FEBS 22092

FEBS Letters 452 (1999) 237^242

K1,3-Fucoslytransferase IX (Fuc-TIX) is very highly conserved between
human and mouse; molecular cloning, characterization and tissue
distribution of human Fuc-TIX
Mika Kanekoa;b , Takashi Kudoa , Hiroko Iwasakia , Yuzuru Ikeharaa , Shoko Nishiharaa ,Satoshi Nakagawac , Katsutoshi Sasakic , Takashi Shiinad , Hidetoshi Inokod , Naruya Saitoub ,Hisashi Narimatsua; *baDivision of Cell Biology, Institute of Life Science, Soka University, 1-236 Tangi-cho, Hachioji-shi, Tokyo 192-8577, Japan
Department of Genetics, School of Life Science, Graduate University for Advanced Studies, National Institute of Genetics, 1111 Yata, Mishima-shi,
Shizuoka 411-8540, Japanc
Tokyo Research Laboratories, Kyowa Hakko Kogyo Company, Limited, 3-6-6 Asahi-machi, Machida-shi, Tokyo 194-8533, JapandSecond Department of Molecular Life Science, School of Medicine, Tokai University, Isehara-shi, Kanagawa 259-1193, Japan

Received 12 April 1999

Abstract The amino acid sequence of Fuc-TIX is very highly
conserved between mouse and human. The number of nonsynonymous nucleotide substitutions of the Fuc-TIX gene
between human and mouse was strikingly low, and almost
equivalent to that of the K-actin gene. This indicates that FucTIX is under a strong selective pressure of preservation during
evolution. The human Fuc-TIX (hFuc-TIX) showed a uniquecharacteristics, i.e. hFuc-TIX was not activated by Mn2 + andCo2 +, whereas hFuc-TIV and hFuc-TVI were activated by thecations. The hFuc-TIX transcripts were abundantly expressed inbrain and stomach, and interestingly were detected in spleen andperipheral blood leukocytes.
z 1999 Federation of European Biochemical Societies.
Key words: Glycosyltransferase; Fucosyltransferase;
K1,3-Fucosyltransferase ; Lewis x; CD15; SSEA-1

1. Introduction
The Lewis x (Lex ) carbohydrate epitope, which is de¢ned as
GalL1,4(FucK1,3)GlcNAc, is synthesized by transferring a fucose (Fuc) to the N-acetylglucosamine (GlcNAc) residue ofthe type 2 chain, GalL1,4GlcNAc, with an K1,3-linkage.
This fucose transfer is catalyzed by K1,3-fucosyltransferase(K1,3Fuc-T). So far, the human genes encoding ¢ve
K1,3Fuc-Ts (Fuc-TIII, IV, V, VI and VII or FUT3, 4, 5, 6
and 7) have been cloned [1^6]. In a previous paper, we reported the molecular cloning of a new member of theK1,3Fuc-T family in the mouse and named the enzyme mouse
Fuc-TIX (mFuc-TIX or mFUT9) [7].
CD15 is one of the di¡erentiation markers of the cells, andthe CD15 epitope has been determined as the Lex carbohydrate structure. Several immunohistochemical studies have de*Corresponding author. Fax: (81) (426) 91-9315.
E-mail: hisashi@scc1.t.soka.ac.jp
Abbreviations: Fuc-T, fucosyltransferase; Lex , Lewis x; Fuc, fucose;
GlcNAc, N-acetylglucosamine; CNS, central nervous system; sLex ,sialyl Lewis x; ORF, open reading frame; PA, pyridylamination;
RT-PCR, reverse transcription-polymerase chain reaction; Ley , Lewis
y; LNnT, lacto-N-neotetraose; LNT, lacto-N-tetraose; sialyl-LNnT,
K2,3-sialyl lacto-N-tetraose; Lea , Lewis a; PBL, peripheral blood leukocytes

