(Received for publication, December 28, 1994)
From the
Differential expression of glycosyltransferases has the
potential to generate functionally distinct glycoforms of otherwise
identical proteins. We have previously demonstrated the presence of
unique oligosaccharides terminating with GalNAc-4-SO on the
pituitary glycoproteins lutropin (LH), thyroid stimulating hormone
(TSH), and pro-opiomelanocortin (POMC). A glycoprotein
hormone:GalNAc-transferase and a GalNAc-4-sulfotransferase are present
in the pituitary and can account for the synthesis of these unique
oligosaccharides on specific glycoproteins. Both transferases are
coordinately expressed in a number of tissues in addition to pituitary,
including submaxillary gland, lacrimal gland, and kidney, suggesting
that additional glycoproteins bearing oligosaccharides terminating with
GalNAc-4-SO
are synthesized in these tissues. In this study
we show that while the glycoprotein hormone:GalNAc-transferase and the
GalNAc-4-sulfotransferase are coordinately expressed in bovine
submaxillary gland, the GalNAc-transferase is expressed in the parotid
gland in the absence of the GalNAc-4-sulfotransferase. The relative
expression of these two transferases in submaxillary and parotid glands
correlates with the presence of unique Asn-linked oligosaccharides on
carbonic anhydrase VI (CA VI) synthesized in each of these tissues. The
majority of Asn-linked oligosaccharides on CA VI synthesized in
submaxillary gland terminate with GalNAc-4-SO
. In contrast,
CA VI which is synthesized in bovine parotid gland bears
oligosaccharides which terminate predominantly with
1,4-linked
GalNAc which is not sulfated. The presence of different terminal
residues on the Asn-linked oligosaccharides of submaxillary and parotid
CA VI thus correlates with the complement of transferases in these
glands and suggests differing biological roles for submaxillary and
parotid CA VI.
Glycoproteins expressed in cells which differ in their
complement of glycosyltransferases will potentially bear
oligosaccharides differing in structure, thus producing distinct
glycoforms of otherwise identical proteins which may differ in their
biologic functions. We demonstrated this to be the case for equine
lutropin (LH) ()and chorionic gonadotropin (CG) which are
synthesized in the anterior lobe of the pituitary and the placenta,
respectively(1) . Equine LH and CG bear Asn-linked
oligosaccharides terminating with
SO
-4-GalNAc
1,4GlcNAc
1,2Man
and sialic acid
2,3Gal
1,4GlcNAc
1,2Man
, respectively, because of
differences in the complement of glycosyltransferases expressed in
pituitary and placenta. We had previously demonstrated that the
pituitary glycoprotein hormones LH and thyrotropin (TSH) from a number
of different mammalian species bear Asn-linked oligosaccharides
terminating with SO
-4-GalNAc
1,4GlcNAc
1,2Man
(2, 3, 4, 5, 6) . We
subsequently determined that the sulfated oligosaccharides play an
important role following release of LH into the blood. Hepatic
reticuloendothelial cells contain a receptor specific for
oligosaccharides terminating with GalNAc-4-SO
(7) ,
which mediates the rapid removal of LH from the circulation (8) thus regulating its circulatory half-life.
Glycoprotein
hormone oligosaccharides terminating with 1,4-linked
GalNAc-4-SO
are synthesized by the sequential action of two
highly specific enzymes. GalNAc is added to the synthetic intermediate
GlcNAc
Man
GlcNAc
Asn by a
protein-specific GalNAc-transferase(9) . In the presence of a
specific protein recognition motif, the K
for GalNAc addition to this synthetic intermediate by the
glycoprotein hormone:GalNAc-transferase is 5-10 µM,
in contrast to a K
of 1-2 mM for addition to the same oligosaccharide intermediate in the
absence of the recognition marker (9, 10, 11) . The specificity of the
transferase accounts for the addition of GalNAc to LH and TSH but not
to other pituitary glycoproteins. Addition of sulfate to the 4-hydroxyl
of terminal GalNAc residues occurs by the action of a
GalNAc-4-sulfotransferase which is not
proteinspecific(12, 13, 14) , requiring only
the terminal sequence GalNAc
1,4GlcNAc
for the transfer of
sulfate from adenosine 3`-phosphate 5`-phosphosulfate (PAPS). (
)The absence of GalNAc-4-SO
on equine and human
CG is accounted for by the lack of expression of either of these
transferases in equine and human placenta.
