(Received for publication, July 20, 1995; and in revised form, October 3, 1995)
From the
Thyroglobulin (Tg) is the substrate for thyroid hormone
biosynthesis, which requires tyrosine iodination and iodotyrosine
coupling and occurs at the apical membrane of the thyrocytes. Tg
glycoconjugates have been shown to play a major role in Tg routing
through cellular compartments and recycling after endocytosis. Here we
show that glycoconjugates also play a direct role in hormonosynthesis.
The N-terminal domain (NTD; Asn-Met
) of
human Tg, which bears the preferential hormonogenic site, brings two N-glycans (Asn
and Asn
). NTD
preparations were purified from Tg with low and mild iodine content in vivo and from poorly iodinated Tg after in vitro iodination and coupling. NTD separated from poorly iodinated Tg
was also submitted to iodination and coupling after desialylation and
deglycosylation. The various NTD isoforms were analyzed for their N-glycan structures and hormone contents. Our results show
that 1) in vivo as well as in vitro unglycosylated
isoforms did not synthesize hormones, whereas fully or partially (at
Asn
) glycosylated isoforms did; 2) high mannose type
structures enhanced the hormone content; and 3) desialylation did not
affect in vitro hormone synthesis. Evidence of a direct
involvement in hormonosynthesis adds to the role of N-glycans
in Tg function and opens the way to new mechanisms for regulation (e.g. TSH modulation of N-glycan) or alteration (e.g. Asn
mutation) of thyroid hormone synthesis.
Thyroglobulin (Tg), ()the prothyroid hormone, is the
major component of the thyroid gland. Tg is a large dimeric
glycoprotein (2
330 kDa) containing about 10% of carbohydrates.
Among all proteins, Tg has the unique ability to form triiodothyronine
(T3) and thyroxine (T4) residues by coupling the iodotyrosine residues.
The iodine content of human Tg varies largely with the iodine intake:
0.05-1.1% (w:w), i.e. 2.5-55 atoms of iodine/mol
of Tg. With as few as four iodine atoms, Tg can form T4, which
indicates that specific mechanisms succeed in iodinating only few of
the Tg molecules. Only four hormonogenic sites have been identified in
human Tg. The preferential site is Tyr
, where most of the
T4 is formed; the other sites are localized in the C terminus of the
molecule(1) .
Human Tg is glycosylated with N-linked and O-linked oligosaccharide
residues(2) . N-Glycosylation of Tg begins after
transfer of the nascent polypeptide chain into the lumen of the
endoplasmic reticulum where a common oligosaccharide
(Glc-Man
-GlcNAc
) is transferred
from a specific lipid carrier to Asn residues present in about 15 of
the 20 potential glycosylation sites of the Tg subunit(3) .
Processing then occurs in the rough endoplasmic reticulum and the Golgi
apparatus, in which the initial oligosaccharide form is gradually
converted into different types of N-glycans: high mannose
type, hybrid type, and bi- or triantennary complex type structures.
During its intracellular transport, Tg is also submitted to other
post-translational modifications such as sulfation, phosphorylation,
and iodination. During the past few years, numerous studies have
focused on the mechanisms and the location of thyroid hormone synthesis (4) . It is generally accepted that the follicular lumen is the
main site of Tg iodination, although it may also take place during the
intracellular transport(5) . Tg is iodinated, and hormone
synthesis occurs in presence of thyroid peroxidase and the
H
O
generating system. Lastly, hormone secretion
requires that hormone-containing Tg be reabsorbed from the colloid by
endocytosis and then degraded in the lysosomes. The hormones are
finally released into the venous flow. In the intracellular movements
of Tg, the physiological role of glycosylation is not fully understood.
It has been demonstrated that in thyroid cell cultures, exocytosis of
Tg is suppressed if glycosylation is totally inhibited by
tunicamycin(6, 7) . It has also been proposed that
sialylation of Tg may operate as an export signal because certain
thyroid pathologies in which sialyltransferases are lacking are
associated with a defect in Tg secretion(8) . However, in
primocultures of porcine thyroid cells, inhibition of the formation of
sialyllactosaminyl structures does not impair Tg secretion(9) .
