Glycosylphosphatidylinositol Anchors: Structure, Biosynthesis and Functions - The Sugar Code - page 164

subunit, PIG-X, forms a complex with PIG-M, thereby stabilizing it. PIG- X
shows 16% identity with the C-terminal part of yeast PBN1. Dol-P-Man is
synthesized from dolichol phosphate ( Dol - P ) and GDP - Man by an enzyme
called Dol-P mannose synthase. The antibiotic amphomycin produced by
Streptomyces canis forms a complex with Dol-P in the presence of Ca 2+ , which
blocks the interaction between the Dol- P - Man synthase and Dol - P, thereby
inhibiting GPI biosynthesis in vitro (for the role of dolichol in N - glycan
biosynthesis, please see Chapter 6, especially Info Box 1). Therefore, Dol- P - Man
biosynthesis could be an important target for the development of specifi c
inhibitors. The specifi city of the

1,4 - mannosyltransferase was investigated in
T. brucei , P. falciparum and mammalian (HeLa) cells using various GlcN-PI
analogs, which may provide a potential target for drug design, particularly
against the malaria parasite P. falciparum. Here, in this organism, posttranslational
modifi cations like N - and O - glycosylation are either absent or present at extremely
low levels and GPIs represent its sole carbohydrate modifi cations.
• Step 5: modifi cation of Man - GlcN - acyl - PI with a phosphoethanolamine side - chain .
The fi rst mannose is subsequently modifi ed by EtN-P and the reaction is mediated
by PIG-N in mammalian cells (MCD4 in yeast). Phosphatidylethanolamine
serves as a donor of the EtN-P group. MCD4 is essential in yeast.
• Step 6: addition of the second mannose . The second mannose is transferred from
Dol-P-Man to position 6 of the fi rst mannose in EtN - P - Man - GlcN - acyl - PI. The
reaction is mediated by PIG-V in mammalian cells (GPI18 in yeast). The GPI18
is an essential gene in S. cerevisiae and gpi18

α

Δ mutants are rescued also by the

expression of mammalian PIG-V.
• Step 7: addition of the third mannose . The third mannose is transferred from Dol-
P-Man to position 2 of the second mannose by PIG-B (Gpi10 in yeast). Gpi10p
requires intermediates containing an EtN-P residue on Man 1 , whereas this is not
important for Gpi10p homologs of most protozoa, since GPI anchors of these
organisms do not have an EtN-P on Man 1 . Deletion of PIG-N does not completely
prevent the addition of GPIs to proteins, explaining also why an addition of
YW3548 does not signifi cantly affect GPI protein expression in mammals or T.
brucei , whereas it blocks the growth of yeast. YW3548 is a natural terpenoid
lactone isolated from Codinea simplex and causes an accumulation of the
intermediate Man 2 - GlcN - (acyl) - PI in yeast, pathogenic fungus Candida albicans
and mammalian (lymphoma) cells, but not in parasitic protozoa. In yeast,
YW3548 blocks also the addition of EtN-P to the fi rst mannose residue, which is
a substrate requirement for the addition of the third but not second mannose,
therefore, the accumulation of Man 2 - GlcN - (acyl) - PI was observed [7] . Similar
effects of YW3548 were observed in yeast and mammalian cells using the
metalloprotease inhibitor phenanthroline. An accumulation of GPI intermediates
that are substrates for EtN-P transferases were identifi ed in cells treated with
phenanthroline. Therefore, both phenanthroline and YW3548 are likely to inhibit
GPI-phosphoethanolamine transferases in mammalian and yeast cells, but
not in protozoa. This selective inhibition is another indication of signifi cant
differences between parasite and mammalian/yeast GPI biosynthesis.