INSERM U504, University of Paris Sud XI, Villejuif, France
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Abstract |
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Introduction |
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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 Asn-X-Ser/Thr of the nascent protein chain. The N-glycosylation process is completed by a series of trimming 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
).
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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.
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Materials and Methods |
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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 5'-CAGGAGTAGGTTCTTGGGCAGTGGC-3' and antisense 5'-AATGTAATTTGAAGACCACCACTGCACC-3' for ALG10, and sense 5'-GGCATTTCCTGGTGTCTGAGCCTG-3' and antisense 5'-CACAGAACTACCCATAGCTGGTCC-3' 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-Pharmacia-Biotech) 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
2/6-mannosyltransferase superfamily; [VL]X[YF]T[DEK][IV]D[YW]X[VIAT] for the
3/4-mannosyltransferase superfamily; W[GT]LDYPP[LF][TF]A[FYW] for the
3-glucosyltransferase family; and LADNRH[YF][TL]FY for the
2-glucosyltransferase family. A combined PHI-PSI-BLAST was also used for the
2/3-glucosyltransferase 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 814 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 1524 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.
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Results and Discussion |
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The 2- and
6-Mannosyltransferase Superfamily Using Dol-P-Man as Donor Substrate
The 6-mannosyltransferases (ALG12) (Burda et al. 1999
) are clustered in the same glycosyltransferase-22 family of the Carbohydrate Active enZyme database (CAZy) (http://afmb.cnrs-mrs.fr/
pedro/CAZY/db.html) together with three
2-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 PHI-BLAST converges and retrieves the 28 enzymes of the
2/6-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 6-mannosyltransferases on one side and among the three
2-mannosyltransferase families on the other side, suggesting the existence of two conserved long peptide motifs, one specific for the
6-mannosyltransferases (ALG12) and another specific for the
2-mannosyltransferases (ALG9, PIG-B and SMP3) (fig. 2A
). The phylogenetic tree of all mannosyltransferases supports the idea that the three
2-mannosyltransferases have a common genetic origin and are more closely related among themselves than to the
6-mannosyltransferase family (fig. 3
).
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Seven of the eleven amino acids, identical among 2-mannosyltransferases, are also shared by the
6-mannosyltransferases (solid circles, fig. 4
). This might be related to the fact that the hydroxyl groups on C2 and C6 of the
-D-mannose acceptor are on the same side of the molecule, and they may offer some similarities of acceptor surface to the corresponding
2- and
6-mannosyltransferases (fig. 5
), although they are not very close in space. We have previously found a similar phenomenon between
2-fucosyltransferases and
6-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
3-fucosyltransferases and
4-fucosyltransferases (Oriol et al. 1999
). This last observation prompted us to ascertain whether the
3- and the
4-mannosyltransferases using Dol-P-Man as donor substrate have also conserved common peptide motifs.
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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 TMD-10 and TMD-11 (fig. 6
). Seven identical amino acid positions are shared by the 3- and the
4-mannosyltransferases (solid circles in fig. 6
). Five of these seven identical positions are in the first large luminal loop, which is equivalent to the first loop of the
2/6-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
2/6-mannosyltransferases and
3/4-mannosyltransferases. In addition, the presence of a small conserved loop, in the cytosolic side of the membrane, between TMD-10 and TMD-11 of
3- and
4-mannosyltransferases, supports the idea of conserved TMD topology between
2/6-mannosyltransferases (fig. 4
) and
3/4-mannosyltransferases (fig. 6
). Six of the eight
3-mannosyltransferases (ALG3) and five of the nine
4-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.
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The 2 and
3-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 3-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
).
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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 -D-mannose and the first
-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
-D-glucose, which is the acceptor substrate for ALG10. Therefore, we expect more common features between ALG6 and ALG8, which share both acceptor and donor substrates, than between the
2-glucosyltransferase (ALG10) and the two
3-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.
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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 2/6-mannosyltransferase (8%19%), the
3/4-mannosyltransferase (21%27%) and the
2/3-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 2/6- and the
3/4-mannosyltransferase superfamilies. A duplication of the
2/6-mannosyltransferase gene may be at the origin of the
6-mannosyltransferase genes (ALG12) and the ancestor of the
2-mannosyltransferase genes. Two further duplications of the
2-mannosyltransferase branch may be at the origin of SMP3, PIG-B, and ALG9 genes. A duplication of the gene of the
3/4-mannosyltransferase superfamily may have originated ALG3 and PIG-M genes. Similar duplication events may have originated the
2-glucosyltransferase genes (ALG10) and the ancestor of
3-glucosyltransferase genes in the glucosyltransferase branch. Finally, a last and more recent duplication must be at the origin of the present two enzymes with
3-glucosyltransferase activity (ALG6 and ALG8) because they display the highest score of sequence identity (30 amino acids, fig. 8
).
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Acknowledgements |
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Footnotes |
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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, Phosphatidyl-Inositol Glycan anchor (GPI-anchor); TMD, transmembrane domain.
Keywords: 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: oriol{at}infobiogen.fr
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