The diversity of dolichol-linked precursors to
Asn-linked glycans likely results from secondary
loss of sets of glycosyltransferases
John Samuelson*†, Sulagna Banerjee*, Paula Magnelli*, Jike Cui*, Daniel J. Kelleher‡, Reid Gilmore‡,
and Phillips W. Robbins*
*Department of Molecular and Cell Biology, Boston University Goldman School of Dental Medicine, 715 Albany Street, Boston, MA 02118-2932; and
‡Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester, MA 01665-0103
Contributed by Phillips W. Robbins, December 17, 2004
The vast majority of eukaryotes (fungi, plants, animals, slime mold,
and euglena) synthesize Asn-linked glycans (Alg) by means of a
lipid-linked precursor dolichol-PP-GlcNAc2Man9Glc3. Knowledge of
this pathway is important because defects in the glycosyltrans-
ferases (Alg1–Alg12 and others not yet identified), which make
dolichol-PP-glycans, lead to numerous congenital disorders of
glycosylation. Here we used bioinformatic and experimental
methods to characterize Alg glycosyltransferases and dolichol-
PP-glycans of diverse protists, including many human patho-
gens, with the following major conclusions. First, it is demon-
strated that common ancestry is a useful method of predicting
the Alg glycosyltransferase inventory of each eukaryote. Second,
in the vast majority of cases, this inventory accurately predicts the
dolichol-PP-glycans observed. Third, Alg glycosyltransferases are
missing in sets from each organism (e.g., all of the glycosyltrans-
ferases that add glucose and mannose are absent from Giardia and
Plasmodium). Fourth, dolichol-PP-GlcNAc2Man5(present in Entam-
oeba and Trichomonas) and dolichol-PP- and N-linked GlcNAc2
(present in Giardia) have not been identified previously in wild-
type organisms. Finally, the present diversity of protist and fungal
dolichol-PP-linked glycans appears to result from secondary loss of
glycosyltransferases from a common ancestor that contained the
complete set of Alg glycosyltransferases.
evolution ? N-glycans ? protist
(Alg) by means of a lipid-linked precursor dolichol-PP-
GlcNAc2Man9Glc3(Fig. 1A) (1–3). Each of the 14 sugars is added
to the lipid-linked precursor by means of a specific glycosyltrans-
ferase (Alg1–Alg12 and others as yet specified), which were num-
bered according to the order of their discovery rather than by the
sequence of enzymatic steps (4–6). Defects in these Alg glycosyl-
which can cause dysmorphic features and mental retardation (7).
We and others used the budding yeast Saccharomyces cerevisiae
mutants to characterize many but not all of the Alg glycosyltrans-
ferases, which are present on the cytosolic aspect of the ER. These
glycosyltransferases include Alg7, which adds phospho-GlcNAc to
dolichol phosphate, and Alg1, Alg2, and Alg11, which add the first,
second, and fifth Man residues, respectively (Fig. 1A). Dolichol-
PP-GlcNAc2Man5is flipped into the lumen of the ER by a flippase
and Alg12 make dolichol-PP-GlcNAc2Man9by using dolichol-P-
Man (made by Dpm1) as the sugar donor (Fig. 1A) (1, 9). Also
within the ER lumen, glucosyltransferases Alg6, Alg8, and Alg10
make dolichol-PP-GlcNAc2Man9Glc3 by using dolichol-P-Glc
(made by Alg5) as the sugar donor (Fig. 1A).
An oligosaccharyltransferase (OST), which contains a catalytic
peptide, STT3, transfers the dolichol-PP-linked oligosaccharide to
‘‘sequon’’ Asn residues (N-X-T?S) on nascent peptides (Fig. 1A)
he majority of eukaryotes studied to date (fungi, plants, ani-
mals, slime mold, and euglena) synthesize Asn-linked glycans
to N-glycans of improperly folded proteins, which are retained in
(13). Although the Alg glycosyltransferases in the lumen of ER
appear to be eukaryote-specific, archaea and Campylobacter sp.
glycosylate the sequon Asn and?or contain glycosyltransferases
with domains like those of Alg1, Alg2, Alg7, and STT3 (1, 14–16).
