(Received for publication, June 29, 1995)
From the
The circulatory half-life of the mammalian glycoprotein hormone
lutropin is controlled by its unique Asn-linked oligosaccharides, which
terminate with the sequence SO-4-GalNAc
1,4GlcNAc. A
cluster of basic amino acids essential for recognition of the
subunit by the glycoprotein
hormone:N-acetylgalactosaminyltransferase is located within
two turns of an
helix (Mengeling, B. J., Manzella, S. M., and
Baenziger, J. U.(1995) Proc. Natl. Acad. Sci. U. S. A. 92,
502-506). The amino acids within this region are virtually
invariant in the
subunits of all vertebrates, indicating that the
recognition determinant utilized by the N-acetylgalactosaminyltransferase has been conserved in
species ranging from teleost fish to mammals. We demonstrate that the
glycoprotein hormone:N-acetylgalactosaminyltransferase and the N-acetylgalactosamine-4-sulfotransferase responsible for the
synthesis of these unique sulfated oligosaccharides are expressed in
the pituitaries of vertebrates ranging from teleost fish to mammals.
Furthermore, we show that Asn-linked oligosaccharides terminating with
SO
-4-GalNAc
1,4GlcNAc are present on the
and
subunits of the salmon glycoprotein hormone GTH II. Asn-linked
oligosaccharides terminating with SO
-4-GalNAc
1,4GlcNAc
are unique structural features of the glycoprotein hormones that have
been conserved during vertebrate evolution, suggesting they are
critical for the expression of hormone biologic activity.
Even though extensive structural diversity and heterogeneity are
characteristic of the oligosaccharides found on glycoproteins, there
are instances in which highly distinctive oligosaccharide structures
are present on specific glycoproteins or types of glycoproteins from
different animal species. The presence of such characteristic
oligosaccharide structures indicates that the glycoproteins bearing
them have biologic functions that are dependent on the structural
features of these oligosaccharides. We (1, 2, 3) and others (4) have
demonstrated that Asn-linked oligosaccharides terminating with
SO-4-GalNAc
1,4GlcNAc are characteristic of the
glycoprotein hormones lutropin (LH) (
)and thyrotropin from a
number of mammalian species, whereas oligosaccharides terminating with
sialic acid-Gal
1,4GlcNAc, commonly found on many glycoproteins,
predominate on follitropin(2, 3) . Oligosaccharides
bearing terminal
1,4-linked GalNAc-4-SO
are recognized
by a receptor in hepatic endothelial cells, resulting in the rapid
removal of glycoproteins bearing these structures from the
blood(5, 6, 7) . The short circulatory
half-life of LH in conjunction with its stimulated release from
gonadotrophs by gonadotropin-releasing hormone produces a periodic rise
and fall in circulating LH levels, which is thought to be essential for
optimal activation of the ovarian LH receptor and fertilization. Thus,
in mammals the presence of terminal GalNAc-4-SO
is
associated with a highly specific function.
The presence of terminal
GalNAc-4-SO reflects the activity of a glycoprotein
hormone:N-acetylgalactosaminyltransferase and a
GalNAc-4-sulfotransferase, which we have shown are expressed in the
anterior lobe of the pituitary from a number of
mammals(8, 9, 10, 11) . In addition
to the oligosaccharide acceptor, the N-acetylgalactosaminyltransferase recognizes a protein
determinant within the
subunit of the glycoprotein
hormones(8, 12) . All of the information required for
recognition of the
subunit by the GalNAc-transferase is contained
within a 23-amino acid glycopeptide fragment(10) . The
recognition determinant consists of a cluster of basic amino acids that
are contained within two turns of an
helix(13) . A number
of studies have demonstrated that the region of the
subunit,
which includes the N-acetylgalactosaminyltransferase
recognition determinant, also contains residues that are critical for
combination with the
subunits (14, 15) and for
binding to and activation of the LH/CG-receptor(16) . Thus, a
number of distinct functions are dependent on interactions of different
proteins with the same region of the
subunit.
Homologues of
the glycoprotein hormones have been identified in vertebrates from
mammals to teleost fish(17) . In mammals, follitropin is
responsible for follicular development, LH drives oocyte maturation and
ovulation, and thyrotropin regulates thyroid function(18) .
