Common Origin and Evolution of Glycosyltransferases Using
Dol-P-monosaccharides as Donor Substrate
Rafael Oriol, Ivan Martinez-Duncker, Isabelle Chantret, Rosella Mollicone,
and Patrice Codogno
INSERM U504, University of Paris Sud XI, Villejuif, France
On the basis of the analysis of 64 glycosyltransferases from 14 species we propose that several successive duplications of a common ancestral gene, followed by divergent evolution, have generated the mannosyltransferases and
the glucosyltransferases involved in asparagine-linked glycosylation (ALG) and phosphatidyl-inositol glycan anchor
(PIG or GPI), which use lipid-related donor and acceptor substrates. Long and short conserved peptide motifs were
found in all enzymes. Conserved and identical amino acid positions were found for the a2/6- and the a3/4mannosyltransferases and for the a2/3-glucosyltransferases, suggesting unique ancestors for these three superfamilies. The three members of the a2-mannosyltransferase family (ALG9, PIG-B, and SMP3) and the two members
of the a3-glucosyltransferase family (ALG6 and ALG8) shared 11 and 30 identical amino acid positions, respectively, suggesting that these enzymes have also originated by duplication and divergent evolution. This model
predicts a common genetic origin for ALG and PIG enzymes using dolichyl-phospho-monosaccharide (Dol-Pmonosaccharide) donors, which might be related to similar spatial orientation of the hydroxyl acceptors. On the
basis of the multiple sequence analysis and the prediction of transmembrane topology we propose that the endoplasmic reticulum glycosyltransferases using Dol-P-monosaccharides as donor substrate have a multispan transmembrane topology with a first large luminal conserved loop containing the long motif and a small cytosolic
conserved loop containing the short motif, different from the classical type II glycosyltransferases, which are
anchored in the Golgi by a single transmembrane domain.
Introduction
Asparagine-linked glycans (N-glycans) are engaged
in a large panel of cellular and tissular functions (Varki
1993). They are involved in (1) quality control of proteins synthesized in the endoplasmic reticulum (ER)
(Helenius and Aebi 2001), (2) intracellular trafficking of
some glycoproteins along the secretory pathway (Hauri
et al. 2000), (3) lysosomal delivery of acid hydrolases
(Kornfeld 1990), (4) membrane targeting of some proteins in polarized cells (Scheiffele, Peränen, and Simons
1995), and (5) they act as recognition signals at the cell
surface and participate in morphogenesis and development (Takahashi, Honda, and Nishikawa 2000).
The process of N-glycosylation starts in the cytosolic face of the ER by the successive assembly of two
N-acetylglucosamines and five mannoses on the lipid
carrier dolichylpyrophosphate. Then the lipid-linked
heptasaccharide is translocated across the membrane to
the lumen of the ER by the RFT1 flippase (Helenius et
al. 2002), where consecutive additions of four residues
of mannose and three glucose residues complete the oligosaccharide, which is in turn transferred en bloc from
the dolichol carrier to asparagine, in the sequence AsnX-Ser/Thr of the nascent protein chain. The N-glycosylation process is completed by a series of trimming
Abbreviations: ALG, Asparagine-Linked Glycosylation; CAZy,
Carbohydrate Active enZyme database; CDG, Congenital Disorder of
Glycosylation; Dol, dolichol; ER, endoplasmic reticulum; Man, mannose; Glc, glucose; GlcNAc, N-acetylglucosamine; PIG, PhosphatidylInositol Glycan anchor (GPI-anchor); TMD, transmembrane domain.
Key words: glucosyltransferase, GPI-anchor, mannosyltransferase,
N-glycan, transmembrane topology, phylogeny.
Address for correspondence and reprints: Rafael Oriol, INSERM
U504, University of Paris Sud XI, 16 Avenue Paul Vaillant-Couturier,
94807 Villejuif Cedex, France. E-mail: [email protected].
Mol. Biol. Evol. 19(9):1451–1463. 2002
q 2002 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038
and processing reactions ending in the late Golgi compartment (Kornfeld and Kornfeld 1985; Burda and Aebi
1999).
The assembly of the initial heptasaccharide on the
cytosolic face of the ER is carried out by glycosyltransferases using UDP-GlcNAc or GDP-Man as donor substrates, whereas the sequential addition in the ER lumen
of the following four mannoses and three glucoses is
carried out by glycosyltransferases using Dol-P-Man
and Dol-P-Glc as donor substrates (fig. 1). These last
two donor substrates are synthesized in the cytosol from
GDP-Man and UDP-Glc. The existence of specific flippases able to transport the dolichol-based donor substrates from the cytosol to the luminal side of the membrane has been proposed but they have not yet been
identified (Schenk, Fernandez, and Waechter 2001), and
it is possible that more than one protein contributes to
these flippase activities (Anand et al. 2001).
The phosphatidyl-inositol glycan anchor (PIG or
GPI) is a posttranslational modification frequently observed in proteins expressed in the plasma membrane
(Cole and Hart 1997). GPI is synthesized in the ER
membrane, where it substitutes the C-terminus of some
newly synthesized proteins. The four mannoses of the
PIG anchor are also added in the ER (Maeda et al.
2001). The first mannose is added by PIG-M which is
an a4-mannosyltransferase, the second mannose is added by an a6-mannosyltransferase which has not yet been
cloned, and the third and fourth mannoses are added by
the PIG-B and SMP3 a2-mannosyltransferases, respectively. These four enzymes use the same Dol-P-Man donor substrate and make linkages similar to those of the
corresponding asparagine-linked glycosylation (ALG)
mannosyltransferases of the N-glycan series (fig. 1). In
spite of these functional similarities, the ALG and PIG
mannosyltransferases have always been considered sep1451
1452
Oriol et al.
