A new superfamily of protein-O-fucosyltransferases, {alpha}2-fucosyltransferases, and {alpha}6-fucosyltransferases: phylogeny and identification of conserved peptide motifs

Ivan Martinez-Duncker2, Rosella Mollicone2, Jean-Jacques Candelier2, Christelle Breton3 and Rafael Oriol1,2

2 Unité de Glycobiologie et Signalisation Cellulaire, INSERM U504, GDR CNRS 2590, Université de Paris Sud XI, 16 Ave Paul Vaillant-Couturier, 94807 Villejuif Cedex France, and 3 CERMAV-CNRS, BP 53, 38041 Grenoble Cedex 9, France

Received on July 9, 2003; accepted on August 14, 2003


    Abstract
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 References
 
The presence of three conserved peptide motifs shared by {alpha}2-fucosyltransferases, {alpha}6-fucosyltransferases, the protein-O-fucosyltransferase family 1 (POFUT1) and a newly identified protein-O-fucosyltransferase family 2 (POFUT2), together with evidence that the present genes encoding for these enzymes have originated from a common ancestor by duplication and divergent evolution, suggests that they constitute a new superfamily of fucosyltransferases.

Key words: {alpha}2-fucosyltransferase / {alpha}6-fucosyltransferase / conserved peptide motifs / phylogeny / protein-O-fucosyltransferase


    Introduction
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 References
 
Like all the glycosyltransferases, the fucosyltransferases (FUTs) need two substrates to catalyze the reaction: a nucleotide-sugar donor and an acceptor. Because all the known FUTs use the same GDP-Fuc donor substrate (Becker and Lowe, 2003Go), they have been classified in four different families according to their acceptor substrate and the linkage made.

  1. The {alpha}2-FUTs FUT1 (Larsen et al., 1990Go), FUT2, and Sec1 in nonhuman mammals (Kelly et al., 1995Go) add fucose in {alpha}1,2 to the terminal Gal of lactosamine to make the H antigens (Apoil et al., 2000Go).
  2. The {alpha}3/4-FUTs FUT3 (Kukowska-Latallo et al., 1990Go), FUT4 (Goelz et al., 1990Go), FUT5 (Weston et al., 1992a), FUT6 (Weston et al., 1992b), FUT7 (Natsuka et al., 1994Go), and FUT9 (Cailleau-Thomas et al., 2000Go; Nakayama et al., 2001Go) add fucose in {alpha}1,4 or {alpha}1,3 to the GlcNAc of type I lactosamine (Lea, sia-Lea, or Leb antigens) or type II lactosamine (Lex, sia-Lex, or Ley antigens), respectively (Watkins, 1995Go). In the particular case of plants, insects (Staudacher et al., 1999Go), and some parasites (Van Die et al., 1999Go) they can add fucose in {alpha}1,3 to the first GlcNAc of the N-glycan core.;
  3. In vertebrates, this last linkage is replaced by an {alpha}1,6 linkage on the same sugar made by the FUT8 {alpha}6-FUTs (Yanagidani et al., 1997Go).
  4. The protein-O-fucosyltransferases (POFUTs) add fucose directly to Ser or Thr in glycoproteins (Wang et al., 2001Go).

The FUT1, FUT2, and Sec1 have a high degree of similarity and constitute a single family of {alpha}2-FUTs. The {alpha}3- and {alpha}4-FUTs are different from the {alpha}2-FUTs and are considered as a single family of {alpha}3/4-FUTs because they also have a high degree of similarity. Furthermore, in some cases it has been demonstrated that both {alpha}1,3 and {alpha}1,4 activities can be carried on by the same enzyme (FUT3 or FUT5) (Costache et al., 1997Go), and the change of a single amino acid can modify their relative {alpha}1,3- or {alpha}1,4-activities (Dupuy et al., 1999Go, 2002Go). The {alpha}2- and {alpha}6-FUTs share a lower degree of similarity between them, and only the highly sensitive bidimensional hydrophobic cluster analysis (HCA) revealed the presence of three common conserved peptide motifs (I, II, and III), which constitute a signature for these enzymes (Breton et al., 1998Go; Chazalet et al., 2001Go). This has led us to consider {alpha}2- and {alpha}6-FUTs as part of the same superfamily originated from a common ancestor by duplication followed by divergent evolution (Oriol et al., 1999Go).

When originally described, the first family of POFUT was thought to be independent from all the other known FUTs (Wang et al., 2001Go), but we show now that there are two families of POFUTs, POFUT1 and POFUT2, which are related to {alpha}2- and {alpha}6-FUTs.


