(Received for publication, June 7, 1995; and in revised form, September 6, 1995)
From the
We used a novel approach to study the role of the Asn-linked
oligosaccharides for human thyrotropin (hTSH) activity. Mutagenesis of
Asn(N) within individual glycosylation recognition sequences to Gln (Q)
was combined with expression of wild type and mutant hTSH in cell lines
with different glycosylation patterns. The in vitro activity
of hTSH lacking the Asn oligosaccharide
(
Q52/TSH
) expressed in CHO-K1 cells (sialylated
oligosaccharides) was increased 6-fold compared with wild type, whereas
the activities of
Q78/TSH
and
/TSH
Q23 were
increased 2-3-fold. Deletion of the Asn
oligosaccharide also increased the thyrotropic activity of human
chorionic gonadotropin, in contrast to previous findings at its native
receptor. The in vitro activity of wild type hTSH expressed in
CHO-LEC2 cells (sialic acid-deficient oligosaccharides), CHO-LEC1 cells
(Man
GlcNAc
intermediates), and 293 cells
(sulfated oligosaccharides) was 5-8-fold higher than of wild type
from CHO-K1 cells. In contrast to CHO-K1 cells, there was no difference
in the activity between wild type and selectively deglycosylated
mutants expressed in these cell lines. Thus, in hTSH, the
oligosaccharide at Asn
and, specifically, its
terminal sialic acid residues attenuate in vitro activity, in
contrast to the previously reported stimulatory role of this chain for
human chorionic gonadotropin and human follitropin activity. The
increased thyrotropic activity of
Q52/CG
suggests that
receptor-related mechanisms may be responsible for these differences
among the glycoprotein hormones. Despite their increased in vitro activity,
Q52/TSH
, and
Q78/TSH
from CHO-K1
cells had a faster serum disappearance rate and decreased effect on
T
production in mice. These findings highlight the
importance of individual oligosaccharides in maintaining circulatory
half-life and hence in vivo activity of hTSH.
Thyrotropin (thyroid-stimulating hormone, TSH) ()is a
member of the glycoprotein hormone family, which also includes
chorionic gonadotropin (CG), lutropin (luteinizing hormone, LH) and
follitropin (follicle-stimulating hormone, FSH). These hormones are
structurally related heterodimers consisting of a common
subunit
and a distinct
subunit that confers the biological specificity
for each hormone (1) . The common
subunit bears two N-linked oligosaccharides, and the
subunit bears either
one (in TSH and LH), or two (in CG and FSH)(2, 3) .
The oligosaccharides, which represent 18-35% of total
weight(2, 3, 4) , have been shown to play a
role in the proper folding, assembly, secretion, metabolic clearance,
and biological activity of these hormones (for recent review, see Refs.
3 and 4). In the case of hCG and hFSH, enzymatic or chemical
deglycosylation led to a decrease or loss of cAMP production and
steroidogenesis, while high affinity binding was
maintained(3, 4, 5) . Sairam et al.(6) reported that the carbohydrates of the common
subunit rather than those of the
subunits were important for the
activity of these hormones. Using site-directed mutagenesis, Matzuk et al.(7) identified the oligosaccharide at position
52 of the
subunit to be critical for the in vitro bioactivity of hCG. Subsequently, this oligosaccharide was shown
to be similarly important for the stimulatory activity of
hFSH(8, 9) .
In contrast to hCG and hFSH, the roles
of the individual oligosaccharides for the activity of TSH or the more
closely related LH are not known. Due to the limited availability of
purified pituitary human TSH (phTSH), studies on the role of the
carbohydrates for TSH have mostly used pituitary bovine TSH. Similar to
the findings for the gonadotropins, these studies have shown by
chemical or enzymatic deglycosylation that the oligosaccharides, and
particularly those of the subunit, were required for full in
vitro activity of pituitary bovine TSH(10, 11) .