FIG. 1.—Comparative structures of the oligosaccharides of ALG and PIG series. Empty symbols of ALG represent the initial heptasaccharide
(GlcNAc2Man5) assembled in the cytosol by the glycosyltransferases using nucleotide-sugar donor substrates. Solid symbols of the ALG and
PIG series are Man and Glc units added by the glycosyltransferases using Dol-P-monosaccharides as donor substrates in the lumen of the ER.
The numbers inside the symbols indicate the order of sequential addition of the cytosolic GlcNAc (1–2), the cytosolic Man (1–5), the luminal
Man (6–9) and the luminal Glc (1–3) of ALG, and the luminal Man (1–4) of PIG. The dashed line represents the a-linkage made by PIG-M,
between the OH of C1 in the first mannose of PIG and the OH of C4 in glucosamine. This linkage has an equatorial orientation similar to that
of the a1→3 linkage made by ALG3 (fig. 9). Enzymes not cloned are represented by a question mark. The same ALG9 enzyme is supposed
to make both branches of N-glycans, but a different ALG9-like enzyme may exist.
arately, and no attempts had been made, in the past, to
find a common evolutionary path for these two series of
enzymes.
The aims of this work were (1) to find conserved
peptide motifs, potential candidates for interaction with
acceptor or donor substrates, (2) to find eventual relations between ALG and PIG mannosyltransferases using
the same donor substrate, and (3) to see if some common characters of these enzymes could be related to
structural features of the linkage to the acceptor substrates and if these could help to understand their evolutionary path.
Materials and Methods
Nomenclature
We consider that the glycosyltransferases which
use the same donor and acceptor substrates and make
the same sugar linkage (i.e., a2-, a3-, a4- or a6-mannosyltransferases and a2- or a3-glucosyltransferases)
belong to the same family and that families sharing
common peptide motifs, identical amino acid positions, and similar transmembrane domain (TMD) topology can be clustered in superfamilies (i.e., a2/6mannosyltransferases, a3/4-mannosyltransferases, or
a2/3-glucosyltransferases).
Sequence Retrieval
Saccharomyces cerevisiae or human (or both)
DNA and protein glycosyltransferase sequences were
retrieved from the literature. Orthologous protein sequences from other species were first searched with
gapped-BLAST. The cloning of human ALG12
(AJ303120) has been reported by Chantret et al.
(2002). The human EST databank was searched with
rat alg10 and with S. cerevisiae rft1 (U15087). Contigs
were built for both series of human ESTs by alignment
with HUCAP (Huang 1992). Primers were designed in
the EST contigs (sense 59-CAGGAGTAGGTTCTTGGGCAGTGGC-39 and antisense 59-AATGTAATTTGAAGACCACCACTGCACC-39 for ALG10, and
sense 59-GGCATTTCCTGGTGTCTGAGCCTG-39
and antisense 59-CACAGAACTACCCATAGCTGGTCC-39 for RFT1). The corresponding human 1,582-bp
ALG10 (AJ312278) and 1,761-bp RFT1 (AJ318099)
cDNA sequences were amplified from an expression
library (Cailleau-Thomas et al. 2000), and they were
cloned in a PCR3.1 expression vector (Eukaryotic TA
cloning kit from Invitrogen), and sequenced in both
strands by the dideoxy chain termination method with
the T7 DNA polymerase (kit Amersham-PharmaciaBiotech) and submitted to EMBL. The cDNA sequences of the coding sequences of Mus musculus alg12,
Drosophila melanogaster alg10 and Caenorhabditis
elegans pig-B were deduced from HUCAP alignments
made with more than two concordant EST overlaps per
position.
Conserved Peptide Motifs
After retrieval of all the sequences, the best-conserved peptide patterns for each family were determined
with CLUSTALW 1.8 (Thomson, Higgins, and Gibson
1994) and used in a final search with the corresponding
human sequence in an iterative PHI-BLAST search with
default parameters, until convergence (Altschul et al.
1997). The seed expression patterns for the PHI-BLAST
were: H[KQ]EXRF[ILMV][IYFLSV][YPLV] for the a2/
6-mannosyltransferase superfamily; [VL]X[YF]T[DEK]
[IV]D[YW]X[VIAT] for the a3/4-mannosyltransferase superfamily; W[GT]LDYPP[LF][TF]A[FYW] for the a3glucosyltransferase family; and LADNRH[YF][TL]FY
for the a2-glucosyltransferase family. A combined PHIPSI-BLAST was also used for the a2/3-glucosyltrans-
Phylogeny of ALG and PIG Glycosyltransferases
Table 1
ALG and PIG Mannosyltransferases and Glucosyltransferases Using
Dol-P-Monosaccharide as Donor Substrate (64 enzymes)
ENZYME (transfer)
Species
DNA
GENBANK/
EBI
PROTEIN
SWISSPROT ALL
(SWALL)
SIZE
Amino
Acid
TMD
ALG3 (a3-mannosyltransferase)
Neurospora crassa . . . . . . . . . . . .
Saccharomyces cerevisiae . . . . . .
Schizosaccharomyces pombe . . . .
Arabidopsis thaliana. . . . . . . . . . .
Caenorhabditis elegans . . . . . . . .
Drosophila melanogaster . . . . . . .
Drosophila virilis . . . . . . . . . . . . .
Homo sapiens . . . . . . . . . . . . . . . .
