongs to a class or not, by looking at the predicted class
value. A predicted value greater then 1 suggests that the specific object
belongs to the class and a value closer to 1 that it doesn’t. By using PLS-DA
for protein family classification, a protein with an unknown function can be
predicted by using the described method to investigate if it has the same
property patterns as other members of the group.
31
Summary of papers
Paper I
The aim of this paper was to use a new approach based on multivariate
data analyses and ACC to separate and classify GTs. With this method, not
only similarity in specific positions is accounted for, but also property
patterns along the sequence. This method would in theory find relationship
between sequences that share no similarity according to BLAST, but that
share the same fold or function. This would be possible since it is believed
that sequences behave far from random and that the protein sequence reflects
both the structure and the function. A data set consisting of GTs classified by
CAZy, with an established function and from families with an established
fold was constructed. The data set was used to divide fold families, and
reaction mechanism, and to predict the function of proteins with unknown
function.
We found that it was possible to separate the three different fold groups
from each other with this method with high scores, a Q(cum)2 value of 0.730
was obtained. It was also possible to separate the inverting from retaining
reaction mechanism within the GT-A and GT-B fold, with a score of
Q(cum)2 values of 0.702 and 0.562 respectively. The GT-C family was not
included in these experiments due the occurrence of only one reaction
mechanism within the family. The fold classification showed that the
occurrence of TM segments within the GTs was easily recognised. GT-A
and GT-B proteins with TMs moved closer to the GT-C family, consisting of
several TM segments and small cytoplasmic domains. Divisions with only
two families were also done, yielding high scores.
The MVD method also gives information about the separating
parameters in the data set. The parameters are visualised on a w*c plots.
Only the separating parameters for the reaction mechanism division within
each fold family were plotted. The important variables for the GT-A family
were (in rank) z(2)1z(3)13 (e.g. size in the first position and polarisability 13
amino acids downstream), z(2)1z(2)5, z(1)1z(1)18, z(2)1z(2)10, z(2)1z(1)4,
and z(1)1z(1)14. The shorter distances variables were size (z(2)) and
hydrophilicity/hydrophobicity (z(1)) patterns, e.g. positions 1 to 4 and 1 to 5.
32
This most likely corresponds to amino acids on the same side of a helix.
Alignments established by pair wise aligning sequences with an established
structure in the following CAZy families, for the retaining enzymes GT6,
GT 8, and for the inverting group GT7, GT43, GT2, and GT13 were
achieved. A stretch of 84 amino acids in the retaining group could
successfully be aligned. The top variables were searched for in the sequences
using the alignment, only the z(1)1z(1)18 variable could easily be visualised.
However, a comparison between the two reaction mechanisms could not be
done since no corresponding alignment for the inverting group could be
established.
When analysing the GT-B fold group the most important variables were
z(2)1z(2)5, z(1)1z(1)16, z(2)1z(2)8, z(1)1z(3)14, z(3)1z(1)11, and
z(2)1z(2)2 for the separation of the retaining and inverting sequences. The
GT-B fold group was also analysed by the same method as described above.
Here, sequences from the GT1 and GT28 families were aligned. The variable
z(1)1z(3)16 was marked out at the sequence level by its z products and
should be positively correlated to the inverting enzyme group. In the
alignment 18 positive and 7 negative pairs were found within the inverting
group, and 3 positive and 7 negative pairs were found for the retaining
families. This confirms the importance of the z(1)1z(3)16 variable. The
alignment was found to coincide with the UDP-binding site (se fig 11), and
also a structural correlation could be made.
Figure 11 Alignment (ClusalW) of selected GTs with the GT-B fold. The
z(1)1z(3)16 variable pairs indicated in the alignment. p, first position in positive
variable pairs, (+) the second position; n, first position in a negative variable pair; (-)
the second position. Conserved amino acids are marked with * and amino acids with
the same properties, (:) and similar properties (.) as marked in Clustalw.
The method could also be used to predict the fold and reaction
mechanism of GTs from E. coli and Synechocystis. The proteins used were
all taken from the CAZy database, however the fold was only known for a
few of the included CAZy families, hence a few proteins. To evaluate the
predictions, multiple fold predictions, made by the MetaServer
(bioinfo.pl/meta) were used. When predicting the fold, with all three fold
33
groups in the reference set, 82% of the E. coli GTs were predicted to have
the right fold, and excluding the GT-C group increased the prediction
correctness to 89%. The reaction mechanism predictions also gave very good
results, with 100% of the E. coli and 86% of the Synechocystis set within the
GT-A fold group, and 60% versus 80% for the GT-B group, being correctly
predicted.
In conclusion, the multivariate data analysis approach could successfully
be used to separate different fold groups from each other and reaction
mechanism. The separating parameters were potentially found in the protein
sequences, however further mutagenic studies need to be made. The method
could also be used predict the fold and function of GTs from E. coli and
Synechocystis and M. pneumoniae.
Paper II
Enzymes acting on membrane bound substrates are known to be
regulated by factors such as lipid charge and the curvature stress. This
indicates that the enzymes must be attached to the membrane in order to
sense these changes, however the binding is not well characterised among
glycosyltransferases of the GT-B fold. When comparing the fold of
membrane bound GTs from the GT-B family with soluble enzymes,
structural differences are difficult to detect. The aim of this paper was to
identify the binding region of these GTs and characterise it.
We used as in the previous paper a multivariate data analysis approach
to detect differences between soluble and membrane bound GTs with the
GT-B fold. The score plot, from the separation of the two different groups
reveales two well separated clusters, and the model has 3 significant PLS
components and a Q(cum)2 value for the model of 0.52. The variables were
in order of importance, pI of N-domain, pI of full enzyme, z21*z39,
z31*z39, z31*z13 and z31*z33. The two first variables were substantially
stronger than all the following ones. To summarise these variables it can be
said that the differences between the two groups are seen in the charge and
amphipathic patterns. Membrane bound GTs have higher pI in the Nterminal domain and amphipathic patterns that we believe are localised in
helices on the surface. These characteristics are believed to mediate the
binding of the protein to the membrane surface, with the positively charged
surface interacting with the negatively charged membrane lipids. The
hydrophobic amino acids in the amphipathic helix will be buried into the
lipid bilayer. Using in silico mutated GTs, where whole domains or just
34
helices with amphipathic characteristics were swapped, showed that the Ndomains had a large impact on the localisation of the protein in its group, but
also the helix. This indicates that the pI of the domain and the amphipathic
helix only about 20 aa long, absent in soluble GTs, are important for the
membrane binding (se fig 12 for localisation within the proteins).
A
B
Figure 12. A; MurG showing the amphipathic helix, involved in membrane binding
in green, B; Fold model of AlMGS with the peptide in green. The figure was made
in PDB wiever and PovRay.
The features of the amphipathic helix found in alMGS, a well studied
GT from A. laidlawii, and that we believe mediates the binding of the
proteins to the bilayer were further investigated by CD, Biacore, and NMR
studies. The experimental study showed that the helix, S65-L87 adopted a αhelix conformation in several membrane mimicking environments, however
not in water. The ability to bind to a lipid surface, investigated by Biacore
studies revealed that the peptide had the same binding affinities as the full
size protein. The usage of spin-labelled phospholipids showed that the Nterminal part of the peptide is interacting with the lipids in the bilayer, with
zwitterionic bicells influencing the binding more than partly negatively
charged bicells. The C-terminus of the peptide was not affected. This
indicates that the charged C-terminus is situated outside the membrane,
while the N-terminus is penetrating into the bilayer. These studies show that
the peptide probably undergoes conformational changes when the full size
protein reaches the membrane, due to the fact that lipids induce the helical
fold. The N-terminal part of the helix will when reaching the membrane fold
and go in into the lipid bilayer and anchoring the protein to the surface. The
cluster of charges found in the C-terminal will further stabilise the binding
by interacting with the charged lipids.