Protists, unicellular eukaryotes, suggest three notable exceptions
to the N-linked glycosylation path described in yeast and animals
(17). First, the kinetoplastid Trypanosoma cruzi (cause of Chagas
myocarditis), fails to glucosylate the dolichol-PP-linked precursor
tid Leishmania mexicana (cause of skin ulcers) lacks the manno-
sylating activities of Alg9 and Alg12 and makes dolichol-PP-
Second, Tetrahymena pyriformis, which is a free-living ciliate, lacks
all of the mannosylating activity in the ER lumen and makes
dolichol-PP-GlcNAc2Man5Glc3(19). Third, it has been difficult to
identify N-glycans from either Giardia lamblia (cause of diarrhea)
or Plasmodium falicparum (cause of severe malaria) (20–22).
Because these observations of unique protist glycans were
made before identification of multiple Alg glycosyltransferases
and?or whole-genome sequencing of these protists, numerous
important questions remain concerning the diversity of N-glycan
precursors among eukaryotes. First, are the same Alg glycosyl-
transferases conserved across all eukaryotes, and are these Alg
glycosyltransferases specific to eukaryotes or are they also
present in prokaryotes? Second, are kinetoplastids [Trypano-
soma cruzi and Trypanosoma brucei (cause of African sleeping
sickness)] missing the genes encoding the glucosylating enzymes
or are the genes present but silent, and similarly, is Tetrahymena
missing the set of genes encoding mannosylating enzymes in the
lumen of the ER? Third, what Alg glycosyltransferases are
missing from Giardia and Plasmodium? Fourth, what is the
diversity of predicted Alg glycosyltransferases of other protists
(e.g., Entamoeba histolytica, Trichomonas vaginalis, Toxoplasma
gondii, and Cryptosporidium parvum, which cause dysentery,
vaginitis, birth defects, and diarrhea, respectively), and what are
the Alg glycosyltransferases of Encephalitozoon cuniculi (an
opportunistic fungus with a dramatically reduced genome) and
Cryptococcus neoformans (an opportunistic fungus that is dis-
tantly related to Saccharomyces)? Fifth, do the predicted Alg
glycosyltransferases correlate with the dolichol-PP-linked gly-
cans of each protist or fungus? Sixth, does the pattern distribu-
tion of Alg glycosyltransferases across protists, fungi, and meta-
zoa suggest whether these glycosyltransferases have been added
to or lost from eukaryotes during their evolution (23, 24)?
Freely available online through the PNAS open access option.
Abbreviations: Alg, Asn-linked glycan; OST; oligosaccharyltransferase; TMH, transmem-
†To whom correspondence should be addressed: E-mail: email@example.com.
© 2005 by The National Academy of Sciences of the USA
February 1, 2005 ?
vol. 102 ?
Materials and Methods
Use of Bioinformatics to Identify Alg Glycosyltransferases from Di-
verse Protists and Fungi. Twelve Saccharomyces Alg glycosyltrans-
ferases, DPM1, and STT3 were used to search predicted proteins
of eukaryotes, which have been sequenced in their entirety or
near entirety (Plasmodium falciparum, Encephalitozoon cuniculi,
Cryptosporidium parvum, Giardia lamblia, Homo sapiens, and
Arabidopsis thaliana) in the NR protein database of the National
Center for Biotechnology Information by using PSI-BLAST (25–
30). BLASTP or TBLASTN searches of Entamoeba histolytica,
Trichomonas vaginalis, Trypanosoma brucei, Trypanosoma cruzi,
and Tetrhymena thermophilia were performed at web sites man-
aged by The Institute for Genomic Research (www.tigr.org).
Predicted proteins of the slime mold Dictyostelium discoideum
and kinetoplastid Leishmania major were performed on the
Sanger Institute GENEDB web site (www.genedb.org), and the
apicomplexan Toxoplasma gondii-predicted proteins were
searched at TOXODB (http:??ToxoDB.org). Transmembrane he-
lices were predicted by using the Phobius combined transmem-
brane topology and signal peptide predictor (31).