    Results and discussion
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 References
 
The PSI-BLAST of human FUT8 converged after five iterations and retrieved six enzymes from different species. Three more sequences were revealed by TBLASTN of EST to complete the nine FUT8 sequences listed in Figure 1.



View larger version (123K):
[in this window]
[in a new window]
 
Fig. 1. CLUSTAL W alignment of the motifs found in the 11 selected {alpha}2-FUTs, and all the {alpha}6-FUTs and POFUTs. White letters with black background are amino acids conserved at 70% in two or more families. Black letters with gray background are conserved amino acids at 70% in only one of the four families. Amino acids: [VILM], [YFW], [TS], [RKH], and [EDQN] were considered equivalent to define conserved positions. When the EMBL entries are ambiguous, SWALL or NCBI protein accession numbers are given in italics.

 
After six iterations the PSI-BLAST of human POFUT1 converged and retrieved six enzymes of the POFUT1 family and seven sequences of a new family that we called POFUT2. Similar results were obtained with a PSI-BLAST made with mouse POFUT2. Three more sequences of POFUT1 and four more sequences of POFUT2 were revealed by TBLASTN of EST with the corresponding human POFUT1 and mouse POFUT2, respectively. This completed the 9 sequences of POFUT1 and the 11 sequences of POFUT2 listed in Figure 1. The original human POFUT2 present in the SWALL data bank (Q9Y2G5) did not have the complete conserved peptide motif III due to a change of reading frame, but during this study we found a new human POFUT2 (AJ575591) with the three complete conserved peptide motifs (Figure 1).

Conserved peptide motifs
The HCA of the human POFUT1 and POFUT2 allowed us to identify the same three conserved peptide motifs (I, II, and III) of {alpha}2- and {alpha}6-FUTs in the POFUT (Figure 2), suggesting that {alpha}2-, {alpha}6- and protein-O-FUTs belong to the same superfamily.



View larger version (85K):
[in this window]
[in a new window]
 
Fig. 2. HCA of human FUT1 (A), FUT2 (B), FUT8 (C), POFUT1 (D), and POFUT2 (E). The amino acids and the contour of the hydrophobic clusters inside the conserved peptide motifs I, II, and III are highlighted in red with yellow background. HCA uses a bidimensional representation of the amino acid sequences duplicated and coiled up in an open virtual cylinder. The clusters of contiguous hydrophobic amino acids are drawn by the program, and each amino acid is represented by the single letter code except: proline (stars), glycine (diamonds), serine (boxes with dots) and threonine (open boxes) (Lemesle-Varloot et al., 1990). The analysis involves the visual comparison of hydrophobic cluster shapes and their distribution to find similarities between protein sequences with low overall sequence similarity.

 
In general, the intermotif distances are relatively well conserved within each family (Figure 1), but the distances between motifs I and II are longer in POFUT1 (57–62) than in the other families (25–34), and the II–III intermotif distances are longer in FUT8 (33–35) and in POFUT2 (27–30) than in the other families (19–23). These intermotif distance differences, secondary to indels of different sizes, constitute a handicap for the classical alignment methods and explain why the common features of these four families remained elusive to observe at the time of their cloning and characterization (Uozumi et al., 1996Go; Wang et al., 2001Go).

At the level of the peptide motifs, the most closely related enzymes are the two {alpha}2-FUTs, FUT1 and FUT2, that share 53 out of the 56 conserved positions (95%). The two POFUTs share 28 conserved positions (50%). FUT1 and FUT8 share 25 conserved positions (45%). FUT1 and POFUT1 share 23 conserved positions (41%), and finally FUT1 and POFUT2 are the less similar with only 21 shared conserved positions (38%). FUT8 and POFUT2 share 28 conserved positions (50%) suggesting that FUT8 has more similarity with POFUTs than with {alpha}2-FUTs (45%) (Figure 1). This is in good agreement with the shapes of the hydrophobic clusters detected in the human conserved motifs by HCA (Figure 2).

Phylogeny
The phylogenetic analysis was carried on the selected 54 amino acid positions of the block of 56 positions, constituted by the juxtaposition of the 3 conserved peptide motifs shown in Figure 1. The expected four families appear clearly, and the roots of each of these four families have highly significant bootstrap values (87–99%) (Figure 3), illustrating that in spite of the presence of numerous conserved positions, there are enough differences among the conserved peptide motifs to clearly cluster the four enzyme families separately.



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 3. Neighbor joining phylogenetic tree made with the selected 54 amino acid positions of the 40 sequences, comprising the 3 conserved peptide motifs (I, II, III) of the {alpha}2-FUTs, {alpha}6-FUTs, and POFUTs shown in Figure 1. Bootstrap values >50% are reported on the left of each divergence point. The four dots are the roots of each individual family composing the new superfamily. The scale bar represents the number of substitutions per site for a unit branch length.

