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Conserved structural features in eukaryotic and prokaryotic fucosyltransferases
Introduction
Results and discussion
Materials and methods
Note added in proof
Acknowledgments
Abbreviations
References
Conserved structural features in eukaryotic and prokaryotic fucosyltransferases
Fucosyltransferases are the enzymes transferring fucose from GDP-Fuc to Gal in an [alpha]1,2-linkage and to GlcNAc in [alpha]1,3-, [alpha]1,4-, or [alpha]1,6-linkages. Since all fucosyltransferases utilize the same nucleotide sugar, their specificity will probably reside in the recognition of the acceptor and in the type of linkage formed. A search of nucleotide and protein databases yielded more than 30 sequences of fucosyltransferases originating from mammals, chicken, nematode, and bacteria. On the basis of protein sequence similarities, these enzymes can be classified into four distinct families: (1) the [alpha]-2-fucosyltransferases, (2) the [alpha]-3-fucosyltransferases, (3) the mammalian [alpha]-6-fucosyltransferases, and (4) the bacterial [alpha]-6-fucosyltransferases. Nevertheless, using the sensitive hydrophobic cluster analysis (HCA) method, conserved structural features as well as a consensus peptide motif have been clearly identified in the catalytic domains of all [alpha]-2 and [alpha]-6-fucosyltranferases, from prokaryotic and eukaryotic origin, that allowed the grouping of these enzymes into one superfamily. In addition, a few amino acids were found strictly conserved in this family, and two of these residues have been reported to be essential for enzyme activity for a human [alpha]-2-fucosyltransferase. The [alpha]-3-fucosyltransferases constitute a distinct family as they lack the consensus peptide, but some regions display similarities with the [alpha]-2 and [alpha]-6-fucosyltranferases. All these observations strongly suggest that the fucosyltransferases share some common structural and catalytic features. Key words: fucosyltransferases/hydrophobic cluster analysis/protein sequence analysis
Introduction
Fucosyltransferases are a group of enzymes catalyzing the transfer of fucose from GDP-Fuc, in [alpha]1,2-, [alpha]1,3-, [alpha]1,4-, and [alpha]1-6-linkages, with inversion of anomeric configuration, to various oligosaccharide acceptors. Fucosylated glycoconjugates may play important roles in physiological and pathological processes such as fertilization, embryogenesis, lymphocyte trafficking, immune response, and cancer metastasis (Schenkel-Brunner, 1995; Staudacher, 1996). All eukaryotic fucosyltransferases cloned to date have the typical domain structure of type II transmembrane proteins, consisting of a short NH2-terminal cytoplasmic tail, a transmembrane domain and a stem region followed by a large globular COOH-terminal catalytic domain (Joziasse, 1992; Kleene and Berger 1993). Since all fucosyltransferases utilize the same nucleotide sugar as donor, their specificity will probably reside in the recognition of the acceptor substrate and in the type of linkage formed.
The [alpha]-2- and [alpha]-3/4-fucosyltransferases have attracted a large interest in recent years as they are involved in the last steps of the biosynthesis of ABH and Lewis related carbohydrate antigens (Henry et al., 1995; Watkins, 1995; Costache et al., 1997). Seven human FUT genes (named FUT1 to FUT7 according to the chronology of cloning) and 14 animal genes (named according to their homology with the human counterparts) have been cloned and sequenced. FUT1 and FUT2 genes encode for the [alpha]-2-fucosyltransferases H and Se (secretor), respectively, while FUT3 to FUT7 genes code for the [alpha]-3-fucosyltransferases Fuc-TIII to Fuc-TVII. The enzymes Fuc-TIII and Fuc-TV also display [alpha]-4-fucosyltransferase activity (Weston et al., 1992a; Wong et al., 1992). A third group of mammalian fucosyltransferases has emerged with the recent cloning of the pig and human [alpha]-6-fucosyltransferases which are implied in the synthesis of N-glycans (Uozumi et al., 1996; Yanagidani et al., 1997). These two enzymes exhibit no significant homology with other cloned fucosyltransferases.