35
Paper III
M. pneumoniae is one of the smallest organisms capable of autonomous
growth. It has only 688 ORF with 25% of the ORFs having an unknown
function. Its ability to synthesise lipids is surprisingly large considering the
down sized-genome. In this paper, we analysed the ability to make
glycolipids and cloned three potential GTs.
We found that the ability to synthesise glycolipids is greater than
previously known. In vivo labelling studies showed that this bacterium can
make at least three glycolipids, five phosphoglycolipids, and six
phospholipids, using glucose and phosphate as the labelled source. In vitro
studies using solubilsed M. pneumoniae cells showed that the bacterium has
a large ability to use a variety of different acceptors as well as both UDPglucose and UDP-galactose as sugar donors, although the latter was
preferred. The opposite was seen in vivo, which is due to the epimerase that
epimerise UDP-glucose to UDP-galactose, which is preferred by the GTs.
The bacterium has the ability to use diacylglycerol, αGalDAG, and
βGalDAG as substrates with UDP-glucose as the donor and diacylglycerol,
αGalDAG, and βGalDAG as well as αGlcDAG, αGlcαGlcDAG, βGlcDAG
with UDP-galactose as donor. Up to three galactose residues could be
attached to a diacylglycerol, and one to two residues to the glycolipids. Most
reactions were stimulated by PG, however one sugar could be added to a
diacylglycerol, but the elongation stopped in the absence of PG. Ceramide,
was used in the assays to investigate the ability to use eukaryotic lipids as
substrates. A similar pattern as for diacylglycerol-based lipids was seen, and
once again, PG stimulated the reaction. When combining these results, we
suspected that all these glycolipids could not be produced by three GTs. To
investigate if the bacteria had more GTs than the three proposed GTs, GTs
with an established function from Gram positive bacteria were used as
search probes against the M.pnumoniae genome. However, no further GTs
genes where found.
The three proposed GTs were cloned and solubilised E. coli expressing
the M. pneumoniae GTs were used in assays using the same substrates as
with solubilsed M. pneumoniae cells. Activity were only seen with MPN483
expressing cells. The cloned MPN483 enzyme showed the largest activity
with β-GalDAG as the acceptor and UDP-galactose as the donor in vitro.
This lipid was used as a reference to compare the activity with other
substrates. Substituting UDP-galactose for UDP-glucose gave only 30% of
the activity. Other substrates yielding a high activity were DAG, βGalDAG,
αGalβGalDAG, βGalCer with UDP-galactose, and DAG, and βGalDAG
with UDP-glucose, respectively. These results indicate that the enzyme
36
prefers a terminal galactose as the acceptor sugar to be able to extend the
sugar chain. PG was once again needed as an activator in all reactions. A
lipid product was also purified from E.coli cells expressing MPN483 and
structurally determined. The product were found to be a βGlcβGalDAG. The
phospholipid could be replaced by cardiolipin, that also is anionic, but not by
the zwitterionic PC or PE. The charge thus seems important. The lipid
environment thus has effects on the GT enzyme, with both the head group
elongation and the total amount being affected.
Glycolipids are known to be important in the infection of M. pneumoniae in
humans and the immune system is known to react to these. We used both
purified lipids from M. pneumoniae and pure glycolipids with established
structures, to investigate the immunological responses towards glycolipids
during a M. pneumoniae infection. ELISA studies of purified M. pneumoniae
lipids showed a highly increased response with an IgM fraction from patient
sera, compared to the IgM negative control for the three different
glycolipids. The di-hexoseDAG variant both purified from M. pneumoniae
and E.coli (synthesised by MPN483) gave the highest response. The
MPN483 enzyme was also used to synthesise different glycolipids to be used
in the ELISA test. These studies showed that the MPN483 produced
βGalCer and especially βGalDAG were reactive towards the positive IgM
serum. None of the above mentioned lipids gave any negative results.
Recently it has been shown that bacterial lipids, such as αGalDAG from
B.burgdorferi as well as αGalCer and βGalCer bind to the CD1d receptor,
presented on antigen-presenting cells (Kinjo et al., 2006). This binding
stimulates iNKT through TCR and further activate factors in the host
defence against bacteria lacking lipopolysaccharide (LPS) (Porcelli and
Modlin, 1999).
In conclusion, glycolipids do not constitute the major membrane lipid
fraction in M. pneumoniae, but their important characteristics are essential to
the cell. The glycolipids are also important in the recognition of the bacteria
by the host immune system. The promiscuous and processive character of
the MPN483-enzyme probably yields bacterial-human lipid hybrids that
might be important for an auto-immune process.
Paper IV
Many membrane associated mechanisms are belived to be regulated by
the curvature stress in the membrane. The proportion of bilayer/nonbilayerprone lipids in the membrane regulates this. The E.coli AD93 strain, lacking
the nonbilayer-prone lipid PE, was used to investigate the ability of different
37
glycolipids to restore cell growth, cell division, stress response, and
membrane permeability malfunctioning here. The membrane in wild type
E.coli normally contains 75 mole% PE, and the depletion of this lipid has a
large effect on many vital cell functions. The membrane of AD93 cells have
a very high level, about 85 mole % of the negatively charged PG and the
lipid mutant needs divalent cation like Mg2+ to survive, by neutrally a
negatively charged membrane, which is critical for the cell to survive. The
neutral glycolipid, αGlcDAG also nonbilayer-prone, (NB-prone) can restore
the cell growth of AD93, however the membrane permeability was not fully
restored (Wikström et al., 2004). This lipid, constituting about 50% of the
membrane lipids, is believed to dilute the high surface charge and restore the
environment of many membrane processes. However, the shape of this lipid
is also similar to PE.
To further investigate what properties can restore cellular processes in the
PE mutant, surface charge or lipid shape, four new GTs were introduced into
this E.coli strain. These were DGS from A. laidlawii making
αGlcαGlcDAG, two Arabidopsis thaliana GTs making βGalDAG,
αGalβGalDAG, and a processive GT, MPN483 from M. pneumoniae
making GalDAG, βGlcβGalDAG, and GalGalGalDAG. The disugar lipids
are bilayer-prone in nature due to their larger uncharged head groups and as
the mono variants also uncharged. The glycolipid content in these strains
reached up to 60 mol%, however the M. pneumoniae GT could only produce
up to 28 mol% glycolipid in total. The βGlcβGalDAG lipid was the major
glycolipid in this strain. These results reflect the maximum levels of
glycolipid produced by the different organisms. The strain producing the
bilayer-prone αGlcαGlcDAG lipid was here found to behave like wt E. coli
in most aspects, and αGlcDAG and βGalDAG performed almost equally
well. However, the βGalβGalDAG and MPN483 strains hardly survived.
Analysis of the acyl chain composition revealed that all strains had an
increased level of unsaturated chains, with the GalGalDAG and MPN483
glycolipid mix having the highest levels. The average chain length was
increased in all but the GlcDAG strains. This is probably a way to
compensate for loss of NB-prone lipids, increasing the curvature. All
glycolipid strains except MPN483, have a major fraction of noncharged
glycolipids, and reduced anionic ones compared to the AD93 host. The wt,
GlcDAG and GalDAG strains have high levels of NB-prone lipids, however
the AD93, GlcGlcDAG and the GalGalDAG strains lack these totally. The
MPN483 clone has low levels of NB-prone lipids, since the GalDAG lipids
is a very minor species, and the bilayer-forming GlcGalDAG and tri-sugarDAG dominate the glycolipids. All glycolipid strains except MPN483, have
a major fraction of noncharged glycolipids. The increased acyl chain
unsaturation and length, increase the curvature stress and potentially
38
compensate for properties lost, and not fully restored by the foreign lipids.