On the other hand, thyrocytes contain GlcNAc receptors (10) that recycle the GlcNAc-bearing Tg back to the colloid and
prevent these molecules from lysosomal degradation(11) . Taken
together, the data point to a major role of glycoconjugates in routing
Tg to the cellular compartments where iodination and hormone synthesis
occur. In contrast, there have been no reports about a direct role of N-glycans in thyroid hormone synthesis.
A major difficulty
in studying Tg is its large size, which precludes detailed analysis of
the various domains in terms of structure-activity relationship. To
circumvent this problem, we focused on the N-terminal domain (NTD) of
Tg. Previously, we had separated NTD from the peptides obtained after
CNBr treatment of Tg(12) . NTD
(Asn-Met
, N-glycans at
Asn
and Asn
) was cleaved at
Met
, and a disulfide bond linked this peptide with
Glu
-Met
. The apparent molecular
weight of NTD varies according to the number of oligosaccharide side
chains, which are known to be structurally heterogeneous(13) .
Note that NTD was able to form T4 in vitro after iodination
and coupling of the acceptor residue (Tyr
) with the donor
residue (Tyr
)(14) . This indicated that NTD
maintained most of its three-dimensional conformation, which was
further demonstrated by surface epitope mapping(15) . NTD thus
offered an interesting opportunity to determine the role of N-glycans in the hormone synthesis process.
Figure 1: Flow chart of the preparation of various NTDs.
Two other types of NTDs were prepared from PI-NTD (Fig. 1C) after desialylation (PI-NTD-dS) or deglycosylation (PI-NTD-dG) as described below. In vitro iodination and coupling of PI-NTD, PI-NTD-dS, and PI-NTD-dG were performed according to Marriq et al.(14) .
The
NTDs and H-labeled oligosaccharide structures were
desialylated by 20 milliunits of neuraminidase (sialidase from Vibrio
cholerae; Boehringer Mannheim). After incubation at 37 °C overnight
in 100 mM acetate buffer, pH 4.5, containing 100 mM CaCl2, samples were heated at 100 °C for 3 min. Each of the
asialo-peptides or asialo-oligosaccharide structures was further
chromatographed on a RCA
-Sepharose column.
Tryptic
hydrolysis of the PI-NTD-dG was performed with
trypsin-tosyl-L-phenylalanine chloromethyl ketone
(Worthington, Freehold, NJ) in 0.1 M ammonium bicarbonate for
4 h at 37 °C with a 1:25 (w/w) enzyme/substrate ratio after
reduction and S-carboxymethylation of the peptide according to
Crestfield et al.(18) . The hydrolysate was
fractionated on a Bio-Gel P-30 column (1.0 70 cm) in 50 mM ammonium bicarbonate.
SDS-PAGE was performed, without reduction of the samples using a 10 or 15% acrylamide and 1% SDS gel system. Protein bands were stained with Coomassie Brilliant Blue. Immunoblotting was performed with a mouse monoclonal antibody directed against T4 residues(15) . The second reagent was peroxidase-conjugated goat antimouse antibody (diluted 1:1000). Detection was performed with 4-chloro-1-naphthol as substrate. The scanning surfaces of the different isoforms identified by SDS-PAGE or by immunoblotting were obtained with a Microtech MSF-300GS. Finally densitometry analysis was performed with the NIH Image 1.47 software.
The ConA affinity chromatography elution profiles
showed that both PI-NTD and T4-NTD separated into three fractions (Fig. 2, A and B). The nonretained (NR)
fractions contained the NTD isoforms unable to bind to ConA. The
isoforms of the weakly retained (WR) fractions bound to ConA and were
eluted with 10 mM -methyl-D-glucopyranoside. The
firmly retained (FR) isoforms also bound to ConA but were eluted with
300 mM
-methyl-D-mannopyranoside. The elution
profiles of PI-NTD and T4-NTD were nearly identical, but the relative
distribution of isoforms in the three fractions differed slightly. The
relative content of the NR fraction was 2-fold lower for PI-NTD than
for T4-NTD (4.9 ± 1.2% and 11.9 ± 2.4%, respectively).