The presence of fucosyltransferase activities has also been demonstrated in plants, insects, snails, parasites, and bacteria (for a review, see Staudacher, 1996). At the present time, two bacterial enzymes (NodZ), from rhizobia species (Azorhizobium caulinodans and Bradyrhizobium japonicum) have been cloned (Stacey et al., 1994; Mergaert et al., 1996). These enzymes catalyze the transfer of fucose from GDP-Fuc in [alpha]1,6 linkage to the GlcNAc at the reducing end of Nod factors. Other protein sequences originating from the random genomic sequencing of Caenorhabditis elegans (Wilson et al., 1994) and the bacterium Yersinia enterocolitica (Zhang et al., 1997) have been considered as putative fucosyltransferases since they display a low but significant homology with some mammalian fucosyltransferases, but their functionality has not yet been demonstrated.
The sensitive hydrophobic cluster analysis (HCA) method (Gaboriaud et al., 1987; Lemesle-Varloot et al., 1990) has been successfully used for the grouping of proteins exhibiting low sequence identity and for prediction of catalytic residues in a number of glycosyl hydrolases (Henrissat et al., 1995). Using HCA and fold recognition methods, we have previously suggested that mammalian [alpha]-2- and [alpha]-3-fucosyltransferases, despite their lack of sequence identity, are related proteins sharing the same type of fold (Breton et al., 1996). We have also predicted the occurrence of a nucleotide binding domain of the Rossman-type in the catalytic domain of these enzymes. The aim of the present study was to extend the protein sequence analysis to all the prokaryotic and eukaryotic fucosyltransferase sequences available in data banks, to provide insight into structure-function relationships of this class of enzymes.
Results and discussion
A search of nucleotide and protein databases yielded 37 sequences of fucosyltransferases originating from mammals, chicken, nematode, and bacteria. Among them, 13 are partial sequences, hypothetical proteins, or pseudogenes. Only the complete sequences, listed in Table I, were considered in this study. The protein sizes are extremely variable ranging from 283 amino acids for the bacterial Yersinia enterocolitica sequence to 575 residues for the mammalian [alpha]-6-fucosyltransferases. On the basis of protein sequence similarities, these sequences can be classified into four families (1) the [alpha]-2-fucosyltransferases, (2) the [alpha]-3-fucosyltransferases, (3) the mammalian [alpha]-6-fucosyltransferases, and (4) the bacterial [alpha]-6-fucosyltransferases. Large homologies in protein sequences were found for human and animal fucosyltransferases within each family, ranging from 50% to more than 80% overall identity for members of the [alpha]-2-fucosyltransferase family and from 40% to 99% for members of the [alpha]-3-fucosyltransferase family. The Y.enterocolitica sequence (an hypothetical fucosyltransferase) exhibits a low but significant homology with other sequences of [alpha]-2-fucosyltransferase (22-27%) and was included in this family. The three Caenorhabditis elegans sequences are also hypothetical fucosyltransferases which can be grouped into the [alpha]-3-fucosyltransferase family as they share 25-31% of identity with the human and animal [alpha]-3-fucosyltransferases. The two recently cloned mammalian [alpha]-6-fucosyltransferases display high homology (96% of identity) and appear to be unrelated with the two bacterial enzymes NodZ.