The few studies on phTSH have yielded conflicting results and the role
of carbohydrates for hTSH action remained
controversial(12, 13) . The recent availability of
recombinant human TSH (rhTSH) (14) allowed further
investigation of the role of the carbohydrates in the action of hTSH.
rhTSH expressed in CHO-K1 cells contains N-linked
oligosaccharides which terminate with
Sia
2-3Gal
1-4GlcNAc
1-2Man
(14, 15, 16) .
By comparison, phTSH, which physiologically occurs in a variety of
glycosylation isoforms, terminates both in
SO
-4GalNAc
1-4GlcNAc
1-2Man
and
Sia
2-3Gal
1-4GlcNAc
1-2Man
as
described by Green and Baenziger(2) . Interestingly, enzymatic
removal of terminal sialic acid residues of rhTSH increased the in
vitro bioactivity of the hormone (17) similar to findings
for recombinant bovine LH(15) , but unlike for hCG, in which
sequential deglycosylation resulted in a stepwise reduction of
activity(18) . These findings suggested that the role of the
oligosaccharides may be different for hTSH compared with hCG and hFSH.
In the present study we have used site-directed mutagenesis to study the role of individual oligosaccharides of hTSH by selectively inhibiting their cotranslational attachment. We combined site-directed mutagenesis with the expression of the selectively deglycosylated hTSH mutants in different cell lines producing hormones with distinct carbohydrate patterns(19, 20, 21) , using our recently developed and optimized transient transfection protocol(22) . This novel approach allowed us to identify unique roles for individual oligosaccharides and their terminal sialic acid residues for the in vitro as well as in vivo activity of hTSH.
hTSH bears three N-linked oligosaccharide chains at
positions Asn and Asn
of the
subunit
and at Asn
of the
subunit(1, 2, 3) . To prevent the
cotranslational attachment of individual oligosaccharides to the hTSH
molecule, we disrupted the respective glycosylation recognition
sequences NX(T/S), widely considered to be an absolute
requirement for glycosylation to occur. In the
subunit
(individually and in a composite mutation) and in the TSH
subunit
we mutated Asn at each position to Gln, thus creating genes coding for
the following mutant subunits:
Q52,
Q78,
Q52.Q78, and
TSH
Q23. The conservative mutation of Asn to Gln is unlikely to
affect protein conformation and has not been reported to influence the
tertiary structure of the related glycoproteins hCG and hFSH, when used
to generate mutants lacking individual oligosaccharide attachment
sites(7, 8, 9) . Thus, the observed effects
on the hTSH expression, binding, and activity should be primarily due
to changes in the carbohydrate chains.
The shift in the elution
profiles of the hTSH mutants observed using gel filtration (Fig. 1A) and the differences in gel migration using Western
blotting (Fig. 1B) were consistent with the absence of
individual oligosaccharides. After N-glycanase digestion, wild
type hTSH, Q52/TSH
, and
Q78/TSH
had similar
profiles (Fig. 1, A and B), further
demonstrating that the differences prior to N-glycanase
treatment were due to differences in glycosylation.
Figure 1:
A,
Superdex 75 elution profiles of the hTSH mutants before (open
symbols) and after (filled symbols, +) N-glycanase digestion. Conditioned media from transfected
CHO-K1 cells were chromatographed using hCG as an internal standard
(the peak of hCG immunoreactivity corresponds to fraction 0 in all
cases). Fraction size was 0.175 ml. hTSH immunoreactivity was monitored
by immunoassay (Nichols Institute). B, Western blotting
analysis of ConA-Sepharose-fractionated hTSH deglycosylation
mutants before(-) and after (+) N-glycanase
digestion (see ``Experimental Procedures''), using an
antibody against the hTSH
subunit. rhTSH (Genzyme Corp.) served
as the internal standard. In the case of N-glycanase-treated
wild type and
Q52/TSH
, presumably due to incomplete enzymatic
digestion, two bands were visible, representing hTSH
subunit
lacking one or two oligosaccharides,
respectively.