Alignments of protein sequences were made by using CLUSTALW
(http:??www.ebi.ac.uk?clustalw), and manual adjustments and
trimming of the alignments were performed with JALVIEW (32).
Phylogenetic trees were constructed from the positional variation
with maximum likelihood by using quartet puzzling (33, 34).
Identification of Dolichol-PP-Linked Precursors and N-Linked Glycans.
Genome project strains of Entamoeba histolytica, Trichomonas
vaginalis, Giardia lamblia and Cryptococcus neoformans were
grown axenically and labeled with 200 ?Ci (1 Ci ? 37 GBq)
[2-3H]Man, [6-3H]GlcN, or [3H]Glc in a Glc-free medium for 10
min in a final volume of 250 ?l (6). Dolichol-PP-linked glycans
were extracted with chloroform?methanol?water, dried, and
hydrolyzed in 0.1 M HCl for 45 min at 90°C. Glycans were
for separation on a 1-m Biogel P-4 superfine column (BioRad).
Standards were GlcNAc2Man5 from a Saccharomyces alg3?
mutant incubated with14C-Man, GlcNAc2Man9from a Saccha-
romyces alg6? mutant, and unlabeled GlcNAc and GlcNAc2.
For identification of N-glycans, Entamoeba, Trichomonas, and
Giardia were labeled with mannose and GlcN for 2 h in medium
containing 0.1% glucose before washing and lyophilization. The
dry-cell pellet was delipidated with chloroform?methanol?water,
glycosylphosphatidylinositol precursors were removed with water-
saturated butanol, and glycogen and other free glycans were
removed with 50% methanol (35). The clean pellet was finely
resuspended by using a manual homogenizer in 500 ?l Tris?HCl
(0.1M, pH 8) and incubated with 50 milliunits of peptide-N-
glycosidase F (PNGaseF) at 37°C for 16 h. Negative controls
omitted the PNGaseF, and the peptide:N-glycanase supernatant
was chromatographed on a P-4 column. Radioactivity was mea-
sured by scintillation counting, and peaks were isolated for treat-
ment with glycosidases. The putative GlcNAc2Man5from Entam-
oeba and Trichomonas and putative GlcNAc2Man7 from
Cryptococcus were treated with ?-1,2-mannosidase, whereas the
putative GlcNAc2of Giardia was cleaved with chitiobiase.
In Vitro Synthesis of Glycopeptides by Using Intact Protist Membranes
as a Source of Dolichol-PP-Glycans.Total cellular membranes were
prepared from cultures of Trichomonas, Entamoeba, and Cryp-
tococcus. The membranes were incubated for 2–90 min at 37°C
with the membrane permeable tripeptide acceptor, 5 ?M
N?-Ac-Asn-[125I]-Tyr-Thr-NH2 (NYT), in the presence of
deoxynojiromycin to ensure that the glycopeptide products
were not degraded by glucosidases I and II (36). Glycopeptide
products were collected by binding to immobilized Con A and
separated on HPLC by using standards from Saccharomyces
that included Man5GlcNAc2-NYT, Man9GlcNAc2-NYT,
Glc3Man5GlcNAc2-NYT, and Glc3Man9GlcNAc2-NYT (37).
Results and Discussion
A Common Ancestor of Eukaryotes and Archaea May Have Contained
STT3 and Alg7, but the Remaining Alg Glycosyltransferases Appear to
Be Eukaryote-Specific. SimilaritiesbetweeneukaryoticcytosolicAlg
glycosyltransferases (Alg1, Alg2, and Alg7) and STT3 and their
Leishmania major, and Cryptococcus neoformans (B), Tetrahymena thermophilia, Toxoplasma gondii, and Cryptosporidium parvum (C), Entamoeba histolytica
and Trichomonas vaginalis (D), Plasmodium falciparum and Giardia lamblia (E), and Encephalitozoon cuniculi (F) (see also Table 1). With the exceptions of
(e.g., Saccharomyces), are indicated in black. Names of organisms, whose dolichol-PP-linked glycans were identified here (e.g., Cryptococcus), are indicated in
red. Names of organisms, whose dolichol-PP-linked glycans have not yet been identified (e.g., Cryptosporidium), are indicated in green.