Table I
| Accession number |
Origin | Gene | Enzyme | Size (aa) | References |
| [alpha]-2-Fucosyltransferases | |||||
| M35531 | Man | FUT1 | H | 365 | Larsen et al., 1990 |
| L50534 | Pig | FUT1 | H | 365 | Cohney et al., 1996 |
| X80226 | Rabbit | FUT1 | H-I | 373 | Hitoshi et al., 1995 |
| Y09883 | Mouse | FUT1 | H | 377 | Hitoshi et al., 1996a |
| U17894 | Man | FUT2 | Se | 343 | Kelly et al., 1995 |
| X91269 | Rabbit | FUT2 | Se-III | 354 | Hitoshi et al., 1996b |
| X80225 | Rabbit | Sec1 | Se-II | 347 | Hitoshi et al., 1995 |
| Y09882 | Mouse | Sec1 | Sec1 | 368 | Hitoshi et al., 1996a |
| U46859 | Y.enterocolitica | ORF11.8 | yeFuc-Ta | 283 | Zhang et al., 1997 |
| [alpha]-3-Fucosyltransferases | |||||
| X53578 | Man | FUT3 | Fuc-TIIIc | 361 | Kukowska-Latallo et al., 1990 |
| Y14033 | Chimpanzee | FUT3 | Fuc-TIIIc | 372 | Costache et al., in press |
| M81485 | Man | FUT5 | Fuc-TVc | 374 | Weston et al., 1992a |
| Y14034 | Chimpanzee | FUT5 | Fuc-TVc | 374 | Costache et al., in press |
| L01698 | Man | FUT6 | Fuc-TVI | 359 | Weston et al. 1992b |
| Y14035 | Chimpanzee | FUT6 | Fuc-TVI | 359 | Costache et al., in press |
| X87810 | Bovine | FUTb | Fuc-Tb | 365 | Oulmouden et al., 1997 |
| M58596 | Man | FUT4 | Fuc-TIV | 405 | Goelz et al., 1990 |
| U33457 | Mouse | FUT4 | Fuc-TIV | 433 | Gersten et al., 1995 |
| U58860 | Rat | FUT4 | Fuc-TIV | 433 | Sajdel-Sulkowska et al., 1997 |
| U73678 | Chicken | FUT4 | Fuc-TIV | 356 | Lee et al., 1996 |
| X78031 | Man | FUT7 | Fuc-TVII | 341 | Sasaki et al., 1994 |
| U45980 | Mouse | FUT7 | Fuc-TVII | 389 | Smith et al., 1996 |
| Z66497 | C.elegans | K08F8.3 | ceFUTAa | 451 | Wilson et al., 1994 |
| U40028 | C.elegans | T05A7.5 | ceFUTBa | 331(C-t)b | Wilson et al., 1994 |
| U40028 | C.elegans | T05A7.5 | ceFUTCa | 450(N-t)b | Wilson et al., 1994 |
| Mammalian [alpha]-6-fucosyltransferases | |||||
| D86723 | Pig | FUT8 | Fuc-TVIII | 575 | Uozumi et al., 1996 |
| D89289 | Man | FUT8 | Fuc-TVIII | 575 | Yanagidani et al., 1997 |
| Bacterial [alpha]-6-fucosyltransferases | |||||
| L18897 | A.caulinodans | nodZ | NodZ | 328 | Mergaert et al., 1996 |
| L22756 | B.japonicum | nodZ | NodZ | 324 | Stacey et al. 1994 |
The degree of identity between proteins belonging to the different families is too low (typically less than 20%) to obtain a reliable global alignment using the classical 1D sequence alignment methods (Bestfit, Gap, or Pileup of the GCG package). Because of its known sensitivity at very low sequence identity, the HCA method was used for the comparison of fucosyltransferase sequences. The effectiveness of HCA comes from its ability to significantly detect the regular secondary structure elements constituting the hydrophobic core of globular proteins (Woodcock et al., 1992). This method uses a bidimensional representation of the protein sequence and the clusters of contiguous hydrophobic residues are drawn. The analysis involves the visual comparison of hydrophobic cluster shapes and their distribution in order to find similarities between protein sequences.
