From the
The carbohydrate moieties found on glycoproteins have long been recognized as having great potential to bear biologically important information. However, actual examples of systems in which oligosaccharides play defined physiological roles have remained limited. These oligosaccharides with known biologic functions typically have distinctive structural features and are generally confined to specific glycoproteins. Synthesis of structurally unique oligosaccharides on specific glycoproteins at defined times is essential if these structures are to fulfill their biologic purpose. Since cells produce many distinct oligosaccharides as newly synthesized glycoproteins pass through the endoplasmic reticulum and the Golgi, mechanisms are required to assure that the correct structures are added to the numerous glycoproteins being synthesized. Determining how synthesis of the vast array of oligosaccharides produced by each cell is regulated is essential for understanding the biologic importance of these complex structures.
Asn-linked oligosaccharides arise by
processing of a common precursor structure, which is transferred en
bloc from dolichol to the nascent peptide chain in the endoplasmic
reticulum(1) . As a result Asn-linked oligosaccharides have a
common core region and differ primarily in the number and location of
their peripheral branches and terminal modifications. Since all newly
synthesized glycoproteins pass through the same subcellular
compartments and are exposed to the same transferases, structural
differences in oligosaccharides on individual glycoproteins and/or at
individual glycosylation sites must in some fashion reflect the
influence of the protein moiety on one or more glycosyltransferases.
This suggests that key glycosyltransferases recognize features encoded
within the peptide as well as the oligosaccharide of the target
glycoprotein. Among the three glycosyltransferases thus far
demonstrated to display peptide as well as oligosaccharide recognition,
UDP-glucose:glycoprotein glucosyltransferase,
UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, and
UDP-GalNAc:glycoprotein hormone
1,4-N-acetylgalactosaminyltransferase (
1,4-GalNAcT, (
)reviewed in (2) ), one of the most extensively
characterized is the
1,4-GalNAcT, which produces the terminal
sequence GalNAc
1,4GlcNAc
1-R on glycoproteins that contain a
specific peptide recognition determinant in addition to an appropriate
oligosaccharide acceptor. The product of the
1,4-GalNAcT may be
further modified by the addition of sulfate, sialic acid, or fucose,
thus producing a range of unique oligosaccharide structures defined by
the presence of
1,4-linked GalNAc as illustrated in Fig. 1.
Each of these structures has the potential to be recognized by a
specific receptor or binding protein and thus mediate a distinct
biological function. As will become apparent below, the
1,4-GalNAcT is a key component of a well characterized system,
which includes unique oligosaccharide structures, highly specific
glycosyltransferases, and oligosaccharide-specific receptors. This is
therefore an excellent model system for understanding protein-specific
glycosylation.
Figure 1:
Synthesis of oligosaccharides
containing 1,4-linked GalNAc or Gal. The product of reaction 1 is a common synthetic intermediate, which can be further modified
by the addition of
1,4-linked Gal, reaction 2, or
1,4-linked GalNAc, reaction 3. The
1,4-GalT does not
display any peptide specificity and will transfer Gal to any
-linked terminal GlcNAc with the same catalytic efficiency. In
contrast the
1,4-GalNAcT recognizes features encoded in the
peptide (i.e. the
1,4-GalNAcT recognition determinant) as
well as the terminal
-linked GlcNAc. In the presence of the
recognition determinant the catalytic efficiency for GalNAc addition to
the same oligosaccharide is as much as 500-fold greater than for
addition of either Gal or addition of GalNAc in the absence of the
recognition determinant(15, 17, 18) .
1,3-Fucosyltransferase will transfer fucose to GlcNAc in the
presence of either
1,4-linked Gal (reaction 8) or GalNAc
(reaction 5)(51) , and
2,6-sialyltransferase will
transfer sialic acid to either
1,4-linked Gal (reaction 7) or GalNAc (reaction 6)(52) . In contrast,
2,3-sialyltransferase will add sialic acid to
1,4-linked Gal
(reaction 9) but not GalNAc (37) , while
GalNAc-4-sulfotransferase will add sulfate to the 4-hydroxyl of
1,4-linked GalNAc (reaction 4) but not to
1,4-linked
Gal(53, 54) . However, all of the oligosaccharides
containing
1,4-linked GalNAc are distinct from those containing
Gal and are largely confined to glycoproteins containing the
1,4-GalNAcT recognition determinant, which are synthesized by
cells expressing the
1,4-GalNAcT.
, GalNAc;
,
GlcNAc;
, Man;
, Gal;
,
2,6-Sia;
,
2,3-Sia;
, Fuc; shaded rectangle, recognition
determinant.