tected CD15 antigens in certain neuronal cells and glial cells
in the central nervous system (CNS) of humans and rodents[8^10]. The expression of CD15 antigens in the CNS is developmentally regulated, and considered to play a role in neuronal development. In our previous study, mFuc-TIX was identi¢ed as the most likely candidate for the enzyme synthesizing
the Lex structure (CD15 epitope) in the mouse CNS [7].
Some K1,3Fuc-Ts are known to be polymorphic in human.
Three kinds of null alleles of the Fuc-TIII (Le) gene, named
le1, le2 and le3 alleles, are widely distributed in the Japanese
population [11^13]. Homozygotes of null Fuc-TVI alleles werealso found in some populations [14]. In bovine, there is only
one gene, named futb, corresponding to the hFuc-TV-hFucTIII-hFuc-TVI gene cluster [15]. In addition, the mouse geneorthologous to the futb gene seems to be a pseudogene [16]. In
this sense, the hFuc-TIII, hFuc-TV and hFuc-TVI genes are
regarded as non-essential for individual ontogeny, and therefore their sequences are not conserved among species.
Mice and humans possess functional Fuc-TIV and FucTVII genes [3^6,16,17]. The distribution of Fuc-TVII is limited to speci¢c tissues and cell populations such as leukocytes
and high endothelial cells of the venule [18]. Fuc-TVII apparently participates in the synthesis of sialyl Lewis x (sLex )epitopes in leukocytes: this epitope is required for leukocyte
tra¤cking [18]. However, Fuc-TVII cannot synthesize theCD15 (Lex ) epitope [4,5,19]. Fuc-TIV is ubiquitously expressed in various tissues, and probably participates in
CD15 epitope synthesis in some tissues, but its biological
function remains to be elucidated [16].
In this study, we cloned a human gene, named hFuc-TIX,
orthologous to the mFuc-TIX gene, and analyzed its characteristics and tissue distribution. Interestingly, the nucleotide
and amino acid sequences of hFuc-TIX were very highly conserved in comparison with those of mFuc-TIX [7]. This indicates that Fuc-TIX has been under a strong selective pressureduring its evolution, suggesting that it is essential for ontogeny.
2. Materials and methods
2.1. cDNA cloning and sequencing of the hFuc-TIX gene
A cDNA library derived from a human gastric mucosa was constructed in our laboratory (Ikehara et al., unpublished). We screened
the human stomach cDNA library with a mFuc-TIX cDNA probe

0014-5793 / 99 / $20.00 ß 1999 Federation of European Biochemical Societies. All rights reserved.
PII: S 0 0 1 4 - 5 7 9 3 ( 9 9 ) 0 0 6 4 0 - 7

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M. Kaneko et al./FEBS Letters 452 (1999) 237^242

encompassing the full-length open reading frame (ORF), and obtained
several clones encoding the hFuc-TIX gene. The inserts were subjected
to nucleotide sequencing.
2.2. Calculation of the numbers of synonymous and non-synonymous
nucleotide substitutions
To calculate the numbers of synonymous and non-synonymous
nucleotide substitutions in Fuc-T genes between mouse (or other species) and human, we employed the ODEN program [20] of Supernig
at the National Institute of Genetics, Japan.
2.3. Flow cytometric analysis of Namalwa cells stably expressing the
hFuc-TIX gene and assaying of K1,3Fuc-T activity
Namalwa cells stably expressing the hFuc-TIX gene were established, and subjected to £ow cytometry analysis, using SSEA-1(anti-Lex ; Developmental Studies Hybridoma Bank, University of
Iowa, Ames, IA, USA) [21], AH6 (anti-Lewis y (Ley )) [22] andCSLEX-1 (anti-sLex ) [23] and assaying of K1,3Fuc-T activity using
pyridylaminated (PA) oligosaccharides, as described previously [7].
2.4. Quantitative analysis of K1,3Fuc-T transcripts in various humantissues using the competitive reverse transcription-polymerase chain
reaction (competitive RT-PCR) method
The competitive RT-PCR methods for the measurement of thetranscripts for the hFuc-T III, IV, V, VI, VII genes have been described in detail in our previous paper [24]. To quantify the hFucTIX transcripts by competitive RT-PCR, a competitor DNA plasmid(pBS-hFT9d) was prepared by deleting the 113-bp Nhe -NsiI fragmentfrom the standard DNA (pBS-hFT9s) which encodes the full-length
ORF of the hFuc-TIX gene. A pair of PCR primers (forward primer,
5P-GGCAAGCATTTGGAGAATATGTCAAT-3P and reverse primer, 5P-ACCAACAGACTTATATTCTTGATGCC-3P) ampli¢ed 398bp and 285-bp fragments from pBS-hFT9s and pBS-hFT9d, respectively. cDNA samples were prepared from various human tissues by
the method described previously [7,24]. The PCR conditions for the
hFuc-TIX gene were as follows: a pre-PCR heating step at 95³C for
11 min, followed by 45 cycles of 1 min at 95³C, 1.5 min at 65³C, and2 min at 72³C. To ascertain the e¤ciency of cDNA preparation fromtotal RNAs, measurement of the L-actin transcripts in each sample
was performed.
2.5. In situ hybridization and immunohistochemical analyses
Surgically resected stomach specimens for in situ hybridization wereprepared as described previously [7]. The antisense and sense RNAprobes, at the nucleotide positions 655^835 of hFuc-TIX cDNA, labeled with digoxigenin were synthesized using a Dig RNA Labeling
Kit (Boehringer Mannheim) according to the supplier's manual. The
hybridization and signal detection procedures were performed as described previously [7].
Immunohistochemistry was performed with monoclonal antibodies,
PM-81 (anti-Lex ) [25] and AH6 (anti-Le y ), by the method described
previously [26].
2.6. Construction of a phylogenetic tree of the K1,3Fuc-T family
We used amino acid sequences for six human [1^6], three mouse[7,16,17], one rat [27], one bovine [15], one chicken [28], two zebra¢sh[29] and four Caenorhabditis elegans [30] genes. These 18 sequenceswere multiply aligned, and all positions with gaps were eliminated,
resulting in the alignment of 356 amino acid residues. Clustal W [31]was used for multiple alignment and the neighbor-joining method [32]was used to construct a tree. We also performed a bootstrap analysis
to gain a statistical measure of con¢dence in the phylogenetic tree. Bybootstrap analysis, a total of 1000 trees were generated from theinitial data set, and the percentage of trees containing a particularclade was measured.