The [alpha]-3-fucosyltransferases constitute a homogenous family since these enzymes display between 30% and more than 90% of identity. The HCA alignment is straightforward and similarities were clearly observed in the catalytic domains of the three C.elegans sequences and the mammalian [alpha]-3-fucosyltransferases. However, we have to keep in mind that the C.elegans proteins are putative fucosyltransferases and that their functionality still needs to be proved. Since the catalytic amino acids are submitted to intense conservation pressure, they are generally located in the best conserved regions. The divergence brought in by the invertebrate sequences can help to identify such regions but the number of conserved amino acids is still too high to predict those which could be important for activity. Nevertheless, two highly homologous regions, named I and II, were detected and they are represented in Figure 1 as a multiple linear sequence alignment deduced from the HCA comparison of all the sequences of this family. Seven residues in region I and 12 in region II are found to be identical. Other highly conserved basic, aromatic, and aliphatic amino acids are also observed in these two regions.
Figure
In contrast, the [alpha]-2-fucosyltransferases, the mammalian [alpha]-6-fucosyltransferases, and the bacterial [alpha]-6-fucosyltransferases constitute a more heterogenous family. As indicated above, these three groups of fucosyltransferases display no significant homology. However, by HCA, a region spanning approximately 100 residues was found conserved in the catalytic domain of all the enzymes belonging to this large family. Figure 2 displays the partial HCA plots of four selected Fuc-Ts. These four protein sequences exhibit 16-25% overall identity. The vertical lines delineate hydrophobic clusters which were found conserved in the C-terminal part of all the [alpha]-2 and [alpha]-6-fucosyltransferases. Homologies were also observed in the N-terminal part of the catalytic domains of the human H enzyme and the putative Y.enterocolitica fucosyltransferase (dotted vertical lines). In addition, a consensus peptide sequence corresponding to the boxed region in Figure 1, has been clearly identified. This peptide motif (V/I - G - V/I - H - V/I - R - R/H - G/T - D/N) is detailed in Figure 3 with all protein members exhibiting [alpha]-2 and [alpha]-6-fucosyltransferase activities. At first sight, the B.japonicum NodZ sequence appeared to be different in the C-terminal end from its counterpart from A.caulinodans, and we were unable to find similarities by HCA in this region. However, a shift in the reading frame in this region restored the similarity with the other sequences and extended the sequence of B.japonicum NodZ by approximately 40 residues. These observations allow the grouping of these enzymes in one superfamily, and one can expect that these proteins share common structural and catalytic features in the concerned region.
Figure
Figure
What could be the significance of such a conserved motif present in prokaryotes and eukaryotes and more strikingly in enzymes catalyzing two very different reactions (i.e,. [alpha]-2-linkage on galactose and [alpha]-6 linkage on N-acetylglucosamine)? The common feature of all the fucosyltransferases is the use of the same nucleotide sugar and one can argue that the consensus peptide sequence is part of the nucleotide binding domain. Although the peptide motif has not been detected in the [alpha]-3-fucosyltransferase family, a closer examination of the HCA plots suggests that the region containing the peptide motif in [alpha]-2 and [alpha]-6-fucosyltransferases could correspond to the region I defined for the [alpha]-3-fucosyltransferases in Figure 1. These two regions display similar hydrophobic cluster shapes as well as conserved basic and acidic residues (Figure 4). Similarly, the region II of the [alpha]-3-fucosyltransferases (i.e., Fuc-TIII230-260) could match with another well conserved region in [alpha]-2 and [alpha]-6-fucosyltransferases (i.e., H300-330) as they share common structural features in the HCA representation. Nevertheless, the correspondences indicated in Figure 4 are still highly speculative especially for the region II.