The inventory of Alg glycosyltransferases and predicted dolichol-linked glycans vary dramatically among protists and fungi. Predicted Alg glycosyl-
Samuelson et al.
February 1, 2005 ?
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no. 5 ?
prokaryotic counterparts suggest their common origin (1, 24), but
phylogenetic methods have not been used to test this idea. STT3 is
present in all eukaryotes examined except Encephalitozoon (Fig. 1,
Table 1, and see below) and is present in multiple copies in some
soma brucei, and four in Leishmania (data not shown)]. Homo-
logues of STT3 are also present in the bacterium Campylobacter
jejuni and both divisions of archaea, euryarchaeota and crenarcha-
eota (16). The hydrophobicity plots of the eukaryotic and prokary-
otic STT3 closely resemble each other, each containing 10 to 15
predicted transmembrane helices (data not shown). In addition,
over a ?700-amino acid (90%) overlap and a 19–23% positional
identity with prokaryotic STT3 over a ?600-amino acid (80%)
overlap. Phylogenetic analyses of STT3 show distinct eukaryotic
and archael clades, although it was not possible to determine
whether eukaryotic STT3 are more similar to homologues of
euryarchaeota or crenarchaeota (data not shown). The Campy-
lobacter STT3 gene appears to have been laterally transferred from
are consistent with the idea that a common ancestor to eukaryotes
and archaea contained STT3.
Alg7, which is a UDP-GlcNAc:dolichol-phosphate GlcNAc-
1-phosphate transferase, is the first enzyme in the synthesis of
dolichol-PP-linked glycans and is present in all eukaryotes
examined except Encephalitozoon (Fig. 1, Table 1, and see
below). Proteins similar to Alg7 are predicted from whole-
genome sequences of some but not all archaea and bacteria. The
hydrophobicity plots of the eukaryotic and prokaryotic Alg7
closely resemble each other, each containing 8 to 12 predicted
transmembrane helices (data not shown). Eukaryotic Alg7 show
a 28–40% positional identity with each other over an ?310-
amino acid (80%) overlap and show an ?19–23% positional
overlap. Phylogenetic analyses of Alg7 show distinct eukaryotic,
archaeal, and bacterial clades (Fig. 2A). Although eukaryote
Alg7 are much more similar to archaeal than bacterial homo-
Alg7 was more similar to those of euryarchaeota or crenarcha-
eota. These results and the presence of dolichol-PP-linked
glycans in archaea (14) are consistent with the idea that a
common ancestor to eukaryotes and archaea contained Alg7.
5th-mannose residues to the dolichol-PP-linked precursor, respec-
tively, ?50% of each eukaryotic protein is alignable with homo-
logues of prokaryotes, and Alg1, Alg2, and Alg11 each have
transmembrane domains that are absent from prokaryotic glyco-
syltransferases (data not shown). Phylogenetic analyses show eu-
karyotic Alg1, Alg2, and Alg11 form distinct clades, which are well
supported by bootstrap values (Fig. 2B). The relationship of eu-
karyotic Alg1, Alg2 and Alg11 to archaeal and bacterial glycosyl-
transferases, however, is unresolved, suggesting that cytosolic man-
nosylating enzymes are eukaryote-specific and that their precise
origins are not clear. These results then do not support the recent
hypothesis that the set of cytosolic Alg glycosyltransferases were
present in an archaeal ancestor of eukaryotes (24).
In the case of Alg5 and Dpm1 that make dolichol-P-Glc and
dolichol-P-Man, respectively (9), phylogenetic analyses show Alg5
and Dpm1 form distinct clades, which are well supported by
into two clades. Dpm1 clade A contains enzymes with a C-terminal
transmembrane helix (TMH) (Saccharomyces, Entamoeba,
Trypanosoma, and Leishmania). Dpm1 clade B contains enzymes
with no TMH (plants, animals, and fungi) or an N-terminal TMH
(Plasmodium). Numerous organisms lacking TMH in their Dpm1
have Dpm2 homologues, which contain two predicted TMH and
Plasmodium, and Entamoeba, which contain TMH in their Dpm1,
no Dpm1 but has multiple copies of Alg5.