3. Results
3.1. A putative amino acid sequence of hFuc-TIX deduced fromthe cDNA sequence which is highly conserved between
mouse and human
The 3019-bp nucleotide sequence of the hFuc-TIX cDNA
was registered by us in DDBJ/EMBL/GenBank with the ac-

Fig. 1. A schematic diagram of the primary structure of hFuc-TIX.
Three amino acid substitutions between hFuc-TIX and mFuc-TIX
are presented at positions 37, 237 and 292. A putative transmembrane domain is indicated with a dark shaded box. The amino acidsequence and position of the K1,3Fuc-T motif conserved in all
K1,3Fuc-Ts, including those of Helicobacter pylori [33], is presented.
A possible N-glycosylation site is indicated with an asterisk.

cession number AB023021. The putative amino acid sequence
predicted from the nucleotide sequence encodes a typical type
II membrane protein, and is very similar to that of mFucTIX, i.e. only three amino acid residues, Val-37 to Ile, Thr237 to Ala and Phe-292 to Tyr, di¡er among 359 amino acids(Fig. 1). The consensus amino acid sequence for the membersof the K1,3Fuc-T family [33], FxL/VxFENS/TxxxxYxTEK, isconserved in the hFuc-TIX sequence.
3.2. Synonymous and non-synonymous substitutions of themembers of the Fuc-T gene family
We calculated the numbers of synonymous (dS ) and nonsynonymous (dN ) nucleotide substitutions between mFuc-TIX
and hFuc-TIX genes to establish if there is some signi¢cantevolutionary pressure for maintaining the primary structure of
the enzyme (dS : number of substitutions per site which cause
no amino acid replacement; dN : number of substitutions persite which cause amino acid replacement) (Table 1). The other
Fuc-T genes, H (FUT1), Fuc-TIII (FUT3), Fuc-TIV (FUT4),
Fuc-TVII (FUT7) and FUT8 genes, were also analyzed todetermine their dS and dN values (Table 1). The mouse FucTIII and FUT8 genes have not been cloned, therefore thebovine orthologue (futb) for hFuc-TIII gene and the porcine
FUT8 gene were compared with the respective human genes.
We considered that it was essentially not a problem to usedi¡erent species to calculate dS and dN , because they started
to diverge more or less at the same time. The Fuc-TIX gene
showed the lowest dN value (0.005), followed by the FUT8
gene (0.021), among the six members of the Fuc-T gene family. The di¡erence in dN values between the Fuc-TIX gene andthe others was almost 40-fold. On the other hand, the dS

Table 1
Synonymous (dS ) and non-synonymous (dN ) substitutions per site of
K1,3Fuc-T genes
Gene

La

dS

dN

Species pairH (FUT1)
Fuc-TIII (FUT3)
Fuc-TIV (FUT4)
Fuc-TVII (FUT7)
FUT8
Fuc-TIX (FUT9)
histone H4b
K-actinb

368
366
408
343
576
360
101
376

0.938
0.516
0.734
0.950
0.331
0.527
0.228
0.587

0.148
0.218
0.159
0.111
0.021
0.005
0.001
0.002

mouse vs. humanbovine vs. humanmouse vs. humanmouse vs. humanporcine vs. humanmouse vs. humanmouse vs. humanmouse vs. humanabNumber of codons compared.
Li et al. [40].