Figure
Some important structural and functional information have been recently reported for several fucosyltransferases. These studies were aimed to identify the amino acids that are possibly involved in nucleotide sugar and acceptor recognition and also to get new insights in the mechanism of these enzymes. Sixty-one and 75 amino acids can be eliminated from the N-terminus of Fuc-TIII and Fuc-TV, respectively, without significant loss of enzyme activity (Xu et al., 1996). These truncation experiments allowed the delineation of the stem region and the catalytic domain. Thus the catalytic domains of these two enzymes begin with a large hydrophobic region in which a conserved Trp (Trp68 in Fuc-TIII) was shown to be essential for activity (Elmgren et al., 1997). In contrast the removal of one or more amino acids from the C-terminus resulted in a dramatic or total loss of enzyme activity (Xu et al., 1996). Domain swapping experiments were also performed to localize the region that account for the difference observed in acceptor specificity between Fuc-TIII, Fuc-TV, and Fuc-TVI (Legault et al., 1995; Xu et al., 1996). As few as 11 nonidentical amino acids, found within a hypervariable peptide segment positioned at the N-terminus of the catalytic domain (region 103-153 in Fuc-TIII), determine whether or not these enzymes can add fucose in [alpha]1,4-linkage to type I acceptor (Legault et al., 1995). Another study has also demonstrated the importance of a cysteine residue probably located in the GDP-Fuc binding pocket of Fuc-TIII, Fuc-TV, and Fuc-TVI enzymes (Holmes et al., 1995). This GDP-fucose-protectable cysteine (Cys143 in Fuc-TIII) is located at the end of the region identified as being involved in acceptor recognition. In a very recent report, the amino acids essential for the activity of Fuc-TVI were investigated through chemical modification of the enzyme with group-selective reagents (Britten and Bird, 1997). Cysteine and histidine residues were shown to be essential for activity, but no inactivation was observed with reagents selective for arginine and lysine. The authors have identified five histidine residues conserved within the human [alpha]-3-fucosyltransferases. From our HCA alignment, His108 and His109 of Fuc-TVI appear to be the most conserved ones. However, a GDP-fucose-protectable lysine residue has been shown to be present in an [alpha]-3-fucosyltransferase isolated from human lung small cell carcinoma NCI-H69 cells (Holmes, 1992).
The recognition of complex carbohydrates by proteins occurred through hydrogen bond interactions with hydroxyl groups on carbohydrate and through stacking of the sugar ring with aromatic amino acids (Vyas, 1991). A series of deoxygenated type I and II acceptors were used to identify key polar groups on acceptors that are essential in enzyme binding (Hindsgaul et al., 1991; deVries et al., 1995; Du et al., 1996). In addition to the reactive hydroxyl at C-3 or C-4 of GlcNAc, Fuc-TIII, FucT-IV, and FucT-V were shown to have an absolute requirement for a hydroxyl group at carbon C-6 of galactose (de Vries et al., 1995; Du et al., 1996). In a previous study, Hindsgaul et al. (1991) have suggested that, for the enzymes Fuc-TIII and Fuc-TVI, the reactive acceptor hydroxyl group is involved in a critical hydrogen bond donor interaction with a base-acting group on the enzyme which removes the developing proton during the glycosyl transfer. This group has not yet been identified and could vary from one enzyme to another, but typical hydrogen bond acceptors include histidines and carboxylates. However, with regard to the mechanism of action of Fuc-TV, it has been recently proposed that the glycosidic bond cleavage occurs prior to the nucleophilic attack (Murray et al., 1997), and this enzyme has been shown to have a catalytic residue with pKa = 4.1, which is presumably a carboxylic acid residue (Murray et al., 1996). Product inhibition studies have also demonstrated that Fuc-TV has an ordered, sequential mechanism with GDP-Fuc binding first and the product GDP releasing last (Qiao et al., 1996).