The saturation and chain length, thus seem to be the major factor in vivo for
E. coli to control the curvature stress of the lipid bilayer. The levels of the
NB-prone CL is increased in all glycolipid strains, and was found to be
highest in the bilayer-prone GlcGlcDAG strain and lowest for the
monosugar-DAGs. The observed increased levels of CL is probably due to
compensation for the loss of nonbilayer-prone PE, hence to regulate the
membrane curvature by other NB-prone lipids. The βGalβGalDAG strain
also requires Mg2+ to grow. Hence, curvature may increase by a large
membrane fraction of CL plus Mg2+, that is making CL NB-prone.
The lateral pressure between the acyl-chains was measured in all glycolipid
strain to especially investigate the difference observed between the
GlcGlcDAG and GalGalDAG lipids. It was seen that GalGalDAG has a
larger lateral headgroup area, that changes the physical state of the
membrane towards less nonbilayer-prone (less curvature) compared to
GlcGlcDAG. This change seems to be compensate for E. coli by increased
CL levels. This decrease of lateral pressure in the membrane decreases and
prevents activities of several membrane functions. Smaller headgroups of
lipids, most likely in combination with dilution of high surface concentration
of anionic lipid charges, seems to bring advantages to a number of
membrane associated proteins in E. coli. This was tested by using the
transmembrane transporter LacY, known to have an altered topology in the
absence of PE, as a marker. The passive downhill transport in this channel
was restored in the presence of both GlcDAG (Wikström et al., 2004) and
GlcGlcDAG, however only active transport was supported in the GlcDAG
clone (Wikström et al., 2004). The topology of LacY was correct in both
clones, still the uphill transport was absent in the GlcGlcDAG clone. The
anionic phospholipid and non-charged glycolipid levels are similar in both
clones, but the GlcGlcDAG one is essentially lacking nonbilayer-prone
lipids species and has a substantially lower chain ordering due to its large
headgroup. This will substantially lower the curvature stress and thus has an
affect on the function of the LacY. Accordingly, LacY needs a certain
curvature stress to function correctly.
39
Concluding remarks
The lipid bilayer has several important functions as described earlier. The
membrane protects and shields the organism from its surrounding and
provides the right properties for many enzymes and membrane proteins. It
has been found that several enzymes are regulated by the membrane
curvature stress or charge, and lipids are also closely associated with many
membrane proteins e.g. bacteriorhodopsin (Luecke et al., 1999). The
membrane is also an immediate contact layer, e.g. in bacteria lacking LPS,
where the membrane is the first barrier against the host immune system.
I have in this thesis shown that a multivariate method can be used to
classify the different glycosyltransferase folds, reaction mechanisms within
each fold family (paper I), and soluble and membrane bound GTs of the GTB type (paper II). The multivariate sequence analysis method used here
(paper I), could conveniently separate the three different fold types from
each other, and divide the two different reaction mechanisms within the A
and B structural fold groups. The method could also be used to predict the
fold and reaction mechanism within E.coli and Synechocystis GTs in the
CAZy database. This indicates that ORFs with unknown function and that
share no sequence homology with known glycosyltransferases could
potentially be identified with this method. When analysing the MPN483
enzyme with the above method, it was classified as a GT-A enzyme
performing an inverting reaction mechanism. This method is easy to use and
a powerful bioinformatic tool.
Amphipathic helices was found to be important for the group separation
of membrane-bound and soluble GTs (paper II), analysed by swapping these
helixes from alMGS and E.coli MurG to a corresponding pair of soluble
enzymes. The pI of the N-domain was also found to be important. In this
work we have also shown that an amphipathic helix, here represented by a
synthetic peptide, probably is unfolded until it reaches the membrane where
it undergoes conformational changes. The helix will also regulate the
enzymatic activity by binding to charged lipids, hence as the lipid content of
the membrane changes it will affect the binding and also the activity. It was
previously known that the MGS activity is regulated by charged lipids. This
is believed to be mediated through the cluster of charges found in the Cterminal of the peptide (in enzyme the N-domain). These properties are most
40
likely found in other types of membrane acting enzymes, such as both GT-A
and GT-B glycosyltransferases. The activity of the processive MPN483 is
regulated by the PG and CL content of the micelle (paper III), indicating that
the enzyme activity is regulated by a similar bilayer properties. It also has
predicted amphipathic helixes, however this needs to be further investigated.
In paper III, I have shown that the MPN483 enzyme from M. pneumoniae
is both processive and promiscuous. The enzyme has the ability to use both
ceramide, an eukaryotic lipid and DAG as precursors in the synthesis of
glycolipids. This ability can be a strategy to make itself look more like the
human host cell that the bacterium is attached to, or just a way to be flexible
in the usage of different substrates (to take what one gets), which is probably
beneficial for a very small cell. The use of both DAG and ceramide would
normally be handled by having several enzymes, however having a
promiscuous enzymes will solve this problem and also be energy saving.
During evolution this bacterium has lost many enzymes, e.g. several GTs
and this loss have been covered by developing of enzymes like MPN483.
This GT has also been found to be essential, emphasising the importance of
processive enzymes and glycolipids. The ability of neutral glycolipids to
replace the neutral PE in the PE-depleted AD93 E. coli strain was analysed
in paper IV. In this work, it was shown that GlcDAG and GalDAG could
replace this lipid and restore many cellular functions, however di-sugar
variants were less suited for this. The mono-sugar variants are all
nonbilayer-prone due to its smaller sugar headgroups and the larger DAG
backbone, this is a similar structural feature to the depleted PE, and
substantially affects the curvature stress, compared to the AD93 strain.
Disugar lipid molecules are instead bilayer-prone, making them less suitable
for this. This was clearly demonstrated with LacY, where the topology of the
protein were correct with both GlcDAG and GlcGlcDAG, but the activity
much reduced in the latter strain. This channel thus needs a certain curvature
stress to function correctly. These lipid molecules can not replace PE in its
cellular function, showing the importance of different lipid molecules. The
MPN483 enzyme preferentially making GlcGalDAG or GalGalDAG is
essential in M pneumoniae showing that the variety of different sugar
molecules are important. The produced glycolipids in M. pneumoniae are
however a target for the immune system and the lipids are recognised by
(early) antibodies during an infection. The exact mechanism for this is
unclear today. Further studies needs to be made to analyse the mechanism by
which this occur. The exact function of glycolipids in cellular processes also
needs to be analysed, as well as mutational studies to investigate the
importance of the separating parameters found in the different multivariate
studies.
41
Varför fett?
Sammanfattning på svenska
Alla bakterier, djur och växter består av celler, och alla celler har minst ett
membran. Detta är cellens väggar och det gör det möjligt för cellen att ha
olika miljö på dess ut- och insida, vilket är en mycket viktig funktion för att
möjliggöra uppkomsten av liv. Väggarna kring cellen består av ca 50% fett
(lipider) och 50% protein, sk äggviteämnen, där fettet är själva
byggmaterialet och proteinerna cellens dörrar och fönster som används för
att transportera in och ut ämnen som cellen behöver. Fett består av ett
vattenälskande huvud (hydrofil) och icke vattenälskande svansar
(hydrofoba), dessa organiserar sig så att svansarna lägger sig mot varandra
och med huvudena utåt (se figuren), för att skyla svansarna från vatten. Det
är därför fett lägger sig som ringar på vatten och inte löser sig.
Det finns många olika typer av lipider, laddade, oladdade, phospholipider
och sockerlipider, koniska och raka, där alla har en speciell funktion. Jag har
i denna avhandling bla sett att formen på lipiden påverkar funktionen av
proteinerna som är insatta i membranet. Om membranet innehåller fel typ av
lipid fungerar inte proteinet som det ska. I papper 4 har vi sett att i ett
membran med för lite koniska lipider blir för hårt packat och vissa proteiner
förlorar sin funktion. Det är alltså viktigt att membranet innehåller rätt sorts
fett. Det är inte bara proteiner som är insatta i membranet som påverkas av
lipidinnehållet, utan även proteiner som binder mot ytan påverkas. I papper 2
har jag undersökt hur dessa proteiner binder mot ytan och hur denna
bindning påverkas av olika lipider.