Figure 2: cAMP induction in CHO cells expressing the rhTSH receptor (JP09) by the hTSH mutants produced in CHO-K1 cells. Increasing concentrations of wild type or mutant hTSH were incubated with JP09 cells, and the cAMP concentration in the resulting supernatants was assayed by radioimmunoassay. The amount of cAMP released from the cells in the presence of concentrated medium from mock-transfected cells was not different from base-line levels (buffer only). A representative experiment, repeated at least twice, is shown. Values from triplicate determinations are depicted as mean ± S.E.
In
FRTL-5 cells expressing endogenous rat TSH receptor(27) , we
observed, relative to wild type (EC = 12.6 ±
2.1 ng/ml), a similar 6-fold decrease in the EC
of
Q52/TSH
(EC
= 2.0 ± 0.7 ng/ml, p < 0.001) (Fig. 3).
Figure 3:
cAMP
induction in FRTL-5 cells. Wild type or Q52/TSH
, expressed in
CHO-K1 cells, was incubated with FRTL-5 cells expressing the endogenous
rat TSH receptor. A representative experiment, repeated at least twice,
is shown. Values from triplicate determinations are depicted as mean
± S.E.
We next expressed mutants
Q52/TSH
,
Q78/TSH
, and
/TSH
Q23 in 293
cells. In contrast to CHO-K1 cells, 293 cells express N-acetylgalactosaminyl-transferase and
GalNAc
1,4GlcNAc
1,2Man
4-sulfotransferase and produce
oligosaccharides terminating in >70% in sulfated N-linked
carbohydrate moieties(19) . Since we previously found that 293
cells produce small amounts of free
subunit(29) , we
initially cotransfected the hTSH
minigene alone to assess whether
significant amounts of wild type hTSH would be produced. This, however,
was not the case, indicating that significant contamination of the
mutants with wild type hTSH did not occur. Wild type hTSH, expressed in
293 cells had increased cAMP inducing activity, evidenced by a 5.6-fold
left shift in the EC
compared with wild type expressed in
CHO-K1 cells (Fig. 4, Table 2) (p < 0.001).
However, in relation to the increased wild type activity, there was no
difference in activity between wild type and the selectively
deglycosylated hTSH mutants (Fig. 4, Table 2).
Figure 4: cAMP induction in JP09 cells by the hTSH mutants produced in 293 cells. Experiments were carried out as described in the legend to Fig. 2and under ``Experimental Procedures.'' For comparison, cAMP induction of wild type expressed in CHO-K1 cells assayed in the same experiment is also shown. A representative experiment, repeated at least twice, is shown. Values from triplicate determinations are depicted as mean ± S.E.
To
further understand which carbohydrate components may be associated with
the increase of bioactivity upon expressing the hTSH mutants in CHO-K1
cells, we also transiently transfected CHO-LEC2 and CHO-LEC1 cells.
These are CHO glycosylation mutant cell lines selected from the wild
type CHO-K1 cell line for resistance to toxic plant
lectins(23) . CHO-LEC2 cells have a defect in the CMP-sialic
acid translocation into the Golgi resulting in the synthesis of
glycoproteins with a > 90% reduction in sialic acid(20) ,
whereas CHO-LEC1 cells lack N-acetylgalactosaminyl-transferase
I and hence give rise to oligosaccharides bearing
ManGlcNAc
intermediates (21) . The
reduction of sialic acid on the CHO-LEC2-expressed hTSH was evidenced
by a decrease in L. flavus agglutinin binding, a lectin from
the slug L. flavus that recognizes terminal sialic acids (30) (53 ± 8.3% compared with 94.5 ± 2.7% in the
case of CHO-K1-expressed hTSH) as well as by an accelerated elution
profile from L. flavus agglutinin minicolumns. Further,
neuraminidase digestion decreased L. flavus agglutinin binding
of hTSH from CHO-K1 cells to levels similar to nondigested
CHO-LEC2-expressed hTSH. As expected, neuraminidase digestion did not
further decrease L. flavus agglutinin binding of hTSH from
CHO-LEC2 cells (data not shown).