Phylogenetic methods also show that Alg glycosyltransferases in
the lumen of the ER, which are unique to eukaryotes but are often
similar to each other (e.g., Alg6 and Alg8 or Alg9 and Alg12), may
Alg3 and Alg10, which are present only in eukaryotes, are not
similar to other Alg glycosyltransferases. In organisms that contain
them, luminal Alg glycosyltransferases are single-copy. These re-
glycosyltransferase repertoire may be used to correctly predict the
dolichol-PP-linked glycans made by each organism.
Sets of Alg Glycosyltransferases Correlate Precisely with Known
Dolichol-PP-Linked Glycans. Alg glycosyltransferases were examined
first from organisms from which dolichol-PP-linked precursors
have been characterized. The slime mold Dictyostelium discoideum,
which makes dolichol-PP-GlcNAc2Man9Glc3, contains all 12 Alg
glycosyltransferases that have been molecularly characterized (Fig.
dolichol-PP-GlcNAc2Man9, and Trypanosoma brucei are missing
the set of genes encoding glucosylating enzymes in the ER lumen
(Fig. 1B) (18). Leishmania major, which causes visceral leishman-
iasis, is also missing the Alg12 gene and likely makes dolichol-PP-
GlcNAc2Man7. The ciliate Tetrahymena thermophilia, which makes
dolichol-PP-GlcNAc2Man5Glc3as described for Tetrahymena pyri-
formis (ref. 19; unpublished data), is lacking the set of genes
encoding mannosylating enzymes in the ER lumen (Fig. 1C).
Plasmodium falciparum, from which it has been difficult to identify
N-glycans, is missing all of the Alg glycosyltransferases except Alg7
and STT3 (Fig. 1E) (21, 22). The absence of the other Alg
glycosyltransferases makes it likely that putative mannosylated
N-glycans of Plasmodium were contaminants from host cells (38).
Table 1. Predicted Alg glycosyltransferases of representative eukaryotes
Cytosol GlcNAcCytosol Man ER lumen ManER lumen GlcOST
Alg7 Alg1 Alg2Alg11Rft1Alg3Alg9Alg12Dpm1 Alg5 Alg6 Alg8Alg10STT3*
Sc, Saccharomyces cerevisiae; Hs, Homo sapiens; Dd, Dictyostelium discoideum; Tb, Trypanosoma brucei; Tc, Trypanosoma cruzi; Lm, Leishmania mexicana;
Cn, Cryptococcus neoformans; Eh, Entamoeba histolytica; Tv, Trichomonas vaginalis; Tt, Tetrahymena thermophilia; Tg, Toxoplasma gondii; Cp, Cryptosp
parvum; Pf, Plasmodium falciparum; Gl, Giardia lamblia; Ec, Encephalitozoon cuniculi.
*No. of STT3 subunits in each organism.
www.pnas.org?cgi?doi?10.1073?pnas.0409460102 Samuelson et al.
(18, 19) is caused by the absence of the gene encoding the enzyme
rather than an inactive gene so that sets of predicted Alg glycosyl-
transferases correlate precisely with experimentally determined
Whole-Genome Sequences of Select Protists and Fungi Predict Addi-
tional Diversity in Alg Glycosyltransferases and Subsequently Doli-
neoformans is lacking the set of glucosylating enzymes in the ER
lumen and is predicted to make dolichol-PP-GlcNAc2Man9(Fig.
1B, Table 1, and see below) (18). Like Tetrahymena to which it is
the set of luminal mannosylating enzymes (Fig. 1C) (19). Crypto-
PP-GlcNAc2Man5Glc (Table 1). Entamoeba histolytica and
ferases that add Man and Glc to lipid-linked precursors and likely
make dolichol-PP-GlcNAc2Man5(Fig. 1D and see below).
