(Received for publication, July 21, 1995; and in revised form, September 21, 1995)
From the
The mammary gland is a unique biosynthetic tissue that produces
a variety of species-specific glycoconjugates, but the factors
regulating the production of specific glycoconjugates are not well
understood. To explore the underlying regulation, a fusion gene
containing a cDNA encoding the human 1,2-fucosyltransferase
(
1,2FT), which generates the H-blood group antigen, flanked by the
murine whey acidic protein promoter and a polyadenylation signal, was
introduced into mice. Milk samples from transgenic animals contained
soluble forms of the
1,2FT, as revealed by Western blots of milk
samples using an anti-
1,2FT antiserum and by the demonstration of
1,2FT enzyme activity. Milk from transgenic animals also contained
large quantities of 2`-fucosyllactose
(Fuc
1-2Gal
1-4Glc) and modified glycoproteins
containing the H-antigen, whereas milk from control animals lacked
these glycoconjugates. Expression levels of 2`-fucosyllactose were high
in most animals and represented to nearly of the total milk
oligosaccharides. These results demonstrate that heterologous
transgenic expression of a glycosyltransferase can result in the
expression of both the transgene and its secondary gene products and
that the structures of milk oligosaccharides can be remodeled depending
on expression of the appropriate enzyme. Furthermore, these results
suggest that the lactating mammary gland may be a unique biosynthetic
reactor for the production of biologically active oligosaccharides and
glycoconjugates.
Despite the fact that animal oligosaccharides are important in many biological and pathological processes (Karlsson, 1989; Kornfeld, 1992; Drickamer and Taylor, 1993; McEver et al., 1995), little is known about the regulation of animal cell glycoconjugate biosynthesis. Biosynthesis is influenced by expression of appropriate glycosyltransferases, correct targeting of the enzymes to the Golgi apparatus, sugar nucleotide levels, competition between enzymes, residence time of glycoconjugates in intracellular compartments, acceptor specificity of the enzymes, and many other factors (Kornfeld and Kornfeld, 1985; Kobata and Takasaki, 1992; Cummings, 1992; Baenziger, 1994). Experimental modulation of these factors has been difficult. One approach that has proven somewhat successful in vitro is to attempt to alter oligosaccharide biosynthesis by genetic manipulation of glycosyltransferases (Lee et al., 1989; Smith et al., 1990; Lowe et al., 1990). This approach has so far been limited to cultured and immortalized cell lines and has been useful in producing neoglycoconjugates for biological studies (Lowe et al., 1990).
One major site for glycoconjugate biosynthesis in animals, and one that has been of enormous historical importance in the field of glycobiology, is the lactating mammary gland. Milk contains, in addition to lactose, numerous glycoproteins (Patton et al., 1990; Fiat and Jollès, 1989) and a variety of free oligosaccharides (Kobata et al., 1972). The structure and composition of the free oligosaccharides in milk differ between mammals, but the human lactating mammary gland synthesizes the most complex mixture of free, reducing oligosaccharides, many of which contain fucose and other determinants for human blood groups, including the ABO and Lewis system. The structures of some of these oligosaccharides in humans are determined genetically by the expression of glycosyltransferases during lactation and the availability of sugar nucleotide substrates (Hill and Brew, 1975; Ginsburg, 1972; Smith et al. 1987), but the precise mechanisms regulating differential expression of individual milk oligosaccharides are not known.
Recent advances in transgenic technology have resulted in
successful expression of transgene-encoded proteins in animal milk
(Wilde et al., 1992; Paleyanda et al., 1991; Archer et al., 1994). Using this methodology, we have begun to
explore factors regulating glycoconjugate biosynthesis. Our analyses of
normal mouse milk demonstrated that it is deficient in
fucose-containing oligosaccharides, and in particular it lacks
oligosaccharides containing the H-blood group antigen. Based on this
information, we chose to use a cDNA encoding a human
1,2-fucosyltransferase (
1,2FT) (
)responsible for
synthesis of the blood group H-antigen
Fuc
1-2Gal
1-3(4)-R (Rajan et al., 1989;
Larsen et al., 1990). Our studies demonstrate that the human
1,2FT can be successfully introduced into transgenic animals
causing the lactating mammary gland to synthesize relatively large
quantities of oligosaccharides containing the H-antigen and at least
one major glycoprotein containing this determinant. These studies
provide new insights into the factors regulating biosynthesis of milk
oligosaccharides within the lactating mammary gland and a new approach
for studying factors controlling glycoconjugate biosynthesis.