Wild type hTSH expressed in both
CHO-LEC2 and CHO-LEC1 was 7-8-fold more active than wild type
from CHO-K1 cells (p < 0.001) (Fig. 5A, Table 2). Compared with this increased wild type activity, there
was no difference in the EC of cAMP induction for the
various selectively deglycosylated mutants expressed in these cells.
Figure 5:
A, cAMP
induction in JP09 cells by the hTSH mutants produced in CHO-LEC2 cells.
Experiments were carried out as described in the legend to Fig. 2. For comparison, cAMP induction of the wild type
expressed in CHO-K1 cells from the same experiment is depicted. A
representative experiment, repeated at least twice, is shown. Similar
results were obtained when wild type and Q52/TSH
were
expressed in CHO-LEC1 cells (Table 2). B, the effects of
neuraminidase digestion on hTSH mutants produced in CHO-K1 cells (filled symbols, +). For comparison, nondigested wild
type and mutants from the same transfection are shown, as well as wild
type hTSH from CHO-LEC2 cells (open symbols). As expected,
neuraminidase digestion did not further increase activity of hTSH from
CHO-LEC2 cells (data not shown). Values from triplicate determinations
are depicted as mean ± S.E.
Direct evidence for the inhibitory roles of terminal sialic acids
was obtained by testing for cAMP stimulation of wild type and mutant
hTSH after neuraminidase digestion (Fig. 5B). As
expected, neuraminidase digestion of wild type hTSH from CHO-K1 cells
increased its in vitro activity to an EC similar
to CHO-LEC2-expressed hTSH. Furthermore, digestion of
Q78/TSH
, which retains the sialylated Asn
oligosaccharide also increased in vitro activity to
similar levels. Conversely, the already maximally increased in
vitro activity of
Q52/TSH
, in which the Asn
sialic acid moieties are absent, was not further increased. As
expected, neuraminidase digestion did not further increase the activity
of hTSH from CHO-LEC2 cells (data not shown).
Figure 6:
Induction of cell growth by
Q52/TSH
from CHO-K1 cells. Increasing concentrations of wild
type or mutant hTSH were incubated with FRTL-5 cells, which were
previously grown in the absence of TSH. After 48 h,
[
H]thymidine was added, and after an additional
24 h, radioactivity incorporated into the DNA was measured. Values are
shown as the mean of triplicate observations ± S.E. The
[
H]thymidine incorporation in the presence of
concentrated medium from mock-transfected cells was not different from
base-line levels.
Figure 7:
Inhibition of I-bTSH binding
by wild type and
Q52/TSH
expressed in CHO-K1, CHO-LEC2, and
293 cells. Increasing doses of wild type or mutant hTSH were incubated
with porcine membranes in the presence of a constant amount of
I-bTSH.
I-bTSH bound to membranes was
precipitated and quantitated in a
counter, and radioactivity
precipitated in the presence of concentrated medium from mock
transfections was defined as 100%. Values are shown as mean ±
S.E. of triplicate determinations. Experiments were repeated two times.
Binding of wild type and
Q52/TSH
expressed in CHO-LEC1 was
not different from that expressed in CHO-LEC2
cells.
Figure 8:
Inhibition of I-bTSH binding
by the hTSH mutants expressed in CHO-K1 cells. The binding assay was
performed as described in the legend to Fig. 7. Values of a
representative experiment, performed at least three times, are shown as
mean ± S.E. of triplicate
determinations.
Figure 9:
Thyrotropic activity of CHO-K1-expressed,
site-specifically -deglycosylated hCG in JPO9 cells. For
experimental details, see the legend to Fig. 2and
``Experimental Procedures.'' Values of a representative
experiment, performed at least three times, are depicted as mean
± S.E. of triplicate determinations.