Oligosaccharides
terminating with GalNAc-4-SO have to date been shown to be
major constituents on two glycoproteins which are not members of the
glycoprotein hormone family: pro-opiomelanocortin (POMC) (15, 16, 17) and recombinant tissue factor
pathway inhibitor (TFPI)(18) . POMC synthesized by AtT-20
cells, a pituitary corticotroph-derived cell line(15) , and the
16-kDa amino-terminal fragment derived from bovine POMC (17, 19) both have oligosaccharides terminating with
GalNAc-4-SO
. TFPI was known to bear sulfated
oligosaccharides when synthesized by endothelial cells (20, 21) and was predicted to be a substrate for the
glycoprotein hormone:GalNAc-transferase based on the presence of a
putative GalNAc-transferase recognition sequence. The Asn-linked
oligosaccharides on recombinant TFPI synthesized in 293 cells, which
express both the glycoprotein hormone:GalNAc-transferase and the
GalNAc-4-sulfotransferase, were subsequently shown to consist almost
exclusively of structures terminating with GalNAc-4-SO
(18) .
We first identified the glycoprotein
hormone:GalNAc-transferase and the GalNAc-4-sulfotransferase in
extracts prepared from pituitary, the site of LH and TSH
synthesis(9, 12, 13, 14) . Both
enzymes were subsequently localized to several other tissues including
submaxillary gland, kidney, and lacrimal gland but were absent in a
number of the tissues surveyed, such as heart and liver(22) .
Expression of the glycoprotein hormone:GalNAc-transferase and the
GalNAc-4-sulfotransferase in tissues other than pituitary suggests that
glycoproteins bearing oligosaccharides terminating with
GalNAc-4-SO are synthesized in these tissues; however,
glycoproteins bearing such structures as major components have not
previously been identified.
We report here that both the
glycoprotein hormone:GalNAc-transferase and the
GalNAc-4-sulfotransferase are expressed in bovine submaxillary gland
and that the majority of Asn-linked oligosaccharides present on the
secreted form of carbonic anhydrase (CA VI) synthesized by bovine
submaxillary gland terminate with GalNAc-4-SO. In addition,
we demonstrate that a similar proportion of Asn-linked oligosaccharides
on CA VI synthesized in the bovine parotid gland terminate with
1,4-linked GalNAc which is not sulfated. The latter structure is
consistent with our determination that the glycoprotein
hormone:GalNAc-transferase but not the GalNAc-4-sulfotransferase is
expressed in the parotid gland. Oligosaccharides terminating with
GalNAc-4-SO
have been described on Tamm-Horsfall
glycoprotein as minor components(23) ; however, this is the
first instance of a non-pituitary glycoprotein in which
oligosaccharides terminating with GalNAc-4-SO
represent the
major component. Our observations suggest that a crucial function
exists for the Asn-linked oligosaccharides on CA VI, and, furthermore,
that submaxillary and parotid CA VI may have distinct biological roles
based on the differences in the structures of their Asn-linked
oligosaccharides.
Glycoprotein
hormone:GalNAc-transferase assays were incubated at 37 °C for 90
min. Each assay of 50 µl contained 25 mM HEPES (pH 7.5),
0.1% Triton X-100, 10 mM ATP, 1 mg/ml BSA, 15% glycerol, 10
mM MnCl, protease inhibitors, 1 mM UDP-GalNAc, 165 ng of agalacto-hCG as acceptor substrate, and
tissue extract. GalNAc incorporation into hCG was quantitated using
biotinylated Wisteria floribunda agglutinin as
described(29) .
GalNAc-4-sulfotransferase reactions (50
µl) were carried out as described (14) at 28 °C for 2 h
and contained 15 mM HEPES (pH 7.4), 1% Triton X-100, 40 mM -mercaptoethanol, 10 mM NaF, 1 mM ATP, 4
mM magnesium acetate, 13% glycerol, protease inhibitors, 2
µM unlabeled PAPS, 1
10
cpm
[
S]PAPS, 20 µM GGnM-MCO, and tissue
extract. [
S]SO
-GGnM-MCO was
separated from [
S]PAPS and from labeled
endogenous acceptors by passage over a Sep-Pak (C
)
cartridge (Waters) as described previously(14) . For each
tissue extract, a control reaction was done in the absence of GGnM-MCO.
For monosaccharide composition analysis, 40 µg of purified submaxillary or parotid CA VI was hydrolyzed at 100 °C in 4 N HCl for 4 h. The hydrolysates were dried down under vacuum and resuspended in distilled water, and the monosaccharides were separated isocratically on a CarboPak PA1 column in 16 mM NaOH at a flow rate of 0.5 ml/min. The column effluent was mixed with an equal volume of 300 mM NaOH before entering a pulsed amperimetric detection cell. Elution positions of monosaccharides were determined by comparison with authentic standards.