Fett har tidigare bara ansett som ett byggmaterial för cellen som utan
någon större funktion, men betydelsen av olika fettstrukturer börjar nu
42
klarna. Forskar värden ansåg tidigare att endast proteiner kändes igen av
kroppens försvar, men även här har detta visats vara fel. Även vissa typer av
lipider känns igen av kroppens försvar. Detta har visat sig vara av stor
betydelse vid autoimmuna sjukdomar, där kroppen börjar brytas ned, istället
för en inkräktare. Detta tros bero på att både bakterier, inkräktare och
kroppsegna celler består av liknande fett molekyler och där försvaret
förvirras av dess likheter. Det finns även bakterier som tros använda detta
som ett tillvägagångssätt att gömma sig i kroppen. De använder fett som är
så pass likt mänskliga varianter att försvaret inte ska kunna se någon
skillnad. När inkräktaren väl upptäcks bryts även kroppens egna celler ner.
Mycoplasma pneumoniae, är en av de minsta bakterierna som hittats på vår
jord och som är en vanlig orsak till lunginflammation tros använda detta
tillväga gångs sätt för att dels kunna undvika att bli upptäckt, men också för
att spara energi. Denna bakterie (papper 3) har utvecklat en förmåga att
använda fettet som finns i sin omgivning och sedan modifiera dessa, för att
själv behöva tillverka fettmolekyler. Detta får till följd att den lättare kan
interagera med mänskliga celler, men det kan även orsaka att den egna
kroppen bryts ner efter en infektion.
Fett är sammanfattningsvis väldigt viktigt för oss, utan fett med rätt form
och innehåll fungerar inte våra proteinmaskiner.
43
Acknowledgment
I would like to express my gratitude to all the people that is making DBB
such a great place to work at. Especially I would like to thank:
Åke, min handledare, tack för att din entusiasm för forskning och att du gett
mig så mycket frihet under alla dess år. Tack för att du överraskade mig med
att flytta till Stockholm när jag trodde att jag skulle få tillbringa ytterligare
många år framöver i Umeå.
Mikael Sjöström, för att du hjälpt mig att förstå ACC, PLS och PLS-DA.
Utan din hjälp hade papper I aldrig blivit till.
Stefan, för att din dörr alltid är öppen och för att du tar dig tid. Du är bra
boss för oss doktorander.
Anki, Kicki, Ann, Charlotta och Sofia för att ni alltid med ett leende hjälpt
mig med allt det praktiska.
Malin W, för att du fått timmarna på labb att gå så mycket fortare. Vi har
lärt känna varandra väldigt väl under alla dessa år, och gått igenom
graviditeter och småbarns år tillsammans och mycket annat. Efter vår tid på
plan 5 kan alla där upp allt om spädbarn och vet vilka underbarn vi har!!!
Tuulia, för att du är en så fantastik person! För att du sprider glädje omkring
dig och förlängt mitt liv med många år av alla goda skratt.
Linda, som helt plötsligt klev in på kontoret och frågade om du inte kunde
få göra D-kurs projekt hos mig. Utan din hjälp hade jag aldrig blivit klar med
M.p lipid pappret i tid.
Alex, for all good laughs at lunch time and for interesting conversations in
the office.
Amélie, for helping me with difficult clones. I value your deep knowledge
very high, you always had an answer for everything.
44
Maria E, för att du introducerade fler dimensioner i mitt liv. Stefan B, och
Patrik för tuffa innebandy matcher. Lu, for all great discussions in the lab.
Hanna, för allt morgonkaffe du kokat det sista året, trevliga (korta) lunch
diskussioner och gott samarbete i doktorandrådet. Kajsa, för den värme du
sprider på plan 5.
Det gamla lunch och fika gänget på plan 4, ni förgyllde tillvaron med många
goda skratt och för att ni alltid lyssnat och peppat.
Elin, för att du är en sådan fantastiks vän. Det finns ingen som känner mig så
väl, och alltid förstår vad jag menar. För att du alltid finns där. För alla
trevliga middagar på ”Haga deli”. Vi fixade det! Jag hoppas att vi hinner
träffas oftare nu när avhandlingarna är klara (nästan).
Karin, Lovisa och Erika, mitt glada tjejgäng. Vi är spridda från norr till
söder, öst till väst men vänskapen består. Jag tänker ofta på de fantastiska
studentåren vi tillbringade tillsammans i Umeå. Tack för att ni alltid finns
där, även om det ibland går lång tid mellan de tillfällen då vi ses.
Mormor och Morfar, för alla trevliga år i Umeå och underbara somrar.
Morfar för att du introducerat jakt för mig, vilket gett oss många fantastiska
timmar i skogen. Mormor för all god mat och fantastiska bullar.
Anna, min underbara syster, för att du alltid finns där och peppar mig och
för att du får mig att tänka på annat än jobb och familj. Det ska bli skönt att
få hem dig igen. Gunnar för ditt intresse för forskning, och för att du som
samhällsvetare försöker förstå lipider.
Mamma och Pappa, utan allt ert stöd hade denna avhandling aldrig blivit
skriven. Tack för att ni alltid trott på mig, uppmuntrat mig och för att ni
alltid finns där. Vad vore jag utan er?
Mina fantastiska tjejer Emmy och Thyra. Emmy för dina oerhörda värme
och den glädje du ger mig, att resonera om varför saker är som det är en
underbar utmaning. Thyra för att du alltid är så glad. Ni har gett livet en ny
mening!!!
Göran, du är mitt allt!!! Jag älskar dig innerligt. Nu ska vi äntligen få mer
tid för varandra!!!
45
References
.
Abdian, P.L., Lellouch, A.C., Gautier, C., Ielpi, L., and Geremia, R.A.
(2000) Identification of essential amino acids in the bacterial alpha mannosyltransferase aceA. J Biol Chem 275: 40568-40575.
Ang, C.W., Jacobs, B.C., Brandenburg, A.H., Laman, J.D., van_der_Meche,
F.G., Osterhaus, A.D., and van_Doorn, P.A. (2000) Cross-reactive
antibodies against GM2 and CMV-infected fibroblasts in GuillainBarré syndrome. Neurology 54: 1453-1458.
Baseman, J.B., Lange, M., Criscimagna, N.L., Giron, J.A., and Thomas, C.
(1995) Interplay between mycoplasmas and host target cells. Microb
Pathog 19: 105-116.
Baseman, J.B., and Tully, J.G. (1997) Mycoplasmas: sophisticated,
reemerging, and burdened by their notoriety. Emerg Infect Dis 3: 2132.
Berg, S., and Wieslander, Å. (1997) Purification of a phosphatase which
hydrolyzes phosphatidic acid, a key intermediate in glucolipid
synthesis in Acholeplasma laidlawii A membranes. Biochim Biophys
Acta 1330: 225-232.
Boggs, J.M. (1987) Lipid intermolecular hydrogen bonding: influence on
structural organization and membrane function. Biochim Biophys
Acta 906: 353-404.
Boix, E., Swaminathan, G.J., Zhang, Y., Natesh, R., Brew, K., and Acharya,
K.R. (2001) Structure of UDP complex of UDP-galactose:betagalactoside-alpha -1,3-galactosyltransferase at 1.53-Å resolution
reveals a conformational change in the catalytically important C
terminus. J Biol Chem 276: 48608-48614.
Borg, N.A., Wun, K.S., Kjer-Nielsen, L., Wilce, M.C., Pellicci, D.G., Koh,
R., Besra, G.S., Bharadwaj, M., Godfrey, D.I., McCluskey, J., and
Rossjohn, J. (2007) CD1d-lipid-antigen recognition by the semiinvariant NKT T-cell receptor. Nature 448: 44-49.