For transgenic studies, the cDNA
fragment encoding the 1,2FT was inserted into a second expression
plasmid, pWAP-
1,2FT (Fig. 1). This plasmid contains bGH
poly(A) and utilizes the murine whey acidic protein (WAP) promoter
(Pittius et al., 1988) to direct gene expression primarily to
lactating mammary gland tissue. An EcoRI-BamHI DNA
fragment containing the WAP-
1,2FT-bGH poly(A) sequences was
isolated from this plasmid and injected into fertilized mouse (B6/SJL)
eggs as described previously (McGrane et al., 1988). The WAP
promoter was generously provided by Dr. Lothar Hennighausen (National
Institutes of Health).
Figure 1:
The ampicillin-resistant (Ap) plasmid pWAP-FT contains a 2.5-kb WAP
5`-flanking region, a 1.2-kb FT protein coding segment, and a 0.6-kb
bGH poly(A) addition site. No introns have been incorporated into this
plasmid. Cleavage with EcoRI and BamHI results in a
linear 5.3-kb WAP-FT-bGH fusion gene, which was used for
microinjection.
Figure 2: Chromatographic profiles of mouse milk oligosaccharides. Oligosaccharide extracts from a control non-transgenic mouse and two different transgenic mice, both of which were descendants of Founder 28, were subjected to Dionex high performance liquid chromatography. A, milk from control mice did not contain any DFL (a2 region) or 2`-FL (a3). A trace of 3-FL (a1) was detected and analyzed as described in the text. B, a second generation transgenic mouse (code 28-28) synthesized a large amount of 2`-FL (b3). Traces of 3-FL (b1) and DFL (b2) were detected. C, a larger amount of 2`-FL was present in milk from a homozygous third generation transgenic animal (code 28-28-37). The elution positions of monosaccharides and standard oligosaccharides are indicated.
Milk samples
from transgenic animals contained detectable 1,2FT activity as
determined by a method modified from Rajan et al.(1989), while
activity was absent in samples from control animals (data not shown).
These results demonstrate that active
1,2FT enzyme was expressed
in milk from transgenic animals.
The presence of 1,2FT
polypeptide was determined by immunoblot analysis of milk protein using
a polyclonal rabbit antibody against
1,2FT, as shown in Fig. 3. No immunoreactive protein was present in milk from
control animals, whereas milk from transgenic animals contained four
immunoreactive species of apparent molecular mass of 46, 32, 30, and 25
kDa (Fig. 3). The
1,2FT cDNA encodes the full-length type-2
transmembrane form of the enzyme that has a predicted size of
approximately 46 kDa when fully glycosylated. Whether the 46-kDa form
of the
1,2FT observed in transgenic milk represents a full-length
form of the enzyme or a partly degraded form with altered glycosylation
is not yet known. Nevertheless, the presence of discrete lower
molecular weight forms of the enzyme in milk indicates discrete
proteolytic cleavages of the polypeptide resulting in the species
observed. These lower molecular weight forms were not found in cell
extracts of
1,2FT transfected mouse L-cells. The potential
proteolytic cleavage sites of the recombinant enzyme are under
investigation.
Figure 3:
1,2FT was present in milk of
transgenic animals. Protein pellets from milk samples were separated by
SDS electrophoresis, blotted, and assayed for binding to
anti-
1,2FT antiserum as described under ``Experimental
Procedures.'' Milk proteins from control animals were not
specifically recognized by the antiserum (lane 1), while some
proteins from transgenic animals (lanes 2 and 3) were
recognized. One of the detected bands (identified by an arrow)
was in the 46-kDa region, which corresponds to the expected molecular
weight of the full-length
1,2FT. The migration positions of
molecular weight markers are indicated.
To confirm that the
neooligosaccharide in the milk of transgenic animals was 2`-FL, the
purified oligosaccharide collected after Dionex-HPAEC was subjected to
FACE analysis and specific enzyme degradation. An electrophoretogram of
the neooligosaccharide before and after treatment with
1,2-specific fucosidase from Corynebacterium sp. is shown
in Fig. 4. The fluorophore-labeled neooligosaccharide (lane
4) comigrated with authentic 2`-FL (lane 2), and, like
the standard 2`-FL, it was susceptible to the action of the
1,2-specific fucosidase (lanes 3 and 5). The
neooligosaccharide was also exhaustively treated with a combination of
the
1,2-specific fucosidase and E. coli
-galactosidase. This combined treatment resulted in the
release of equimolar amounts of fucose, galactose, and glucose, as
determined by FACE analysis (data not shown). These results demonstrate
that the neooligosaccharide present in the milk of transgenic animals
expressing the human
1,2FT is 2`-FL.
Figure 4:
The major neooligosaccharide found in milk
of transgenic mice comigrated with authentic 2`-FL in FACE analysis and
was susceptible to a specific 1,2-fucosidase. Fractions
corresponding to the major neooligosaccharide (region b3, Fig. 2, panel B) were pooled. An aliquot was directly
labeled with 8-aminonapthalene-2,3,6-trisulfonic acid and subjected to
FACE analysis (lane 4). A second aliquot was treated with 20
milliunits of Corynebacterium sp.