Similar functions have been attributed to glycoprotein hormone
homologues in other vertebrate species. Features of both the common
subunit and the hormone-specific
subunits have been
conserved in vertebrate glycoprotein hormones including locations of
the Cys and Asn glycosylation sites(17, 18) .
Furthermore, crystallographic studies of human chorionic gonadotropin
have revealed that both the
and
subunits have a
cysteine-knot motif like that found on nerve growth factor,
transforming growth factor-
, and platelet-derived growth
factor(19, 20) . The residues that are critical for
recognition of the
subunit by the N-acetylgalactosaminyltransferase fall within a region of the
subunit that is virtually invariant among vertebrate species.
Since the recognition determinant utilized by the mammalian N-acetylgalactosaminyltransferase is present within the
subunits of virtually all vertebrate species, we wished to determine if
the glycoprotein
hormone:N-acetylgalactosaminyltransferase(8) , the
GalNAc-4-sulfotransferase(9) , and the sulfated oligosaccharide
structures, which we have described on LH, thyrotropin, and the free,
uncombined
subunits of mammalian species(2, 3) ,
are also conserved. The presence of these same sulfated
oligosaccharides on glycoprotein hormones of other vertebrates would
strongly support the biologic importance of these structures, which
have been found to control the circulatory half-life of LH in
mammals(6, 7) .
Transfer of GalNAc by the glycoprotein
hormone:N-acetylgalactosaminyltransferase (GalNAc-transferase)
to identical oligosaccharide acceptors on human chorionic gonadotropin
(hCG) and transferrin (Trf), which do and do not contain the
GalNAc-transferase recognition motif, respectively, was compared using
the assay previously described(11) . Each 50-µl transferase
reaction consisted of 25 mM HEPES (pH 7.5), 0.1% (w/v) Triton
X-100, 10 mM ATP, 15% (w/v) glycerol, 10 mM MnCl, protease inhibitors (5.75 millitrypsin inhibitor
units of aprotinin, 1 µg each of leupeptin, antipain, pepstatin,
and chymostatin), 1 mM UDP-GalNAc, 200 ng of agal-hCG or 420
ng of agal-Trf (hCG and Trf, which had been digested with neuraminidase
and
-galactosidase), and 10-50 µg of pituitary extract
protein. Reactions were carried out at 37 °C for 90 min.
The
amount of 1,4-linked GalNAc incorporated was determined as
described(11, 21) . Individual wells of a Dynatech
96-well microplate were coated with sufficient monoclonal anti-hCG
(Ventrex Laboratories Inc., Clone 19, 5012191) to bind 8 ng of hCG/well
or with sufficient rabbit anti-mouse IgG Fc (Pierce) followed by
monoclonal anti-Trf (Medix Biotech, CA, MIT 0603) to bind 5 ng of
Trf/well. Biotinylated Wisteria floribunda, which is specific
for
1,4-linked GalNAc(22, 23) , was used in
combination with streptavidine and biotinylated Aequorin (Sealite,
Athens, GA) to quantitate the amount of GalNAc
incorporated(21) . hCG and Trf fully substituted with
1,4-linked GalNAc were prepared as described (11) and used
to construct standard curves. Activities are expressed as pmol of
GalNAc transferred/mg of protein/h.
GalNAc-4-sulfotransferase
activity was assayed as described(9, 11) . Each
50-µl assay contained 15 mM HEPES, pH 7.3, 1% Triton X-100
(w/v), 40 mM 2-mercaptoethanol, 6 mM magnesium
acetate, 10 mM NaF, 1 mM ATP, 166 mg/ml glycerol,
protease inhibitors (5.75 millitrypsin inhibitor units of aprotinin, 1
µg each of leupeptin, antipain, pepstatin, and chymostatin), 2.0
µM PAPS, 1 10
cpm of
PAPS, 20 µM GGnM-MCO, and
10-50 µg of tissue extract. Following incubation for 3 h at
28 °C, 1 ml of H
O was added to each reaction, which was
then loaded onto a Sep-Pak (C18) cartridge (Millipore/Waters). The
Sep-Pak was sequentially developed with 20 ml of H
O, 10 ml
of 15% (v/v) methanol, and 5 ml of 30% (v/v) methanol. The
[
S]SO
-GGnM-MCO product was present
in the 30% methanol eluate and quantitated by liquid scintillation
counting.