Like Plasmodium, Giardia lamblia is missing all Alg glycosyl-
transferases except Alg7 and STT3 (Fig. 1E, Table 1, and see
below). The presence of Rft1 in all eukaryotes except Giardia,
Plasmodium, and Encephalitozoon (see below) supports the idea
that Rft1 flips dolichol-PP-GlcNAc2Man5into the lumen of the
ER (8). Why Toxoplasma is also missing Rft1 is not clear.
Encephalitozoon cuniculi, whose genome has been sequenced
in its entirety, lacks all Alg glycosyltransferases and is missing
STT3 (Fig. 1F and Table 1) (28). Consistent with the absence of
N-glycans, Encephalitozoon, like Plasmodium and Giardia, is
missing UDP-Glc:glycoprotein glucosyltransferase, glucosidases
I and II, calreticulin?calnexin, ERGIC-53, and ?-1,2-
mannosidases, which operate on N-linked glycans in the ER and
Golgi apparatus (unpublished data) (13, 24).
Entamoeba and Trichomonas Make the Predicted Dolichol-PP-
GlcNAc2Man5, Whereas Cryptococcus Makes Dolichol-PP-GlcNAc2-
Man7–9. The major dolichol-PP-linked glycan of Entamoeba his-
tolytica and Trichomonas vaginalis contains GlcNAc2Man5,
which was predicted from the Alg glycosyltransferases of these
protists (Fig. 3 A and B). Each peak digests with ?-1,2-
mannosidase to GlcNAc2Man3and mannose (data not shown).
To show that the labeled dolichol-PP-linked precursor is the
same as that transferred to nascent peptides by the OST,
membranes of Entamoeba and Trichomonas were incubated with
an iodinated tripeptide NYT. As expected, the products of
Entamoeba and Trichomonas in vitro comigrate with the
GlcNAc2Man5standard (Fig. 3 D and E). Finally, when Enta-
moeba and Trichomonas are briefly labeled with3H-Man in vivo
and N-glycans are released with PNGaseF, a major product is
GlcNAc2Man5(data not shown).
have not shown its glycosyltransferase activity in vitro, correct
predictions of previously uncharacterized dolichol-PP-linked gly-
cans of Entamoeba and Trichomonas strongly suggest each Alg
glycosyltransferase is functioning as expected. The presence of
UDP-Glc:glycoprotein glucosyltransferase, glucosidase II, calreti-
culin?calnexin, and ERGIC-53 in Entamoeba and Trichomonas
(unpublished data) suggests these enzymes and?or lectins function
with N-glycans built on GlcNAc2Man5 rather than the usual
When the fungus Cryptococcus neoformans is labeled with
3H-Man, the major peak on P-4 runs with GlcNAc2Man7–8,
whereas a minor peak runs with GlcNAc2Man9, which was
predicted from their complement of Alg genes (Fig. 3C). How-
ever, GlcNAc2Man9is the most abundant glycan transferred to
the iodinated peptide in vitro (Fig. 3F).
maximum likelihood method of representative eukaryotic and prokaryotic Alg7 (A) and Alg1, Alg2, and Alg11 (B). Branch lengths are proportionate to
differences between sequences, and numbers at nodes indicate bootstrap values for 100 replicates. Eukaryotes include Arabidopsis thaliana (At), Cryptococcus
major (Lm), Plasmodium falciparum (Pf), Saccharomyces cerevisiae (Sc), Schizosaccharomyces pombe (Sp), Tetrahymena thermophilia (Tt), Toxoplasma gondii
acidarmanus (Fa), Methanococcoides burtonii (Mb), Methanococcus jannaschii (Mj), Methanopyrus kandleri (Mk), Methanosarcina mazei (Mm), Picrophilus
torridus (Pt), Pyrococcus abyssi (Pab), Pyrococcus furiosus (Pfu), Pyrococcus horikoshii (Ph), and Crenarchaeota Pyrobaculum aerophilum (Pae), Sulfolobus
solfataricus (Ss), and Sulfolobus tokodaii (St). Bacteria include Actinobacillus actinomycetemcomitans (Aa), Bifidobacterium longum (Bl), Borrelia garinii (Bg),
(Spn), Synechococcus elongates (Se), Thermus thermophilus (Tth), and Tropheryma whipplei (Tw). Not all organisms are present in each tree.