AF309686
X79489
Z99532
AC005309
Z83234
X95241
X77635
Y09022
Q9C1K8
P38179
Q9Y714
O82244
Q9NAP5
Q27333
Q24332
Q92685
442
458
406
438
604
510
526
438
10
11
14
12
8
11
12
11
ALG6 (a3-glucosyltransferase)
Saccharomyces cerevisiae . . . . . .
Schizosaccharomyces pombe . . . .
Arabidopsis thaliana. . . . . . . . . . .
Caenorhabditis elegans . . . . . . . .
Drosophila melanogaster . . . . . . .
Homo sapiens . . . . . . . . . . . . . . . .
U43491
AL022019
AB005248
Z46676
AE003628
AF102851
Q12001
O43053
BAB09358
Q09226
Q9VKX7
Q9Y672
544
506
533
503
461
507
13
13
9
12
13
10
ALG8 (a3-glucosyltransferase)
Saccharomyces cerevisiae . . . . . .
Schizosaccharomyces pombe . . . .
Arabidopsis thaliana. . . . . . . . . . .
Caenorhabditis elegans . . . . . . . .
Drosophila melanogaster . . . . . . .
Homo sapiens . . . . . . . . . . . . . . . .
X75929
Z73099
AC003672
Z54342
AE003438
AJ224875
P40351
Q10479
O80505
P52887
Q9W3V8
O60860
577
501
383
682
511
526
12
13
11
9
10
12
ALG9 (a2-mannosyltransferase)
Saccharomyces cerevisiae . . . . . .
Schizosaccharomyces pombe . . . .
Arabidopsis thaliana. . . . . . . . . . .
Caenorhabditis elegans . . . . . . . .
Drosophila melanogaster . . . . . . .
Leishmania major . . . . . . . . . . . . .
Mus musculus . . . . . . . . . . . . . . . .
Homo sapiens . . . . . . . . . . . . . . . .
X96417
AL157734
AC051629
Z49909
AE003751
AL390114
BC021791
AK025498
P53868
Q9P7Q9
Q9FZ49
P54002
Q9VBV8
Q9GY50
AAH21791
Q9H6U8
555
577
570
653
623
822
611
618
10
9
12
10
10
10
11
10
ALG10 (a2-glucosyltransferase)
Saccharomyces cerevisiae . . . . . .
Schizosaccharomyces pombe . . . .
Arabidopsis thaliana. . . . . . . . . . .
Caenorhabditis elegans . . . . . . . .
Drosophila melanogaster . . . . . . .
Ratus norvegicus. . . . . . . . . . . . . .
Homo sapiens . . . . . . . . . . . . . . . .
X87941
Z69728
AL162874
Z81131
AJ431376
U78090
AJ312278
P50076
Q10254
Q9LZ85
O02332
CAD24126
O88788
CAC41349
525
445
498
438
499
474
473
12
12
12
12
10
13
13
ALG12 (a6-mannosyltransferase)
Saccharomyces cerevisiae . . . . . .
Schizosaccharomyces pombe . . . .
Caenorhabditis elegans . . . . . . . .
Drosophila melanogaster . . . . . . .
Mus musculus . . . . . . . . . . . . . . . .
Homo sapiens . . . . . . . . . . . . . . . .
Z71645
AL031856
U53155
AE003684
AJ429133
AJ303120
P53730
O74753
Q23361
Q9VH78
CAD22101
Q9BV10
551
547
492
678
483
488
9
9
10
10
12
12
PIG-B (a2-mannosyltransferase)
Zimomonas mobilis. . . . . . . . . . . .
Saccharomyces cerevisiae . . . . . .
Schizosaccharomyces pombe . . . .
Arabidopsis thaliana. . . . . . . . . . .
Oryza sativa . . . . . . . . . . . . . . . . .
Caenorhabditis elegans . . . . . . . .
Drosophila melanogaster . . . . . . .
Trypanosoma brucei . . . . . . . . . . .
Mus musculus . . . . . . . . . . . . . . . .
Homo sapiens . . . . . . . . . . . . . . . .
AF179611
X99960
AL109957
AL391149
AP002908
AJ431373
AE003479
AB033824
D84436
D42138
Q9RNP1
P30777
Q9USN0
Q9LEQ5
Q9AWW0
CAD24083
Q9VZM5
Q9NKZ7
Q9JJQ0
Q92521
509
616
506
498
506
496
512
558
542
554
10
10
9
12
8
8
9
12
10
8
ER RETENTION
SIGNAL
KKLN
KKA
KK
KKLQ
KKSK
KKHA
KKEA
KKQ1
KKE
KKIS
KRKTK
KTKKQ
KTKKK
KTK
KKSGG
KKL
KK
KMKF
1453
1454
Oriol et al.
Table 1
Continued
ENZYME (transfer)
Species
DNA
GENBANK/
EBI
PROTEIN
SWISSPROT ALL
(SWALL)
SIZE
Amino
Acid
TMD
PIG-M (a4-mannosyltransferase)
Saccharomyces cerevisiae . . . . . .
Schizosaccharomyces pombe . . . .
Arabidopsis thaliana. . . . . . . . . . .
Caenorhabditis elegans . . . . . . . .
Drosophila melanogaster . . . . . . .
Trypanosoma brucei . . . . . . . . . . .
Mus musculus . . . . . . . . . . . . . . . .
Ratus norvegicus. . . . . . . . . . . . . .
Homo sapiens . . . . . . . . . . . . . . . .
Z49513
AL354632
AL589883
Z49907
AE003454
AB050105
AK018560
AB028126
AB028127
P47088
Q9P6R5
Q9C575
Q17515
Q9W2E4
BAB20994
Q9D315
Q9EQY6
Q9H3S5
403
411
417
417
466
431
423
423
423
11
12
11
11
9
11
11
10
12
SMP3 (a2-mannosyltransferase)
Saccharomyces cerevisiae . . . . . .