Isomers of SGGnM-MCO and
sulfated monosaccharides were separated on a CarboPac PA1 column
(Dionex, 4 250 mm) as described(9) . The
S-labeled SGGnM-MCO assay product was treated with 1.0 N NaOH for 1 h at 80 °C to determine stability to
alkali(9, 24) . The sulfated monosaccharide was
released from
S-labeled SGGnM-MCO assay product using 40
mM HCl at 100 °C for 2 h. Sulfated monosaccharides
released by mild acid hydrolysis were separated from other degradation
products by gel filtration on Sephadex G-10 in 126 mM NH
HCO
prior to analysis by HPLC as
described(9, 24) .
Figure 1:
Alignment of
glycoprotein hormone subunit sequences for different species
between the two pairs of double cysteines. The glycoprotein hormone
subunits have been aligned in the region between the two pairs of
double cysteines (Cys
-Cys
of human
) for examples of each vertebrate class. Dots indicate
identities. Amino acids previously identified as essential for
recognition by the glycoprotein hormone:GalNAc-transferase (13) are underlined. The essential basic residues are
found within two turns of an
helix (13) . The position of
the glycosylated Asn within this region is indicated by the asterisk. The sequences have been obtained from the NBRF and
EMBL data banks: human, Homosapiens (N-TTHUAP); rat, Rattusnorvegicus (N-JT0408): frog, Ranacatesbeiana (N-S20287); chicken, Gallusdomesticus(37) ; fish, Cyprinuscarpio1 (N-JK0024) and Cyprinuscarpio2 (N-A40554).
GalNAc-transferase activity with the appropriate specificity was detected in pituitary extracts from representatives of each vertebrate class (Table 1). GalNAc was added to oligosaccharide acceptors on hCG, which contains the recognition motif, but not Trf, which does not contain the recognition motif, at the equimolar concentrations of acceptor protein. Thus, a GalNAc-transferase, which specifically modifies the oligosaccharides on glycoprotein hormones, is present in extracts from all the pituitaries examined. GalNAc-4-sulfotransferase activity was detected in the same extracts (Table 1), indicating that GalNAc added to oligosaccharide acceptors would likely be further modified by sulfate addition.
The specific activities (pmol/mg/h) of the
GalNAc-transferase and GalNAc-4-sulfotransferase differed by as much as
6-fold among the extracts from different classes of vertebrates. At
least three cell types (gonadotrophs, thyrotrophs, and corticotrophs)
express GalNAc-transferase and sulfotransferase in mammalian
pituitaries(1, 2, 3, 11, 24) ;
furthermore, the levels of both transferase activities in gonadotrophs
are modulated by the hormonal state of the animal(28) . As a
result, differences in specific activity may reflect the proportion of
cells expressing GalNAc-transferase and GalNAc-4-sulfotransferase, the
levels within specific cell types, hormonal state, and/or other
factors. Nonetheless, the levels of both transferases are sufficient to
account for the presence of oligosaccharides terminating with
GalNAc-4-SO on one or more glycoproteins synthesized within
the pituitaries of all vertebrate classes.
Salmon is representative of the lowest vertebrate class in which glycoprotein hormone homologues have been identified. We therefore characterized the GalNAc-transferase and sulfotransferase products produced by salmon pituitary extracts to verify that the salmon transferases synthesize the same structures as their mammalian counterparts.
Agal-hCG and
UDP-[H]GalNAc were incubated with salmon
pituitary extract to produce a radiolabeled product for
characterization. The
H label, which had been incorporated
into hCG, was released upon digestion with
peptide:N-glycosidase F, which releases Asn-linked
oligosaccharides intact. The
H-labeled oligosaccharides
produced by peptide:N-glycosidase F digestion were then
isolated by gel filtration. The
H-labeled oligosaccharides
were bound by immobilized W. floribunda and specifically
eluted with 50 mM GalNAc (Fig. 2, panelA), indicating that the oligosaccharide products
contained terminal
1,4-linked GalNAc(22, 23) .