A Peptide Recognition Determinant, Which Directs Synthesis of
Unique Oligosaccharides
Lutropin (LH), a glycoprotein hormone produced in the
anterior lobe of the pituitary, was the first glycoprotein shown to
have Asn-linked oligosaccharides with one or two branches terminating
with the sequence
SO-4GalNAc
1,4GlcNAc
1,2Man
(3) .
Oligosaccharides on LH, thyrotropin (TSH), and the free glycoprotein
hormone
subunit from a number of different animal species were
subsequently shown to terminate with the same
sequence(4, 5, 6, 7) . A growing
number of glycoproteins, which are unrelated to the glycoprotein
hormones but which bear oligosaccharides terminating with the sequence
SO
-4GalNAc
1,4GlcNAc
1,2Man
(6, 7, 8, 9, 10, 37, 39, 42) ,
are synthesized in the pituitary as well as other tissues.
Glycoproteins that bear oligosaccharides terminating with
GalNAc
1,4GlcNAc
1-R(6, 7, 8, 9, 37, 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50) ,
Sia
2,6GalNAc
1,4GlcNAc
1-R(7, 40, 42, 47, 48, 49, 50) , (
)and GalNAc
1,4(Fuc
1,3)GlcNAc
1-R (40, 42, 44, 45, 46) have
also been described from a number of sources. Furthermore,
pro-opiomelanocortin, which contains Asn-linked oligosaccharides
terminating with SO
-4GalNAc
1,
4GlcNAc
1-R(8, 9) , is the first instance of a
glycoprotein bearing Ser/Thr-linked oligosaccharides terminating with
this same sequence(10) .
Even though a number of
glycoproteins bearing oligosaccharides with the sequence
GalNAc1,4GlcNAc
1-R have been described, this carbohydrate
structural motif is not common in vertebrates. The vast majority of
other glycoproteins produced by the tissues or cells, which synthesize
glycoproteins bearing
1,4-linked GalNAc, do not contain
1,4-linked GalNAc but instead contain
1,4-linked Gal,
indicating that the addition of
1,4-linked GalNAc is generally a
highly protein-specific process. A
1,4-GalNAcT, which can account
for the specific modification of Asn-linked oligosaccharides, is
present in a limited number of tissues and cell lines, including those
which are known to produce oligosaccharides with the
1,4-linked
GalNAc motif (11) . In contrast,
1,4-galactosyltransferase
(
1,4-GalT), which transfers Gal in
1,4-linkage to virtually
any terminal GlcNAc, is expressed at relatively high levels in
virtually all vertebrate tissues and cell
lines(12, 13) . Since the
1,4-GalNAcT and
1,4-GalT compete for the same oligosaccharide acceptors (Fig. 1), preferential addition of GalNAc to an oligosaccharide
must reflect recognition of the protein bearing the oligosaccharide.
An in vitro model system was established for examining the
protein specificity of the 1,4-GalNAcT using human chorionic
gonadotropin (hCG). hCG, which is closely related to LH but is
synthesized in the placenta(14) , binds to the same receptor as
LH. hCG contains the peptide recognition determinant utilized by the
1,4-GalNAcT but bears oligosaccharides that terminate with
Sia-
2,3Gal because neither the
1,4-GalNAcT nor the
GalNAc-4-sulfotransferase (reactions 3 and 4,
respectively, in Fig. 1) are expressed in human
placenta(15) . We established the existence of the peptide
recognition determinant by comparing glycoproteins and glycopeptides
bearing the identical oligosaccharide,
GlcNAc
Man
GlcNAc
Asn (the product of
reaction 1 in Fig. 1), as acceptors for the addition of
either Gal or GalNAc by transferases present in pituitary extracts. Gal
is added to each of the glycoproteins tested with the same apparent K
of 1-2 mM and the same
catalytic efficiency (V
/K
). In
contrast, the apparent K
for addition of
GalNAc is markedly influenced by the protein moiety. For example,
GalNAc is transferred to oligosaccharides on glycoproteins such as
transferrin with an apparent K
of
1-2 mM, whereas transfer to the same oligosaccharides on
hCG occurs with an apparent K
of
5-10 µM. Thus, the catalytic efficiency for addition
of GalNAc to oligosaccharides on certain glycoproteins is
100-500-fold greater than for transfer to the same
oligosaccharides on other glycoproteins, indicating the presence of a
specific
1,4-GalNAcT recognition determinant on a particular
subset of glycoproteins including
hCG(15, 16, 17) . We have located two
recognition determinants on hCG, one on the
subunit and one on
the
subunit(17) , and have established a number of
features that are critical for recognition by the
1,4-GalNAcT(18) .