Common ancestry is a useful method of predicting the Alg glycotransferase inventory of each eukaryote. Phylogenetic reconstructions by using the
Samuelson et al.
February 1, 2005 ?
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no. 5 ?
Giardia Dolichol-PP- and N-Linked Glycans Contain GlcNAc1–2. No
3H-Man, consistent with the absence of cytosolic Alg glycosyl-
transferases that add Man to dolichol-PP-linked precursors (Fig.
1E and Table 1). In contrast, when Giardia is labeled with
3H-GlcN, dolichol-PP- and N-linked glycans include GlcNAc
and GlcNAc2(diacetylchitobiose) (solid lines in Figs. 4A and
4B). As expected, Giardia dolichol-PP- and N-linked GlcNAc2
are cleaved with chitobiase to GlcNAc (dotted lines in Fig. 4 A
and B). These results, which suggest there is no further modifi-
cation of N-glycans in the Golgi apparatus of Giardia, are
consistent with (i) binding of wheat germ agglutinin to the
surface of Giardia (39), and (ii) the prediction that ?90 secreted
proteins of Giardia have ?10 predicted sites for N-linked
glycosylation (unpublished data) (10). These results suggest that
the Alg enzyme that makes dolichol-PP-GlcNAc2, which has not
yet been molecularly characterized, is present in Giardia (1).
Origins of Eukaryotic Alg Glycosyltransferase Diversity. The present
diversity of fungal and protist precursors for N-glycosylation may
have resulted from development of an increasingly complex
series of Alg glycosyltransferases with eukaryotic evolution (Fig.
5A) or from secondary loss of sets of Alg glycosyltransferases
(Fig. 5B) (23, 24). Fig. 5A is drawn so that organisms with the
least complex N-glycans are at the bottom, whereas those with
the most complex glycans are at the top. Each step in Fig. 5A
indicates the addition of a particular set of sugars to the N-glycan
precursor. For example, an ancestral eukaryote like Encephali-
tozoon lacked all dolichol-PP-linked precursors (step 1) until
STT3 and Alg7 were obtained to make organisms like Giardia
and Plasmodium (step 2). Subsequently cytosolic mannosylating
Trichomonas vaginalis (A), Entamoeba histolytica (B), and Cryptococcus neoformans (C), as well as in vitro OST assays by using membranes from Trichomonas
(D), Entamoeba (E), and Cryptococcus (F). In A–C, glycans were labeled in vivo with [3H]Man and separated on a P-4 column, whereas in D–F, glycans were
transferred to a radio-iodinated tripeptide NYT in vitro, captured with Con A, and separated by HPLC.
Predicted Trichomonas, Entamoeba, and Cryptococcus dolichol-PP-glycans were identified in vivo and in vitro. Dolichol-PP-linked precursors from
GlcNAc2. Dolichol-PP-linked glycans (A) and N-linked glycans (B) of Giardia
lamblia, each labeled in vivo with [3H]GlcN and separated on a P-4 column.
digestion of the excised GlcNAc2peak with chitobiase. Giardia dolichol- and
N-linked glycans are composed of GlcNAc and GlcNAc2.
Giardia dolichol- and N-linked glycans are composed of GlcNAc and Fig. 5.
appears to result from secondary loss glycosyltransferases from a common
ancestor that contained the complete set of Alg glycosyltransferases. Models
suggesting sequential addition (A) or secondary loss (B) of Alg glycosyltrans-
ferases during eukaryotic evolution are shown. Nodes, which are labeled
numerically in A and alphabetically in B, are explained in the text.
The present diversity of protist and fungal dolichol-PP-lined glycans
www.pnas.org?cgi?doi?10.1073?pnas.0409460102Samuelson et al.
enzymes were added to make ancestors resembling Entamoeba
and Trichomonas (step 3), followed by the addition of luminal
mannosylating enzymes as in Trypanosoma and Cryptococcus
(step 4) (18) or of luminal glucosylating enzymes as in Tetrahy-
mena and Cryptosporidium (step 5) (19). The final result was
organisms with an entire set of Alg glycosyltransferases and a
complete 14-sugar dolichol-PP-linked precursor (step 6) as in
Saccharomyces, Euglena, Dictyostelium, animals, and plants (1).