The linkage and identity of the
H-sugar was confirmed by
digestion with jack bean
-hexosaminidase and analysis on an Aminex
HPX-87H column (Fig. 2, panelB). The
H-labeled oligosaccharides from a mock digestion eluted
with the column void. Digestion with jack bean
-hexosaminidase
resulted in the disappearance of the
H-labeled material in
the void and the appearance of a single peak, which comigrated with
GalNAc (Fig. 2, panelB). Thus, the
GalNAc-transferase in salmon pituitary transfers GalNAc from UDP-GalNAc
to Asn-linked oligosaccharide acceptors in
1,4-linkage. Since the
salmon GalNAc-transferase displays the same protein specificity as the
GalNAc-transferase from mammalian species, adding GalNAc to
oligosaccharides on hCG with a much greater catalytic efficiency than
to the same oligosaccharides on transferrin (Table 1), it most
likely utilizes the same recognition motif as the mammalian enzyme.
Figure 2:
The salmon pituitary GalNAc-transferase
product binds to W. floribunda-agarose and is released by jack
bean -hexosaminidase. Agal-hCG oligosaccharides were labeled with
H by incubating agal-hCG with
UDP-[
H]GalNAc and an extract of salmon pituitary.
The
H oligosaccharides were released from hCG by digestion
with peptide:N-glycosidase F and separated from other products
by gel filtration on Sephadex G-15. PanelA, the
H oligosaccharides were applied to W.
floribunda-agarose in 20 mM Tris, pH 7.4, 150 mM NaCl. The column was eluted with 20 mM Tris, pH 7.4, 150
mM NaCl to remove unbound material and with the same buffer
containing 50 mM GalNAc (arrow) to specifically elute
bound material. PanelB, the
H
oligosaccharides were analyzed on Aminex HPX-87H column eluted with
0.01 N H2SO4 before (solidline) and
following digestion with jack bean
-hexosaminidase (brokenline). The elution positions of GlcNAc and GalNAc are
indicated.
Pituitary extracts from mammals contain a sulfotransferase, which
adds sulfate to the 4-hydroxyl of terminal GalNAc-linked 1,4 to an
underlying GlcNAc(9, 11) . Salmon pituitary extracts
were incubated with [
S]PAPS and GGnM-MCO.
[
S]SO
-labeled GGnM-MCO was separated
from other reaction products by passage over Sep-Pak C
cartridges and elution with 30% MeOH(9, 25) . A
single peak was obtained, which comigrated with authentic
SO
-4-GalNAc
1,4GlcNAc
1,2Man
(CH
)
CO
CH
when analyzed on a CarboPac PA1 carbohydrate analysis column
(Dionex) (Fig. 3, panelA). Treatment of the
same sulfated product with 1.0 N NaOH at 80 °C for 1 h
does not alter its mobility when analyzed on a CarboPac PA1 column (Fig. 3, panelB). Only sulfate esters at the
3- or 4-hydroxyl of GalNAc or the 3-hydroxyl of GlcNAc in the
GalNAc
4GlcNAc
2Man trisaccharide would be predicted to be
alkali stable(9) . Mild acid hydrolysis, under conditions which
cleave glycosidic bonds more rapidly than sulfate esters, yielded a
[
S]SO
-labeled peak that comigrated
with authentic GalNAc-4-SO
as the predominant species (Fig. 3, panelC). Since no
SO
-3-GalNAc
1,4GlcNAc
1,2Man
was detected (Fig. 3, panelA) and no GlcNAc-3-SO
was detected after acid hydrolysis (Fig. 3, panelC), these results indicate that the sulfate is added
exclusively to the 4-position of terminal GalNAc in the sequence
GalNAc
1,4GlcNAc
1,2Man
by the salmon pituitary
sulfotransferase. Thus, the salmon GalNAc-4-sulfotransferase has the
same specificity as the mammalian transferase.
Figure 3:
Characterization of salmon pituitary
sulfotransferase reaction product. The S-labeled product
obtained upon incubation of a salmon pituitary extract with
[
S]PAPS and GGnM-MCO was analyzed on CarboPac
PA1 column as described(9) . PanelA,
[
S]SO
-GGnM-MCO; panelB, [
S]SO
-GGnM-MCO
after treatment with alkali; and panelC,
[
S]SO
-GGnM-MCO after partial acid
hydrolysis. The elution positions of authentic standards are indicated: 1, SO
; 2, SO
-3-GGnM-MCO; 3, SO
-4-GGnM-MCO; 4,
GlcNAc-3-SO
; 5, GalNAc-4-SO
; 6, GlcNAc-6-SO
; 7,
GalNAc-6-SO
.
