(Received for publication, August 1, 1995; and in revised form, October 18, 1995)
From the
The lumen of the Golgi apparatus is the subcellular site where
galactose is transferred, from UDP-galactose, to the oligosaccharide
chains of glycoproteins, glycolipids, and proteoglycans. The nucleotide
sugar, which is synthesized in the cytosol, must first be transported
into the Golgi lumen by a specific UDP-galactose transporter.
Previously, a mutant polarized epithelial cell (MDCKII-RCA)
with a 2% residual rate of transport of UDP-galactose into the lumen of
Golgi vesicles was described (Brandli, A. W., Hansson, G. C.,
RodriguezBoulan, E., and Simons, K.(1988) J. Biol. Chem. 263,
16283-16290). The mutant has an enrichment in glucosyl ceramide
and cell surface glycoconjugates bearing terminal N-acetylglucosamine, as well as a 75% reduction in sialylation
of cell surface glycoproteins and glycosphingolipids.
We have now
studied the biosynthesis of galactose containing proteoglycans in this
mutant and the corresponding parental cell line. Wild-type Madin-Darby
canine kidney cells synthesize significant amounts of chondroitin
sulfate, heparan sulfate, and keratan sulfate, while the above mutant
synthesizes chondroitin sulfate and heparan sulfate but not keratan
sulfate, the only proteoglycan containing galactose in its
glycosaminoglycan polymer. The mutant also synthesizes chondroitin
6-sulfate rather than only chondroitin 4-sulfate as wild-type cells.
Together, the above results demonstrate that the Golgi membrane
UDP-galactose transporter is rate-limiting in the supply of
UDP-galactose into the Golgi lumen; this in turn results in selective
galactosylation of macromolecules. Apparently, the K for galactosyltransferases involved in the synthesis of
linkage regions of heparan sulfate and chondroitin sulfate are
significantly lower than those participating in the synthesis of
keratan sulfate polymer, glycoproteins, and glycolipids. The results
also suggest that the 6-O-sulfotransferases, in the absence of
their natural substrates (keratan sulfate) may catalyze the sulfation
of chondroitin 4-sulfate as alternative substrate.
Proteoglycans are complex macromolecules consisting of a protein core to which glycosaminoglycans are covalently linked. Their strategic localization in the plasma membrane and extracellular matrix makes them important intermediates between cells and their environment(1, 2) . They have been implicated to play a role in cell-cell (3) and cell-matrix interactions(4) , organization of basement membranes(5) , control of macromolecules' diffusion(6) , and also interactions with a variety of ligands such as growth factors, hormones, and neurotransmitters(7) .
In most GAGs, ()the repeating disaccharide units are
composed of one amino sugar and one uronic acid, the only exception
being keratan sulfate in which galactose replaces the sugar acid. Most
GAGs are attached to serine of the core protein by a tetrasaccharide of
xylose-galactose-galactose-glucuronic acid(8, 9) .
Keratan sulfate is an exception; keratan sulfate I, from cornea, is N-linked to proteins and keratan sulfate II, from skeletal
tissues, is O-linked via serine or threonine to N-acetylgalactosamine(10) .
The biosynthesis of proteoglycans is a post-translational event and takes place in the lumen of the endoplasmic reticulum and Golgi apparatus(11, 12) . Most sugar nucleotides involved in these glycosylation steps are synthesized in the cytoplasm of the cell and require specialized transporters to translocate them to their site of synthesis within the lumen of these organelles(13) .
Because nucleotide sugar transporters may play important roles in the control of biosynthesis of many glycoconjugates, we searched for a model where the in vivo relevance of these transporters could be demonstrated. A Madin-Darby canine kidney strain II cell line resistant to Ricinus communis agglutinin was previously isolated and characterized(14) . The in vitro biochemical defect leading to the altered phenotype was determined to be a 98% deficiency in the rate of transport of UDP-galactose into Golgi vesicles; transport of UDP-GlcNAc and CMP-sialic acid was similar to that into wild-type cells. The levels of activity of galactosyltransferases and sialyltransferases were the same in mutant and wild-type cells. The defect resulted in enrichment of cell surface glycoconjugates bearing terminal N-acetylglucosamine and of glucosylceramides, both endogenous acceptors for galactose. UDP-galactose is also crucial in the assembly of the linkage regions of virtually all proteoglycans; therefore this MDCK II cell mutant can be used as a tool to investigate the consequences of limited availability of UDP-Gal within the Golgi lumen in the biosynthesis of different glycosaminoglycan chains. We found that limiting the availability of UDP-galactose selectively inhibits the biosynthesis of keratan sulfate; this in turn results in changes in the sulfation pattern of chondroitin sulfate.