For analysis by
anion exchange HPLC and by ion suppression amine adsorption, Asn-linked
oligosaccharides were released from purified bovine submaxillary and
parotid carbonic anhydrase VI by digestion with
peptide:N-glycosidase F, purified by passage over a Sep-Pak
C18 cartridge in water, and desalted by passage over a Bio-Gel P-2
column (Bio-Rad). The oligosaccharides were labeled at their reducing
termini by reduction with [H]NaBH
(American Radiochemical) as described previously(3) . The
H-labeled oligosaccharides were fractionated by anion
exchange chromatography on MicroPak AX-5 (Varian Associates) at pH 4.0
using a gradient of KH
PO
as described
previously(34, 35) . Ion suppression amine adsorption
HPLC on MicroPak AX-5 (Varian Associates) was carried out as described
previously(35, 36) .
High levels of glycoprotein
hormone:GalNAc-transferase activity are detected in bovine submaxillary
and parotid glands (Table 1). In contrast,
GalNAc-4-sulfotransferase levels which are significantly higher than
those in pituitary extracts are detected in submaxillary gland
extracts, whereas little or no activity is detected in bovine parotid
gland extracts (Table 1). Little or no activity for either
transferase is detected in bovine thyroid gland, in agreement with
previous results in rat tissue (22) . The high levels of
glycoprotein hormone:GalNAc-transferase and GalNAc-4-sulfotransferase
in submaxillary gland suggest the presence of endogenous glycoproteins
bearing 1,4-linked GalNAc-4-SO
. Likewise, the presence
of GalNAc-transferase but not GalNAc-4-sulfotransferase activity in
parotid gland extracts makes it likely that this gland synthesizes
glycoproteins which terminate in
1,4-linked GalNAc. The high
levels of transferase activity in bovine submaxillary glands make it an
excellent tissue source for purification of the
GalNAc-4-sulfotransferase.
Figure 1:
An endogenous 45-kDa glycoprotein from
bovine submaxillary gland incorporates
[S]SO
into its Asn-linked
oligosaccharides. A partially purified preparation of
GalNAc-4-sulfotransferase from bovine submaxillary glands was incubated
with [
S]PAPS as outlined under
``Experimental Procedures.'' Duplicate reactions were either
not digested (lane 1), mock-digested (lane 2), or
digested with PNGase F (lane 3) and analyzed by SDS-PAGE (10%)
and autoradiography. The 45-kDa species is indicated with an arrow.
Sulfate incorporation into Asn-linked oligosaccharides has been
described in at least five different linkages (Man-6-SO,
Man-4-SO
, GalNAc-4-SO
, Gal-3-SO
,
and
GlcNAc-6-SO
)(37, 38, 39, 40, 41) .
Since the GalNAc-4-sulfotransferase preparation used in the in
vitro labeling reaction was only partially purified, we determined
the location and linkage of the sulfate on the Asn-linked
oligosaccharides of the endogenous acceptor. The
[
S]SO
-labeled 45-kDa protein was
separated from other endogenous sulfate acceptors by SDS-PAGE, eluted
from the polyacrylamide gel, and treated with PNGase F. The labeled
oligosaccharides were fractionated on concanavalin A (ConA)-Sepharose
into bound (51% of the incorporated counts) and unbound (49% of the
incorporated counts) fractions. The ConA-Sepharose bound and unbound
oligosaccharides were each subjected to mild acid hydrolysis under
conditions that cleave glycosidic bonds more rapidly than sulfate
esters(14) . The labeled monosaccharides were separated from
incompletely hydrolyzed oligosaccharides and from free
[
S]SO
by chromatography on Sephadex
G-10 and were analyzed by HPLC on a CarboPak PA1 column (Dionex). The
only sulfated monosaccharide product detected in the ConA-bound (Fig. 2) and unbound (data not shown) fractions co-migrated with
authentic GalNAc-4-SO
. This suggested that at least a
fraction of the 45-kDa glycoprotein bears Asn-linked oligosaccharides
terminating with
1,4-linked GalNAc which are subject to sulfation
by the GalNAc-4-sulfotransferase which transfers sulfate exclusively to
structures terminating in GalNAc in a
1,4 linkage(14) .