Schizosaccharomyces pombe . . . .
Drosophila melanogaster . . . . . . .
Homo sapiens . . . . . . . . . . . . . . . .
X58121
Z56276
AE003464
AK022830
Q04174
Q09837
Q9W176
Q9H9G6
516
533
830
579
8
10
12
10
ferase superfamily with human ALG10 and the initial
PHI-BLAST seed pattern: [WP]X[LI][DT][YT][PFL]
PX[TFIL][AY]. Only complete sequences comprising
both the long and the short conserved peptide motifs
were used for this study, but the presence of numerous
EST and partial sequences in other species suggests that
these enzymes are widely distributed among other animals and plants and even some bacteria.
Transmembrane Domains
Helical TMDs were predicted by PHD-htm for each
enzyme (table 1) (Rost, Fariselli, and Casadio 1996).
This analysis gave a similar, but not identical multispan
TMD structure for all individual sequences (ranging
from 8–14 TMDs, table 1). Then, because the best computer-assisted TMD prediction methods have about 86%
accuracy (Rost, Fariselli, and Casadio 1996), we
searched for consensus TMD topologies. Individual
TMDs predicted by PHD-htm were added to each species sequence on the CLUSTALW alignments for each
enzyme, and TMDs detected at marginal significance
were added or deleted, when needed, to get the best
overall consensus TMD fit with the other species. Helical segments of 15–24 amino acids were considered as
single helical TMDs, and longer hydrophobic helical domains of more than 25 amino acids were considered as
double TMDs entering and leaving the membrane from
the same side. This artificial consensus TMD prediction
of all species for each enzyme was drawn, and the loop
invariant amino acids were added to the figures. The
high stringent criteria of total identity for each individual amino acid position was selected on purpose for visualization, but the less stringent analysis based on conserved amino acid positions at 50% leads to similar conclusions. The analysis of conserved amino acid positions
and the phylogeny were derived independently from the
transmembrane topology.
ER RETENTION
SIGNAL
KKNN
KPKTD
RIKYD
RIKYD
RIKYD
Phylogeny
Peptide sequences of mannosyltransferases and
glucosyltransferases were aligned with CLUSTALW.
Two strategies for selection of informative positions
were used to make the trees: (1) selection of the two
conserved peptide motifs of each sequence (fig. 2), followed by CLUSTALW alignment and global gap removal, which gave blocks of 43 of the original 53 sites
(81%) for the 45 species of mannosyltransferases and
48 of the 57 original sites (84%) for the 17 species of
glucosyltransferases; and (2) computer selection of conserved blocks with GBLOCKS (Castresana 2000) with
parameters adjusted for maximum block retrieval in the
CLUSTALW alignments of each of the three superfamilies, which gave 106 sites (9% of the original 1,132)
for the 28 species of the a2/6-mannosyltransferase superfamily, 150 sites (25% of the original 616) for the
17 species of the a3/4-mannosyltransferase superfamily,
and 137 sites (20% of the original 668) for the 17 species of the a2/3-glucosyltransferase superfamily. Both
selection methods gave trees with similar topology,
showing that the additional sites recruited by
GBLOCKS do not bias the phylogeny. Distance matrices were generated with the observed difference of the
neighbor-joining method from the PHYLOpWIN package (Galtier, Gouy, and Gautier 1996). The sequence of
alg8 of C. elegans, which has a frameshift and lacks the
short motif, and the sequence of alg10 of A. thaliana,
which lacks half of the short motif, were excluded from
the phylogeny study. Five hundred sets of data were
used for bootstrap calculations.
Results and Discussion
Conserved Peptide Motifs
We found two highly conserved peptide motifs in
each of the mannosyltransferases and glucosyltransferases, by protein sequence alignment with CLUSTALW.
Phylogeny of ALG and PIG Glycosyltransferases
1455
FIG. 2.—Long and short conserved peptide motifs of the a2/6-mannosyltransferase superfamily (A) the a3/4-mannosyltransferase superfamily (B), and the a2/3-glucosyltransferase superfamily (C). Black letters on a gray background correspond to amino acid positions conserved
at 50% in one enzyme, and white letters on a black background are positions conserved at 50% in two or more enzymes. The size, the relative
positions of the long and the short conserved motifs, and the inter motif distance , . are well conserved. For the evaluation of conserved
amino acid positions, ST, KR, DENQ, VILM, and WFY were considered equivalent.
1456
Oriol et al.
The long motif (41–44 amino acids) is located close to
the amino terminus and the short motif (9–10 amino
acids) is located in the carboxy terminal side of the enzymes. The distance between these two motifs is relatively well conserved in the three superfamilies (median
252, range 215–381) (fig. 2A–C).
The a2- and a6-Mannosyltransferase Superfamily
Using Dol-P-Man as Donor Substrate
The a6-mannosyltransferases (ALG12) (Burda et
al. 1999) are clustered in the same glycosyltransferase22 family of the Carbohydrate Active enZyme database
(CAZy) (http://afmb.cnrs-mrs.fr/;pedro/CAZY/db.
html) together with three a2-mannosyltransferases, one
belonging to the same asparagine-linked glycosylation
series (ALG9) and two enzymes involved in the synthesis of the PIG anchor (PIG-B and SMP3) (Ferguson
1992). These four enzymes were known to have a short
common peptide pattern: H[KQ]EXRF (Canivenc-Gansel et al. 1998), which is part of the short conserved
peptide motif shown in figure 2A. After five iterative
runs with the human ALG12 protein sequence, the PHIBLAST converges and retrieves the 28 enzymes of the
a2/6-mannosyltransferase superfamily, including all the
ALG12, ALG9, PIG-B, and SMP3 enzymes (table 1).