Figure 10:
In vivo activity of
site-specifically hTSH mutants from CHO-K1 cells in
T-suppressed mice. T
values of nonsuppressed
mice ranged from 6 to 8 µg/ml. Mock injection (n =
3) did not significantly increase T
values of suppressed
mice (control, n = 3). For each concentration, 100,
200, and 400 ng, a total number of five mice were injected
intraperitoneally with wild type as well as with each hTSH mutant.
After 6 h, blood was obtained from the orbital sinus for T
determinations. Data were compiled from individual injections. *, p < 0.05 compared with an equal dose of wild
type.
Figure 11:
Serum
disappearance rate of hTSH mutants from CHO-K1 cells in male rats.
After bolus injection of 200-300 ng of wild type or mutant hTSH
into the femoral vein, blood for hTSH determinations was obtained over
120 min at equal time points. An IRMA without cross-reactivity to rat
TSH (Nichols Institute), was used. Immunoreactivity was expressed as
percentage remaining, and serum concentration at 0 min was defined as
100%. Wild type, n = 4; Q52/TSH
, n = 4;
Q78/TSH
, n = 3. Data were
compiled from individual experiments.
The glycoprotein hormones hTSH, hCG, hLH, and hFSH share a
common signal transduction pathway involving interaction with a
hormone-specific G protein-coupled receptor and the generation of cAMP
as the initial event in the signal transduction
cascade(3, 4) . Previous studies on hCG (7) and hFSH (8, 9) had indicated a
site-specific requirement of the oligosaccharide at Asn for their in vitro activity, but similar studies on hTSH
and the more closely related hLH have not been performed. Moreover,
previous studies using recombinant DNA techniques to study
oligosaccharides in glycoprotein hormones had been limited to using
either site-directed mutagenesis of glycosylation attachment sites or
glycosylation mutant cell lines to synthesize these hormones.
Therefore, in a novel approach to study the role of carbohydrates for
hTSH, we combined site-specific deletion of individual oligosaccharides
with the expression of recombinant hormones in a variety of cell lines
that differentially process the carbohydrate moieties.
In this study
we provide evidence for a different role of the oligosaccharides for
hTSH bioactivity, distinct from their role for hCG and hFSH.
Site-specific disruption of each of the three individual carbohydrate
attachment sites led to an increase in the in vitro activity
of hTSH expressed in CHO-K1 cells producing sialylated
carbohydrates(14, 15, 16) . Our findings
using a sialic acid-binding lectin (30) as well as enzymatic
desialylation show that the hTSH transiently expressed in CHO-K1 cells
was indeed sialylated. Further, its in vitro bioactivity as
well as its circulatory half-life was indistinguishable from that of
rhTSH (Genzyme Corp.), ()which contains 1.8-2.2 sialic
acid residues/chain(14, 16) . Interestingly, this
increase of in vitro activity upon site-specific
deglycosylation was most pronounced upon deletion of the carbohydrate
at Asn
. In contrast, this Asn
oligosaccharide had previously been shown to be essential for
receptor stimulation of hCG and hFSH, which were also expressed in
CHO-K1 cells (7, 8, 9) . The increase of
cAMP-inducing activity in our study occurred to a similar degree with
the recombinant hTSH receptor and the endogenous rat receptor,
indicating that these effects were neither system-dependent nor
species-specific. Furthermore, in addition to the immediate induction
of cAMP synthesis, site-specific deglycosylation of hTSH also affected
long term effects, including DNA synthesis by and growth of target
cells in a similar fashion.