Figure 2:
The
[S]SO
-labeled Asn-linked
oligosaccharides released from the 45-kDa endogenous substrate in
submaxillary glands contain GalNAc-4-SO
. The Asn-linked
oligosaccharides from bovine submaxillary 45-kDa glycoprotein were
labeled with [
S]SO
as in Fig. 1. The labeled protein was subjected to SDS-PAGE,
electroeluted, and treated with PNGase F. Released Asn-linked
oligosaccharides were purified and passed over a ConA-Sepharose column.
The ConA-Sepharose-bound fraction was subjected to mild acid hydrolysis
and analyzed by HPLC on a CarboPak PA1 column (Dionex). Elution
positions for authentic standards are
shown.
Since this 45-kDa glycoprotein was relatively abundant in preparations of partially purified GalNAc-4-sulfotransferase, we purified sufficient material for amino acid sequence determination by preparative separation on SDS-PAGE followed by elution of the 45-kDa band from the gel. Amino-terminal sequence could not be obtained due to blockage of the amino terminus; therefore, internal sequences were obtained on fragments generated by digestion of the protein with chymotrypsin or trypsin. These sequences were used to search the Swiss protein data base and were found to be homologous to sequences within sheep and human CA VI. This suggested that the 45-kDa endogenous substrate in submaxillary gland is CA VI, originally identified in rat (42) and human (43) saliva as a glycosylated form of carbonic anhydrase which is thought to function in the pH regulation of saliva.
Sulfonamides are specific inhibitors of carbonic anhydrases which have been used extensively in the purification of a number of these enzymes (30, 42, 43) . Passage of partially purified GalNAc-4-sulfotransferase over p-aminomethylbenzene sulfonamide-agarose resulted in the removal of the 45-kDa endogenous substrate, further supporting the identity of the endogenous substrate as CA VI (not shown). We therefore characterized the Asn-linked oligosaccharides present on CA VI which had been purified from bovine submaxillary and parotid glands by affinity chromatography on p-aminomethylbenzene sulfonamide-agarose using a modification of the procedure of Fernley et al.(30) . Purified CA VI from both tissues migrated at 45 kDa by SDS-PAGE, which was reduced to 35 kDa by digestion with PNGase F suggesting the presence of two Asn-linked oligosaccharides (Fig. 3). This is consistent with the molecular weights and number of Asn-linked oligosaccharides observed on CA VI isolated from sheep, rat, and human(30, 42, 43) . The specific activity of CA VI purified from bovine submaxillary and parotid glands, as measured by a phenol red pH indicator assay(31) , was 1933 units/mg and 1590 units/mg, respectively, which is similar to that reported for homogeneous sheep CA VI(30) .
Figure 3:
Purification of carbonic anhydrase VI from
bovine submaxillary and parotid salivary glands. CA VI was purified
from bovine submaxillary and parotid glands by affinity chromatography
on p-aminomethylbenzene sulfonamide-agarose and elution in 0.4 M NaN as indicated under ``Experimental
Procedures.'' 2 µg of purified protein from each source was
subjected to SDS-PAGE (10%), before(-) and after (+)
treatment with PNGase F. Proteins were visualized by staining with
Coomassie Blue.
[S]SO
was
transferred from [
S]PAPS to both submaxillary
and parotid CA VI in vitro by GalNAc-4-sulfotransferase which
had been partially purified from bovine pituitaries and was itself free
of the 45-kDa endogenous substrate (Fig. 4, lanes 2, 3, 5, and 6). The incorporated
[
S]SO
was quantitatively released
from both tissue forms by digestion with PNGase F, indicating that the
sulfate was transferred exclusively to the Asn-linked oligosaccharides (Fig. 4, lanes 4 and 7).
[
S]SO
-labeled submaxillary CA VI was
purified on p-aminomethylbenzene sulfonamide-agarose, digested
with PNGase F, and the purified Asn-linked oligosaccharides were
subjected to mild acid hydrolysis and analysis on a CarboPak PA1 column
as before. A single peak was obtained which co-migrated with authentic
GalNAc-4-SO
, indicating that
[
S]SO
was incorporated into CA VI
Asn-linked oligosaccharides as GalNAc-4-SO
(data not
shown). Since both submaxillary and parotid CA VI are in vitro substrates for GalNAc-4-sulfotransferase, some fraction of the
Asn-linked oligosaccharides from CA VI from both tissues must contain
Asn-linked oligosaccharides with terminal
1,4-linked GalNAc.