The HCA method also permits the identification of strictly conserved amino acids which are of interest because they can be involved in nucleotide donor and acceptor recognition and in catalysis. Invariant residues such as aspartic or glutamic acid could be of particular importance for catalysis (Murray et al., 1996), whereas basic residues could interact with the phosphate groups of the nucleotide sugar donor (Vrielink et al., 1994), and aromatic and polar residues are frequently found in protein-carbohydrate interactions (Vyas et al., 1991). Among the [alpha]-3-fucosyltransferases, more than 20 basic, acidic, and polar residues are found conserved in the catalytic domains of these enzymes, and thus it is not possible to predict which ones are the most important for activity. Only two cysteine residues both located in the N-terminal part of the catalytic domain were found highly conserved in all the [alpha]-3-fucosyltransferases (Cys81 and Cys91 in Fuc-TIII). In contrast, only a few amino acids are strictly conserved in the [alpha]-2 and [alpha]-6-fucosyltransferase superfamily, and they are displayed in Figure 2 on a black background. The peptide motif contains three conserved basic (Arg, His) and one aspartic acid (or Asn in NodZ sequences) residues. Other invariant amino acids, including two acidic residues were also found. Two cysteine residues are found conserved among all the [alpha]-2-fucosyltransferases including the Y.enterocolitica sequence (corresponding to Cys169 and Cys268 in the human H enzyme).
More than 20 sporadic nonprevalent inactivating mutations in FUT1 have been reported among H deficient individuals worldwide (Kelly et al., 1994; Kaneko et al., 1997; WWagner and Flegel 1997). Three types of mutation can give the H deficient phenotype: missense, nonsense, or microdeletions resulting in frameshifts. The single point missense mutations occurring in the best conserved part of the catalytic domain of the human H enzyme are listed in Table II. All the amino acids concerned by these mutations are strictly conserved among the mammalian [alpha]-2-fucosyltransferases. The corresponding amino acids in the Y.enterocolitica protein, pig [alpha]-6-Fuc-T and A.caulinodans NodZ sequences were deduced from the HCA alignment. Identical or conservative residues were found at the same positions in the Y.enterocolitica sequence. Two of these mutations (Arg220Cys and Asp278Asn) are indeed interesting as they involve amino acids which are found conserved in all the [alpha]-2 and [alpha]-6-fucosyltransferases. The residue Arg220 is located in the consensus peptide motif which we propose to be part of the nucleotide binding domain. The isosteric replacement Asp278Asn completely abolishes enzyme activity. These observations strongly suggest a role for these two amino acids in the catalytic process. Table II
Combining HCA and fold recognition methods, we have previously suggested that the mammalian [alpha]-2 and [alpha]-3-fucosyltransferases have a similar fold consisting of alternating [alpha]-helices and [beta]-strands (Breton et al., 1996). One of the most probable folds could be that of the [beta]-glucosyltransferase from phage T4, an enzyme catalyzing the transfer of glucose from UDP-Glc to hydroxymethylcytosine in DNA (Vrielink et al., 1994). In the crystal structure, the fold comprises two [alpha]/[beta] domains separated by a wide cleft constituting the binding pocket and the C-terminal domain is a typical nucleotide binding motif, the so-called Rossman fold (Rossman et al., 1975). In the present study, the HCA analysis was extended to all the eukaryotic and prokaryotic fucosyltransferase sequences and it allowed us to refine our previous predictions. Based on the assumption that fucosyltransferases possess a nucleotide binding domain, it is highly probable that it is located in the C-terminus part of these enzymes, starting approximately in the region preceding the peptide motif in [alpha]-2- and [alpha]-6-fucosyltransferases and in the corresponding region I in [alpha]-3-fucosyltransferases, with a minimal size estimated to approximately 100 residues. Consequently, we can assign the acceptor binding region in the N-terminus part of the catalytic domain. These observations are in agreement with the proposed location of the acceptor region of Fuc-TIII, Fuc-TV, and Fuc-TVI reported by Legault et al. (1995) and Xu et al.(1996). Conclusion
The present work demonstrates the presence of conserved structural features as well as a peptide motif in prokaryotic and eukaryotic [alpha]-2 and [alpha]-6-fucosyltransferases. We have also identified a few invariant amino acids which are probably involved in the catalytic process of these enzymes and which constitute potential targets for site-directed mutagenesis studies. Despite the lack of the peptide motif in the [alpha]-3-fucosyltransferases, some regions could be aligned by HCA with the [alpha]-2 and [alpha]-6-fucosyltranferases. These similarities occur in a region of the catalytic domain of these enzymes that could correspond to the nucleotide binding domain, by analogy to the crystal structure of the [beta]-glucosyltransferase of phage T4. All these observations suggest that the fucosyltransferases have evolved from a common ancestor even if we cannot totally exclude a case of convergent evolution.