Wild type hTSH expressed in 293,
CHO-LEC2, or CHO-LEC1 cells had a higher in vitro activity
than wild type from CHO-K1 cells, and this increased activity was not
further augmented by site-specific deglycosylation. CHO-LEC mutant cell
lines lack a defined glycosylation activity and accumulate specifically
truncated carbohydrates typical of intermediates in the biosynthetic
pathway(23) . In contrast to CHO-K1 cells, which produce
terminally sialylated oligosaccharides, Golgi vesicle membranes from
CHO-LEC2 cells translocate CMP-sialic acid at only 2% of the rate of
vesicles from CHO-K1 cells and thus produce carbohydrates with a
greater than 90% decrease in sialic acid content(20) . CHO-LEC1
cells lack N-acetylgalactosaminyl-transferase I and hence
terminate in ManGlcNAc
intermediates(21) . 293 cells express Nacetylgalactosaminyl-transferase and
GalNAc
1,4GlcNAc
1, 2Man
4-sulfotransferase and produce N-linked carbohydrate moieties terminating in sulfate in more
than 70% of the chains(19) . The fact that the increase of the in vitro activity upon site-specific deglycosylation was
confined to hTSH expressed in CHO-K1 cells therefore indicates that the
terminal sialic acid residues attenuate signal transduction of hTSH.
This was further supported by our present findings after neuraminidase
digestion and is in accord with recent reports on sequential
deglycosylation of rhTSH (17) and recombinant bovine
LH(15) . Importantly, our current approach allowed us to
identify site-specific differences in the role of the terminal sialic
acid residues and showed that the residues at the Asn
oligosaccharide attenuated TSH receptor activation to a much
higher degree than those at the Asn
or
Asn
carbohydrate chain. Further, our data demonstrate
that hTSH expressing Man
GlcNAc
intermediates
retained full receptor coupling. In contrast to our findings for hTSH,
Keene et al.(32) showed that the activity of wild
type hCG expressed in these CHO glycosylation mutant cells was lower
compared with hCG from CHO-K1 cells. The same group reported that the in vitro activity of hFSH did not change upon expression in
these cell lines(33) . These studies, however, did not assess
the effects of site-specific deglycosylation on the activity of hCG or
hFSH expressed in these cells. Taken together, these findings indicate
that the terminal sialic acid residues can differently modulate
glycoprotein hormone activity in a hormone-dependent manner.
Our
study has further demonstrated a unique role of the carbohydrate at
Asn for hTSH receptor binding. In previous reports
for hCG and hFSH, deletion of the oligosaccharide at Asn
had no effect (7, 8) or led to only a minor
increase in receptor binding(9) . The nonessential role of
oligosaccharides Asn
and Asn
in
hTSH receptor binding observed in this study is similar to that for the
gonadotropins(7, 8, 9) . In contrast to the
findings on bioactivity, the increased binding of
Q52/TSH
was
independent of the carbohydrate pattern, suggesting that the increase
of activity of
Q52/TSH
from CHO-K1 cells was not a direct
result of the increased binding of this mutant. The molecular mechanism
by which carbohydrates activate the receptor is unknown, but in accord
with our present findings, is believed to occur at a postreceptor
binding step(3, 4) . An indirect mechanism involving a
conformational change of the hormone (35) appears more likely
than a direct interaction of the oligosaccharide with the receptor,
since a lectin-like component identified in the hCG receptor (34) is not present in the hTSH receptor. Recent reports on the
crystal structure of hydrofluoric acid-treated hCG (36, 37) located the oligosaccharide at position
Asn
to be in a putative receptor binding region.
Since the hormone-specific
subunit influences the conformation of
the
subunit in a hormone-specific manner(38) ,
differences in spatial orientation of this oligosaccharide may
contribute to its differential role in hTSH compared with hCG or hFSH.
This speculation, however, awaits confirmation by structural analysis,
since, among the glycoprotein hormones, only hCG has been crystallized
to date.