Figure 4:
The Asn-linked oligosaccharides on
purified submaxillary and parotid carbonic anhydrase VI incorporate
[S]SO
. 1 µg of purified CA VI
from bovine submaxillary (lanes 2, 3, and 4)
and parotid (lanes 5, 6, and 7) glands was
incubated with [
S]PAPS and a partially purified
GalNAc-4-sulfotransferase preparation from bovine pituitary as outlined
under ``Experimental Procedures.'' Identical reactions were
analyzed by SDS-PAGE (10%) and autoradiography either after no
digestion (lanes 2 and 5), after mock digestion (lanes 3 and 6), or after digestion with PNGase F (lanes 4 and 7). The reaction in lane 1 contains no exogenous CA VI.
CA VI isolated from submaxillary and parotid glands, respectively, have nearly identical monosaccharide compositions (Table 2). There is sufficient mannose to account for two complex Asn-linked oligosaccharides with three mannose residues each. CA VI from both glands contains GalNAc as well as Gal, indicating that there are Asn-linked oligosaccharides terminating with GalNAc and/or that there are O-glycosidically linked oligosaccharides as well as Asn-linked oligosaccharides present. If there are no O-glycosidically linked structures present, the amounts of GalNAc present would indicate that a majority of the Asn-linked oligosaccharides present on both submaxillary and parotid CA VI bear one or more GalNAc moieties.
Figure 5:
Western blot analyses of submaxillary and
parotid carbonic anhydrase VI using an anti-GalNAc-4-SO monoclonal antibody and W. floribunda agglutinin. The
indicated amounts of affinity-purified submaxillary and parotid CA VI
were subjected to SDS-PAGE (10%) followed by electroblotting onto
polyvinylidene difluoride. Blots were probed with biotinylated
anti-GalNAc-4-SO
monoclonal antibody or with biotinylated W. floribunda agglutinin (WFA), which specifically
recognizes terminal
1,4-linked GalNAc residues. Blots were
detected with streptavidin-peroxidase and
chemiluminescence.
The structures of the Asn-linked oligosaccharides from
submaxillary and parotid CA VI were examined in greater detail to
determine more precisely the proportion of oligosaccharides terminating
with GalNAc and GalNAc-4-SO, as well as other structural
features. Asn-linked oligosaccharides were released from CA VI from
both tissues by digestion with PNGase F and were labeled at their
reducing termini by reduction with
[
H]borohydride. When fractionated by anion
exchange HPLC, oligosaccharides from the two tissue forms of CA VI
displayed markedly different distributions of anionic species (Fig. 6, A and B) with oligosaccharides from
submaxillary CA VI yielding a more complex pattern than those from
parotid CA VI. Oligosaccharides from submaxillary CA VI co-migrated
with authentic standards containing no anionic species(N0), 1 sialic
acid(N1), 1 sulfate (S1), 2 sialic acids(N2), 1 sulfate and 1 sialic
acid (SN), 2 sulfates (S2), 3 sialic acids(N3), 1 sulfate and 2 sialic
acids (S1N2), 2 sulfates and 1 sialic acid (S2N1), and 4 anionic
moieties (SxNy where x + y = 4) (Fig. 6A). Treatment with 2 N acetic acid at 100 °C for 15 min will release sialic acid but
not sulfate from oligosaccharides(2) . Following treatment with
2 N acetic acid, the proportion of oligosaccharides from
submaxillary gland CA VI which migrated as neutral species increased
from 13% to 44% of the total. Those oligosaccharides not migrating as
neutral species after treatment with 2 N acetic acid migrated
as oligosaccharides with 1 sulfate (35% migrate as S1) or with 2
sulfate (17% migrate as S2) moieties (not shown). Digestion of the
oligosaccharides from submaxillary CA VI with Newcastle disease virus
neuraminidase, which will release sialic acid in an
2,3-linkage
but not sialic acid in an
2,6-linkage(28) , yielded a
pattern identical with that obtained with 2 N acetic acid.
Thus, 35% and 17% of the oligosaccharides on submaxillary CA VI contain
1 or 2 sulfate moieties, respectively, and sialic acid, when present,
is exclusively in an
2,3-linkage.
Figure 6:
Anion exchange HPLC of Asn-linked
oligosaccharides from submaxillary and parotid carbonic anhydrase VI.
Asn-linked oligosaccharides from purified CA VI were released by
treatment with PNGase F, purified, and labeled at their reducing
termini with [H]borohydride as outlined under
``Experimental Procedures.'' Labeled oligosaccharides from
submaxillary (A) and parotid (B) CA VI were subjected
to anion exchange HPLC on an AX-5 column. The elution positions of
authentic oligosaccharides bearing no charged residues (N0),
one (N1), two (N2), or three (N3) sialic
acid residues; one (S1) or two (S2) sulfate residues;
one sulfate and one sialic acid residue (SN), one sulfate and
two sialic acid residues (S1N2), two sulfate and one sialic
acid residue (S2N1), or a heterogeneous combination of sulfate
and sialic acid residues (SxNy) producing four
anionic charges are indicated.