The alignment of the sequences of the homologous
ALG12 enzymes from four different species (S. cerevisiae, Schizosaccharomyces pombe, D. melanogaster,
and C. elegans) showed another highly conserved peptide pattern (TKVEESF) in the long conserved peptide
motif of ALG12 (fig. 2A), which helped us to clone the
mouse and the human ALG12 genes. Unlike the short
motif, which is highly conserved in the four enzymes
of this superfamily, the long motif has few positions
conserved in all four families, but it has a large proportion of amino acid positions conserved among the
a6-mannosyltransferases on one side and among the
three a2-mannosyltransferase families on the other side,
suggesting the existence of two conserved long peptide
motifs, one specific for the a6-mannosyltransferases
(ALG12) and another specific for the a2-mannosyltransferases (ALG9, PIG-B and SMP3) (fig. 2A). The phylogenetic tree of all mannosyltransferases supports the
idea that the three a2-mannosyltransferases have a common genetic origin and are more closely related among
themselves than to the a6-mannosyltransferase family
(fig. 3).
The enzymes of this a2/6-mannosyltransferase superfamily have a consensus multispan TMD structure
with an average of 12 spans. The long motif is flanked
by TMD-1 and TMD-2, whereas the short motif is
flanked by TMD-10 and TMD-11 (fig. 4). PIG-B (Takahashi et al. 1996) and ALG9 (Burda et al. 1996) glycosyltransferases add an a2-mannose to a sterically similar mannose acceptor (fig. 1), and they share eleven
identical amino acid positions (solid and gray circles in
fig. 4). Three of the eight ALG9 and three of the ten
PIG-B enzymes have an ER retention signal (table 1)
(Teasdale and Jackson 1996), suggesting that their
COOH terminus is in the cytosol. The SMP3 enzymes
FIG. 3.—Neighbor-joining phylogenetic tree of mannosyltransferases (based on the 43 selected sites of the long and the short motifs).
ALG12 are a6-mannosyltransferases. ALG9, PIG-B and SMP3 are a2mannosyltransferases (a2-mt). ALG3 are a3-mannosyltransferases and
PIG-M are a4-mannosyltransferases. Bootstrap values from 500 data
sets are indicated at the divergence points. The scale bar represents the
number of substitutions per site for a unit branch length.
make a further elongation of a fourth mannose (Grimme
et al. 2001) also linked in a1→2 in PIG (fig. 1). They
share the same eleven identical amino acids with the two
other a2-mannosyltransferases (fig. 4), but none of the
four known SMP3 enzymes has ER retention signals
(table 1).
It is interesting to note that a large proportion of
the conserved amino acids in the a2-mannosyltransferases are located in the long loop between the first two
TMDs, where we found the ALG12-specific highly conserved pattern (TKVEESF). In addition, the four identical amino acids, which are specific of the a2-mannosyltransferase activity (gray circles in fig. 4), are also in
this first loop, suggesting that it might be involved in
a2- and a6-mannosyltransferase active sites.
Seven of the eleven amino acids, identical among
a2-mannosyltransferases, are also shared by the a6mannosyltransferases (solid circles, fig. 4). This might
be related to the fact that the hydroxyl groups on C2
and C6 of the a-D-mannose acceptor are on the same
side of the molecule, and they may offer some similarities of acceptor surface to the corresponding a2- and
a6-mannosyltransferases (fig. 5), although they are not
very close in space. We have previously found a similar
phenomenon between a2-fucosyltransferases and a6-fucosyltransferases which share three conserved peptide
motifs (Breton, Oriol, and Imberty 1998; Chazalet et al.
2001). In addition, conserved peptide motifs were also
found between a3-fucosyltransferases and a4-fucosyltransferases (Oriol et al. 1999). This last observation
Phylogeny of ALG and PIG Glycosyltransferases
1457
FIG. 4.—Consensus TMD topology of the a6-mannosyltransferases (six ALG12) and the a2-mannosyltransferases (eighth ALG9, ten PIGB, and four SMP3). Vertical empty rectangles are helical TMDs across the ER membrane bilayer (gray horizontal band). Empty circles with
black letters are identical amino acid positions in all members within each of the four series of enzymes. Gray solid circles with black letters
represent the four identical amino acids in the three enzymes of the a2-mannosyltransferase family, and solid circles with white letters are the
seven amino acids identical in the four series of enzymes constituting the a2/6-mannosyltransferase superfamily. The boxed KK represents a
putative ER retention signal (table 1). The solid star in TMD-4 of ALG12 identifies the inactivating mutation F142→V (AJ290427) of human
ALG12, inducing the CDG of type Ig (Chantret et al. 2002). C, cytosol; L, lumen of the ER.
prompted us to ascertain whether the a3- and the a4mannosyltransferases using Dol-P-Man as donor substrate have also conserved common peptide motifs.
The a3- and a4-Mannosyltransferase Superfamily
Using Dol-P-Man as Donor Substrate
FIG. 5.—Structure of the a-D-mannose acceptor for: a6-mannosyltransferases (ALG12), a2-mannosyltransferases (ALG9, PIG-B, and
SMP3), and a3-mannosyltransferases (ALG3). The empty ovals contain the OH on C2 and C6 of the mannose acceptor (top). Structure of
the a-D-glucosamine acceptor for the a4-mannosyltransferase (PIGM) (bottom). The two oval shaded symbols represent the OH on C3
of the mannose (top) and on C4 of the glucosamine (bottom). Both
the 2 and the 6 positions of the mannose are on the upper side (empty
ovals) and both the 3 and the 4 positions are on the equatorial left side
of the mannose and the glucosamine (shaded ovals).
