GLYCOSYL PHOSPHATIDYL INOSITOL

1. Synthesis and Structure of Major Glycan
Classes
1/24/05

2. Large O-linked
Glycosaminoglycans and poly-
lactosamine structures
Glycoprotein N-linked and O-
linked oligosaccharides
Glycolipid oligosaccharides

3. The building blocks
4
4
 4 2
9Ac
3
3
6  4 2

6
3

6
9Ac
4
-
4
4 2
9Ac
3
4 2
3
3
6
6
3
Symbolic
Representation
Fuc

3
Sia
3Gal4GlcNAc 2Man Fuc

6 6
Man
4GlcNAc4GlcNAc 
3
9Ac-Sia6Gal4GlcNAc2Man
Simplified
Traditional

4. Glycan synthesis
in a cellular context

5. Overview
From ER
through
Trans-Golgi and
points inbetween

6. ER processing of N-linked glycans

7. Major Classes of N-Glycans
“High-Mannose”
(oligo-mannose)
“Complex”
“Hybrid”

8. Biosynthesis of N-Glycans:
Production of GlcNAc-P-P-Dolichol
Adapted from Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79
GlcNAc
Man
Gal
Sia
Fuc
Glc
Dolichol
Tunicamycin
Blocks - not
very specific!

9. Biosynthesis of the N-Glycan
Precursor on the Cytosolic Leaflet of the
Endoplasmic Reticulum (ER)
Adapted from Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79
GlcNAc
Man
Gal
Sia
Fuc
Glc
CDG = Congenital Disorder of Glycosylation in Humans

10. Biosynthesis of the N-Glycan
Precursor on Lumenal Leaflet of ER
Adapted from Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79
GlcNAc
Man
Gal
Sia
Fuc
Glc

11. Completion of Biosynthesis of N-Glycan
Precursor on Lumenal Leaflet of ER
- and Transfer to Protein
Adapted from Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79
GlcNAc
Man
Gal
Sia
Fuc
Glc

12. Oligosaccharyltransferase complex (OST) in the ER membrane transfers
the dolichol N-glycan precursor to asparagine residues on nascently
translated proteins
Target “sequon” for N-glycosylation
• Necessary but not sufficient
• X = any amino acid except proline
• Rarely can be Asn-X-Cys
• Transfer co-
translational/immediate
post-translational before folding
• ~2/3 of proteins have sequons
• ~ 2/3 sequons actually occupied
(some variably)
Yeast OST complex contains nine membrane-bound subunits

13. Initial Processing of N-Glycans in
the ER and Golgi
Adapted from Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79
GlcNAc
Man
Gal
Sia
Fuc
Glc
ER Golgi

14. Calnexin (and Calcireticulin) function during
glycoprotein
folding in the endoplasmic reticulum
3 Glucose
Residues
Improperly folded proteins are
re-glucosylated by
glucosyltransferase which acts
as “sensor” for improper folding

15. ER glycolipid synthesis

16. Biosynthesis of Ceramide and
Glucosylceramide

17. ER glycolipid synthesis

18. Basic Glycosylphosphatidylinositol (GPI) Anchor
Phospholipid

19. Examples of GPI-Anchored Proteins
Cell surface hydrolases
alkaline phosphatase
acetylcholinesterase
5’ nucleotidase
Adhesion molecules
neural cell adhesion molecule
heparan sulfate proteoglycan
Others
decay accelerating factor
scrapie prion protein
folate receptor
Protozoal antigens
trypanosome VSG
leishmanial protease
plasmodium antigens
Mammalian antigens
Thy-1
carcinoembryonic antigen

20. Structure of the Basic GPI Anchor
Etn
P
INOSITOL
P
= Mannose (Man)
= Glucosamine
Etn = Ethanolamine
P = Phosphate
NH 2
NH 2
Pig-A
PNH Defect
GPI-Linked Protein
Cell Surface
Membrane

21. Structural Analysis
of the GPI Anchor
Enzymatic and chemical
cleavage sites
are useful in identifying
GPI anchored
membrane proteins