Figure 4:
Salmon GTH II bears oligosaccharides
terminating with SO-4-GalNAc and
1,4-linked GalNAc. Panel A, Western blot probed with
anti-SO
-4-GalNAc
1,4GlcNAc
1,2Man
(0.1
µg/ml) after separation by SDS-PAGE on a 15% acrylamide gel in the
presence of 2-mercaptoethanol. Lane1, hCG (2
µg); lane2, bLH (2 µg); lane3, intact coho salmon GTH II dimer (4 µg); lane4, GTH II
subunit (4 µg); and lane5, GTH II
subunit (4 µg). Panel B,
autoradiogram of
S-labeled proteins after incubation of
glycoprotein hormones with partially purified bovine submaxillary
GalNAc-4-sulfotransferase and [
S]PAPS and
separation by SDS-PAGE on a 15% acrylamide gel in the presence of
2-mercaptoethanol. Lane1, no exogenous substrate
added; lane2, asialo-hCG (2 µg); lane3, GalNAc-hCG (2 µg); lane4,
intact coho salmon GTH II dimer (4 µg); lane5,
GTH II
subunit (4 µg); and lane6, GTH
II
subunit (4 µg).
The and
subunits of GTH II
were reactive with the lectin W. floribunda (not shown),
indicating that terminal
1,4-linked GalNAc is also present.
Digestion with recombinant human GalNAc-4-sulfatase abolished staining
with anti-S4GGnM while increasing staining with W. floribunda (not shown), consistent with the selective release of sulfate from
terminal GalNAc.
The GalNAc-4-sulfotransferase is highly specific
for the terminal sequence GalNAc1,4GlcNAc
, and incorporation
of [
S]SO
into oligosaccharides by
partially purified GalNAc-4-sulfotransferase can be used to identify
glycoproteins bearing oligosaccharides that terminate with this
structure. GalNAc-4-sulfotransferase purified from bovine submaxillary
glands was used for this purpose (Fig. 4, panelB). Sulfate was not incorporated into hCG bearing
Asn-linked oligosaccharides with terminal
1,4-linked Gal (lane2), whereas sulfate was transferred to hCG bearing
Asn-linked oligosaccharides with terminal
1,4-linked GalNAc (lane3), demonstrating the specificity of the
sulfotransferase preparation. [
S]SO
was transferred to dimeric GTH II (lane4) as
well as to the separated
(lane5) and
(lane6) subunits of GTH II.
The results obtained
with the monoclonal antibody, W. floribunda, digestion with
GalNAc-4-sulfatase, and transfer of sulfate with
GalNAc-4-sulfotransferase all indicate that some fraction of the
Asn-linked oligosaccharides on the and
subunits of GTH II
bear terminal
1,4-linked GalNAc-4-SO
and/or
1,4-linked GalNAc. These studies did not indicate what proportion
of these oligosaccharides have this modification or their underlying
structure.
Figure 5:
Anion exchange HPLC profile of Asn-linked
oligosaccharides from salmon GTH II subunit. Asn-linked
oligosaccharides from salmon GTH II
subunit were released by
treatment with peptide:N-glycosidase F and radiolabeled at
their reducing termini with [
H]borohydride as
described under ``Materials and Methods.''
H-Labeled oligosaccharides were subjected to anion exchange
HPLC on an AX-5 column. Shown are
H-labeled oligosaccharide
elution positions corresponding to authentic standards bearing no
charged residues (N0), one (N1), or two (N2)
sialic acid residues; one (S1), two (S2), or three (S3) sulfate residues; one sulfate and one sialic acid residue (SN), one sulfate and two sialic acid residues (S1N2), or two sulfates and one sialic acid (S2N1).
