(Received for publication, June 23, 1995; and in revised form, August 11, 1995)
From the
Partial amino acid sequence of
GDP-L-fucose:-D-galactoside
-2-L-fucosyltransferase purified from porcine
submaxillary glands was determined. Amino acid sequence analysis
yielded 100, 93.3, and 84.2%, and 75, 46.6, and 84.2% sequence identity
between 12-, 15-, and 19-amino acid tryptic peptides generated from
porcine enzyme and amino acid residues 61-72, 111-125, and
308-326 and 89-100, 139-153, and 338-356 of the
human Secretor and H type
-2-fucosyltransferases, respectively. Higher amino acid sequence
homology of the porcine enzyme with the predicted sequence for the
human Secretor locus as compared with H gene-encoded
blood group
-D-galactoside
-2-L-fucosyltransferase suggests that porcine
-2-fucosyltransferase highly corresponds to the human Secretor gene-encoded enzyme.
The minimum structure of the H antigenic determinant is
a terminal Fuc1
2Gal. Two distinct
GDP-L-fucose:
-D-galactoside
-2-L-fucosyltransferases, encoded by the H and Secretor (Se) genes, are known to control the
biosynthesis of the mono- and difucosylated lactoseries structures in
normal tissues and in epithelial carcinomas in humans(1) ,
although evidence from normal and tumor tissues suggests that the human
genome contains additional structural genes encoding
-2-fucosyltransferases(2, 3, 4, 5) .
A two-loci model for expression of
-2-fucosyltransferase was
proposed based on the presence of
-2-fucosyltransferase enzymatic
activity in the serum of Bombay, Se-positive individuals, who
lack H gene expression(6) . In this model, both Se and H represent structural genes encoding
-2-fucosyltransferases, and the enzymes under the control of the Se and H genes could preferentially use type 1, 2, 3,
and 4 precursors or type 2 structures and are expressed in cells of
endodermal and mesodermal origin, respectively(7) . The
two-loci model for expression of human
-2-fucosyltransferases was
firmly established by cloning H(8) and a candidate
for human Secretor locus-encoded
-2-fucosyltransferases(9) . The Se gene-encoded
enzyme shares 68% identity with the human H
-2-fucosyltransferase protein sequence, which confirms the
hypothesis that H and Secretor loci represent two
distinct but closely linked
-2-fucosyltransferase genes.
Evolutionary evidence has suggested that the Se gene is ancestral in mammals, with the evolutionarily newer H gene present only in humans and anthropoid apes. Indeed, ABO blood group antigens are present in red blood cells, vascular endothelium, and secretions in man and anthropoid apes but only in secretions in lower mammals(10) .
The evidence that the
-D-galactoside
-2-fucosyltransferase from porcine
submaxillary glands is equivalent to the human serum Se-type
and not H-type enzyme is based on striking similarities with
the Se enzyme with respect to the kinetic pattern, specificity
toward various oligosacchride acceptors and physicochemical properties.
Both, the porcine and human Se
-2-fucosyltransferase
display the preference for type 1 and 3 lactoseries oligosaccharide
acceptors, are represented by 55-kDa glycoproteins, and demonstrate
binding properties to
S-Sepharose(11, 12, 13, 14, 15, 16) .
We postulated that porcine
-2-fucosyltransferase is equivalent to
the human Se gene-encoded enzyme and different from the H blood group
-2fucosyltransferase.
Tryptic peptides
generated from the porcine submaxillary gland
-D-galactoside
-2-fucosyltransferase, share higher
amino acid sequence homology with human Se blood group enzyme
as compared with human H gene-encoded enzyme and results
confirm the hypothesis that porcine submaxillary gland enzyme is
equivalent to the human Se
-2-fucosyltransferase.
The partial amino acid sequence of the
-D-galactoside
-2-fucosyltransferase from porcine
submaxillary glands, which was purified to homogeneity, was determined.
Pooled active fractions eluted from S-Sepharose were subjected to three consecutive steps of affinity chromatography on GDP-hexanolamine-Sepharose columns. GDP-hexanolamine-Sepharose was synthesized, and a matrix with the same ligand concentration of 5.4-6.6 µmol/ml was used for all steps of affinity purification.