22. Examples of C-Terminal Sequences Signaling
the Addition of GPI-Anchors
Protein GPI-Signal Sequence
Acetylcholinesterase (Torpedo) NQFLPKLLNATAC DGELSSSGTSSSKGIIFYVLFSILYLIFY
Alkaline Phosphatase (placenta) TACDLAPPAGTTD AAHPGRSVVPALLPLLAGTLLLLETATAP
Decay AcceleratingFactor HETTPNKGSGTTS GTTRLLSGHTCFTLTGLLGTLVTMGLLT
PARP (T. Brucei) EPEPEPEPEPEPG AATLKSVALPFAIAAAALVAAF
Prion Protein (hamster) QKESQAYYDGRRS SAVLFSSPPVILLISFLIFLMVG
Thy-1 (rat) KTINVIRDKLVKC GGISLLVQNTSWLLLLLLSLSFLQATDFISI
Variant Surface Glycoprotein (T. Brucei) ESNCKWENNACKD SSILVTKKFALTVVSAAFVALLF
Bold AA is site of GPI attachment Sequence to right is
cleaved by the transpeptidase upon Anchor addition

23. Golgi processing of N-linked glycans

24. Completion of Processing of
N-Glycans in ER and Golgi
Adapted from Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79
GlcNAc
Man
Gal
Sia
Fuc
Glc
Final products often show
“microheterogeneity” at each
N-Glycosylation site

25. GlcNAc-Transferases Determine Number of
“Antennae” of N-glycans

26. Some representative examples of
mammalian complex-type N-glycans

27. Evolutionary Variations of the N-glycan
Processing Pathway
N
Asn
N
Asn
6
Vertebrates
3
4
4
 4
N
Asn
Yeast
3
Insects
6

2
Plants
3
Slime
Mold
N
Asn
3 6
“Pauci-
mannose”

28. Golgi processing of O-linked glycans

29. Plasma membrane proteins are either peripheral proteins or integral membrane proteins.
The latter include proteins that span the lipid bilayer once or several times, and another class that are
covalently attached to lipids. Proteins attached to glycosylphosphatidylinositol (GPI) via their
carboxyl termini are generally found in the outer leaflet of the lipid bilayer and face the extracellular
environment.
The GPI membrane anchor may be conveniently thought of as an alternative to the single
transmembrane domain of type-1 integral membrane proteins.

30. BACKGROUND AND DISCOVERY
The first data suggesting the existence of protein-phospholipid anchors appeared in 1963 with the
finding that crude bacterial phospholipase C (PLC) selectively releases alkaline phosphatase from
mammalian cells.
Phosphatidylinositol-protein anchors were first postulated in the mid-1970s based on the ability of
highly purified bacterial phosphatidylinositol-specific PLC enzymes to release certain proteins, such
as alkaline phosphatase and 5′-nucleotidase, from mammalian plasma membranes.
By 1985, these predictions were confirmed by compositional and structural data from studies
on Torpedo acetylcholinesterase, human and bovine erythrocyte acetylcholinesterase, rat Thy-1, and
the sleeping sickness parasite Trypanosoma brucei variant surface glycoprotein (VSG).
The first complete glycosylphosphatidylinositol (GPI) structures, which were those for T.
brucei VSG and rat Thy-1, were solved in 1988

31. DIVERSITY OF PROTEINS WITH GPI ANCHORS
To date, hundreds of GPI-anchored proteins (GPI-APs) have been identified in many eukaryotes, ranging from
protozoa and fungi to humans.
The range of described GPI-APs and the distribution of putative GPI biosynthesis genes suggests that (1) GPI
anchors are almost ubiquitous among eukaryotes; (2) GPI-APs are functionally diverse and include hydrolytic
enzymes, adhesion molecules, complement regulatory proteins, receptors, protozoan coat proteins, and prion
proteins; and (3) in mammals, alternative messenger RNA (mRNA) splicing may lead to the expression of
transmembrane and/or soluble and GPI-anchored forms of the same gene product.
These variants may be developmentally regulated. For example, neural cell adhesion molecule (NCAM) exists
in GPI-anchored and soluble forms when expressed in muscle and in GPI-anchored and two transmembrane
forms when expressed in brain.