1-2 fucosidase prior to
labeling (lane 5). Authentic 2`-FL was also labeled (lane
2) and treated with the enzyme (lane 3). Lane 1 contains labeled oligosaccharide
standards.
A minor oligosaccharide
that coelutes with the human milk tetrasaccharide difucosyllactose
(DFL, Fuc1-2Gal
1-4(Fuc
1-3)Glc) was
also detected in the chromatographic profiles from transgenic animals (Fig. 2, panels B and C). Fractions
corresponding to the elution time of this oligosaccharide were pooled
and analyzed by FACE. The resulting band obtained from transgenic
animal milk comigrated with authentic DFL (data not shown). Results
were consistent with the presence of this oligosaccharide only in milk
from transgenic animals. It is expected that this product is generated
by the sequential action of the
1,2FT and the endogenous
1,3FT.
Figure 5:
Glycoproteins containing the novel
Fuc1-2 linkages were detected in milk from transgenic
animals. Protein pellets from mouse milk samples were resuspended in a
volume of SDS-polyacrylamide gel electrophoresis sample buffer equal to
that of the original volume of milk. Aliquots of these extracts (5
µl) were resolved by electrophoresis and blotted in nitrocellulose
membranes. The membranes were then incubated with a preparation of
labeled UEA I, as described under ``Experimental
Procedures.'' Samples obtained from transgenic animals (lanes
2-5) were specifically recognized by UEA I. Fucose
1,2
linkages were not detected in samples from control animals (lanes 1 and 6). The migration positions of molecular weight
markers are indicated.
Results presented in this report demonstrate that it is
possible to dramatically alter the structure of the milk
oligosaccharides and glycoproteins by the transgenic introduction of a
heterologous glycosyltransferase under the control of a lactogenically
induced expression of the enzyme. We chose the human 1,2FT as a
model enzyme to explore its expression within the mammary gland and the
consequences of its expression on biosynthesis of milk glycoconjugates.
We found that 2`-FL, which is lacking in normal mouse milk, was a major
oligosaccharide in the milk of transgenic animals and was present at
levels rivaling that of lactose. In addition, active human
1,2FT
and an oligosaccharide that coelutes with difucosyllactose were found
in the milk, and at least one milk glycoprotein was a major substrate
for the transgenic
1,2FT.
Biosynthesis of milk oligosaccharides
from lactose would be expected to be directly related to the expression
of particular glycosyltransferases, but other factors, such as sugar
nucleotide availability, lactose availability, enzyme localization,
enzyme secretion, etc., could profoundly alter biosynthesis. We
reasoned that the experimental introduction of the transgenic
1,2FT would test many of these factors. For example, we considered
the possibility that the low levels of 3-FL, the absence of 2`-FL, and
the low amount of fucose in mouse milk might be due to a low expression
of GDP-Fuc. The high level expression of 2`-FL we observed, however,
indicates that the Golgi apparatus of the lactating mammary gland must
be sufficiently adaptable in regard to GDP-Fuc uptake to accommodate
the need of the transgenic
1,2FT. We also considered the
possibility that the transgenic
1,2FT might be proteolytically
processed within the lactating mammary gland and be either inactivated
or quantitatively secreted without being functionally present in the
Golgi apparatus. It is conceivable that some other glycosyltransferase
genes expressed as transgenes within the lactating mammary gland will
not be functional and be unable to cause the efficient remodeling of
milk glycoconjugates. Further studies with other genes are required to
address the generality of this phenomenon.
All samples from homozygous animals analyzed so far contained more 2`-FL than those of hemizygous descendants of the same founder. There were, however, variations of 2`-FL content among milk samples from hemizygous animals descending from the same founder. We have found that in humans, milk oligosaccharide content varies between samples from the same donor (data not shown). In mice, lactation stage, number of pups, age, and nutritional status are some of the factors that may contribute to the observed differences.
The functions of milk oligosaccharides are not known, but free oligosaccharides are known to influence the adhesion of pathogenic bacteria to human tissue in vivo (Andersson et al., 1986), suggesting that milk oligosaccharides may have a protective function in the infant. The ability to remodel milk oligosaccharides by the heterologous expression of glycosyltransferase genes should facilitate future experiments aimed at defining the biological functions of the lactose-derived oligosaccharides.
In addition, the transgenic expression of glycosyltransferases in lactating mammary glands presents a unique opportunity to develop technology that may result in the synthesis of large amounts of neooligosaccharides and substantially modified glycoproteins with therapeutic or industrial value. The findings described in this report may constitute a new method for the production of custom designed homologous or heterologous glycoconjugates.