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M. Kaneko et al./FEBS Letters 452 (1999) 237^242

239

cDNA, were analyzed by £ow cytometry. Namalwa-hFT9cells showed a positive peak with SSEA-1 (anti-Lex ) andAH6 (anti-Ley ), but not with CSLEX-1 (anti-sLex ) (data notshown). In contrast, Namalwa-hFT4 cells (stable transformant with the hFuc-TIV gene) showed positive peaks with all
three antibodies, SSEA-1, AH6 and CSLEX-1, although thepositive peak with CSLEX-1 was very weak (data not shown).
The K1,3Fuc-T activities of hFuc-TIX were measured
against PA oligosaccharides, i.e. lacto-N-neotetraose-PA(LNnT-PA), lacto-N-tetraose-PA (LNT-PA) and K2,3-sialyl
lacto-N-tetraose-PA (sialyl-LNnT-PA). hFuc-TIX exhibited
activity against LNnT-PA, resulting in the synthesis of Lex
epitope, but no activity against LNT-PA or sialyl-LNnTPA, resulting in no synthesis of the Lewis a (Lea ) or sLex
epitope, respectively (data not shown).
Thus, hFuc-TIX showed almost the same results of £ow
cytometry analysis and K1,3Fuc-T activities as mFuc-TIX [7].
3.4. Activation of K1,3Fuc-Ts in the presence of Mn2 + or Co2 +Three human K1,3Fuc-Ts, i.e. hFuc-TIX, hFuc-TIV andhFuc-TVI, were examined for activity against LNnT-PA in
the presence of Mn2 or Co2 (Fig. 2). Interestingly, hFucTIX was not activated at any concentration of Mn2 or Co2 ,moreover it was markedly suppressed by Mn2 and Co2(Fig. 2A). In contrast, hFuc-TIV was markedly activated in
the presence of Mn2 , but less so in that of Co2 (Fig. 2B).
hFuc-TVI was also markedly activated by both Mn2 andCo2 (Fig. 2C).

Fig. 2. Activation of hFuc-TIX, hFuc-TIV and hFuc-TVI by Mn2or Co2 . The activities toward LNnT-PA were measured at various
concentrations of Mn2 (E) or Co2 (F). The activity in the absenceof the dications is presented as 100% activity.

values for Fuc-TIX gene (0.527) and the others (0.331-0.950)
were similar.
3.3. Flow cytometry analysis of Namalwa cells transfected withthe hFuc-TIX gene and measurement of K1,3Fuc-T activityof hFuc-TIX towards oligosaccharides
Namalwa-hFT9 cells, stably expressing the hFuc-TIX

3.5. Tissue distribution and quantitative measurement ofsix human K1,3Fuc-T transcripts
The amounts of six human K1,3Fuc-T transcripts expressedin various tissues were measured by competitive RT-PCR(Table 2). hFuc-TIII was abundantly expressed in gastrointestinal tissues, i.e. stomach, jejunum and colon, and faintly expressed in spleen, lung, kidney and cervix uteri. hFuc-TIII was
not detected in brain, liver, adrenal cortex or peripheral blood
leukocytes (PBL). hFuc-TIV was found to be ubiquitously
expressed in all tissues examined and it was abundantly expressed in cervix uteri. hFuc-TV was not expressed in any
tissues examined, except for spleen in which it was faintly
expressed. The tissue distribution of hFuc-TVI was similar
to that of hFuc-TIII, except that hFuc-TVI was expressed
positively in liver whereas hFuc-TIII was not. hFuc-TVI
was much more abundant in kidney than hFuc-TIII. hFuc-

Table 2
Quantitative analysis of transcripts of six K1,3Fuc-T genes in various human tissues by competitive RT-PCR
Expression levels of K1,3Fuc-Ts relative to the level of L-actin (%)
Forebrain
Stomach (antrum)
Stomach (corpus)
Jejunum
Colon
Liver
SpleenLung
KidneyAdrenal cortex
Cervix uteri
PBL

Fuc-TIII

Fuc-TIV

Fuc-TV

Fuc-TVI

Fuc-TVII

Fuc-TIX

6 0.001
2.42
1.87
13.66
2.90
6 0.001
0.02
0.01
0.34
6 0.001
0.12
6 0.001

0.58
1.83
1.07
2.33
0.43
0.39
0.25
0.01
1.13
0.35
5.14
0.29

6 0.001
6 0.001
6 0.001
6 0.001
6 0.001
6 0.001
0.02
6 0.001
6 0.001
6 0.001
6 0.001
6 0.001