To further characterize GAGs from wild-type and mutant MDCK cells, cultures were incubated with tritiated glucosamine for 48 h; GAGs were obtained and separated as described for the sulfate-labeled cells. As can be seen in Fig. 1, one broad radioactive band, comigrating with standard heparan sulfate, was observed in both wild-type and mutant cells. In addition, a broad band, migrating slower than standard heparan sulfate was observed in wild-type but not mutant cells.
Figure 1:
Labeling of glycosaminoglycans with
[H]glucosamine. Approximately 50,000 cpm of total
cellular GAGs labeled with [
H]glucosamine were
applied to an agarose gel slab in 1,3-diaminepropaneacetate buffer, pH
9.0. After electrophoresis, GAGs were precipitated in the gel with 0.2%
cetyltrimethylammonium bromide for 1 h. The gel was then incubated with
autoradiography enhancer and water, each for 30 min. Standard GAGs were
visualized with toluidine blue. The gel was dried and labeled GAGs were
visualized on x-ray film after exposure for 5 days at -70 °C. Lanes 1 and 2, intracellular GAGs from wild-type
cells and RCA
-mutant cells, respectively. Or,
origin.
The experiments
described below were designed to demonstrate that the above described
slower migrating GAG, containing glucosamine and sulfate, is indeed
keratan sulfate. Because standard keratan sulfate from cornea partially
comigrates with heparan sulfate in the above agarose gel system, S-GAGs from the medium and cells were treated with a crude
fraction of enzymes from F. heparinum as described under
``Materials and Methods.'' This fraction contains enzymes
which degrade chondroitin sulfate, heparan sulfate, heparin, dermatan
sulfate, and hyaluronic acid. Keratan sulfate is resistant (20) and can be adequately separated from the others GAGs. As
can be seen in Fig. 2, both medium and cells from wild-type MDCK
cells contained GAGs which appeared to be resistant to enzymes from F. heparinum. These GAGs were absent in mutant cells (Fig. 2). The broad migration pattern of KS after treatment with
extract from F. heparinum is probably not the result of
incomplete digestion of HS because the pattern did not change following
repeated addition of extract. We hypothesize that there are two
distinct populations of KS; by analogy to KS from cornea, regions that
are highly fucosylated or sulfated and are closer to the linkage region
appear to be more resistant to keratanases(30) .
Figure 2:
Digestion of S-glycosaminoglycans with a crude mixture of enzymes from F. heparinum. Total
S-GAGs (20,000-80,000
cpm) were digested with (+) or without(-) a crude fraction
of enzymes from F. heparinum (see ``Materials and
Methods''). After incubation at 30 °C for 18 h the mixture was
applied to an agarose gel slab in 1,3-diaminepropaneacetate buffer, pH
9.0. After electrophoresis, GAGs were precipitated with 0.2%
cetyltrimethylammonium bromide for 1 h. The gel was dried and standard
GAGs were stained with toluidine blue. Radioactive GAGs were visualized
on x-ray film after 9 days at room
temperature.
Conclusive
evidence that the component resistant to the action of crude extract of F. heparinum is indeed keratan sulfate was obtained with two
specific keratanases. Keratanase from Pseudomonas sp.(21) specifically cleaves keratan sulfate yielding
saturated disaccharides with galactose at the reducing end. No cleavage
occurs when galactose is sulfated in position 6. Keratanase from Bacillus sp.(22) specifically cleaves keratan sulfate
by hydrolyzing 1,3--glucosaminidic residues linked to galactose.
For cleavage the enzyme requires that glucosamine be sulfated in the
6-O-position but acts independently of the sulfate at the
6-O-position of the galactose linked to glucosamine.
[S]Keratan sulfate, synthesized by wild-type
cells, was obtained as described under ``Materials and
Methods.'' Aliquots from the medium and intracellular material
were then treated separately with or without keratanases from Pseudomonas sp. and Bacillus sp.; samples were then
analyzed by agarose gel electrophoresis and paper chromatography. As
seen in Fig. 3a (medium) and 4a (intracellular
material), keratanases degraded approximately 50-60% of GAGs
comigrating with keratan sulfate, compared to controls incubated
without enzymes.