Conversely, the relative content of the WR fraction was higher for
PI-NTD than for T4-NTD (41.7 ± 4.6% and 35.0 ± 3.8%,
respectively). Finally, the FR fractions of PI-NTD and T4-NTD did not
significantly differ (53.4 ± 4.8% and 54.1 ± 6.2%,
respectively).
Figure 2:
ConA-Sepharose chromatography of PI-NTD (A) and T4-NTD (B). 3 mg of each type of NTD was
chromatographed on a ConA-Sepharose column (1 7 cm) with the
equilibration buffer and then successively with 10 mM
-methyl-D-glucopyranoside (
MG, first
arrow) and 300 mM
-methyl-D-mannopyranoside (
MM, second arrow). Flow rate, 10 ml/h.
Fractions of 1 ml were collected, and protein absorbance was monitored
at 210 nm. The insets show SDS-PAGE with 15% acrylamide for
PI-NTD (A) and 10% acrylamide for T4-NTD (B). Lanes 1 are the material prior to chromatography, lanes 2 are the nonretained fractions, lanes 3 are the weakly
retained fraction, lanes 4 are the firmly retained fractions,
and lanes 5 are low molecular weight standards (Bio-Rad).
Proteins were detected by Coomassie Brilliant Blue
staining.
The fractions obtained by ConA affinity
chromatography of PI-NTD and T4-NTD preparations were labeled with
tritium and analyzed for H-labeled oligosaccharide side
chains. As expected (Table 1), both NR fractions presented only
triantennary complex type structure. The WR fractions contained both
types of complex structures; the amount of biantennary was over 50%.
This indicated that all WR isoforms presented at least one biantennary
structure, which reflected the weak binding of these isoforms to ConA.
On the other hand, PI-NTD and T4-NTD differed in their relative amounts
of triantennary oligosaccharide side chains (46 and 25%, respectively).
All the isoforms bearing high mannose type structures were found in the
FR fraction, which was anticipated from ConA elution conditions. High
mannose type side chains of each NTD may or may not be associated with
bi- or triantennary structures. It is noteworthy that PI-NTD contained
less high mannose type structure (62%) than T4-NTD did (71%). The
difference can be explained by the higher amount of triantennary
complex type oligosaccharides (15 and 8% for PI-NTD and T4-NTD,
respectively). Taking this into account, we then calculated the overall
percentage of different N-glycan structures (Table 2).
Electrophoresis study of the three fractions obtained by ConA affinity chromatography showed (Fig. 2, A and B, insets) that the NR isoforms were consistently separated into two bands (lanes 2): one migrating in the 25-kDa region and the second one in the 19-kDa region. WR isoforms migrated in only one band in the 25-kDa region (lanes 3). FR isoforms showed two bands: one band in the 25-kDa region, as already observed with NR and WR isoforms, and one band in the 22-kDa region.
Taken together, these results pointed to a rather large heterogeneity of NTD isoforms. Considering that the molecular weight of NTD, deduced from its amino acid composition, was 18,000, variations in the molecular weight of NTD isoforms may be accounted for by differences in the number of oligosaccharide side chains. On the basis of the conditions of elution of ConA affinity chromatography, the data gathered by analysis of the carbohydrate structure, and the molecular weight of each isoform, we reasoned that the FR fractions contained isoforms with at least one high mannose type structure associated or not with any of the three carbohydrate structures identified. FR fractions would therefore contain four NTD isoforms: high mannose type/high mannose type, high mannose type/biantennary complex type, high mannose type/triantennary complex type, and high mannose type/O. The first three isoforms migrated in the 25-kDa region, whereas the fourth isoform migrated in the 22-kDa region. Effectively, such bands were observed. The same reasoning would apply to the other fractions. The WR fractions were thus considered to contain two 25-kDa isoforms (biantennary complex type/biantennary complex type and biantennary complex type/tri-antennary complex type), and the NR fraction as containing one 25-kDa isoform (triantennary complex type/triantennary complex type). Moreover, the presence of a peptide migrating in the 19-kDa region of the NR fraction suggested that unglycosylated NTD might be present. Accordingly we further separated the NR fraction and characterized the 19-kDa NTD peptide.