0.01
0.27
0.06
0.86
0.41
0.14
0.004
6 0.001
3.18
6 0.001
0.07
6 0.001

6 0.001
6 0.001
6 0.001
6 0.001
6 0.001
6 0.001
0.13
6 0.001
6 0.001
6 0.001
6 0.001
0.15

0.53
0.73
0.37
6 0.001
6 0.001
6 0.001
0.004
6 0.001
6 0.001
6 0.001
6 0.001
0.01

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Fig. 3. Immunohistochemical staining with PM-81 (anti-Lex ) and AH6 (anti-Ley ) and in situ hybridization with the hFuc-TIX probe on the antrum portion of human gastric mucosa. Serial sections were immunohistochemically stained with PM-81 (A) and AH6 (B). C shows the positive
signals detected with the antisense probe of segment 655^835, and D shows the negative signals with the sense probe of segment 655^835. The
scale bar represents 10 Wm.

TVII was detected in spleen and PBL. hFuc-TIX was markedly expressed in forebrain and stomach, i.e. in both the antrum and corpus of the stomach, and, interestingly, was detected in spleen and PBL. hFuc-TIX was not expressed in
kidney at all. In this regard, hFuc-TIX di¡ers from mFucTIX which is abundantly expressed in mouse kidney [7].
3.6. Immunohistochemical analysis of Lex and Ley expression
and in situ hybridization analysis of hFuc-TIX transcripts
in the antrum portion of human gastric mucosa
A stomach specimen of a secretor individual, whose secretor (Se) genotype is Se1/Se1 [26], was subjected to immunohistochemical analysis for Lex and Ley expression and in situ
hybridization with the hFuc-TIX probe. As can be seen in
Fig. 3A,B, the Lex antigens were mainly positive in theglandular compartments of the pyloric mucosa. In contrast,
the Ley antigens were detected both in the glandular compartments and in the pit of the gastric mucosa. The hFuc-TIX
transcripts were localized in the glandular compartments
alone, being well co-localized with the Lex antigens (Fig.
3C).

3.7. A phylogenetic tree of the K1,3Fuc-T gene family
We performed a FASTA search [34], and BLAST search[35] of the DDBJ/EMBL/GenBank International Nucleotide
Sequence Database to identify the genes homologous to the
hFuc-TIX gene. We used two speci¢c search programs, tfastx
and tblastn, which compare amino acid sequences with a
DNA database. Mammalian, chicken, zebra¢sh, C. elegans,
Helicobacter pylori, Drosophila melanogaster, and Vibrio cholerae genes were retrieved, while no members of the K1,2 orK1,6Fuc-T gene families were found to show homology withthe hFuc-TIX gene.
The root was determined by assuming that C. elegans genes
are located as outgroups (Fig. 4). The phylogenetic tree indicates that the Fuc-T genes evolved by independent gene duplication between vertebrates and C. elegans. There are fourclusters in the vertebrate Fuc-T gene family, the Fuc-TIX genesubfamily being the ¢rst to diverge from the ancestral gene.
Two zebra¢sh K1,3Fuc-Ts, zFT1 and zFT2, which are developmentally regulated in embryogenesis, form a cluster withmFuc-TIX and hFuc-TIX.

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M. Kaneko et al./FEBS Letters 452 (1999) 237^242

Fig. 4. Phylogenetic tree of K1,3Fuc-Ts. The branch lengths indicate
the evolutionary distances between di¡erent genes. 7 indicates thegene duplication and b the speciation. Four cosmid clone names
containing C. elegans genes are presented.