32. STRUCTURE OF GPI ANCHORS
The substructure Manα1-4GlcNα1–6myo-inositol-1-P-lipid is a universal hallmark of GPI anchors and
related structures. All but one protein-linked GPI anchors share a larger common core structure.
The structural arrangements of GPI anchors are unique among protein–carbohydrate associations in that
the reducing terminus of the GPI oligosaccharide is not attached to the protein. Rather, the reducing
terminal glucosamine residue is α1-6 linked to the D-myo-inositol head group of a phosphatidylinositol
(PI) moiety.
A distal, non-reducing mannose residue is attached to the protein via an ethanolamine phosphate (EtNP)
bridge between the C-6 hydroxyl group of mannose and the α-carboxyl group of the carboxy-terminal
amino acid. GPIs are one of the rare instances in which glucosamine is found without either an N-acetyl
or N-sulfate moiety (as in proteoglycans)

33. Beyond the common core, the structures of mature GPI anchors are quite
diverse, depending on both the protein to which they are attached and the
organism in which they are synthesized.
Modifications to the core include additional EtNP and a wide variety of
linear and branched glycosyl substituents of largely unknown function.
There is considerable variation in the PI moiety. Indeed, GPI is a rather
loose term because, strictly speaking, PI refers specifically to D-myo-
inositol-1-P-3(sn-1,2-diacylglycerol) (i.e., diacyl-PI), whereas many GPIs
contain other types of inositolphospholipids, such as lysoacyl-PI, alkylacyl-
PI, alkenylacyl-PI, and inositolphosphoceramide.
Another variation, termed inositol acylation, is characterized by the
presence of an ester-linked fatty acid attached to the C-2 hydroxyl of the
inositol residue.
The presence of this modification makes the anchor inherently resistant to
the action of bacterial PI-specific PLC.

34. The available lipid structural data suggest that (1) inositolphosphoceramide-based protein-linked
GPIs are only found in “lower” eukaryotes, such as Saccharomyces cerevisiae, Aspergillus
niger, Dictyostelium discoideum, and Trypanosoma cruzi; (2) the lipid structures of GPIs generally
do not reflect those of the general cellular PI or inositolphosphoceramide pool; and (3) the lipid
structures of some (e.g., trypanosome) GPI-APs are under developmental control.
The factors that control the synthesis of a mature GPI anchor found on a given protein appear to be
similar to those for other posttranslational modifications such as N- and O-glycosylation. Thus,
primary control is at the cellular level, whereby the levels of specific biosynthetic and processing
enzymes dictate the final repertoire of structures.
Secondary control is at the level of the tertiary/quaternary structure of the protein bearing the GPI
anchor, which affects accessibility to processing enzymes.
Examples of primary control include (1) differences in GPI glycan side chains in human versus
porcine membrane dipeptidase and brain versus thymocyte rat Thy-1 and (2) differences in glycan
side chains and lipid structure when T. brucei VSG is expressed in bloodstream and insect life-cycle
stages of the parasite.
An example of secondary control is the difference in VSG glycan side chains when VSGs with
different carboxy-terminal sequences are expressed in the same trypanosome clone.

35. Non-Protein-Linked GPI Structures
In mammalian cells, some free GPIs (GPI-anchor biosynthetic intermediates) are found at the
cell surface, but their functional significance is unknown.
On the other hand, several protozoa (particularly trypanosomatids) express high numbers
(>10
7
copies per cell) of free GPIs on their cell surface as metabolic end products.
These include the so-called glycoinositol phospholipids (GIPLs) and lipophosphoglycans
(LPGs) of the Leishmania. Some protozoan (type-1) GIPLs conform to the Manα1-6Manα1-
4GlcNα1-6PI sequence common to protein-linked GPIs, whereas others contain a (type-2)
Manα1-3Manα1-4GlcNα1-6PI motif, and still others are hybrid structures containing the
branched motif (Manα1-6)Manα1-3Manα1-4GlcNα1-6PI.

36. IDENTIFICATION OF GPI-APs
The presence of a GPI anchor may be inferred by the identification of an amino-terminal signal
peptide and a carboxy-terminal GPI signal peptide from the predicted primary amino acid
sequence of a given gene. Such predictions can be verified by structural analysis, described
below, or by indirect methods: the shift of proteins from the pellet to the supernatant after
treatment of whole cells with PI-PLC is one such simple procedure.
Variations on this theme use Triton X-114 (a nonionic detergent) phase separation whereby GPI-
APs partition into the detergent-rich phase before, but not after, PI-PLC treatment. An additional
criterion is the appearance of an epitope known as the “cross-reacting determinant” following PI-
PLC cleavage.
Some GPI anchors are acylated at C-2 of inositol and therefore resistant to PI-PLC but all are
sensitive to serum GPI-phospholipase D (GPI-PLD).
However, GPI-PLD cleavage generally requires detergent solubilization of the substrate and does
not generate a “cross-reacting determinant.”