 
The neighbor joining tree (Figure 3) suggests in addition the following.
  1. The duplication event in the {alpha}2-FUTs at the origin of the present FUT1 and FUT2 is relatively recent, after the separation of worms (Caenorhabditis elegans) and amphibians (Xenopus laevis), from the main evolutionary pathway but before the great mammalian radiation.
  2. Unlike this, the two duplication events at the origin of the separation of the ancestors of {alpha}6-FUTs and POFUTs are relatively more ancient events, which occurred before the separation of worms, insects, and urochordates from the main evolutionary path, because sequences of C. elegans, Drosophila melanogaster, and Ciona intestinalis are present in the three branches of FUT8, POFUT1, and POFUT2.
  3. The POFUTs are more closely related to the {alpha}6-FUTs than to {alpha}2-FUTs, in good correlation with their degree of motif conservation.

Recently two new putative human FUTs with the common conserved peptide motifs of {alpha}3-FUTs have been described and called FUT10 (AJ512465) and FUT11 (BC036037) (Roos et al., 2002Go). Although they are not yet fully characterized, these last two enzymes have been added to the GDB list of FUTs that now has 11 members, from FUT1 to FUT11. Following the same line of thought, human POFUT1 and POFUT2 should receive the following numbers in this series of human FUTs (FUT12 and FUT13, respectively), because the phylogenetic evidence suggests that they have a common genetic origin, they belong to the same superfamily as the {alpha}2- and {alpha}6-FUTs, and they use the same GDP-Fuc donor substrate as does every other FUT (Figure 3).


    Materials and methods
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 References
 
Only eukaryote animal sequences were considered for this study, orthologous proteins from other animal species were searched in EMBL by PSI-BLAST (Altschul et al., 1997Go) with default parameters using the corresponding human or mouse proteins. In addition, the other EST database (nonhuman nonmouse) was searched by TBLASTN with each of the human or mouse enzymes. Contigs of the different ESTs of each gene were made with CAP (Huang, 1992Go). New complete open reading frames identified in this EST-CAP searches with more than two identical amino acids overlapping in each position were annotated and submitted to EMBL as putative {alpha}6-FUTs (C. intestinalis clone 2, AJ515151; C. intestinalis clone 5, AJ515152; X. laevis, AJ514872), putative POFUTs related to POFUT1 (X. laevis, AJ514425; Gallus gallus, AJ535754; Sus scrofa, AJ567917) and putative POFUTs related to POFUT2 (Homo sapiens, AJ575591; Bos taurus, AJ575655; C. intestinalis, AJ575656; and G. gallus, AJ575657).

The conserved peptide motifs I, II, and III of human FUT1, FUT2, FUT8, POFUT1, and POFUT2, were visually identified by HCA (Lemesle-Varloot et al., 1990Go) (http://smi.snv.jussieu.fr/hca/hca-form.html) (Figure 2). Then the amino acid conserved positions within the peptide motifs of all the enzymes were identified by CLUSTAL W alignment (Thomson et al., 1994Go).

We have previously identified 18 putative {alpha}2-FUTs in C. elegans (Oriol et al., 1999Go); one of them (P91200) has been certified to have {alpha}2-FUT activity (Zheng et al., 2002Go). We selected only two of the C. elegans {alpha}2-FUTs, because all the C. elegans {alpha}2-FUTs appear in a single cluster, branching out from the main evolutionary path, before the separation of the vertebrate FUT1 and FUT2 enzymes (Oriol et al., 1999Go). In addition, the X. laevis {alpha}2-FUT and four species with known FUT1 and FUT2 enzymes (human, murine, porcine, and bovine) were added to complete the {alpha}2-FUT family. The {alpha}2-FUTs have not been detected among insects (Roos et al., 2002Go), urochordates, or birds (this study).

Phylogeny was made with Neighbor Joining (PHYLOWIN package) (Galtier et al., 1996Go), with G-BLOCKS (Castresana, 2000Go), and observed distances. Five hundred sets of data were used for bootstrap calculations (Figure 3).


    Acknowledgements
 
This work was partially supported by Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS), and Association de la Recherche contre le Cancer (ARC) grant RM-5348.