It had previously been reported that the weak thyrotropic
activity of hCG (for review, see (39) ), which has recently
been linked to a direct interaction with the rhTSH
receptor(31, 40, 41) , increased upon
desialylation(42) , in contrast to a decrease at its native
receptor (18) . When we tested the thyrotropic activity of
CHO-K1-expressed hCG site-specifically deglycosylated at the
subunit, we observed an increase of activity of
Q52/hCG
but
not of
Q78/hCG
. This leads to the conclusion that the
deletion of the oligosaccharide at Asn
can have
opposite effects on hCG activity, depending on the receptor with which
the hormone interacts. Therefore, these findings point to the
intriguing possibility that the observed differences in the role of
individual oligosaccharides for glycoprotein hormone action may be
related, at least in part, to differences in glycoprotein hormone
receptor structure and/or to receptordependent differences in
receptor-ligand interaction.
The carbohydrate moieties are known to
be important for the circulatory half-life and hence the in vivo bioactivity of the glycoprotein hormones (for review, see (43) ). However, the role of individual oligosaccharides for
the in vivo activity of hTSH has not, to our knowledge, been
investigated previously. Since the availability of a hTSH superagonist
may have potential clinical applications(44) , we were
interested to determine whether the increased in vitro activity of sialylated Q52/TSH
from CHO-K1 cells could
be maintained in vivo, despite the known protective effect of
terminal sialic acid for the plasma half-life of rhTSH(16) .
However, we found a significant decrease in the in vivo activity of
Q52/TSH
at the highest dose tested, as well
as a slightly increased serum disappearance rate. By comparison, the
significantly greater relative loss in the in vivo activity of
the mutant
Q78/TSH
was correlated with a significant increase
in its serum disappearance rate. This significantly greater decrease in
circulatory half-life upon deletion of the oligosaccharide at
Asn
compared with Asn
may be
related to its peripheral, surface-exposed location, whereas the
oligosaccharide at Asn
appears to be buried at the
dimer interface(36, 37) . These results are comparable
with recent findings on the site-specific role of the carbohydrates for
the in vivo activity of hFSH(45) . The lack of
correlation between the in vitro and in vivo activities illustrates the importance of carbohydrates in
determining hTSH activity in the whole organism. Further, our findings
emphasize that the circulatory half-life, and not the in vitro activity, appears to be the primary determinant of the in vivo bioactivity of these hormones(16, 45) . It will
be interesting to study whether further modifications of the
Q52/TSH
mutant, e.g. fusion of the hCG
carboxyl-terminal peptide to the TSH
carboxyl terminus (24, 46) , can compensate for the increased clearance
rate and hence help to generate hTSH mutants with increased in vivo bioactivity.
In conclusion, we have demonstrated that the roles
of the terminal sialic acids as well as of individual oligosaccharides
are different for hTSH compared with hCG and hFSH. Consistent with our
recent observations of the unique importance of the
carboxyl-terminal Ser
for hTSH action(22) , these
findings indicate that conserved structures within the context of a
given ligand receptor complex may contribute to signal transduction in
different ways. In conjunction with previous findings on the
glycoprotein
hormones(15, 16, 17, 18, 32, 33) ,
we conclude that in hCG, which is exclusively sialylated, sialic acid
is required for full expression of in vitro activity. In hFSH,
which is predominantly sialylated, sialic acid residues appear to be
dispensable for in vitro activity. However, in the case of bLH
or phTSH, which bear sialic acids and are predominantly sulfated,
expression of terminally sialylated oligosaccharides attenuates
effective in vitro receptor activation. In particular, whereas
the oligosaccharide at Asn
is necessary for hCG and
hFSH action, the same chain, and specifically its terminal sialic acid
residues markedly attenuate TSH receptor binding and activation. As
posttranslational modifications of carbohydrates regulate glycoprotein
hormone activity in normal physiology, modulation of terminal
sialylation of the Asn
oligosaccharide, which appears
more heterogeneous than other side chains(47) , may hence be
important in regulating activity in a hormone-specific manner. Our
observation that
Q52/CG
also had an increased activity at the
hTSH receptor, opposite to the effect at its native receptor, points to
differences in receptor-dependent ligand receptor interactions as a
possible explanation for these distinct roles that may have evolved
during evolution to maintain the specificity of receptor ligand
interactions within the glycoprotein hormone family.