37. The GPI-PLD reaction also leaves one fatty acid attached to the protein (the inositol acyl group)
and, depending on the protein, this may prevent complete Triton X-114 phase separation after
GPI-PLD digestion.
Finally, GPI anchors may be labeled biosynthetically with [H]myo-inositol. Certain pore-forming
bacterial toxins such as aerolysin have been shown to bind to GPI anchors, and these may be used
to probe one- and two-dimensional gel western blots.

38. THE CHEMISTRY OF GPI ANCHORS
GPI anchors are complex molecules that include amide, glycosidic, phosphodiester, and
hydroxyester linkages between their various components and the challenge of their organic
synthesis has been met by several groups.
Analogs of GPI substructures have been instrumental in probing the comparative enzymology
of GPI biosynthesis in “lower” and “higher” eukaryotes and methods to ligate synthetic GPIs to
proteins, to make fluorescent GPI membrane probes and GPI glycan microarrays are now well
established.
The GPI-anchor structure lends itself to selective cleavage by several chemical and enzyme
reagents.
These were originally used to help determine GPI structures and are now applied to confirm the
presence of a GPI anchor and/or obtain partial structural information from native or
[
3
H]mannose-, [
3
H]glucosamine-, [
3
H]inositol-, or [
3
H]fatty-acid-radiolabeled GPI-APs or GPI-
biosynthetic intermediates. Some of these reactions and their applications for GPI structure.

39. A key reaction is nitrous acid deamination of the glucosamine residue. This gentle (room
temperature, pH 4.0) reaction is dependent on the free amino group of the glucosamine residue
and gives a highly selective cleavage of the glucosamine-inositol glycosidic bond.
The reaction liberates the PI moiety, which can be isolated by solvent partition and analyzed
by mass spectrometry, and generates a free reducing terminus on the GPI glycan in the form of
2,5-anhydromannose. This reducing sugar can be reduced to [1-
3
H]2,5-anhydromannitol
(AHM) by sodium borotritide reduction to introduce a radiolabel, or it may be attached to a
fluorophore such as 2-aminobenzamide (2-AB) by reductive amination.
Once the GPI glycan is radioactively or fluorescently labeled and dephosphorylated with
aqueous hydrogen fluoride, the glycan can often be conveniently sequenced using
exoglycosidases.
Partial structural information may also be obtained by tandem mass spectrometry of GPI-
peptides generated by trypsin, proteinase K, or Pronase digestion of GPI-APs, or by tandem
mass spectrometry of GPI glycans released by aqueous hydrogen fluoride dephosphorylation
and permethylated before analysis.

40. GPI BIOSYNTHESIS AND TRAFFICKING
The biosynthesis of GPI anchors occurs in three stages: (1) preassembly of a GPI precursor in the
endoplasmic reticulum (ER) membrane, (2) attachment of the GPI to newly synthesized protein in
the lumen of the ER with concomitant cleavage of a carboxy-terminal GPI-addition signal peptide,
and (3) lipid remodeling and/or carbohydrate side-chain modifications in the ER and after
transport to the Golgi.
Analysis of GPI precursor biosynthesis was first made possible by the development of a cell-free
system in T. brucei. Each trypanosome has 1×10
7
molecules of GPI-linked VSG on its surface.
Therefore, enzymes and intermediates in the GPI-biosynthetic pathway are relatively abundant in
microsomal membrane preparations produced from this organism.
The sequence of events underlying GPI biosynthesis has been studied in T. brucei, T.
cruzi, Toxoplasma gondii, Plasmodium falciparum, Leishmania major, Paramecium, S.
cerevisiae, Cryptococcus neoformans, and mammalian cells.
The emphasis on eukaryotic microbes reflects the abundance of GPI-APs in these organisms and
the potential of GPI inhibition for chemotherapeutic intervention.
This notion has been genetically validated in the bloodstream form of T. brucei, in yeast, and
in Candida albicans.