Homologues of the glycoprotein hormones have been described
in representatives of each class of vertebrate(17) . The
glycoprotein hormones are dimeric proteins consisting in each case of a
common subunit and a hormone-specific
subunit. Fish are the
only known example in which two forms of
subunit differing in
amino acid sequence are present(17) . Recent crystallographic
studies have revealed that both the
and
subunits of the
glycoprotein hormones have cysteine knot motifs, placing them in a
family of proteins that includes nerve growth factor, transforming
growth factor-
, and platelet-de-rived growth
factor(19, 20) . A number of structural features have
been highly conserved among the glycoprotein hormone homologues
including 1) the cysteines that form five and six disulfide bonds in
and
subunits, respectively, 2) the location and number of
Asn-glycosylation sites, and 3) individual amino acids that are thought
to be involved in dimer formation, receptor binding, and receptor
activation.
The current studies demonstrate that yet another
structural feature of the glycoprotein hormones is highly conserved
through evolution from fish to mammals: the presence of Asn-linked
oligosaccharides terminating with the sequence
SO-4-GalNAc
1,4GlcNAc
1,2Man
. We have
determined that GalNAc-transferase and GalNAc-4-sulfotransferase
activities with the same specificities and properties as the mammalian
enzymes are expressed in representatives of each class of vertebrate.
In addition, we have shown that salmon GTH II bears Asn-linked
oligosaccharides with this terminal sequence. Taken together with the
fact that the amino acids that we have shown are critical for
recognition of the
subunit by the glycoprotein
hormone:GalNAc-transferase are conserved in the
subunits of each
class of vertebrate, it is highly likely that individual members of the
glycoprotein hormone family in each class of vertebrate bear Asn-linked
oligosaccharides terminating with
SO
-4-GalNAc
1,4GlcNAc
1,2Man
.
The central
region of the subunit, a stretch of 30 amino acids between two
sets of double cysteines (see Fig. 1) and the carboxyl-terminal
region of the
subunit are highly conserved through evolution from
fish to mammals(17) . Various residues within the central
region have been shown to be important for interaction with and
activation of glycoprotein hormone receptors such as the LH receptor (16) and for dimer formation(14, 15) . The
remarkable degree of sequence conservation in this region may thus
reflect the requirement to mediate a number of interactions with
different
subunits and receptors. The requirement for recognition
of specific basic residues within this region by the glycoprotein
hormone:GalNAc-transferase adds yet another restriction to the changes
in amino acid sequence, which would be tolerated in this region.
We
have previously shown that the presence of oligosaccharides terminating
with SO-4-GalNAc
1,4GlcNAc
1,2Man
on LH plays
an important role in mammals by reducing the circulatory half-life of
LH in the blood(6) . LH bearing oligosaccharides terminating
with SO
-4-GalNAc
1,4GlcNAc
1,2Man
is bound by
a SO
-4-GalNAc-specific receptor, which is expressed on
hepatic endothelial cells and removed from the circulation (5) . This rapid removal is essential to produce the pulsatile
rise and fall characteristic of circulating LH and is thought to be
critical for maximal receptor activation. The presence of the sulfated
oligosaccharides on glycoprotein hormones from all vertebrate classes
suggests that these structures play a similar and critical biologic
role in all vertebrate classes. Notably, Fontaine et al.(36) observed that the metabolic clearance rate for hCG,
which bears oligosaccharides terminating with sialic acid-Gal, was 20
times slower than for carp GTH. If carp GTH, like salmon GTH, bears
oligosaccharides terminating with
SO
-4-GalNAc
1,4GlcNAc
1,2Man
, this rapid rate
of clearance could reflect the presence of a
SO
-4-GalNAc-specific receptor in the liver of fish similar
to that found in mammals.
The presence of terminal
SO-4-GalNAc
1,4GlcNAc
1,2Man
is characteristic
of vertebrate glycoprotein hormones in species ranging from fish to
mammals. Thus, expression of glycoprotein hormone biologic activity
must at some juncture require the presence of oligosaccharides
terminating with SO
-4-GalNAc
1,4GlcNAc
1,2Man
.
Our previous studies indicate the most likely role of these structures
is to regulate the circulatory half-life of individual glycoprotein
hormones through the GalNAc-4-SO
receptor; however, other
equally critical functions are also possible. In addition, the presence
of these same structures on other glycoproteins such as carbonic
anhydrase synthesized in salivary glands (25) and
proopiomelanocortin (24) raises the possibility that these
structures will serve other roles on other glycoproteins.