Chemistry Reference
In-Depth Information
saprotrophic mold fungus
Aspergillus fumigatus
and soil-living amoeba
Dictyoste-
lium discoideum
, sometimes as the sole anchor lipid. The
S. cerevisiae
Cwh43p,
whose N-terminal region contains a sequence homologous to mammalian PGAP2,
is involved in the remodeling of the lipid moiety of GPI anchors to ceramides (for
further details on ceramides, please see Chapters 10 and 30 ). The remodeling
reactions involve the exchange of the fatty acid components of the lipid moiety for
different fatty acids or whole-lipid components.
T. brucei
GPI - anchored VSGs
contain exclusively myristate as their lipid components. Fatty acid biosynthesis is
peculiar in
T. brucei
, since the major product is myristate and is mainly destined
to be incorporated into the GPI anchor of VSG protein. This fatty acid pathway
could be inhibited with thiolactomycin, a natural product produced by both
Nocar-
dia
and
Streptomyces
spp. that acts as a fatty acid synthesis inhibitor, leading to
parasite death. Studies using myristic acid analogs such as 10- (propoxy)decanoic
acid or its derivatives showed toxicity towards
T. brucei
(but not to mammalian
cells), another target for drug design, here focusing on membrane anchor synthe-
sis, not the glycan part. Next, we turn to the chemical synthesis - a tool to obtain
pure material for functional analysis and vaccination.
9.3
Chemical Synthesis of GPI s
The fi rst total synthesis of a GPI was achieved in 1991 by Chikara Murakata and
Tomoya Ogawa in Japan, who synthesized the membrane GPI anchor of
T. brucei.
Since then, a number of groups have synthesized several high- profi le GPIs (synthetic
strategies for glycan synthesis are outlined in Chapter 3 ). The fi rst automated chemi-
cally synthesized glycan of a GPI used for vaccination trials was reported 2002 (please
see Chapter 3.9 for further example of synthesis for vaccination) in a study of a GPI-
based antimalaria vaccine, in which the malarial GPI toxin was linked to a carrier
protein to form a glycoconjugate that has been reported to induce protective immune
responses in mice against rodent malaria (for review see [8]). The chemical synthesis
is not only helpful for immunological but also for biochemical studies. For instance,
a fi rst generation of functional fl uorescent GPI probes was used to demonstrate that
the ER possesses transporters capable of fl ipping GPIs in an ATP-independent
manner. In the next section we will discuss how mutant cells can help to clone genes
involved in GPI biosynthesis (please see above), leading us to present information on
natural mutants causing diseases.
9.3.1
Mutant Cells Lead the Way to Identifi cation of Complementation Classes Involved
in GPI Biosynthesis
Genes known to be necessary for the biosynthesis of GPI anchors have been
cloned in the mammalian system mainly by the group of Taroh Kinoshita in
Osaka, Japan, through complementation and/or coimmunprecipitation strategies
[9]. The isolation of mutants with complete defects in various reactions in GPI