Bourne, Y., and Henrissat, B. (2001) Glycoside hydrolases and
glycosyltransferases: families and functional modules. Curr Opin
Struct Biol 11: 593-600.
Breton, C., Mucha, J., and Jeanneau, C. (2001) Structural and functional
features of glycosyltransferases. Biochimie 83: 713-718.
46
Breton, C., Snajdrova, L., Jeanneau, C., Koca, J., and Imberty, A. (2006)
Structures and mechanisms of glycosyltransferases. Glycobiology
16: 29R-37R.
Brigl, M., van den Elzen, P., Chen, X., Meyers, J.H., Wu, D., Wong, C.H.,
Reddington, F., Illarianov, P.A., Besra, G.S., Brenner, M.B., and
Gumperz, J.E. (2006) Conserved and heterogeneous lipid antigen
specificities of CD1d-restricted NKT cell receptors. J Immunol 176:
3625-3634.
Buehner, M., Ford, G.C., Moras, D., Olsen, K.W., and Rossman, M.G.
(1973) D-glyceraldehyde-3-phosphate dehydrogenase: threedimensional structure and evolutionary significance. Proc Natl Acad
Sci U S A 70: 3052-3054.
Campbell, J.A., Davies, G.J., Bulone, V., and Henrissat, B. (1997) A
classification of nucleotide-diphospho-sugar glycosyltransferases
based on amino acid sequence similarities. Biochem. J. 326: 929939.
Chambaud, I., Wroblewski, H., and Blanchard, A. (1999) Interactions
between mycoplasma lipoproteins and the host immune system.
Trend Microbiol 7: 493-499.
Chambaud, I., Heilig, R., Ferris, S., Barbe, V., Samson, D., Galisson, F.,
Moszer, I., Dybvig, K., Wroblewski, H., Viari, A., Rocha, E.P., and
Blanchard, A. (2001) The complete genome sequence of the murine
respiratory pathogen Mycoplasma pulmonis. Nucl Acid Res 29:
2145-2153.
Charnock, S.J., and Davies, G.J. (1999) Structure of the nucleotidediphospho-sugar transferase, SpsA from Bacillus subtilis, in native
and nucleotide-complexed forms. Biochemistry 38: 6380-6385.
Charnock, S.J., Henrissat, B., and Davies, G.J. (2001) Three-dimensional
structures of UDP-sugar glycosyltransferases illuminate the
biosynthesis of plant polysaccharides. Plant Physiol 125: 527-531.
Chiu, C.P., Watts, A.G., Lairson, L.L., Gilbert, M., Lim, D., Wakarchuk,
W.W., Withers, S.G., and Strynadka, N.C. (2004) Structural analysis
of the sialyltransferase CstII from Campylobacter jejuni in complex
with a substrate analog. Nat Struct Mol Biol 11: 163-170.
Cornell, R.B., and Northwood, I.C. (2000) Regulation of
CTP:phosphocholine cytidylyltransferase by amphitropism and
relocalization. Trends Biochem Sci 25: 441-447.
Cornell, R.B., and Taneva, S.G. (2006) Amphipathic helices as mediators of
the membrane interaction of amphitropic proteins, and as modulators
of bilayer physical properties. Curr Protein Pept Sci 7: 539-552.
Coutinho, P.M., and Henrissat, B. (1999) Carbohydrate-active enzymes: an
integrated database approach. In Recent Advances in Carbohydrate
Bioengineering. Gilbert, H.J., Davies, G., Henrissat, B. and
Svensson, B. (eds). Cambridge: The Royal Society of Chemistry, pp.
3-12.
47
Coutinho, P.M., Deleury, E., Davies, G.J., and Henrissat, B. (2003) An
evolving hierarchical family classification for glycosyltransferases. J
Mol Biol 328: 307-317.
Cronan, J.E. (2003) Bacterial membrane lipids: where do we stand? Annu
Rev Microbiol 57: 203-224.
Curatolo, W. (1987a) Glycolipid function. Biochim Biophys Acta 906: 137160.
Curatolo, W. (1987b) The physical properties of glycolipids. Biochim
Biophys Acta 906: 111-136.
Dahlqvist, A., Andersson, S., and Wieslander, Å. (1992) The enzymatic
synthesis of membrane glucolipids in Acholeplasma laidlawii.
Biochim. Biophys. Acta. 23: 131-140.
Davies, G., and Henrissat, B. (1995) Structures and mechanisms of glycosyl
hydrolases. Structure 3: 853-859.
Davies, G.J., Gloster, T.M., and Henrissat, B. (2005) Recent structural
insights into the expanding world of carbohydrate-active enzymes.
Curr Opin Struct Biol 15: 637-645.
DeChavigny, A., Heacock, P.N., and Dowhan, W. (1991) Sequence and
inactivation of the pss gene of Escherichia coli.
Phosphatidylethanolamine may not be essential for cell viability. J
Biol Chem 266: 5323-5332.
Dimitrov, D.S., Franzoso, G., Salman, M., Blumenthal, R., Tarshis, M.,
Barile, M.F., and Rottem, S. (1993) Mycoplasma fermentans
(incognitus strain) cells are able to fuse with T lymphocytes. Clin
Infect Dis 17 Suppl 1: S305-308.
Dowhan, W. (1997) Molecular basis for membrane phospholipid diversity:
why are there so many lipids? Annu Rev Biochem 66: 199-232.
Dowhan, W., and Bogdanov, M. (2002) Functional roles of lipids in
membranes. In Biochemistry of lipids, lipoproteins and membranes.
Vance, D.E.V.a.J.E. (ed). Amsterdam: Elsevier, pp. 1-35.
Drin, G., Casella, J.F., Gautier, R., Boehmer, T., Schwartz, T.U., and
Antonny, B. (2007) A general amphipathic alpha-helical motif for
sensing membrane curvature. Nat Struct Mol Biol 14: 138-146.
Edman, M., Jarhede, T., Sjöström, M., and Wieslander, A. (1999) Different
sequence patterns in signal peptides from mycoplasmas, other grampositive bacteria, and Escherichia coli: a multivariate data analysis.
Proteins 35: 195-205.
Edman, M. (2001) Detection of sequence patterns in membrane proteins : a
story of many dimensions. Umeå: Univ.
Flint, J., Taylor, E., Yang, M., Bolam, D.N., Tailford, L.E., Martinez-Fleites,
C., Dodson, E.J., Davis, B.G., Gilbert, H.J., and Davies, G.J. (2005)
Structural dissection and high-throughput screening of
mannosylglycerate synthase. Nat Struct Mol Biol 12: 608-614.
Franzoso, G., Dimitrov, D.S., Blumenthal, R., Barile, M.F., and Rottem, S.
(1992) Fusion of Mycoplasma fermentans strain incognitus with Tlymphocytes. FEBS Letters 303: 251-254.
48
Fraser, C.M., Gocayne, J.D., White, O., Adams, M.D., Clayton, R.A.,
Fleischmann, R.D., Bult, C.J., Kerlavage, A.R., Sutton, G., Kelley,
J.M., and et al. (1995) The minimal gene complement of
Mycoplasma genitalium. Science 270: 397-403.
Fritz, T.A., Hurley, J.H., Trinh, L.B., Shiloach, J., and Tabak, L.A. (2004)
The beginnings of mucin biosynthesis: the crystal structure of UDPGalNAc:polypeptide alpha-N-acetylgalactosaminyltransferase-T1.
Proc Natl Acad Sci U S A 101: 15307-15312.
Galli, G., Nuti, S., Tavarini, S., Galli-Stampino, L., De Lalla, C., Casorati,
G., Dellabona, P., and Abrignani, S. (2003) Innate immune
responses support adaptive immunity: NKT cells induce B cell
activation. Vaccine 21 Suppl 2: S48-54.