A sharp peak of enzymatic activity was eluted from the GDP-hexanolamine-Sepharose affinity column I at 0.6-0.8 M NaCl using a 0.4-2.0 M linear NaCl gradient. The active, pooled fractions from the GDP-hexanolamine-Sepharose-I column were desalted on a Sephadex G-50 (fine). The enzyme was recovered quantitatively (97%), and some degree of purification (1.4-fold) in addition to the desalting effect of the Sephadex G-50 gel filtration column was achieved.
A second large GDP-hexanolamine-Sepharose
column and an NaCl pulse for nonspecific elution was introduced into
the procedure to combine and concentrate fractions from four batches of
approximately 5 kg of submaxillary gland tissue processed separately on
GDP-hexanolamine-Sepharose column I into the total pool from 21.5 kg of
tissue. To minimize nonspecific interactions with the gel matrix,
cacodylate buffer with higher pH 7.0 than that for S-Sepharose and
GDP-hexanolamine-Sepharose I columns (pH 6.0) was chosen for
GDP-hexanolamine-Sepharose column II. With the 2 M NaCl pulse,
60% of the -2-fucosyltransferase eluted from this column resulted
in 10-fold purification, as compared with nonspecific elution with NaCl
gradient from GDP-hexanolamine-Sepharose column I, from which enzyme
was purified 26-fold with a smaller loss of enzymatic activity (76%).
This may be due to the low concentration of protein eluting from
GDP-hexanolamine-Sepharose II as compared with column I. Again, the
active fractions eluted with 2 M NaCl were desalted using a
Sephadex G-50 (fine) column, which removes approximately 50% of the
total protein at this point without loss of enzymatic activity.
Desalted active fractions were directly applied to a third affinity
purification step, which consisted of small GDP-hexanolamine-Sepharose
III column. The enzyme was specifically eluted using 0.5 mM GDP in 25 mM sodium cacodylate buffer (pH 7.0) containing
0.035 M NaCl. A 14-fold purification and considerable loss of
enzymatic activity (60%) due to a low levels of protein occurred at
this step (Table 1). SDS-PAGE analysis of the pooled fractions
eluted from the third affinity column upon reduction with
-mercaptoethenol showed major protein bands of 60 and 55 kDa and a
band at 18 kDa (not shown).
This fraction was submitted for tryptic digestion and amino acid sequence analysis of the major 60-kDa protein at the Wistar Institute Mass Spectrometry/Protein Microchemistry Laboratory. Amino acid sequencing of two tryptic peptides generated from this protein band demonstrated 98 and 94% homology with bovine catalase, suggesting that the 60-kDa protein purified by affinity chromatography on GDP-hexanolamine-Sepharose III represents catalase (Table 2).
Final purification of the porcine
-2-fucosyltransferase was achieved by HPLC size-exclusion
chromatography. The pooled enzymatic fractions eluted from
GDP-hexanolamine column III were subjected to HPLC size-exclusion
chromatography in 25 mM sodium cacodylate buffer (pH 7.0)
containing 0.035 M NaCl. Enzymatic activity was monitored by
standard assay using phenyl-
-D-galactoside as an acceptor
and absorption at 223 nm (Fig. 1). The HPLC size-exclusion
enzymatic activity profile showed a broad peak corresponding to
molecular size of 55 kDa. SDS-PAGE analysis of the active fractions in
the presence of
-mercaptoethanol revealed the 55-kDa protein
representing
-2-fucosyltransferase (Fig. 1, inset).
Figure 1:
HPLC size-exclusion chromatography of
porcine -D-galactoside
-2-L-fucosyltransferase. One hundred µl of
concentrated fractions eluted from GDP-hexanolamine-Sepharose column
III was injected into an HPLC column equilibrated with 25 mM sodium cacodylate buffer, pH 7.0, containing 0.035 mM NaCl. Eluted fractions were monitored by standard enzymatic assay
using phenyl-
-D-galactoside as an acceptor (open
boxes) and absorption at 223 nm (closed circles). Inset represents
-2-fucosyltransferase protein eluted
from HPLC column as determined by SDS-PAGE in the presence of
-mercaptoethanol after silver staining. The fractions correspond
to those in the graph.