Figure 3:
Digestion of
[S]keratan sulfate from the medium with specific
keratanases. Approximately 70,000 cpm of
[
S]keratan sulfate, isolated as described under
``Materials and Methods,'' together with standard keratan
sulfate, were digested with either 0.04 unit of keratanase from Pseudomonas sp. (lanes 1) or 0.002 unit of keratanase
II from Bacillus sp. (lanes 2) or buffer alone (lanes 3). 4,000 cpm from each incubation were subjected to
agarose gel electrophoresis (a). The remaining sample was
subjected to descending paper chromatography on Whatman No. 1 in
isobutyric acid, 1 M NH
OH 5:3 (v/v) for 24 h (b and c). Labeled products released by keratanase
from Pseudomonas sp. (b, lane 1) and Bacillus sp. (b, lane 2) were visualized on x-ray film after 5 days at
room temperature. b, lane 3, control incubation with labeled
KS and buffer alone. S:
[
S]SO
; in this solvent system,
sulfate and N-acetylglucosamine-6-sulfate comigrate. A, putative GlcNAc,6S; Di6S, GlcNAc,6S-Gal; B, putative; GlcNAc,6S-Gal,6S-GlcNAc,6S-Gal; C,
unknown; D, putative
GlcNAc,6S-Gal,6S-GlcNAc,6S-Gal,6S-GlcNAc,6S-Gal. Products from standard
keratan sulfate were detected with silver nitrate (c).
Treatment of radiolabeled keratan sulfate from
wild-type cells and medium with keratanase from Pseudomonas sp. showed three main products following paper chromatography (Fig. 3b and 4b, lane 1). The fastest moving
product comigrates with a degradation product from bovine cornea
keratan sulfate, previously identified as GlcNAc,6S-Gal(21) .
The second fastest migrating radioactive product (B),
apparently not present in bovine cornea keratan sulfate, is
hypothesized to be a trisulfated tetrasaccharide,
GlcNAc,6S-Gal,6S-GlcNAc,6S-Gal, containing one internal galactose
6-sulfate, which renders it resistant to the action of this keratanase.
The slowest migrating product (D) is hypothesized to be
GlcNAc,6S-Gal,6S-GlcNAc,6S-Gal6S-GlcNAc,6S-Gal based on its resistance
to the above keratanase and previously described mobility in this
system(20) . The identity of the above oligosaccharides was not
further established. Radioactive products comigrating with
GlcNAc,6S-Gal and product B were also observed when
[H]glucosamine-labeled KS was digested with the
above keratanases (not shown).
Digestion of the above radiolabeled keratan sulfate with keratanase from Bacillus sp. (Fig. 3b and Fig. 4b, lane 2), resulted in formation of two major products, with the same migration as those found when standard keratan sulfate from bovine cornea was treated with this enzyme. The action of this keratanase on intracellular keratan sulfate generated, besides the products found in cornea, additional products (Fig. 4b, lane 2). The radioactivity at the origin is most likely undigested keratan sulfate also observed previously in the gel of Fig. 3a and 4a.
Figure 4:
Digestion of intracellular
[S]keratan sulfate with specific keratanases.
Approximately 70,000 cpm of [
S]keratan sulfate
was incubated with keratanase from Pseudomonas sp. (lanes
1), Bacillus sp. (lanes 2), or buffer alone (lanes 3) and then subjected to electrophoresis (a)
or chromatography (b) as described in the legend of Fig. 3. Or, origin; A, putative GlcNAc,6S; Di6S, GlcNAc,6S-Gal; B, putative
GlcNAc,6S-Gal,6S-GlcNAc,6S-Gal; C, unknown. S,
[
S]SO
.
Figure 5:
Digestion of S-glycosaminoglycans with chondroitinase ABC.
Approximately 30,000 cpm of total intracellular or medium
S-GAGs, together with standard chondroitin 4-sulfate, were
incubated with (+) or without(-) 0.02 units of
chondroitinase ABC as described by the manufacturer. After incubation,
the mixture was subjected to paper chromatography as described in the
legend of Fig. 3. S,
[
S]SO
.
U-GalNAc4S,
unsaturated disaccharide 4-sulfate;
U-GalNAc6S,
unsaturated disaccharide 6-sulfate; A, putative unsaturated
tetrasaccharide; Or, origin.