Garinot-Schneider, C., Lellouch, A.C., and Geremia, R.A. (2000)
Identification of essential amino acid residues in the Sinorhizobium
meliloti glucosyltransferase ExoM. J Biol Chem 275: 31407-31413.
Gastinel, L.N., Cambillau, C., and Bourne, Y. (1999) Crystal structures of
the bovine beta4galactosyltransferase catalytic domain and its
complex with uridine diphosphogalactose. EMBO 18: 3546-3557.
Gibson, R.P., Turkenburg, J.P., Charnock, S.J., Lloyd, R., and Davies, G.J.
(2002) Insights into trehalose synthesis provided by the structure of
the retaining glucosyltransferase OtsA. Chem Biol 9: 1337-1346.
Gibson, R.P., Tarling, C.A., Roberts, S., Withers, S.G., and Davies, G.J.
(2004) The donor subsite of trehalose-6-phosphate synthase: binary
complexes with UDP-glucose and UDP-2-deoxy-2-fluoro-glucose at
2 A resolution. J Biol Chem 279: 1950-1955.
Gilbert, M., Brisson, J., Karwaski, M.F., Michniewicz, J., Cunningham,
A.M., Wu, Y., Young, N.M., and Wakarchuk, W.W. (2000)
Biosynthesis of ganglioside mimics in Campylobacter jejuni
OH4384. Identification of the glycosyltransferase genes, enzymatic
synthesis of model compounds, and characterization of nanomole
amounts by 600-mhz (1)h and (13)c NMR analysis. J Biol Chem
275: 3896-3906.
Glass, J.I., Assad-Garcia, N., Alperovich, N., Yooseph, S., Lewis, M.R.,
Maruf, M., Hutchison, C.A., 3rd, Smith, H.O., and Venter, J.C.
(2006) Essential genes of a minimal bacterium. Proc Natl Acad Sci
U S A 103: 425-430.
Gordon, R.D., Sivarajah, P., Satkunarajah, M., Ma, D., Tarling, C.A.,
Vizitiu, D., Withers, S.G., and Rini, J.M. (2006) X-ray crystal
structures of rabbit N-acetylglucosaminyltransferase I (GnT I) in
complex with donor substrate analogues. J Mol Biol 360: 67-79.
Grizot, S., Salem, M., Vongsouthi, V., Durand, L., Moreau, F., Dohi, H.,
Vincent, S., Escaich, S., and Ducruix, A. (2006) Structure of the
Escherichia coli heptosyltransferase WaaC: binary complexes with
ADP and ADP-2-deoxy-2-fluoro heptose. J Mol Biol 363: 383-394.
Guerin, M.E., Buschiazzo, A., Kordulakova, J., Jackson, M., and Alzari,
P.M. (2005) Crystallization and preliminary crystallographic
analysis of PimA, an essential mannosyltransferase from
49
Mycobacterium smegmatis. Acta Crystallograph Sect F Struct Biol
Cryst Commun 61: 518-520.
Ha, S., Walker, D., Shi, Y., and Walker, S. (2000) The 1.9 Å crystal
structure of Escherichia coli MurG, a membrane-associated
glycosyltransferase involved in peptidoglycan biosynthesis. Protein
Sci 9: 1045-1052.
Ha, S., Gross, B., and Walker, S. (2001) E. coli MurG: a paradigm for a
superfamily of glycosyltransferases. Curr Drug Targets Infect
Disord 1: 201-213.
Hanzal-Bayer, M.F., and Hancock, J.F. (2007) Lipid rafts and membrane
traffic. FEBS Lett 581: 2098-2104.
Hauksson, J.B., Lindblom, G., and Rilfors, L. (1994a) Structures of
glucolipids from the membrane of Acholeplasma laidlawii strain AEF22. II. Monoacylmonoglucosyldiacylglycerol. Biochim. Biophys.
Acta 1215: 341-345.
Hauksson, J.B., Lindblom, G., and Rilfors, L. (1994b) Structures of
glucolipids from the membrane of Acholeplasma laidlawii strain AEF22. I. Glycerophosphoryldiglucosyldiacylglycerol and
monoacylbisglycerophosphoryldiglucosyldiacylglycerol. Biochim.
Biophys. Acta. 1214: 124-130.
Hayashi, S., and Wu, H.C. (1990) Lipoproteins in bacteria. J Bioenerg
Biomembr 22: 451-471.
Hellberg, S., Sjöström, M., Skagerberg, B., and Wold, S. (1987) Peptide
quantitative structure-activity relationships, a multivariate approach.
J Med Chem 30: 1126-1135.
Henrissat, B., and Davies, G. (1997) Structural and sequence-based
classification of glycoside hydrolases. Curr Opin Struct Biol 7: 637644.
Henrissat, B., and Davies, G.J. (2000) Glycoside hydrolases and
glycosyltransferases. Families, modules, and implications for
genomics. Plant Physiol 124: 1515-1519.
Himmelreich, R., Hilbert, H., Plagens, H., Pirkl, E., Li, B.C., and Herrmann,
R. (1996) Complete sequence analysis of the genome of the
bacterium Mycoplasma pneumoniae. Nucl Acid Res 24: 4420-4449.
Himmelreich, R., Plagens, H., Hilbert, H., Reiner, B., and Herrmann, R.
(1997) Comparative analysis of the genomes of the bacteria
Mycoplasma pneumoniae and Mycoplasma genitalium. Nucl Acid
Res 25: 701-712.
Hu, Y., and Walker, S. (2002) Remarkable structural similarities between
diverse glycosyltransferases. Chem Biol 9: 1287-1296.
Hu, Y., Chen, L., Ha, S., Gross, B., Falcone, B., Walker, D., Mokhtarzadeh,
M., and Walker, S. (2003) Crystal structure of the MurG:UDPGlcNAc complex reveals common structural principles of a
superfamily of glycosyltransferases. Proc Natl Acad Sci U S A 100:
845-849.
Johansson, E.K., and Pettersson, B. (2002) Taxonomy of Mollicutes. In
Molecular biology and phatogenicity of Mycoplasmas. Razin,
50
S.a.H.R. (ed). New York: Kluwer Academic/Plenum Puplishers, pp.
1-30.
Karlsson, O.P., Dahlqvist, A., and Wieslander, Å. (1994) Activation of the
membrane glucolipid synthesis in Acholeplasma laidlawii by
phosphatidylglycerol and other anionic lipids. J Biol Chem 269:
23484-23490.
Karlsson, O.P., Dahlqvist, A., Vikstrom, S., and Wieslander, Å. (1997) Lipid
dependence and basic kinetics of the purified 1,2-diacylglycerol 3glucosyltransferase from membranes of Acholeplasma laidlawii. J
Biol Chem 272: 929-936.
Keenleyside, W.J., Clarke, A.J., and Whitfield, C. (2001) Identification of
residues involved in catalytic activity of the inverting glycosyl
transferase WbbE from Salmonella enterica serovar borreze. J
Bacteriol 183: 77-85.
Kikuchi, N., Kwon, Y.D., Gotoh, M., and Narimatsu, H. (2003) Comparison
of glycosyltransferase families using the profile hidden Markov
model. Biochem Biophys Res Commun 310: 574-579.
Kinjo, Y., Tupin, E., Wu, D., Fujio, M., Garcia-Navarro, R., Benhnia, M.R.,
Zajonc, D.M., Ben-Menachem, G., Ainge, G.D., Painter, G.F.,
Khurana, A., Hoebe, K., Behar, S.M., Beutler, B., Wilson, I.A.,
Tsuji, M., Sellati, T.J., Wong, C.H., and Kronenberg, M. (2006)
Natural killer T cells recognize diacylglycerol antigens from
pathogenic bacteria. Nat Immunol 7: 978-986.