Anion exchange HPLC of the
labeled oligosaccharides from parotid CA VI (Fig. 6B)
yielded only 3 peaks corresponding to oligosaccharides with 0, 1, or 2
sialic acid moieties (N0, N1, and N2, respectively). Treatment with 2 N acetic acid or digestion with Newcastle disease virus
neuraminidase converted all of the oligosaccharides to neutral species,
indicating that these oligosaccharides were devoid of sulfate and that
the sialic acid moieties were exclusively in an 2,3-linkage.
The oligosaccharides from submaxillary and parotid CA VI were
fractionated by affinity chromatography on WFA-agarose to determine the
proportion of each oligosaccharide species which bear terminal
1,4-linked GalNAc. In the case of submaxillary CA VI, the neutral
oligosaccharide fraction had the greatest proportion of structures
bound by WFA-agarose (45%). The proportion of oligosaccharides bound by
WFA-agarose declined as species with a greater number of anionic
moieties were examined (Table 3). No change in the proportion of
each oligosaccharide species bound by WFA-agarose was seen following
digestions with neuraminidase indicating that the sialic acid present
is not linked to GalNAc. In contrast, following digestion of those
oligosaccharides identified as having one or more sulfate moieties on
the basis of their elution time on anion exchange HPLC (Fig. 6A, species S1, SN, S2, S1N2, and S2N1) with recombinant
GalNAc-4-sulfatase(32, 33) ,
84% of each species
was bound by WFA-agarose (Table 3). The presence of some S1N2 in
the fraction designated N3 accounts for the increase in WFA-agarose
binding of this fraction upon treatment with sulfatase. We have
previously shown that terminal GalNAc in
1,4-linkage to GlcNAc can
be released by digestion with jack bean
-hexosaminidase but not by
digestion with diplococcal
-hexosaminidase(2) . Digestion
of the desulfated oligosaccharides from submaxillary gland CA VI with
jack bean
-hexosaminidase, but not diplococcal
-hexosaminidase, abolished binding to WFA-agarose confirming that
the GalNAc exposed by digestion with GalNAc-4-sulfatase is in
1,4-linkage.
The results described above indicate that 55% of
the oligosaccharides released from submaxillary CA VI contain one or
more branches terminating with the sequence
SO-4-GalNAc
1,4GlcNAc
. Among the oligosaccharides
which are neutral or bear only sialic acid moieties, an additional 9%
of the total have one or more branches terminating with
GalNAc
1,4GlcNAc
. Therefore, at least 64% of the Asn-linked
oligosaccharides obtained from submaxillary CA VI have either sulfated
or nonsulfated termini containing
1,4-linked GalNAc. As was seen
for the glycoprotein hormones(3, 4) , the addition of
GalNAc to the Asn-linked oligosaccharides of submaxillary CA VI is a
highly efficient process.
The oligosaccharides from parotid CA VI
were also fractionated into species that were bound and not bound to
WFA-agarose. The bound fractions represented 52% of N0, 43% of N1, and
18% of N2 (Table 4). Digestion of the unbound species with
Newcastle disease virus neuraminidase did not convert them to species
which could bind WFA indicating that the sialic acid was not linked to
GalNAc. Digestion with jack bean -hexosaminidase but not with
diplococcal
-hexosaminidase abolished WFA binding by the
previously bound species. The properties of the WFA-bound species
indicates that they contain at least one branch which terminates with
the sequence GalNAc
1,4GlcNAc
. Thus, 40% of the Asn-linked
oligosaccharides from parotid CA VI contains one or more branches which
terminate with GalNAc
1,4GlcNAc
.
A striking feature of the
oligosaccharides from both submaxillary and parotid CA VI when examined
by ion suppression amine adsorption-HPLC, which fractionates
oligosaccharides on the basis of size as well as
charge(36, 46) , was their large size. Furthermore,
the anionic oligosaccharides which contained either GalNAc-4-SO or terminal GalNAc from both submaxillary and parotid CA VI were
not bound by ConA (not shown). This suggests that the Asn-linked
oligosaccharides on submaxillary and parotid CA VI are more highly
branched structures than the dibranched oligosaccharides typical of the
glycoprotein hormones LH and TSH(3, 4) . Due to their
heterogeneity and complexity, the detailed structures of these
oligosaccharides have not yet been examined.