After three iterative runs with the corresponding
pattern and either of the human ALG3 (CAZy glycosyltransferase-58 family) or PIG-M (CAZy glycosyltransferase-50 family), the PHI-BLAST reaches convergence and retrieves the 17 enzymes of the a3/4-mannosyltransferase superfamily (table 1). Several conserved amino acid positions were found for both the
long and the short motifs of the a3/4-mannosyltransferase superfamily (fig. 2B). The phylogenetic tree of all
mannosyltransferases show that the ALG3 family and
the PIG-M family constitute separate clusters (fig. 3) and
are different enough from the cluster of a2/6-mannosyltransferases to be considered as an outgroup defining
the position of a possible root for the tree between the
two superfamilies of a2/6- and a3/4-mannosyltransferases.
The TMD prediction also suggests a multispan
structure with an average of 12 spans, with the long
motif in the first loop and the short motif between TMD10 and TMD-11 (fig. 6). Seven identical amino acid positions are shared by the a3- and the a4-mannosyltransferases (solid circles in fig. 6). Five of these seven iden-
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Oriol et al.
FIG. 7.—Neighbor-joining phylogenetic tree of glucosyltransferases (based on the 160 computer selected sites with GBLOCKS).
ALG6 and ALG8 are a3-glucosyltransferases and ALG10 are a2-glucosyltransferases (a2-glu-tr). Bootstrap values from 500 data sets are
indicated at the divergence points. The scale bar represents the number
of substitutions per site for a unit branch length.
FIG. 6.—Consensus TMD topology of the a4-mannosyltransferases (nine PIG-M, top) and the a3-mannosyltransferases (eighth
ALG3, bottom). Empty circles with black letters indicate the identical
amino acids among all members of each of the two families of enzymes. Solid circles with white letters represent the seven amino acids
identical in the two enzymes of the a3/4-mannosyltransferase superfamily. The boxed KK indicates a putative ER retention signal (table
1). The solid star in the second loop of ALG3 represents the inactivating mutation G118→N of the human ALG3 enzyme inducing the
CDG of type Id (Körner et al. 1999; Sharma, Knauer, and Lehle 2001).
C, cytosol; L, lumen of the ER.
tical positions are in the first large luminal loop, which
is equivalent to the first loop of the a2/6-mannosyltransferase superfamily, suggesting again that this first luminal loop is particularly well conserved and may play
a role in the active site of the enzymes and that there
are some similarities between the consensus TMD topology of the two superfamilies of a2/6-mannosyltransferases and a3/4-mannosyltransferases. In addition, the
presence of a small conserved loop, in the cytosolic side
of the membrane, between TMD-10 and TMD-11 of a3and a4-mannosyltransferases, supports the idea of conserved TMD topology between a2/6-mannosyltransferases (fig. 4) and a3/4-mannosyltransferases (fig. 6). Six
of the eight a3-mannosyltransferases (ALG3) and five
of the nine a4-mannosyltransferases (PIG-M) have an
ER retention signal (table 1), in good agreement with
the consensus TMD topology prediction (fig. 6), which
suggests that both NH2 and COOH terminus are cytosolic. PIG-M belongs to the PIG-anchor and ALG3 to
the N-glycan series, suggesting that these two series of
enzymes have also a common genetic origin.
The hydroxyls on C3 of the a-D-mannose and C4
of the a-D-glucosamine acceptors have the same equa-
torial orientation and offer a similar equatorial approach
to the corresponding a3-mannosyltransferases (ALG3)
and a4-mannosyltransferases (PIG-M), especially if the
glucosamine is rotated 1808 around its horizontal axis
(fig. 5). Consequently, we expected more common features between a3- and a4-mannosyltransferases, which
share the same equatorial approach to the acceptor and
the same donor and less common features between the
two superfamilies of a2/6- and a3/4-mannosyltransferases because they only share the donor substrate (DolP-Man).
The a2 and a3-Glucosyltransferase Superfamily Using
Dol-P-Glc as Donor Substrate
After two iterative runs, the PHI-BLAST with either of human ALG6 or ALG8 converges and retrieves
the 12 sequences of the a3-glucosyltransferase family,
comprising the ALG6 and the ALG8 enzymes. These
two enzymes are closely related as shown in the phylogenetic tree of figure 7. Their close similarity is also
illustrated by 30 identical amino acid positions among
ALG6 and ALG8 (fig. 8). Four out of the six ALG6 and
two out of the six ALG8 enzymes (Stagljar, Hessen, and
Aebi 1994; Stanchi et al. 2001) have a putative ER retention signal (table 1), suggesting that their COOH-end
is in the cytosol, as predicted by the consensus TMD
topology (fig. 8).