An
unexpected product was detected in the mutant but not in the wild-type
cells. It represents 13% of the mutant disaccharides and comigrates
with a degradation product from standard chondroitin 6-sulfate,
previously characterized as U(1-3)GalNAc6S. The disaccharide
was also a substrate for chondro-6-sulfatase but not for
chondro-4-sulfatase (not shown). The slowest migrating radioactive
product (A) is a tetrasaccharide, disaccharide disulfate, or a
mixture of these, based on previous degradation structures of
chondroitin sulfate from whale cartilage(23) .
Three important conclusions can be drawn from the above
studies: (a) wild-type MDCK cells synthesize keratan sulfate
in culture; (b) in mutant MDCK cells that are highly deficient
in transport of UDP-galactose into the Golgi lumen, the biosynthesis of
keratan sulfate as well as glycoproteins and glycolipids are highly
reduced while that of heparan sulfate and chondroitin 4-sulfate is
virtually intact; and (c) the above mutant cells show a change
in the sulfation pattern of chondroitin 4-sulfate with additional
sulfation in the 6-O-position. The above shown selective
biosynthesis of galactose containing glycoconjugates, together with
previous
studies(14, 24, 25, 26, 27) ,
demonstrate that limited availability of UDP-galactose in the lumen of
the Golgi results in biosynthesis of chondroitin sulfate and heparan
sulfate, both glycoconjugates with galactose in the linkage region; at
the same time there is a marked decrease in the biosynthesis of
glycoproteins, glycolipids, and keratan sulfate, the latter a
proteoglycan which contains galactose in its glycosaminoglycan polymer.
One likely explanation for this is that the K for
galactosyltransferases involved in the biosynthesis of the linkage
region of proteoglycans such as chondroitin sulfate and heparan sulfate
is significantly lower than other galactosyltransferases resulting in
preferential synthesis of these proteoglycans over other galactose
containing glycoconjugates. Unfortunately, direct proof for this
hypothesis is difficult to establish as physiologic apparent K
measurements require the use of physiologic
substrate intermediates, a problem in GAG biosynthesis. In addition,
reactions in vitro require exogenous detergents, another
source of difficulties in interpreting physiologic parameters. The
possibility that galactosylation of different galactose-containing
proteoglycans occurs in different regions of the Golgi apparatus and
that there is differential transport of UDP-galactose into these
hypothetical compartments cannot be ruled out although there is no
current evidence that would support such hypothesis.
Mutant Chinese hamster ovary cells with a 90-98% deficiency in transport of UDP-galactose into Golgi vesicles have been described (24, 25, 26) ; these cells showed a corresponding decrease in galactose and sialic acid containing glycoproteins and glycosphingolipids. Heparan sulfate and chondroitin sulfate biosynthesis appeared to be normal (27) . Keratan sulfate was not detected in wild-type or mutant Chinese hamster ovary cells(27) .
The occurrence of keratan sulfate in wild-type MDCK cells and its absence in the above mutants deserves some attention. This particular proteoglycan does not occur widely as heparan sulfate and chondroitin sulfate, for example. Depending on the tissue it originates, significant structure variability has been reported including chain length, degree of branching and sulfation, as well as content of fucose, sialic acid, and N-acetylgalactosamine(28, 29) . Keratan sulfate of wild-type MDCK cells appear to have similarity with that from cornea. Significant portions of both of these were resistant to keratanase I and II. In a previous study, it was described that keratanase from Pseudomonas sp. is inactive when galactose is sulfated in the 6-position(21) , and also when N-acetylglucosamine is fucosylated (30) in both cases preventing digestion of adjacent non-sulfated galactose. It is possible that the resistance to keratanase in keratan sulfate from MDCK cells may not solely be due to sulfation of galactose, but also to fucosylation of N-acetylglucosamine, or both.
The
additional sulfation of chondroitin 4-sulfate in position 6, in
RCA-mutant MDCK II cells, was a major surprise, but one may
speculate that in the absence of galactose incorporation into the
keratan sulfate polymer, the galactose-6-O-sulfotransferase
for keratan sulfate, without a substrate in the Golgi lumen, may
utilize chondroitin sulfate galactosamine as an acceptor. This
hypothesis is supported by the fact that purified chondroitin
6-sulfotransferase can sulfate not only chondroitin sulfate but also
keratan sulfate(31) .
Together, the above results strongly suggest that nucleotide sugar transporters in the Golgi membrane are limiting in their supply of substrates to the Golgi lumen; this has not only quantitative effects as to the types of macromolecules being synthesized, but also qualitative. Therefore, it will be important to further understand the regulatory role of these transporters in the biosynthesis and modifications of Golgi lumenal glycoproteins, proteoglycans, and glycosphingolipids.