Figure 3:
RCA-Sepharose chromatography
of the asialo-NR fractions. The fractions nonretained on ConA-Sepharose
from PI-NTD (A) and T4-NTD (B) were desialylated and
then chromatographed on a RCA
-Sepharose column (0.5
3 cm) with the equilibration buffer and then with the same
buffer containing 200 mM lactose (Lac, arrow). Flow rate, 5 ml/h. Fractions of 0.5 ml were collected,
and protein absorbance was monitored at 210 nm. The insets show SDS-PAGE (10% acrylamide). Lanes 1, material prior
to chromatography; lanes 2, the material nonretained on
RCA
, lanes 3, material retained on
RCA
.
Western blot analysis with an anti-T4 monoclonal antibody was performed on the initial preparation and the ConA affinity fractions of T4-NTD. As shown in Fig. 4, most of the T4 residues were detected in the 25-kDa region whatever the sample analyzed (lanes 1-4). T4 residues, however, were also detected in the 22-kDa bands in the initial preparation of T4-NTD (lane 1) and its FR fraction (lane 4). Once again, no T4 residues were observed in the 19-kDa region of the initial preparation (lane 1) and the NR fraction (lane 2).
Figure 4: Immunoblotting of T4-NTD and of the different fractions separated by ConA-Sepharose chromatography. Separation was performed on SDS-PAGE (10% acrylamide) under nonreducing conditions and then transfer to polyvinylidene difluoride. Immunoblot detection was probed with anti-T4 monoclonal antibody as described inder ``Experimental Procedures.'' Gels were calibrated with Rainbow(TM) low molecular weight markers (Amersham Corp.). Lane 1, T4-NTD, lane 2, NR fraction; lane 3, WR fraction; lane 4, FR fraction.
Taken together, our results showed the absence of T4 residues in the unglycosylated isoform and, equally intriguing, the higher amount of T4 residues in isoforms bearing at least one high mannose side chain than in those with only complex type oligosaccharide structure. To gain insight into the relationship between the presence of high mannose type side chain and the amount of T4 residues, we conducted experiments aimed at measuring T4 residues in the three different isoforms present in the FR fraction.
RIA of the T4 residues showed that the first fraction exclusively comprising isoforms with high mannose type structure contained 85 mmol T4/mol peptide. The second fraction with isoforms bearing mixed oligosaccharide structures (high mannose type associated with complex type structure) contained 64 mmolT4/mol peptide.
Western blot analysis of RCA nonretained and
retained fractions was performed with a monoclonal antibody to T4. It
showed that T4 residues were present in the three isoforms (Fig. 5) and that T4 residues were present mostly in isoforms
containing only high mannose side chains (Fig. 5, lane
1).
Figure 5:
Immunoblotting of the three isoforms
present in the FR fraction of the T4-NTD. The FR fraction obtained
after ConA-Sepharose chromatography of the T4-NTD was desialylated and
then chromatographed on a RCA-Sepharose column. Isoforms
bearing only high mannose type (lane 1) and the isoforms
bearing high mannose type associated with a bi- or triantennary complex
type (lane 2) were analyzed by immunoblotting as described in
the legend of the Fig. 4.
These results further pointed to a tight relationship between the glycosylation of NTD isoforms and their hormone contents. Unglycosylated NTD did not present hormone, whereas glycosylated NTD did; T4 content was the highest in NTD isoforms with only one or two high mannose type structures and lower those with one or two complex type structures. We thus studied in vitro T4 formation by NTD isoforms to assess the direct involvement of N-glycans borne by NTD.