4. Discussion
hFuc-TIX can synthesize the Lex and Ley epitopes, but not
the Lea or sLex epitopes, in contrast to hFuc-TIV's low, but
apparently positive activity of sLex synthesis. These characteristics of hFuc-TIX were almost the same as those of mFucTIX. In the present study, we found that Mn2 and Co2 ionsdid not activate the hFuc-TIX activity at all.
Mollicone et al. reported that human brain homogenates
exhibited activity for Lex synthesis, but not for sLex synthesis[36,37]. The enzymes expressed in brain and leukocytes had
similar levels of activity, i.e. both showed similar Km values
for GDP-fucose and H-type 2 compounds, the same optimum
pH, and were resistant to N-ethylmaleimide. However, theydi¡ered in the enzyme activity when activated by dications.
The enzyme(s) in leukocytes exhibited only 10^15% of theactivity in the presence of Co2 as compared to Mn2 , whereas the activity of brain enzyme(s) did not di¡er in the presenceof Mn2 or Co2 [36,37]. Foster et al. indicated that the
enzyme puri¢ed from human neuroblastoma cells exhibiteda level of activity in the presence of 10 mM Co2 that was
75% of that in the presence of 10 mM Mn2 [38]. The characteristics of hFuc-TIX determined in the present study weremost similar to those of the enzyme(s) in adult brain [36].
However, there was some discrepancy between previous studies [36^38] and our study as to the characteristics of K1,3FucT activities which can be explained as follows. As demonstrated in Table 2 in the present study, many tissues containmultiple K1,3Fuc-Ts. The previous studies measured the activities directed by mixed enzymes in tissue homogenates,241

while we measured the activities of the recombinant enzymes.
In tissue homogenates, there may still be unknown K1,3FucTs, the genes of which have not been cloned.
Cameron et al. [39] reported the tissue distribution of three
hFuc-Ts, i.e. hFuc-TIII, V and VI, on Northern analysis andsemi-quantitative RT-PCR. We employed the competitive
RT-PCR method in the present study because of the following
advantages. Competitive RT-PCR enabled us for the ¢rst time
to compare all six K1,3Fuc-Ts in terms of the level of relative
amounts of transcripts in various human tissues. The small
amounts of transcripts, which could not be detected by Northern analysis, were quantitatively detected by this sensitive
method. Fuc-TIV is believed to be a single enzyme capableof Lex (CD15) synthesis in myeloid tissues. It is worth notingthat hFuc-TIX was also expressed in spleen and PBL almost
at the same level as hFuc-TIV. We will examine whether
hFuc-TIX and hFuc-TIV are expressed in cells of di¡erent
subpopulations in PBL or together in the same cells. hFucTIX was not expressed in human kidney at all, whereas
mFuc-TIX was most abundantly expressed in mouse kidneyas demonstrated in our previous study [7]. There may be species-speci¢c and tissue-speci¢c regulation of the Fuc-TIX gene.
In this study, we detected both hFuc-TIX and hFuc-TIV expression almost at the same level in forebrain, while we did
not detect mFuc-TIV in a previous study [7]. This discrepancy
may be attributed to the di¡erent areas of brain examined, or
to a species-speci¢c regulation as in kidney.
The co-localization of hFuc-TIX transcripts with the Lex
antigens in the glandular compartments of the stomach
strongly suggested that hFuc-TIX is involved in the synthesisof Lex antigens in stomach. However, the absence of hFucTIX in the foveolar cells in the gastric pits indicated that someother K1,3Fuc-T(s) are involved in Ley synthesis, because gastric mucosa expressed four kinds of K1,3Fuc-Ts, i.e. hFucTIII, IV, VI and IX, as shown in Table 2.
The position of Fuc-TIX in the phylogenetic tree indicatesthat the Fuc-TIX subfamily was the ¢rst to diverge in vertebrates. CFT1, a chicken homologue (probably orthologue) of
Fuc-TIV, is clustered with the mammalian Fuc-TIV subfamily. This suggests that a chicken orthologue of Fuc-TIX may
be found in the Fuc-TIX subfamily. It is of interest to know
to what extent the Fuc-TIX sequence is evolutionarily conserved in non-mammal vertebrates. The numbers of non-synonymous substitutions (dN ) of protein coding gene regions are
known to vary considerably. Genes for conservative proteinssuch as histones and actins show very low dN values (0.001
and 0.002, respectively, between mouse and human [40]), because selective constraints are strong. It should be emphasizedthat the dN value of the Fuc-TIX gene is as low as those ofhistone and K-actin genes. The very low dN value of the FucTIX gene clearly indicates a strong selective pressure for thepreservation of the Fuc-TIX amino acid sequence during the
mammalian evolution. Recently, two zebra¢sh K1,3Fuc-T
genes, zFT1 and zFT2, were found to be expressed in embryogenesis [29]. hFuc-TIX and mFuc-TIX form a cluster withzFT1 and zFT2 in the phylogenetic tree. Fuc-TIX may possibly be involved in mammalian embryogenesis, and play anessential role for ontogeny.
Acknowledgements: This work was supported in part by a Grant-inAid for Scienti¢c Research on Priority Areas, No. 10178104, from the
Ministry of Education, Science, and Culture of Japan.

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