Either of PHI-BLAST or PSI-BLAST alone with
ALG10 retrieved only the ALG10 enzymes of the different species, but the combined PHI-PSI-BLAST with
human ALG10 retrieves ALG10, ALG8, and ALG6 enzymes of the a2/3-glucosyltransferase superfamily. Few
Phylogeny of ALG and PIG Glycosyltransferases
1459
FIG. 8.—Consensus TMD topology of the a3-glucosyltransferases (six ALG6 and five ALG8) and the a2-glucosyltransferases (six
ALG10), constituting the a2/3-glucosyltransferase superfamily. Empty circles with black letters indicate identical amino acid positions
among all members within each of the three enzymes. Gray solid circles with black letters represent 27 amino acids identical in both a3glucosyltransferases (ALG6 and ALG8), and solid circles with white letters are the five ALG10 identical amino acids with ALG6 or
ALG8 (or both). The solid stars identify inactivating mutations in human ALG6: S170→I, TMD-4 (de Lonlay et al. 2001); deletion of I
299, TMD-7 (Hanefeld et al. 2000); F304→S, TMD-7 (Imbach et al. 2000); A333→V, TMD-8 (Imbach et al. 1999); deletion of L 444,
TMD-11 (de Lonlay et al. 2001); and S478→P, TMD-12 (Imbach et al. 2000), which can induce the CDG of type Ic (Grünewald et al.
2000). C, cytosol; L, lumen of the ER.
FIG. 9.—Structure of the a-D-mannose (top), acceptor for the
first glucose (added by ALG6). The first a-D-glucose (bottom), acceptor for the second glucose (added by ALG8) or the second aD-glucose, acceptor for the third glucose (added by ALG10). The
shaded oval areas correspond to the OH on C3 of the mannose and
the first glucose. The empty oval area shows the OH on C2 of the
second glucose. Identical equatorial orientation is illustrated by the
two shaded areas, in spite of being on different monosaccharides
(mannose and glucose), whereas different orientations are shown
by the two oval shaded areas on the left side and the oval empty
area below the glucose.
similarities exist between ALG10 and the a3-glucosyltransferases, but the short and long motifs are well identified, among all the different species for ALG10 (gray
and black areas of the ALG10 family in fig. 2C). The
phylogenetic tree confirms this impression, showing a
cluster of ALG10 enzymes at high distance from the two
a3-glucosyltransferases, ALG6 and ALG8 (fig. 7). The
alg10 gene was first described in yeast (Burda and Aebi
1998), then in rat (Hoshi et al. 1998), and we have
cloned the Drosophila (AJ431376) and the human
(AJ312278) ALG10 orthologous genes. None of the
ALG10 enzymes has ER retention signals, but all of
them have three identical amino acid positions with
ALG6 and ALG8, plus a glutamic acid (E) identical
with ALG6 in the long motif, and a histidine (H) identical with ALG8 in the short motif (solid circles fig. 8).
The transmembrane topology of the last three glucosyltransferases suggests a consensus TMD organization similar to the aforementioned TMD structure of
mannosyltransferases with about 12 TMD. A large conserved luminal loop between TMD-1 and TMD-2 and a
small conserved loop, in the cytosolic side of the membrane, between TMD-8 and TMD-9 are present (fig. 8).
The scheme of figure 9 illustrates identical equatorial orientations of the hydroxyls on C3 of the last aD-mannose and the first a-D-glucose, which are acceptors for ALG6 and ALG8, respectively. Unlike this, a
different spatial orientation can be seen for the hydroxyl
on C2 of the second a-D-glucose, which is the acceptor
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Oriol et al.
substrate for ALG10. Therefore, we expect more common features between ALG6 and ALG8, which share
both acceptor and donor substrates, than between the
a2-glucosyltransferase (ALG10) and the two a3-glucosyltransferases (ALG6 and ALG8) because they have
different acceptor substrates and they only share the donor substrate (Dol-P-Glc). Comparison of the OH on C3
(acceptors for ALG6 and ALG8) and the OH on C2
(acceptor for ALG10, fig. 9) with the OH on C3, C4,
C2, and C6 (acceptors for mannosyltransferases, fig. 5),
suggests that the acceptor OH of the two families of
glucosyltransferases are as different from each other as
the corresponding acceptors for the two mannosyltransferase superfamilies.
Potential Functions of TMD and Conserved Loops
The transmembrane topology of the glycosyltransferases, using Dol-P-monosaccharides as donor substrate, suggests a common consensus multispan transmembrane topology. The enzymes have an average of
12 putative helical TMD, and both the NH2 and COOHends are in the cytosolic side of the membrane (figs. 4,
6, and 8). But they still are a statistical prediction, and
we cannot be certain that all the putative TMDs span
the membrane in vivo, for example only 7 of the 11
TMDs predicted for the protein O-mannosyltransferase
(Pmt1p) were shown to span the membrane (StraahlBolsinger and Scheinost 1999). However, if some of the
putative a-helical TMDs do not span the membrane, the
second most probable topology with both extremes in
the cytosol would be a 10 TMD, and if there is an odd
number of TMD, the NH2 and the COOH terminus
would be on different sides of the membrane. None of
these possibilities can be formally ruled out for individual sequences because the consensus topology is only
an average artificial prediction. Nevertheless, we can say
that all these enzymes have a multispan TMD topology,
similar to the general structure of sugar transport proteins such as glucose-6-P-transporter (G6PT) (Pan, Lin,
and Chou 1999) or glucose transporter 1 (Glut1) (Sato
and Mueckler 1999). In fact, the great majority of sugar
transporters have a multispan structure with 10 or 12
TMDs (Maiden et al. 1987; Marger and Saier 1993). It
is possible that each of the enzymes under study contributes also to the translocation across the membrane
of the Dol-P-monosaccharide, which is synthesized in
the cytosol and has to reach the acceptor substrate oligosaccharides in the lumen of the ER (Rush and Waechter 1998; Rush et al. 1998).
A similar distribution of the putative TMD along
the peptide chain is an additional structural feature in
favor of a common genetic origin for a series of related
enzymes. Our consensus TMD topology suggests that
the first, large and highly conserved loop, is on the luminal side of the membrane and contains identical amino acid positions specific for each linkage. These properties are compatible with the contribution of this first
loop to the glycosyltransferase active site.