We originally described oligosaccharides terminating with the
sequence SO-4-GalNAc
1,4GlcNAc
1,2Man
on the
pituitary glycoprotein hormones LH and TSH and the uncombined
glycoprotein hormone
subunit(2, 3, 4, 5, 6) .
Synthesis of these unique structures is accounted for by a
GalNAc-transferase which displays peptide as well as oligosaccharide
specificity (9, 10, 11, 47) and by a
GalNAc-4-sulfotransferase(14, 48) . Using highly
specific GalNAc-transferase and GalNAc-4-sulfotransferase assays, we
have detected both transferase activities in a number of tissues and
cell lines(15, 22) , suggesting that oligosaccharides
terminating with GalNAc-4-SO
may be present on
glycoproteins unrelated to the glycoprotein hormones. Oligosaccharides
bearing GalNAc linked
1,4 to an underlying GlcNAc have been
subsequently described on glycoproteins from a number of different
sources(45, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59) ;
however, in addition to the glycoprotein hormones LH and TSH,
oligosaccharides terminating with GalNAc-4-SO
have been
described only on POMC(15, 16, 17) ,
recombinant TFPI(18) , and Tamm-Horsfall glycoprotein purified
from urine(23) . In contrast to POMC, TFPI, and the
glycoprotein hormones, where one or more termini with GalNAc-4-SO
are present on the majority of oligosaccharides isolated from
these glycoproteins, less than 2% of the Asn-linked oligosaccharides
present on Tamm-Horsfall glycoprotein terminate with
GalNAc-4-SO
, making it a relatively minor component on this
glycoprotein(23) .
The present study demonstrates that
Asn-linked oligosaccharides terminating with GalNAc-4-SO are the predominant structure on CA VI synthesized in
submaxillary glands. High levels of GalNAc-transferase and
GalNAc-4-sulfotransferase activities may be required in the
submaxillary gland because the substrate, CA VI, is a major synthetic
product. We have determined that Asn-linked oligosaccharides
terminating with
1,4-linked GalNAc which is neither sulfated nor
sialylated are the predominant structures present on CA VI synthesized
in bovine parotid glands. CA VI is also a major product of the parotid
gland which expresses high levels of GalNAc-transferase but no
GalNAc-4-sulfotransferase. The absence of sulfate on parotid CA VI and
the absence of GalNAc-4-sulfotransferase activity in parotid gland
extracts indicate that this sulfotransferase accounts for sulfate
addition to oligosaccharides on CA VI in the submaxillary gland. The
results obtained with CA VI establish that there are endogenously
synthesized proteins in tissues other than pituitary and unrelated to
the glycoprotein hormones which bear oligosaccharides terminating with
GalNAc-4-SO
. Furthermore, we expect that additional
endogenous glycoproteins bearing this structure will be found in other
tissues such as lacrimal gland and kidney which express relatively high
levels of GalNAc-transferase and GalNAc-4-sulfotransferase.
We have
shown that digestion of submaxillary and parotid CA VI oligosaccharides
with Newcastle disease virus neuraminidase results in the quantitative
release of sialic acid from both forms. Since this neuraminidase is
specific for sialic acid in 2,3 linkage, we conclude that all of
the sialic acid found on the oligosaccharides from both tissue forms of
CA VI is linked
2,3 to the underlying sugar. In addition, removal
of sialic acid from both CA VI tissue forms does not expose any
underlying GalNAc, indicating that sialic acid is linked to sugars
other than GalNAc, predominantly Gal
1,4GlcNAc
. This is
consistent with our observation that
2,3-sialyltransferase, in
contrast to
2,6-sialyltransferase(60) , does not transfer
sialic acid to terminal
1,4-linked GalNAc in an in vitro assay. (
)Since
2,6-sialyltransferase is able to
transfer sialic acid to either
1,4-linked GalNAc or Gal (60) , the absence of detectable
2,6-linked sialic acid on
either submaxillary or parotid CA VI oligosaccharides implies that
2,6-sialyltransferase is not expressed in cells synthesizing CA VI
in either tissue. The absence of GalNAc-4-sulfotransferase and
2,6-sialyltransferase can thus account for the presence of
terminal GalNAc, which is neither sulfated nor sialylated, on CA VI
from bovine parotid glands.