The results gathered after in vitro iodination and coupling of Tg were close to those observed with Tg iodinated and coupled in vivo. Also, they showed that unglycosylated NTD was unable to form hormones and that NTD with high mannose type structure would be the best substrate for T4 synthesis. To confirm these results we devised experiments with purified NTD isoforms.
Figure 6:
Effect of
peptide-N-(acetyl-
-glucosaminyl)asparagine
amidase on the PI-NTD. PI-NTD was incubated at 37 °C with 1.5 units
of peptide-N
-(acetyl-
-glucosaminyl)asparagine
amidase for 1 and 24 h. After 1 h of incubation, SDS-PAGE (A)
identified two bands corresponding to molecular masses of 25 and 22
kDa, respectively (lane 1). After 24 h of incubation, the
PI-NTD was identified as peptides of 22 and 19 kDa, respectively (lane 2). Proteins were detected by Coomassie Brilliant Blue
staining. PI-NTD submitted to deglycosylation for 1 (lane 1)
or 24 h (lane 2) was iodinated, coupled in vitro, and
then analyzed by immunoblotting (B) with an anti-T4 monoclonal
antibody as described under ``Experimental
Procedures.''
These results confirmed that unglycosylated NTD was unable to form thyroid hormone. They also provided direct evidence for thyroid hormone synthesis in the presence of only one oligosaccharide side chain. Note that T4 formation was equally efficient in the presence of one or two chains, desialylated or not. This alluded to a conformational role of the oligosaccharides in thyroid hormone synthesis.
Figure 7:
Bio-Gel P-30 elution profile of the
tryptic digest of NTD. About 2 mg of NTD was reduced, alkylated, and
hydrolyzed by trypsin at 37 °C for 4 h. The hydrolysate was applied
on a Bio-Gel P-30 column (1.0 70 cm) and eluted in 50
mM NH
CO
. Flow rate, 8 ml/h. Fractions
of 0.8 ml were collected. Protein absorbance was monitored at 210 nm.
Fractions I, II, III, and IV were pooled, as indicated by the arrows, then concentrated, and desalted on a Bio-Gel P-2
column before lyophilization.
Most proteins and peptides are efficient substrates for tyrosine iodination. Tg is unique in that its iodotyrosine coupling leads to thyroid hormone formation, a process that does not occur at random(1) . Among the numerous tyrosines of Tg that can be iodinated, only a few are involved in hormone synthesis. The strict specificity of the four hormone-forming sites obviously requires not only consensus sequences (21) but also stringent spatial organization of the Tg molecule. In turn, the three-dimensional structure of Tg is modified during the process of tyrosine iodination and tyrosine coupling(22) . Moreover, it has been demonstrated that glycosylation of Tg was also able to modify the conformational structure of this molecule (see below). Consequently the tight relationship between the structure of Tg and its unique ability to form hormones could point to a direct role of Tg oligosaccharide moieties in hormone synthesis. Up to now, this had not been confirmed. Confirmation was offered by the observation that the N-terminal part of Tg presents up to two N-linked oligosaccharide side chains and that it is able to form T4 in vitro after being separated from the core molecule(14) .