    Footnotes
 
1 To whom correspondence should be addressed; e-mail: oriol{at}vjf.inserm.fr Back


    Abbreviations
 
FUT, fucosyltransferase; HCA, hydrophobic cluster analysis; POFUT, protein-O-fucosyltransferase


    References
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 References
 
Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D.J. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res., 25, 3389–3402.[Abstract/Free Full Text]

Apoil, P.A., Roubinet, F., Despiau, S., Mollicone, R., Oriol, R., and Blancher, A. (2000) Evolution of {alpha}2-fucosyltransferase genes in primates: relation between an intronic Alu-Y element and red cell expression of ABH antigens. Mol. Biol. Evol., 17, 337–351.[Abstract/Free Full Text]

Becker, D.J. and Lowe, J.B. (2003) Fucose: biosynthesis and biological function in mammals. Glycobiology, 13, 41R–53R.[Abstract/Free Full Text]

Breton, C., Oriol, R., and Imberty, A. (1998) Conserved structural features in eukaryotic and prokaryotic fucosyltransferases. Glycobiology, 8, 87–94.[Abstract/Free Full Text]

Cailleau-Thomas, A., Couillin, P., Candelier, J.J., Balanzino, L., Mennesson, B., Oriol, R., and Mollicone, R. (2000) FUT4 and FUT9 genes are expressed early in human embryogenesis. Glycobiology, 10, 789–802.[Abstract/Free Full Text]

Castresana, J. (2000) Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol. Biol. Evol., 17, 540–552.[Abstract/Free Full Text]

Chazalet, V., Uehara, K., Geremia, R., and Breton, C. (2001) Identification of essential amino acids in the Azorhizobium caulinodans fucosyltransferase NodZ. J. Bacteriol., 183, 7067–7075.[Abstract/Free Full Text]

Costache, M., Apoil, P.A., Cailleau, A., Elmgren, A., Larson, G., Henry, S., Blancher, A., Iordachescu, D., Oriol, R., and Mollicone, R. (1997) Evolution of fucosyltransferase genes in vertebrates. J. Biol. Chem., 272, 29721–29728.[Abstract/Free Full Text]

Dupuy, F., Petit, J.M., Mollicone, R., Oriol, R., Julien, R., and Maftah, A. (1999) A single amino acid in the hypervariable stem domain of vertebrate {alpha}1,3/1,4-fucosyltransferases determines the type 1 type 2 transfer—characterization of acceptor substrate specificity of the Lewis enzyme by site-directed mutagenesis. J. Biol. Chem., 274, 12257–12262.[Abstract/Free Full Text]

Dupuy, F., Germot, A.M., Oriol, R., Blancher, A., Julien, R., and Maftah, A. (2002) {alpha}1,4-Fucosyltransferase activity: a significant function in the primate lineage has appeared twice independently. Mol. Biol. Evol., 19, 815–824.[Abstract/Free Full Text]

Galtier, N., Gouy, M., and Gautier, C. (1996) Two graphic tools for sequence alignment and molecular phylogeny. Comput. Appl. Biosci., 12, 543–548.[Abstract]

Goelz, S.E., Hession, C., Goff, D., Griffiths, B., Tizard, R., Newman, B., Chi-Rosso, G., and Lobb, R. (1990) ELFT: a gene that directs the expression of an ELAM-1 ligand. Cell, 63, 1349–1356.[ISI][Medline]

Huang, X. (1992) A cloning assembly program based on sensitive detection of fragment overlaps. Genomics, 14, 18–25.[ISI][Medline]

Kelly, R.J., Rouquier, S., Giorgi, D., Lennon, G.G., and Lowe, J.B. (1995) Sequence and expression of a candidate for the human secretor blood group {alpha}(1,2)fucosyltransferase gene (FUT2)—homozygosity for an enzyme-inactivating nonsense mutation commonly correlates with the non-secretor phenotype. J. Biol. Chem., 270, 4640–4649.[Abstract/Free Full Text]

Kukowska-Latallo, J.F., Larsen, R.D., Nair, R.P., and Lowe, J.B. (1990) A cloned human cDNA determines expression of a mouse stage-specific embryonic antigen and the Lewis blood group {alpha}(1,3/1,4)fucosyltransferase. Gen. Develop, 4, 1288–1303.[ISI]

Larsen, R.D., Ernst, L.K., Nair, R.P., and Lowe, J.B. (1990) Molecular cloning, sequence and expression of a human GDP-L-fucose: ß-D-galactoside {alpha}2-L-fucosyltransferase cDNA that can form the H blood group antigen. Proc. Natl Acad. Sci. USA, 87, 6674–6678.[Abstract]

Lemesle-Varloot, L., Henrissat, B., Gaboriaud, C., Bissery, V., Morgat, A., and Mornon, J.P. (1990) Hydrophobic cluster analysis: procedures to derive structural and functional information from 2-D-representation of protein sequences. Biochimie, 72, 555–574.[CrossRef][ISI][Medline]