For the sake of clarity, only identical amino acid
positions located in the extramembrane loops are shown
in figures 4, 6, and 8, but between 30% and 42% of the
total amino acids of these enzymes is located inside the
putative helical TMD. The comparative analysis of identical amino acid positions in the loops and in the helical
TMD shows that they are not randomly distributed. In
general, there are fewer identical amino acid positions
inside the TMD (8%–35%) than in the loops (65%–
92%), and the proportion of identical amino acids in
TMDs are characteristic and different for the a2/6-mannosyltransferase (8%–19%), the a3/4-mannosyltransferase (21%–27%) and the a2/3-glucosyltransferase (35%),
in favor of a common origin for each of these three
superfamilies of glycosyltransferases.
The mutations of these ER glycosyltransferases,
known to be responsible of congenital disorders of glycosylation (CDG), are also not randomly distributed.
Only one out of eight mutations is in a short loop and
seven are in TMDs (solid stars in figs. 4, 6, and 8). A
similar difference in favor of a TMD location has been
reported for the mutations inactivating the Golgi transporter of CMP-sialic acid (Eckhardt, Gotza, and Gerardy-Schahn 1999), and this protein is also a candidate for
establishing a new CDG of type II (Mollicone et al.,
personal communication).
Divergent Evolutionary Model
All together the common structural features of the
enzymes using Dol-P-monosaccharides as donor substrate suggest that they might have followed a common
evolutionary path. The duplication of a hypothetical ancestral gene, encoding for a protein with the long and
short conserved peptide motifs and the consensus multispan TMD topology, followed by divergent evolution,
might have generated the two main mannosyltransferase
and glucosyltransferase genes (fig. 10). A second duplication within the mannosyltransferases might have originated the a2/6- and the a3/4-mannosyltransferase superfamilies. A duplication of the a2/6-mannosyltransferase gene may be at the origin of the a6-mannosyltransferase genes (ALG12) and the ancestor of the
a2-mannosyltransferase genes. Two further duplications
of the a2-mannosyltransferase branch may be at the origin of SMP3, PIG-B, and ALG9 genes. A duplication
of the gene of the a3/4-mannosyltransferase superfamily
may have originated ALG3 and PIG-M genes. Similar
duplication events may have originated the a2-glucosyltransferase genes (ALG10) and the ancestor of a3glucosyltransferase genes in the glucosyltransferase
branch. Finally, a last and more recent duplication must
be at the origin of the present two enzymes with a3glucosyltransferase activity (ALG6 and ALG8) because
they display the highest score of sequence identity (30
amino acids, fig. 8).
The compilation of the data reported previously
and the identical and conserved amino acid positions in
each of the superfamilies, families, and enzymes, constitute indirect evidence for a common genetic origin
followed by divergent evolution of these enzymes. The
existence of the more ancestral precursor genes (the two
empty circles in fig. 10) is supported by the relative
Phylogeny of ALG and PIG Glycosyltransferases
1461
FIG. 10.—Divergent evolutionary model, based on the consensus TMD topology (figs. 4, 6, and 8), phylogeny (figs. 3 and 7), and
conserved peptide motifs (fig. 2) of all the glycosyltransferases using Dol-P-monosaccharides as donor substrate. The model indicates
the duplication events needed to obtain the six a-mannosyltransferase genes (ALG12, ALG9, PIG-B, SMP3, ALG3, and PIG-M) and the
three a-glucosyltransferase genes (ALG6, ALG8, and ALG10) from a hypothetical common ancestral gene having the consensus multispan
TMD topology. The three superfamilies of a2/6-mannosyltransferases (solid circles), a3/4-mannosyltransferases (dark gray circles), and
a2/3-glucosyltransferase (light gray circles) are supported by the existence of an increasing number of identical and conserved amino
acids, at each divergence point, from left to right, and a conserved proportion of identical amino acids in TMD, within each of the three
enzyme superfamilies. The empty circles represent hypothetical ancestors based on the similar size and location of the conserved peptide
motifs, the multispan TMD topology, and their common use of Dol-P-monosaccharides as donor substrates. This model suggests that the
glycosyltransferases of the N-glycan series (ALG) and of the PIG anchor series (PIG-B, PIG-M, and SMP3), using Dol-P-monosaccharides
as donor substrates, have a common genetic origin.
positions and sizes of the long and the short conserved
peptide motifs, but no identical or conserved amino acid
positions were found between mannosyltransferases and
glucosyltransferases, nor between the a2/6 and the a3/
4-superfamilies of mannosyltransferases. The relative
positions of the duplications reflect the increasing proportion of identical and conserved amino acids from left
to right. All the enzymes on the right side of the tree
have been cloned and experimentally tested, whereas the
existence of a single common ancestor on the left is only
a working hypothesis. However, if the single common
ancestor hypothesis is not confirmed, a very similar evolutionary model with two non-related ancestors, one for
the a-mannosyltransferases and another for the a-glucosyltransferases, or three ancestors, one for each superfamily, can be imagined. Irrespective of considering
one, two, or three ancestors, the tree depicted in figure
10 must be quite ancient because these nine glycosyltranferases have been found in plants, yeasts, worms,
insects, and vertebrates.
Acknowledgments
The research was partially supported by the Association for Research on Cancer (ARC) grant 5348, the
French network for Recombinant Glycosyltransferases
(GT-rec), and the French INSERM/AFM network
4MR29F for CDG. We are grateful to Stuart E. H.
Moore for critical reading of the manuscript and helpful
discussions.
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PIERRE CAPY, reviewing editor
Accepted April 17, 2002