CNBr treatment and separation of the fragments of Tg and then lectin affinity chromatography of NTD provided several isoforms that differed in molecular weight and oligosaccharide composition. The oligosaccharide side chains contained three structures: biantennary and triantennary complex types as well as high mannose type structures. The peptides migrating in the 25-kDa regions all brought two oligosaccharide side chains, but they differed in chain structure. Six 25-kDa isoforms were identified depending on the combination of the three different types of oligosaccharide structures present on the two available glycosylation sites. Conversely, the 19-kDa isoform was shown to be the unglycosylated NTD isoform. Regarding the 22-kDa form, which brings only one oligosaccharide side chain, one would have expected to find three isoforms presenting high mannose, biantennary, or triantennary structure; only the 22-kDa form with one high mannose type structure was detected. These data are in general agreement with our previous results on the heterogeneity of glycosylation in this part of the human Tg molecule(13) . They, however, are at variance with those of Rawitch et al.(23) , who reported that high mannose type structures were limited to the C terminus in bovine Tg. The present study clearly demonstrates the existence of totally (19 kDa) or partially (22 kDa) unglycosylated isoforms. Furthermore, it provides evidence that NTD isoforms may bring only high mannose type structures, one for the 22-kDa form and two for the 25-kDa form. The presence of such isoforms was not expected because Tg prepared from human goiters issues mainly if not totally from the follicular lumen, which is expected to contain mature molecules. Indeed, it is generally accepted that a secreted protein bears mainly mature complex type structures. The presence of high mannose type structures in a mature protein may be explained by the folding of the protein before it enters the Golgi apparatus.
Many proteins possess one or more Asn-Xaa-Ser or Asn-Xaa-Thr
consensus sequences, which are potential sites for N-glycosylation (24) . Glycosylation at some of these
sites has been demonstrated to play a role in the structure, function,
expression, or stability of glycoproteins(25, 26) .
Regarding Tg, N-glycans have been reported to be involved in
its intracellular transport and iodination in relationship with
recycling after endocytosis and sialylation of the protein(8) .
This process was recently explained by the presence of a GlcNAc
receptor in the apical membrane and also in the subapical compartments
of thyroid epithelial cells(11) . The GlcNAc receptor would
play a major role in the processing of internalized Tg molecules
bearing GlcNAc and be recycled through the Golgi apparatus via the
apical membrane to the colloid. During this transit, Tg molecules would
complete their glycosylation of complex type oligosaccharide side
chains in the Golgi apparatus and would increase their iodination
levels at the apical membrane by contacting thyroperoxidase.
Glycosylation has also been shown to be modulated by TSH in cultured
thyroid cells(27, 28) . In primary culture of porcine
thyroid cells, TSH increased the number of oligosaccharide side chains
borne by Tg without modifying the relative distribution of the various
types of oligosaccharide structures(29) . In FRTL-5 cells it
appeared rather that TSH decreased (30) or increased (31) the number of oligosaccharide side chains. For the latter
group, TSH stimulation resulted in the addition of one high mannose
type structure at the N-terminal part of Tg and led to maturation of
pre-existing high mannose type side chains, forming complex type
structures(32) . Discrepancies in the data on cultured thyroid
cells may be explained by differences in cultured cells and
experimental conditions, notably cell culture media(33) . It
has also been observed that Tg obtained from thyroid tissue differed
with regard to N-glycosylation from that obtained from
cultured cells(34) . Nevertheless, a general consensus might be
derived from these and other studies: whatever the effect of TSH or
modifications in experimental conditions on the number and composition
of oligosaccharide side chains, the tridimensional structure and
antigenic properties of Tg molecules changed, which ultimately affects
the ability of Tg to form thyroid hormones(35, 36) .
This conclusion is in close agreement with ours. The modification in
the structure of the N-terminal part of Tg induced by changes in N-glycan structure and number has an important impact on
hormone formation at the major site of T4 synthesis: unglycosylated NTD
does not form hormone; the presence of a single N-glycan side
chain at Asn allows NTD to form T4; as compared with
complex type structures, high mannose type structures enhance the
ability of NTD to form hormones. Taking into account that TSH may
modify N-glycans at the NTD, it appears that TSH may modulate
thyroid hormones more directly than we think. Considering the
relationship between NTD oligosaccharide structure and its ability to
form T4 residues, we speculate that Tg gene abnormality involving the
potential glycosylation sites of NTD might induce abnormality in the
thyroid status of patients(37) . The present results open a new
way to apprehend the physiology and pathology of the thyroid gland.
This has been made possible by the ability of the Tg NTD, separated
from the core molecule, to form thyroid hormone residues. This stresses
the potential interest of molecular dissection in establishing the
structure-activity relationship of proteins.