Nakayama, F., Nishihara, S., Iwasaki, H., Kudo, T., Okubo, R., Kaneko, M., Nakamura, M., Karube, M., Sasaki, K., and Narimatsu, H. (2001) CD15 expression in mature granulocytes is determined by {alpha}1,3-fucosyltransferase IX, but in promyelocytes and monocytes by {alpha}1,3-fucosyltransferase IV. J. Biol. Chem., 276, 16100–16106.[Abstract/Free Full Text]

Natsuka, S., Gersten, K.M., Zenita, K., Kannagi, R., and Lowe, J.B. (1994) Molecular cloning of a cDNA encoding a novel human leukocyte {alpha}-1,3-fucosyltransferase capable of synthesizing the sialyl Lewis x determinant. J. Biol. Chem., 269, 16789–16794.[Abstract/Free Full Text]

Oriol, R., Mollicone, R., Cailleau, A., Balanzino, L., and Breton, C. (1999) Divergent evolution of fucosyltransferase genes from vertebrates, invertebrates, and bacteria. Glycobiology, 9, 323–334.[Abstract/Free Full Text]

Roos, C., Kolmer, M., Mattila, P., and Renkonen, R. (2002) Composition of Drosophila melanogaster proteome involved in fucosylated glycan metabolism. J. Biol. Chem., 277, 3168–3175.[Abstract/Free Full Text]

Staudacher, E., Altmann, F., Wilson, I.B.H., and Marz, L. (1999) Fucose in N-glycans: from plant to man. Biochem. Biophys. Acta, 1473, 216–236.[ISI][Medline]

Thomson, J.D., Higgins, D.G., and Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res., 22, 4673–4680.[Abstract]

Uozumi, N., Yanagidani, S., Miyoshi, E., Ihara, Y., Sakuma, T., Gao, C.X., Teshima, T., Fujii, S., Shiba, T., and Taniguchi, N. (1996) Purification and cDNA cloning of porcine human GDP-L-Fuc:N-acetyl-ß-D-glucosaminide {alpha}6-fucosyltransferase. J. Biol. Chem., 271, 27810–27817.[Abstract/Free Full Text]

Van Die, I., Gomord, V., Kooyman, F.N., Van der Berg, T.K., Cummings, R.D., and Vervelde, L. (1999) Core {alpha}1,3-fucose is a common modification of N-glycans in parasitic helminths and constitutes an important epitope for IgE from Haemonchus contortus infected sheep. FEBS Lett., 463, 189–193.[CrossRef][ISI][Medline]

Wang, Y., Shao, L., Shi, S., Harris, R.J., Spellman, M.W., Stanley, P., and Haltiwanger, S. (2001) Modification of epidermal growth factor-like repeats with O-fucose: molecular cloning and expression of a novel GDP-fucose: protein O-fucosyltransferase. J. Biol. Chem., 276, 40338–40345.[Abstract/Free Full Text]

Watkins, W.M. (1995) Biosynthesis. Molecular basis of antigenic specificity in the ABO, H and Lewis blood group systems. In Montreuil, J., Schachter, H., and Vligenhart, J.F.G. (Eds.), Glycoproteins. Elsevier, Amsterdam, pp. 313–390.

Weston, B.W., Nair, R.P., Larsen, R.D., and Lowe, J.B. (1992a) Isolation of a novel human {alpha}(1,3)fucosyltransferase gene and molecular comparison to the human Lewis blood group {alpha}(1,3/1,4)fucosyltransferase gene. J. Biol. Chem., 267, 4152–4160.[Abstract/Free Full Text]

Weston, B.W., Smith, P.L., Kelly, R.J., and Lowe, J.B. (1992b) Molecular cloning of a fourth member of a human {alpha}(1,3)fucosyltransferase gene family. J. Biol. Chem., 267, 24575–24584.[Abstract/Free Full Text]

Yanagidani, S., Uozumi, N., Ihara, Y., Miyoshi, E., Yamaguchi, N., and Taniguchi, N. (1997) Purification and cDNA cloning of GDP-L-Fuc:N-acetyl-ß-D-glucosaminide: {alpha}1–6 fucosyltransferase ({alpha}1-6 FucT) from human gastric cancer MKN45 cells. J. Biochem., 121, 626–632.[Abstract]

Zheng, Q.L., Van Die, I., and Cummings, R.D. (2002) Molecular cloning and characterization of a novel {alpha}1,2-fucosyltransferase (CE2FT-1) from Caenorhabditis elegans. J. Biol. Chem., 277, 39823–39832.[Abstract/Free Full Text]