Human H
mutationyeFuc-T
p[alpha]6Fuc-T
NodZ
References
Asp148->Tyr
Asn
-
-
Kaneko et al., 1997
Tyr154->His/Cys
Tyr
-
-
Kaneko et al., 1997; Wang et al., 1997;
Yu et al., 1997, Wagner et al., 1997
Leu164->His
Ile
-
-
Kelly et al., 1994
Trp171->Cys
Tyr
-
-
Wagner et al., 1997
Arg220->Cys
Arg
Arg
Arg
Yu et al., 1997
Tyr241->His
Tyr
Leu
Leu
Kaneko et al., 1997
Val259->Glu
Val
Leu
Leu
Wagner et al., 1997
Trp267->Cys
Trp
Leu
Val
Johnson et al., 1994
Asp278->Asn
Asp
Glu
Glu
Johnson et al., 1994
Ala315->Val
Ala
-
-
Wagner et al., 1997
Asn327->Thr
Asn
-
-
Yu et al., 1997
Materials and methods
Protein sequences were retrieved from GenBank or Swiss-Prot databanks. Pairwise sequence comparison were done using Bestfit or Gap programs (GCG package) and sequence similarity searches using Blast (Altschul et al., 1990).
HCA is a graphical protein sequence comparison method based on the detection and comparison of hydrophobic clusters which are presumed to correspond to the regular secondary structure elements constituting the hydrophobic core of globular proteins (Gaboriaud et al., 1987; Lemesle-Varloot et al., 1990). The protein sequences are represented on a duplicated [alpha]-helical net, and the clusters of contiguous hydrophobic (V, I, L, F, M, W, Y) residues are drawn. The HCA plots were obtained using the program HCA-Plot V2.0 (Doriane S.A., Le Chesnay, France). Graphical manipulation of the HCA plots were carried out using IslandDraw V3.0 (Island Graphics Corp., Hoofddorp, The Netherlands).
Note added in proof
During the review of the manuscript, the sequence of a new [alpha]-3-fucosyltransferase from Helicobacter pylori was published in GenBank under accession number AF006039. The nucleotide-deduced protein sequence is composed of 333 amino acids. This enzyme appears to have a different domain organization and display very low overall sequence identity with the other members of the [alpha]-3-fucosyltransferase family. However, we can properly align by HCA the region 1-220 of the H.pylori sequence with the region 100-361 of Fuc-TIII. Interestingly, this sequence contains almost all the highly conserved residues of the region II and four of the conserved residues of the region I. No cysteine residue was found conserved between the eukaryotic [alpha]-3-fucosyltransferase and the H. pylori sequence.
Acknowledgments
The work was supported in part by Grant 9514111 Action Concertée Coordonnée des Sciences du Vivant (ACCSV14) from Ministère de l'Education Nationale de l'Enseignement Supérieur et de la Recherche (MENESR, France), and the Immunology concerted action 3026PL950004 of the Biotechnology program from the European Union (EU). C.B. is a staff member of Institut National de la Recherche Agronomique.
Abbreviations
Fuc-T, fucosyltransferase; GDP-Fuc, guanosine diphosphate-[beta]-l-fucose; Gal, galactose; GlcNAc, N-acetylglucosamine; HCA, hydrophobic cluster analysis; UDP-Glc, uridine diphosphate-[alpha]-d-glucose.
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