We have purified a -D-galactoside
-2-fucosyltransferase to homogeneity from porcine submaxillary
glands, using a modification of a previously published
procedure(15) . Our procedure included additional
GDP-hexanolamine-Sepharose affinity chromatography and a 2 M NaCl pulse elution, and HPLC size-exclusion steps instead of
Sephadex G-150 gel filtration and chromatography on SP-Sephadex steps
of the original procedure.
Three proteins, 60, 55, and 18 kDa, were
eluted from the third GDP-hexanolamine-Sepharose affinity column by
specific elution using 5 mM GDP. Based on a previous analysis
suggesting that a 60- and 55-kDa doublet represented
-D-galactoside
-2-L-fucosyltransferase
purified from porcine submaxillary glands(15) , we analyzed the
amino acid sequence of the 60-kDa protein isolated after the last
affinity chromatography step. Amino acid sequence analysis of two
tryptic peptides of this protein revealed high sequence homology with
bovine catalase, consistent with the previous observation that catalase
for undetermined reasons shows high binding capacity to
GDP-hexanolamine-Sepharose and copurifies with
-2-fucosyltransferase from the affinity column(15) .
Therefore, to further purify the
-2-fucosyltransferase protein,
HPLC size-exclusion chromatography was used as a purification step
following the affinity chromatography steps. The HPLC step resulted in
the isolation of a single major protein of 55 kDa, which coeluted with
enzymatic activity, strongly suggesting that the 55-kDa protein
represents porcine submaxillary gland
-D-galactoside
-2-fucosyltransferase. In this purification scheme, catalase was
separated from fucosyltransferase as a tetrameric protein in native
conditions by the HPLC size-exclusion step.
The isolated porcine
enzyme as determined by amino acid sequence analysis is highly
homologous to human Se and H type blood group
-D-galactoside
-2-fucosyltransferases(8, 9) . As expected, a
higher level of homology was observed between the three tryptic
peptides generated from the porcine enzyme and a recently cloned human
candidate for Se type
-2-fucosyltransferase (100, 93.3,
and 84.2%) as compared with the human H gene-encoded enzyme
(75, 46.6, and 84.2%). The amino acid sequence of the HP 94266 peptide
is identical with the Se type
-2-fucosyltransferase,
whereas 75% of homology was observed with respective sequences of H enzyme. Thus, the 75% homology found between the porcine and H type
-2-fucosyltransferases also reflects the level of
homology between both human Se and H enzymes. Porcine
peptide HP 94278 shares the same degree of homology (84.2%) with both
the Se and H type
-2-fucosyltransferases. The
amino acid substitutions are in exactly the same positions in both
human and porcine
-2-fucosyltransferases, although they are
represented by different amino acids in all three enzymes(9) .
The most striking differences between the protein sequence of porcine
and human H enzyme were observed in the amino acid sequence
corresponding to HP 94244 peptide, where only 46.6% identity was
observed. On the other hand, this peptide shares 93.3% identity with
the corresponding amino acid sequence of Se
-2-fucosyltransferase. These results support the hypothesis
that porcine
-D-galactoside
-2-fucosyltransferase is
equivalent to the human Se type enzyme and different from the
human H blood group fucosyltransferase. Very high sequence
homology between the peptides from the porcine enzyme and recently
cloned rabbit
-2-fucosyltransferase, RFT-II(19) , which
likely corresponds to rat FTB enzyme(20) , also suggests that
they represent equivalent enzymes.
Porcine peptide HP 94244, which
shares the lowest degree of sequence homology with the human H enzyme, is derived from the catalytic domain region where the
least sequence identity between corresponding amino acid sequences of Se and H type enzymes was determined(9) .
These results suggest that this region may determine the
oligosaccharide substrate specificity, in particular this region may be
involved in -D-galactose binding since H and Se enzymes greatly differ in affinity to
-D-galactose and its derivatives, as determined by K
values(11, 12, 13, 14) . Nine
amino acid sequences similar to those of HP 94244 were also found in
bacterial phospho-
- and
-galactosidases, sharing 55.6 and
62.5% homology with the peptide(21, 22, 23) .
The availability of the DNA primary structure of the human H and Se gene-encoded -D-galactoside
-2-fucosyltransferases (9, 10) and their animal
counterparts (19, 20) will enable study of the
molecular basis of
-2-fucosylated glycoconjugates expression in
different species.