Kumada, S., Kusaka, H., Okaniwa, M., Kobayashi, O., and Kusunoki, S.
(1997) Encephalomyelitis subsequent to mycoplasma infection with
elevated serum. Pediatr Neurol 16: 241-244.
Kusunoki, S., Chiba, A., Hitoshi, S., Takizawa, H., and Kanazawa, I. (1995)
Anti-Gal-C antibody in autoimmune neuropathies subsequent to
mycoplasma infection. Muscle. Nerve 18: 409-413.
Kusunoki, S., Shiina, M., and Kanazawa, I. (2001) Anti-Gal-C antibodies in
GBS subsequent to mycoplasma infection: evidence of molecular
mimicry. Neurology 57: 736-738.
Ladbrooke, B.D., Williams, R.M., and Chapman, D. (1968) Studies on
lecithin-cholesterol-water interactions by differential scanning
calorimetry and X-ray diffraction. Biochim. Biophys. Acta. 150: 333340.
Ladokhin, A.S., and White, S.H. (2001) Protein chemistry at membrane
interfaces: non-additivity of electrostatic and hydrophobic
interactions. J Mol Biol 309: 543-552.
Lapinsh, M., Gutcaits, A., Prusis, P., Post, C., Lundstedt, T., and Wikberg,
J.E. (2002) Classification of G-protein coupled receptors by
alignment-independent extraction of principal chemical properties of
primary amino acid sequences. Protein Sci 11: 795-805.
Lee, A.G. (2000) Membrane lipids: it's only a phase. Curr Biol 10: R377380.
Leipold, M.D., Kaniuk, N.A., and Whitfield, C. (2007) The C-terminal
Domain of the Escherichia coli WaaJ glycosyltransferase is
51
important for catalytic activity and membrane association. J Biol
Chem 282: 1257-1264.
Li, X.M., Momsen, M.M., Smaby, J.M., Brockman, H.L., and Brown, R.E.
(2001) Cholesterol decreases the interfacial elasticity and detergent
solubility of sphingomyelins. Biochemistry 40: 5954-5963.
Liu, J., and Mushegian, A. (2003) Three monophyletic superfamilies account
for the majority of the known glycosyltransferases. Protein Sci 12:
1418-1431.
Lobsanov, Y.D., Romero, P.A., Sleno, B., Yu, B., Yip, P., Herscovics, A.,
and Howell, P.L. (2004) Structure of Kre2p/Mnt1p: a yeast
alpha1,2-mannosyltransferase involved in mannoprotein
biosynthesis. J Biol Chem 279: 17921-17931.
Luecke, H., Schobert, B., Richter, H.T., Cartailler, J.P., and Lanyi, J.K.
(1999) Structure of bacteriorhodopsin at 1.55 A resolution. J Mol
Biol 291: 899-911.
Manillof, J. (2002) Phylogeny and Evolution. In Molecular biology and
phatogenicity of Mycoplasmas. Razin, S.a.H.R. (ed). New York:
Kluwer Academic/Plenum Puplishers, pp. 31-44.
Martinez-Fleites, C., Proctor, M., Roberts, S., Bolam, D.N., Gilbert, H.J.,
and Davies, G.J. (2006) Insights into the synthesis of
lipopolysaccharide and antibiotics through the structures of two
retaining glycosyltransferases from family GT4. Chem Biol 13:
1143-1152.
Matsumoto, K., Kusaka, J., Nishibori, A., and Hara, H. (2006) Lipid
domains in bacterial membranes. Mol Microbiol 61: 1110-1117.
McElhaney (1992) Membrane structure. In Mycoplasma; Molecular biology
and pathogenesis. Maniloff, J., McElhaney, R.N., Finch, L.R. and
Baseman , J.B. (eds). Washington D.C.: ASM, pp. 113-157.
Mulichak, A.M., Losey, H.C., Walsh, C.T., and Garavito, R.M. (2001)
Structure of the UDP-glucosyltransferase Gtfb that modifies the
heptapeptide aglycone in the biosynthesis of vancomycin group
antibiotics. Structure 9: 547-557.
Mulichak, A.M., Losey, H.C., Lu, W., Wawrzak, Z., Walsh, C.T., and
Garavito, R.M. (2003) Structure of the TDP-epivancosaminyltransferase GtfA from the chloroeremomycin
biosynthetic pathway. Proc Natl Acad Sci U S A 100: 9238-9243.
Mulichak, A.M., Lu, W., Losey, H.C., Walsh, C.T., and Garavito, R.M.
(2004) Crystal structure of vancosaminyltransferase GtfD from the
vancomycin biosynthetic pathway: interactions with acceptor and
nucleotide ligands. Biochemistry 43: 5170-5180.
Murzin, A.G., Brenner, S.E., Hubbard, T., and Chothia, C. (1995) SCOP: a
structural classification of proteins database for the investigation of
sequences and structures. J Mol Biol 247: 536-540.
Mushegian, A.R., and Koonin, E.V. (1996) A minimal gene set for cellular
life derived by comparison of complete bacterial genomes. Proc
Natl Acad Sci U S A 93: 10268-10273.
52
O'Toole, C., and Lowdell , M. (1990) Infection of human T-cells with
mycoplasma , inhibition of CD4 expression and HIV-1 gp 120
glycoprotein binding and infectivity. Lancet 336: 1067.
Pak, J.E., Arnoux, P., Zhou, S., Sivarajah, P., Satkunarajah, M., Xing, X.,
and Rini, J.M. (2006) X-ray crystal structure of leukocyte type core
2 beta1,6-N-acetylglucosaminyltransferase. Evidence for a
convergence of metal ion-independent glycosyltransferase
mechanism. J Biol Chem 281: 26693-26701.
Parekh, V.V., Singh, A.K., Wilson, M.T., Olivares-Villagomez, D.,
Bezbradica, J.S., Inazawa, H., Ehara, H., Sakai, T., Serizawa, I., Wu,
L., Wang, C.R., Joyce, S., and Van Kaer, L. (2004) Quantitative and
qualitative differences in the in vivo response of NKT cells to
distinct alpha- and beta-anomeric glycolipids. J Immunol 173: 36933706.
Patenaude, S.I., Seto, N.O., Borisova, S.N., Szpacenko, A., Marcus, S.L.,
Palcic, M.M., and Evans, S.V. (2002) The structural basis for
specificity in human ABO(H) blood group biosynthesis. Nat Struct
Biol 9: 685-690.
Pedersen, L.C., Tsuchida, K., Kitagawa, H., Sugahara, K., Darden, T.A., and
Negishi, M. (2000) Heparan/chondroitin sulfate biosynthesis.
Structure and mechanism of human glucuronyltransferase I. J Biol
Chem 275: 34580-34585.
Persson, K., Ly, H.D., Dieckelmann, M., Wakarchuk, W.W., Withers, S.G.,
and Strynadka, N.C. (2001) Crystal structure of the retaining
galactosyltransferase LgtC from Neisseria meningitidis in complex
with donor and acceptor sugar analogs. Nat Struct Biol 8: 166-175.
Porcelli, S.A., and Modlin, R.L. (1999) The CD1 system: antigen-presenting
molecules for T cell recognition of lipids and glycolipids. Annu Rev
Immunol 17: 297-329.
Qasba, P.K., Ramakrishnan, B., and Boeggeman, E. (2005) Substrateinduced conformational changes in glycosyltransferases. Trends
Biochem Sci 30: 53-62.
Rackovsky, S. (1998) "Hidden" sequence periodicities and protein
architecture. Proc Natl Acad Sci U S A 95: 8580-8584.
Ramakrishnan, B., and Qasba, P.K. (2001) Crystal structure of lactose
synthase reveals a large conformational change in its catalytic
component, the beta1,4-galactosyltransferase-I. J Mol Biol 310: 205218.