The difficulties with the Fig. 5A model are (i) Alg7 and STT3
appear to have been present in an ancestor to both eukaryotes
and prokaryotes and should be present in Encephalitozoon (16),
(ii) it is impossible to determine whether luminal mannosylation
(step 4) came before or after luminal glucosylation (step 5), and
(iii) the model is in disagreement with rRNA and protein
phylogenies, which do not place Encephalitozoon at the base of
the phylogenetic tree and do not pair Giardia with Plasmodium,
Entamoeba with Trichomonas, Trypanosoma with Cryptococcus,
or Saccharomyces with Euglena (40–42).
In the Fig. 5B model, which groups organisms according to
rRNA and protein phylogenies, the distribution of Alg glyco-
syltransferases is best rationalized by secondary loss (23). At
node a (fungi), there is loss of luminal glucosylating enzymes in
Cryptococcus and all Alg glycosyltransferases in Encephalito-
zoon, whereas at node b (amebozoa), all luminal Alg glycosyl-
transferases are lost from Entamoeba. At node c (ciliates and
apicomplexa), some luminal glucosylating enzymes are lost from
Cryptosporidium, whereas all luminal glucosylating enzymes and
all cytosolic mannosylating enzymes are lost from Plasmodium.
At node d (kinetoplastids and Euglena), there is loss of luminal
glucosylating enzymes from Trypanosoma and an additional loss
of one mannosylating enzyme from Leishmania major. If one
assumes that Trypanosoma, Giardia, and Trichomonas all
branched at the same time from the base of the tree (node e),
there are additional secondary losses from Giardia and
Trichomonas. Finally, if one assumes the ‘‘big bang’’ hypothesis
for eukaryotic origins (43), the common ancestor must have had
all of the Alg glycosyltransferases, and all of the differences
among extant eukaryotes are due to secondary loss.
A hybrid of the Fig. 5 models, which we cannot rule out
because of poor resolution at the base of the eukaryotic phylo-
genetic tree, suggests that a common eukaryotic ancestor con-
tained Alg7 and STT3. In this hybrid model, Giardia branched
off before acquisition of mannosylating and glucosylating en-
a–d remain a major factor in the diversity of N-glycan precursors
among protists and fungi. Similarly, secondary loss explains the
absence of most mitochondrial function in microaerophilic pro-
tists such as Giardia, Entamoeba, and Trichomonas (44, 45).
and fungus, which we confirmed for free-living organisms (Giardia,
Entamoeba, Trichomonas, and Cryptococcus) and have yet to con-
firm for intracellular pathogens (Plasmodium, Toxoplasma, Cryp-
assumed that the diversity of eukaryotic N-glycans results primarily
from differential modification of a common GlcNAc2Man9Glc3
precursor in the ER and Golgi apparatus (1, 24), these results
suggest that there are major differences in the dolichol-PP-glycans
transferred to the nascent peptide. Secondary losses of Alg glyco-
syltransferases and mitochondrial function (44, 45) suggest all
extant eukaryotes may derive from a relatively complex last com-
mon ancestor, and that simple, deeply branching eukaryotes with a
primary absence of important biochemical pathways may no longer
exist (23). It remains to be determined how the diversity of
dolichol-PP-glycans effects OST function, protein-folding in the
ER, and modification of glycans in the Golgi apparatus, as well as
the antigenicity of glycoproteins on surfaces of these important
We thank Ann-Marie Surette and Charles Specht for help with protist
and fungal cultures; Kosuke Hashimoto for help with collection and
alignment of Alg sequences; Prashanth Vishwanath for advice on
phylogenetic methods; investigators at The Institute for Genomic Re-
search and The Sanger Institute for release of preliminary sequence data
for numerous protists and fungi; and Temple Smith and Armando Parodi
for their comments on this manuscript. This work was supported in part
by National Institutes of Health Grants AI44070 and AI48082 (to J.S.),
GM43768 (to R.G.), and GM31318 (to P.W.R.).
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