41. The essential events in GPI precursor biosynthesis are, like the core structure, highly conserved.
There are, however, variations on the theme, and T. brucei and mammalian cell GPI pathways are
used here to represent these differences. In all cases, GPI biosynthesis involves the transfer of N-
acetylglucosamine from UDP-GlcNAc to PI to give GlcNAc-PI via an ER-membrane-bound
multiprotein complex. This step occurs on the cytoplasmic face of the ER, as does the second step
of the pathway, the de-N-acetylation of GlcNAc-PI to GlcN-PI.
Notable differences between the T. brucei and mammalian GPI-biosynthetic pathways occur from
GlcN-PI onward. Thus, inositol acylation (the transfer of fatty acid to the C-2 hydroxyl group of
the D-myo-inositol residue) of GlcN-PI strictly follows the action of the first mannosyltransferase
in T. brucei, whereas these steps are temporally reversed in mammalian cells.
In the mammalian pathway, inositol acylation and inositol deacylation are discrete steps that occur
only at the beginning and end of the pathway, respectively, whereas in T. brucei these reactions
occur on multiple GPI intermediates.

42. acid is never removed and the mature GPI protein retains three fatty chains.
Fatty-acid remodeling in T. brucei occurs at the end of the pathway, but before transfer to VSG
protein, and involves exchanging the sn-2 fatty acids (a mixture of C18–C22 species) and
the sn-1 fatty acid (C18:0) exclusively for C14:0 myristate.
In contrast, the lipid remodeling in mammalian cells is more complex. Many protein-linked
GPIs contain sn-1-alkyl-2-acyl-PI with two saturated fatty chains, whereas major cellular PI is
predominantly sn-1-stearoyl-2-arachidonoyl-PI (i.e., with C18:0 and C20:4 fatty acids and few,
if any, alkyl or alkenyl species).
Two processes are involved in these structural changes. First, remodeling from the diacyl-PI to
the 1-alkyl-2-acyl form having unsaturated fatty acid at the sn-2-position occurs in the ER to
GlcN-aPI. The reaction that mediates this remodeling is yet to be determined.
Second, fatty-acid remodeling occurs after GPI is transferred to proteins, the inositol-linked
acyl chain is removed, and GPI-APs are transported to the Golgi. This is accomplished by
exchanging the unsaturated sn-2 fatty acid with saturated fatty acid, mainly stearate (C18:0).
Lipid remodeling of GPIs in yeast also involves two processes but they occur in the ER.
The first is fatty-acid remodeling that exchanges the unsaturated sn-2 fatty acid with the C26:0
chain. The second process involves the exchange of diacylglycerol for ceramide on many, but
not all, GPI proteins.

43. MEMBRANE PROPERTIES OF GPI-APs
GPI-APs with two long hydrocarbon chains (i.e., those containing diacylglycerol,
alkylacylglycerol, alkenylacylglycerol, or ceramide) provide a stable association with the lipid
bilayer. It follows that inositol-acylated GPI proteins with three fatty-acid chains should be
more stably associated.
There are many examples of transmembrane signaling via the cross-linking of GPI-APs with
antibody and clustering with a second antibody on various cells, particularly leukocytes.
Cellular responses include an increase in intracellular Ca
++
, tyrosine phosphorylation,
proliferation, cytokine induction, and oxidative burst.

44. BIOLOGICAL FUNCTIONS OF GPI ANCHORS
GPI anchors are essential for life in some, but not all, eukaryotic microbes. In the yeast S.
cerevisiae, and probably most fungi, the presence of a GPI anchor is used to target certain
mannoproteins for covalent incorporation into the β-glucan cell wall.
Cross-linking occurs via a transglycosylation reaction, whereby a mannose residue within the GPI-
anchor core is transferred to the β-glucan polymer. Defects in cell wall biosynthesis are known to be
detrimental to yeast and this may be why GPI biosynthesis is essential to this organism.
Gene knockout studies have shown that GPI biosynthesis is also essential for the bloodstream form
of T. brucei, even in tissue culture. This may be due to nutritional stress because this parasite uses
an essential GPI-anchored transferrin receptor. On the other hand, surprisingly, GPI biosynthesis
and/or transfer to protein are not essential for the insect-dwelling forms of T. brucei or Leishmania.
The availability of GPI-deficient mammalian cell lines shows that GPI-APs are not essential at a
cellular level. However, mouse knockouts and tissue-specific conditional knockouts of
the PIGA gene (the catalytic subunit of the UDP-GlcNAc:PI α1-6 N-acetylglucosaminyltransferase)
clearly show that GPI-APs are essential for early embryo and tissue development, respectively.
In the plant Arabidopsis, GPI biosynthesis is required for cell wall synthesis, morphogenesis, and
pollen tube development. GPI anchors impart to their attached proteins the ability to be shed in
soluble form from the cell surface through the action of cellular or serum GPI-cleaving enzymes.
Mammalian sperm acquire an ability to fuse with oocytes after GPI-anchored TEX101 is released
by a sperm-associated GPI-cleaving enzyme tACE (testis form angiotensin converting enzyme).
Certain proliferating motor neurons initiate differentiation after a GPI-anchored proteinase inhibitor
RECK is released by a GPI-cleaving enzyme GDE2.