Expression of the glycoprotein
hormone:GalNAc-transferase in bovine submaxillary and parotid glands in
conjunction with the observation of 1,4-linked GalNAc in CA VI
Asn-linked oligosaccharides suggests that the GalNAc-transferase in
salivary glands is the same as the GalNAc-transferase we previously
characterized as responsible for GalNAc addition to Asn-linked
oligosaccharides on the glycoprotein hormones in pituitary(9) .
The requirements for peptide recognition by this GalNAc-transferase
have been extensively characterized. In the case of the glycoprotein
hormone
subunit, basic amino acids within the sequence
Pro-Leu-Arg-Ser-Lys-Lys, which is amino-terminal to the first of two
Asn-linked glycosylation sites, are found within an
-helix, thus
forming a cluster of basic residues which is essential for recognition
by the GalNAc-transferase(47) . The amino acid sequences of
human and sheep CA VI have been established by cDNA (61) and
protein (62) sequencing, respectively. CA VI from both species
contains good candidate sequences for recognition by the glycoprotein
hormone:GalNAc-transferase. The sequence Pro-Lys-Arg-Lys-Lys is present
59 residues carboxyl-terminal to the nearest predicted Asn-linked
glycosylation site (Asn
) in sheep CA VI, and the sequence
Pro-Leu-Lys-His-Arg is present 12 residues carboxyl-terminal to a
potential Asn-linked glycosylation site (Asn
) in human CA
VI. The presence of good candidate sequences for recognition by the
glycoprotein hormone:GalNAc-transferase along with the high levels of
GalNAc-transferase activity found in the submaxillary and parotid
glands strongly suggest that the glycoprotein
hormone:GalNAc-transferase accounts for the presence of
1,4-linked
GalNAc on CA VI. However, further studies will be required to determine
whether these candidate sequences account for recognition of CA VI by
the GalNAc-transferase.
Submaxillary CA VI represents the first
example of a naturally occurring glycoprotein not synthesized in the
pituitary in which a major fraction of the Asn-linked oligosaccharides
terminate with SO-4-GalNAc
1,4GlcNAc
. The presence
of the same terminal structure which is not sulfated on parotid CA VI
suggests that critical biologic function(s) are associated with these
oligosaccharides. In the case of LH, a receptor in hepatic endothelial
cells recognizes oligosaccharides with terminal
SO
-4-GalNAc
1,4GlcNAc
and rapidly removes LH from
the circulation(7, 8) . As a result, LH has a
shortened circulatory half-life which produces the pulsatile rise and
fall of this hormone in the blood. Since CA VI is released into the
saliva (42, 43, 63, 64, 65) and
not the bloodstream, it is not likely to encounter the hepatic receptor
for GalNAc-4-SO
. The unique oligosaccharide structures on
CA VI may have an antibacterial function as has been hypothesized for
the heterogeneous oligosaccharides on mucins(66) .
Alternatively, receptors similar to the GalNAc-4-SO
receptor in hepatic endothelial cells (7, 8) and
the Gal/GalNAc-specific receptor in hepatocytes (67) may reside
in different regions of the oral mucosa and selectively immobilize
submaxillary and parotid CA VI, respectively. Immobilization in
specific regions of the mouth could play a critical role in the
localized regulation of oral pH.
Even though the biologic function
of the oligosaccharides on CA VI is not yet established, the different
oligosaccharide structures found on submaxillary and parotid CA VI in
conjunction with the pattern of transferase expression in these two
tissues establish that: 1) glycoproteins bearing Asn-linked
oligosaccharides terminating with
SO-4-GalNAc
1,4GlcNAc
are synthesized outside the
pituitary; 2) the glycoprotein hormone:GalNAc-transferase and the
GalNAc-4-sulfotransferase are not always coordinately expressed in
other tissues as they are in the pituitary; 3) the glycoprotein
hormone:GalNAc-transferase, when expressed in the absence of
GalNAc-4-sulfotransferase, can account for the synthesis of
glycoproteins bearing oligosaccharides with terminal
GalNAc
1,4GlcNAc
; and 4) glycoproteins not destined to enter
the bloodstream may bear SO
-4-GalNAc
1,4GlcNAc
or
GalNAc
1,4GlcNAc
. In addition, the selective transfer of
different terminal sugars to oligosaccharides bearing
GalNAc
1,4GlcNAc
may result in the synthesis of other unique
oligosaccharides with yet other functions. The presence of glycoprotein
hormone:GalNAc-transferase and/or GalNAc-4-sulfotransferase activity in
a number of tissues and cell lines suggests that oligosaccharides
related to those originally described on the glycoprotein hormones will
be found on other glycoproteins of diverse functions.