The recently solved crystal structure
of hCG (19, 20) ()including four Asn-linked
oligosaccharides, illustrated in Fig. 2, has greatly enhanced
our understanding of the
1,4-GalNAcT recognition determinant. It
is immediately apparent that each of the four Asn-linked
oligosaccharides on hCG is nearly as large as the peptide portion of
either the
or the
subunit, and only the innermost 2 or 3
sugars are in direct contact with the peptide. Since the
oligosaccharides are highly mobile rather than being fixed in space,
their molecular dimensions are even greater relative to the peptide
when considered in real time. It is also apparent that the peripheral
sugars, Sia-Gal or SO
-GalNAc, are distant from the peptide,
mobile, and highly exposed, as has been recently been established for
at least one of the
subunit oligosaccharides by high resolution
multinuclear NMR.
The structures of LH, TSH, and
follitropin (FSH), which have the same
subunit and highly
homologous
subunits, are likely to be similar to that of hCG. The
regions of the
subunit (Fig. 2, A and B)
and the
subunit (Fig. 2B only), which include
residues essential for recognition, have been highlighted in yellow. Residues in the
subunit critical for recognition
by the
1,4-GalNAcT include the basic amino acids in the sequence
Pro
-Leu
-Arg
-Ser
-Lys
-Lys
(18) . These residues are present within two turns of an
-helix, forming a cluster of basic residues that projects out from
the
subunit. The region of the
subunit, which we have
proposed to be critical for recognition, is found at the N terminus and
consists of the sequence
Pro
-Leu
-Arg
-Pro
-Arg
-Cys
-Arg
(17) . We have shown that the Arg
is
essential for recognition and are currently examining the contribution
of other residues within this sequence.
Figure 2:
Three-dimensional structure of human
chorionic gonadotropin showing the relationship of the Asn-linked
oligosaccharides to the 1,4-GalNAcT recognition determinants.
Orthogonal views of hCG and its Asn-linked oligosaccharides are shown (19, 20) .
The
subunit and its
oligosaccharides are colored blue and green,
respectively. The
subunit and its oligosaccharides are colored gray and magenta, respectively. The residues
constituting the recognition determinant on the
subunit,
Pro
-Leu
-Arg
-Ser
-Lys
-Lys
,
are found within two turns of an
helix and are shown in yellow in both views (panels A and B) while
the residues proposed to constitute the recognition determinant present
on the
subunit,
Pro
-Leu
-Arg
-Pro
-Arg
-Cys
-Arg
,
are highlighted in yellow only in the view shown in panel
B. Residues 94-115 (21, 38) of the
subunit are colored red and have been proposed to dictate
receptor specificity for the different glycoprotein hormones.
Differences in this region may influence
1,4-GalNAcT interaction
with the peptide recognition determinant in FSH as compared with LH/CG
and TSH. Because the oligosaccharides at Asn
of the
subunit project from the same surface as the recognition determinant on
the
subunit while those at Asn
and Asn
of the
subunit project from the opposite surface, the
second recognition determinant on the
subunit may be required to
mediate efficient GalNAc addition to the oligosaccharides on both
subunits of LH/CG.
Recognition of the peptide
determinants in hCG by the 1,4-GalNAcT does not require the
maintenance of tertiary structure since the separated native or reduced
and alkylated
and
subunits of CG are recognized by the
1,4-GalNAcT(16) . GalNAc is transferred to the
oligosaccharides on glycopeptide fragments containing as few as 23
amino acids with the same apparent K
as
the native glycoprotein hormone subunits from which they were
derived(17) . Each peptide recognition determinant is capable
of directing transfer of GalNAc to multiple oligosaccharides since both
oligosaccharides on the separated native
and
subunits are
modified with GalNAc in vitro. The
1,4-GalNAcT most
likely simultaneously binds to the peptide recognition determinant and
the oligosaccharide being modified, thereby reducing the apparent K
. Even though the recognition
determinants on both the
and
subunits of hCG are in close
proximity within the linear protein sequence to a glycosylation site,
this is not the case for the more distal glycosylation sites on either
subunit. It is the relationship of the recognition determinant to the
oligosaccharide acceptor in three-dimensional space that is critical
for determining which oligosaccharides will or will not be modified
with GalNAc in the native protein. Although the recognition
determinants on the isolated
and
subunits are both
functional, we have not yet established if both contribute to
recognition by the
1,4-GalNAcT in the dimeric form of the hormone.