45. When the activity of ADAM10 metalloproteinase suppressed by RECK is expressed, Notch-
ligand is degraded by ADAM10, resulting in Notch signaling termination and a switch from
proliferation to differentiation.
In “lower” eukaryotes, GPI anchors may be useful for assembling particularly dense cell-surface
protein coats, such as the VSG coat of T. brucei. In this case, each parasite expresses five million
VSG dimers on the cell surface to protect it against complement-mediated lysis.
If each VSG monomer had, instead of a GPI anchor, a single transmembrane domain, there
would be little room for other integral membrane proteins such as hexose and nucleoside
transporters.
Generally, GPI-APs do recycle through intracellular compartments but, compared with typical
transmembrane proteins, they reside in higher proportion on the cell surface and have longer
half-lives.
There are several examples of the exchange of GPI-APs from one cell surface to another. Some
GPI-APs are incorporated into exosomes, suggesting a possibility of exosome-mediated cell-to-
cell transfer. Sperm acquire some GPI-APs such as CD52 from epididymis most likely mediated
by exosomes.

46. GPI ANCHORS AND DISEASE
Paroxysmal nocturnal hemoglobinuria (PNH) is a human disease in which patients suffer from
hemolytic anemia.
The condition arises from loss of expression of several GPI-APs that protect their blood cells from
lysis by the complement system (e.g., decay accelerating factor and CD59).
The defect in PNH cells is a somatic mutation in the X-linked PIGA gene and appears to occur in a
bone marrow stem cell. Unlike other enzymes in the pathway, which are encoded by autosomal
genes, PNH caused by PIGA mutations is thought to arise at a higher frequency because of X
inactivation.
Both in male and female stem cells, somatic mutation in the one active allele of PIGA results in the
complete loss of a functional UDP-GlcNAc:PI α1–6 N-acetylglucosaminyltransferase.
Inherited GPI deficiencies (IGDs) are caused by germline mutations in genes involved in GPI
biosynthesis, protein transfer, and remodeling.
Because complete GPI deficiency causes embryonic lethality, mutations in IGDs are hypomorhic,
causing partial deficiency. Mutations in genes involved in GPI remodeling such as PGAP1 can be
null and cause GPI-APs with abnormal structure.
Patients with IGDs caused by mutations in 14 genes in the GPI-biosynthetic pathway have been
reported. Most of these mutations were identified by whole exome sequencing of patients’ cells.
Major symptoms of IGDs are neurological problems such as developmental delay/intellectual
disability, seizures, cerebral and/or cerebellar progressive atrophy, hearing loss, and visual
impairment. Other symptoms include hyperphosphatasia; brachytelepharangy; typical facial
features such as hypertelolism and tented mouth; cleft palate; anorectal, renal, and heart anomalies;
and Hirschsprung disease.

47. GPI biosynthesis and transfer to protein are essential for yeast, for pathogenic fungi, and for the
African sleeping sickness parasite T. brucei as mentioned above.
Several key surface molecules of the apicomplexan
parasites Plasmodium (malaria), Toxoplasma, and Cryptosporidium are GPI-anchored,
and it is thought that the GPI pathway is likely to be essential to these pathogens. Thus,
pathogen-specific GPI pathway inhibitors are being actively sought as potential drugs.
In addition, there is evidence that some parasite GPI anchors have a direct role in modulating the
host immune response to infection.

48. Like other glycoconjugates, GPI-APs can be exploited by pathogens. For example, the GPI
anchors themselves are receptors for hemolytic pore-forming toxins such as aerolysin
from Aeromonas hydrophilia, which causes gastroenteritis, deep wound infections, and
septicemia in humans.
In addition, the GPI-AP CD55/DAF is the principal cell-surface ligand for enterovirus and
several echoviruses.
Finally, the endogenous prion protein is GPI-anchored and it is thought that the
conformational changes that it undergoes to become the aberrant spongiform-
encephalopathy (“mad cow disease” or scrapie in sheep)-causing form may be associated
with a clathrin-independent endocytic pathway followed by GPI-anchored prion protein in
neurons.