Ramakrishnan, B., Balaji, P.V., and Qasba, P.K. (2002a) Crystal structure of
beta1,4-galactosyltransferase complex with UDP-Gal reveals an
oligosaccharide acceptor binding site. J Mol Biol 318: 491-502.
Ramakrishnan, B., Boeggeman, E., and Qasba, P.K. (2002b) Beta-1,4galactosyltransferase and lactose synthase: molecular mechanical
devices. Biochem Biophys Res Commun 291: 1113-1118.
Ramakrishnan, B., Ramasamy, V., and Qasba, P.K. (2006) Structural
snapshots of beta-1,4-galactosyltransferase-I along the kinetic
pathway. J Mol Biol 357: 1619-1633.
53
Rietveld, A., van Kemenade, T.J., Hak, T., Verkleij, A.J., and de Kruijff, B.
(1987) The effect of cytochrome c oxidase on lipid polymorphism of
model membranes containing cardiolipin. Eur J Biochem 164: 137140.
Rilfors, L., Wieslander, Å., and Lindblom, G. (1993) Regulation and
physicochemical properties of the polar lipids in Acholeplasma
laidlawii. Subcell Biochem 20: 109-166.
Rottem, S., Adar, L., Gross, Z., Ne'eman, Z., and Davis, P.J. (1986)
Incorporation and modification of exogenous Phosphatidylcholines
by mycoplasmas. J Bacteriol 167: 299-304.
Ruuth, E., and Praz, F. (1989) Interactionbetween mycoplamsa and the
immune system. Immun Rev 112: 133-160.
Ruvolo, P.P. (2003) Intracellular signal transduction pathways activated by
ceramide and its metabolites. Pharmacol Res 47: 383-392.
Seelig, J. (2004) Thermodynamics of lipid-peptide interactions. Biochim
Biophys Acta 1666: 40-50.
Shah, D.O., and Schulman, J.H. (1967) Influence of calcium, cholesterol,
and unsaturation on lecithin monolayers. J Lipid Res 8: 215-226.
Shao, H., He, X., Achnine, L., Blount, J.W., Dixon, R.A., and Wang, X.
(2005) Crystal structures of a multifunctional triterpene/flavonoid
glycosyltransferase from Medicago truncatula. Plant Cell 17: 31413154.
Simidjiev, I., Stoylova, S., Amenitsch, H., Javorfi, T., Mustardy, L.,
Laggner, P., Holzenburg, A., and Garab, G. (2000) Self-assembly of
large, ordered lamellae from non-bilayer lipids and integral
membrane proteins in vitro. Proc Natl Acad Sci U S A 97: 14731476.
Simons, K., and Vaz, W.L. (2004) Model systems, lipid rafts, and cell
membranes. Annu Rev Biophys Biomol Struct 33: 269-295.
Smith, P.F. (1992) Membrane Lipid and Lipopolysaccharide Structures. In
Mycoplasma; Molecular biology and pathogenesis. Maniloff, J.,
McElhaney, R.N., Finch, L.R. and Baseman , J.B. (eds). Washington
D.C.: ASM, pp. 79-93.
Snajdrova, L., Kulhanek, P., Imberty, A., and Koca, J. (2004) Molecular
dynamics simulations of glycosyltransferase LgtC. Carbohydr Res
339: 995-1006.
Susuki, K., Odaka, M., Mori, M., Hirata, K., and Yuki, N. (2004) Acute
motor axonal neuropathy after Mycoplasma infection: Evidence of
molecular mimicry. Neurology 62: 949-956.
Tarbouriech, N., Qasba, P.K., and Davies, G.J. (2001) Three-dimensional
structures of the Mn and Mg dTDP complexes of the family GT-2
glycosyltransferase SpsA: a comparison with related NDP-sugar
glycosyltransferases. J Mol Biol 314: 655-661.
Tarshis, M., Salman, M., and Rottem, S. (1993) Cholesterol is required for
the fusion of single unilamellar vesicles with Mycoplasma
capricolum. Biophys J 64: 709-715.
54
Tsiodras, S., Kelesidis, I., Kelesidis, T., Stamboulis, E., and Giamarellou, H.
(2005) Central nervous system manifestations of Mycoplasma
pneumoniae infections. J Infect 51: 343-354.
Tvaroska, I. (2004) Molecular modeling insights into the catalytic
mechanism of the retaining galactosyltransferase LgtC. Carbohydr
Res 339: 1007-1014.
Uemura, K., Loomes, L.M., Childs, R.A., and Feizi, T. (1988) Evidence for
occurrence of passively adsorbed I antigen activity on a cultured
strain of Mycoplasma pneumoniae. Infect. Immun. 56: 3015-3020.
Unligil, U.M., Zhou, S., Yuwaraj, S., Sarkar, M., Schachter, H., and Rini,
J.M. (2000) X-ray crystal structure of rabbit Nacetylglucosaminyltransferase I: catalytic mechanism and a new
protein superfamily. EMBO J. 19: 5269-5280.
Waites, K.B., and Talkington, D.F. (2004) Mycoplasma pneumoniae and its
role as a human pathogen. Clin Microbiol Rev 17: 697-728
van den Brink-van der Laan, E., Boots, J.W., Spelbrink, R.E., Kool, G.M.,
Breukink, E., Killian, J.A., and de Kruijff, B. (2003) Membrane
interaction of the glycosyltransferase MurG: a special role for
cardiolipin. J Bacteriol 185: 3773-3779.
van den Brink-van der Laan, E., Killian, J.A., and de Kruijff, B. (2004)
Nonbilayer lipids affect peripheral and integral membrane proteins
via changes in the lateral pressure profile. Biochim Biophys Acta
1666: 275-288.
van Meer, G. (2005) Cellular lipidomics. Embo J 24: 3159-3165.
Wieslander, Å., Ulmius, J., Lindblom, G., and Fontell, K. (1978) Water
binding and phase structures for different Acholeplasma laidlawii
membrane lipids studied by deuteron nuclear magnetic resonance
and x-ray diffraction. Biochim Biophys Acta 512: 241-253.
Wikström, M., Xie, J., Bogdanov, M., Mileykovskaya, E., Heacock, P.,
Wieslander, A., and Dowhan, W. (2004)
Monoglucosyldiacylglycerol, a foreign lipid, can substitute for
phosphatidylethanolamine in essential membrane-associated
functions in Escherichia coli. J Biol Chem 279: 10484-10493.
Vikström, S., Li, L., Karlsson, O.P., and Wieslander, Å. (1999) A Key role
of the diglucosyldiacylglycerol synthase for the nonbilayer-bilayer
lipid balance of Acholeplasma laidlawii membranes. Biochemistry
38: 5511-5520.
Wold, S., and Sjöström, M. (1998) Chemometrics and its roots in physical
organic chemistry. Acta Chemi Scand 52: 517-523.
Wolf, M., Muller, T., Dandekar, T., and Pollack, J.D. (2004) Phylogeny of
Firmicutes with special reference to Mycoplasma (Mollicutes) as
inferred from phosphoglycerate kinase amino acid sequence data
Int J Syst Evol Microbiol 54: 871-875.
Xie, J., Bogdanov, M., Heacock, P., and Dowhan, W. (2006)
Phosphatidylethanolamine and monoglucosyldiacylglycerol are
interchangeable in supporting topogenesis and function of the
55
polytopic membrane protein lactose permease. J Biol Chem 281:
19172-19178.
Yamamoto, F., Clausen, H., White, T., Marken, J., and Hakomori, S. (1990)
Molecular genetic basis of the histo-blood group ABO system.
Nature 345: 229-233.
Zhang, W., Campbell, H.A., King, S.C., and Dowhan, W. (2005)
Phospholipids as determinants of membrane protein topology.
Phosphatidylethanolamine is required for the proper topological
organization of the gamma-aminobutyric acid permease (GabP) of
Escherichia coli. J Biol Chem 280: 26032-26038.
56