The recognition determinant on the
subunit (see Fig. 2) is
in close proximity to the oligosaccharide at Asn
of the
subunit and to both Asn-linked oligosaccharides on the
subunit. However, the oligosaccharides on the
subunit are found
on the opposite surface of CG and may not be fully accessible to the
1,4-GalNAcT when it is bound to the recognition determinant on
subunit. As a result, it is the recognition determinant near the
N terminus of the
subunit (Fig. 2B) that may
mediate addition of GalNAc to one or both
subunit
oligosaccharides. The oligosaccharide at Asn
of the
subunit is less extensively substituted with GalNAc than the one at
Asn
(22) . This oligosaccharide is more distant
from either recognition determinant (Fig. 2), suggesting the
GlcNAc termini of this oligosaccharide are not in sufficiently close
proximity to the recognition determinant in three-dimensional space to
be efficiently modified.
The evidence is compelling that the peptide
recognition determinants on the glycoprotein hormones reduce the
apparent K for GalNAc addition to the
oligosaccharide acceptor by enhancing binding of the
1,4-GalNAcT.
It is likely that peptide recognition determinants will be found for
other glycosyltransferases that will enhance transfer of sugars to
oligosaccharide acceptors on specific glycoproteins and/or at
individual glycosylation sites. Since it is possible to alter the
catalytic efficiency of GalNAc transfer in vitro by altering
specific residues within the region recognized by the
1,4-GalNAcT,
it will soon be possible to determine the impact of such changes in
vivo.
The Unique Oligosaccharide Structural Motif and the
Peptide Recognition Determinant Are Conserved among Glycoprotein
Hormones from All Classes of Vertebrates
All classes of vertebrates are known to produce glycoprotein
hormones closely related to those found in mammals(23) .
Furthermore, it is the region of the subunit that contains the
recognition determinant for the
1,4-GalNAcT, which is the most
highly conserved among vertebrates. As a result residues critical for
recognition by the
1,4-GalNAcT are found in
subunits from
all classes of vertebrates(24) . This same region of the
subunit is essential for activation of adenylate cyclase activity
following binding to the respective G-protein-coupled hormone
receptor(25) . We have found that the
1,4-GalNAcT and the
GalNAc-4-sulfotransferase, which together account for the synthesis of
SO
-4GalNAc
1,4GlcNAc
1-R termini, are expressed in
the pituitaries of vertebrates ranging from fish to humans.
Furthermore, the oligosaccharides on glycoprotein hormones from all
classes of vertebrates terminate with
GalNAc-4-SO
(24) . This is the first instance in
which a specific oligosaccharide structural motif has been shown to be
maintained on a family of glycoproteins from different classes of
vertebrates. Thus, the unique carbohydrate structural motif, like the
sequence and structure of the glycoprotein hormone peptides, has been
conserved during the evolution of vertebrate species.
LH is a major
product of the gonadotroph and is one of only a few proteins produced
by gonadotrophs or other cells in the pituitary that terminate with
GalNAc-4-SO. The expression of
1,4-GalNAcT and
GalNAc-4-sulfotransferase in the gonadotroph is modulated in parallel
to LH levels in response to circulating levels of
estrogen(26) . As estrogen levels fall, the expression of
1,4-GalNAcT and GalNAc-4-sulfotransferase increases in concert
with increased synthesis of LH. The coordinate regulation of LH
synthesis and
1,4-GalNAcT expression assures that the
oligosaccharides on LH, but not other glycoproteins produced in the
gonadotroph, always terminate with GalNAc-4-SO
. In
contrast,
1,4-GalNAcT activity in other tissues including the
submaxillary gland and kidney is not responsive to estrogen levels.
Conservation of these sulfated oligosaccharide structures during
evolution in conjunction with hormonal regulation of
1,4-GalNAcT
expression in gonadotrophs but not other cells further supports the
view that these sulfated oligosaccharides play a central role in the
biology of the glycoprotein hormones.
The Biological Significance of GalNAc-4-SO for the Glycoprotein Hormones
Consistent with the high degree of regulation seen for the
synthesis of sulfated oligosaccharides on LH, these oligosaccharides
have been found to mediate a crucial biological function. We have shown
that the sulfated oligosaccharides on LH regulate its circulatory
half-life following release into the blood(27) . These
oligosaccharides are recognized by a receptor in hepatic endothelial
cells and Kupffer cells, which is specific for the terminal sequence
SO-4GalNAc
1,4GlcNAc
1,2Man
1-R(28) .
Upon binding the GalNAc-4-SO
-receptor the hormone is
rapidly internalized and transported to lysosomes where it is degraded.
The receptor is plentiful with 500,000 binding sites detectable at the
surface of each endothelial cell and has an apparent K
of 1.63
10
M for LH. The rapid and specific clearance of LH from
the circulation on the basis of its terminal glycosylation was
initially unexpected since rapid clearance of the hormone reduces its
potency to induce ovulation following a single intravenous
injection(27) . This seeming contradiction is resolved upon
consideration of the properties of the LH/CG receptor in the ovary and
the hormonal cycle. The LH/CG receptor is a member of the
seven-transmembrane domain G-protein-coupled receptor family. Upon
hormone binding the receptor is activated and cAMP is produced;
however, at the same time, hormone binding causes rapid inactivation
and internalization of the receptor(29) . As a result,
continuous stimulation would result in the entire population of LH/CG
receptors becoming refractory to further activation. Thus, during the
24-48-h pre-ovulatory surge in circulating LH levels, the LH/CG
receptor would not be maximally activated due to down-regulation.
However, circulating LH levels rise and fall in a pulsatile manner.
During the preovulatory surge it is the frequency and amplitude of
these pulses that increases(30, 31) . This pulsatile
rise and fall reflects both the episodic release of LH from granules
and its rapid clearance from the blood. Other hormones such as FSH are
also released episodically but have a long half-life and do not display
this pulsatile rise and fall in blood levels. We have therefore
proposed that this pulsatile rise and fall in LH levels is essential to
obtain maximal stimulation of the LH/CG receptor. Key to the pulsatile
appearance of LH in the circulation is its rapid clearance from the
bloodstream mediated by its oligosaccharide component.
The crucial
role mediated by these oligosaccharides is highlighted by the fact that
a number of animal species, including humans and horses, synthesize a
glycoprotein hormone, CG, in their placenta during the early stages of
pregnancy. Equine CG and LH arise for the same gene and have identical
peptide sequences(32, 33) . The Asn-linked
oligosaccharides on equine CG terminate with
Sia-2,3Gal
1,4GlcNAc
1-R (34, 35) while
those on equine LH terminate with
SO
-4GalNAc
1,4GlcNAc
1-R(34, 36) .
Consistent with the presence of sialic acid-bearing oligosaccharides,
CG has a long circulatory half-life. Thus equine CG and LH are
different glycoforms of the same protein, which we have shown differ in
their rate and site of clearance from the circulation(34) .
Furthermore, LH is stored in granules and released episodically into
the circulation in response to a releasing factor while CG, which is
not stored in granules, is released continuously from placental
trophoblasts. Thus, the major difference between LH, the hormone of the
ovulatory cycle, and CG, the hormone of pregnancy, is the difference in
their circulatory half-lives, which results in episodic and continual
stimulation of the LH/CG receptor, respectively.
Other Roles for Oligosaccharides Containing
1,4-Linked GalNAc
The sulfated oligosaccharides on LH illustrate how unique oligosaccharide structures can play crucial physiologic roles. We have defined and characterized many of the components of the physiological system involving these oligosaccharides, including 1) their precise structures; 2) the transferases responsible for the synthesis of these structures; and 3) a receptor that specifically recognizes these sulfated oligosaccharides and mediates a specific biological function. Our results, furthermore, demonstrate that this system involves a high degree of regulation and specificity.
Not surprisingly many of these
same elements are used at different times and under different
circumstances for other biologic purposes. As was noted above, the
number of glycoproteins known to contain oligosaccharides terminating
with the sequence GalNAc1,4GlcNAc
1-R has increased. The
addition of SO
,
1,3-linked fucose, or
2,6-linked
sialic acid (Fig. 1, products of reactions 4, 5, and 6, respectively) has the potential to produce
three additional distinct and unique oligosaccharide structures, each
of which can potentially be recognized by a specific receptor similar
to the hepatic GalNAc-4-SO
specific receptor. Different
glycoforms of the same protein may also arise at different times during
development
or in response to hormonal
status(26, 41) . Thus, these glycoforms may fulfill a
variety of biological purposes.
The demonstration of
protein-specific glycosylation by the 1,4-GalNAcT, which acts on
the glycoprotein hormones, is particularly exciting because it provides
a model for understanding how these and other distinct oligosaccharide
structures are synthesized in a protein- and even site-specific manner.
It also exemplifies a mechanism for the addition of unique structures
at precise times to specific glycoproteins. This is essential for
oligosaccharides having biological roles, which involve encoding of
specific information. Oligosaccharides are ideally suited for such a
purpose because they are highly exposed and accessible at the surface
of the proteins which bear them and because of their enormous
structural diversity. It is likely that we have only gained a glimpse
of the potential functions of carbohydrates thus far and that many new
discoveries await those willing to embark on the study of this form of
post-translational modification.