(Received for publication, August 31, 1994)
From the
Transthyretin (TTR) is involved in the transport of thyroid
hormones and, due to its interaction with serum retinol-binding
protein, also of vitamin A. The importance of both ligands in
vertebrate embryonic development has prompted us to investigate the
molecular details of TTR transport function in a powerful germ cell
system, the rapidly growing chicken oocytes. Yolk TTR is derived from
the circulatory system, since biotinylated TTR was recovered by
immunoaffinity chromatography of yolk obtained from a hen previously
infused with invitro biotinylated chicken serum
proteins. In concordance with the intraoocytic localization in an
endosomal compartment, ligand blotting and chemical cross-linking
experiments revealed the presence of a 115-kDa TTR-binding oocyte
membrane protein. This putative TTR receptor was not detected in
chicken ovarian granulosa cells or embryonic fibroblasts and was
different from the previously described oocyte-specific receptor for
two estrogen-induced chicken serum lipoproteins, vitellogenin and very
low density lipoprotein (Barber, D. L., Sanders, E. J., Aebersold, R.,
and Schneider, W. J.(1991) J. Biol. Chem. 266,
18761-18770). Furthermore, in contrast to the serum levels of the
yolk precursor lipoproteins, those of TTR were not significantly
changed by estrogen; thus, TTR represents a newly defined,
estrogen-independent class of yolk precursor proteins. These data
strongly suggest that oocytic TTR is derived from the circulation,
where it is a constitutive component, and deposited into yolk as a
result of endocytosis mediated by a specific receptor.
Transthyretin (TTR, ()previously called prealbumin)
is a
60-kDa homotetrameric protein present in serum and
cerebrospinal fluid of vertebrates. The three-dimensional crystal
structure is known for normal human TTR (1) as well as for a
mutant TTR involved in familial amyloidic polyneuropathic
disease(2) . Based on the known primary structure of chicken
TTR(3) , the tertiary and quaternary structures of the human
and chicken proteins are predicted to be highly similar(3) .
The amino acid sequences of TTRs of chicken (130 amino acids) and man
(127 amino acids) are 78% identical overall and completely identical in
a central channel formed by the tetrameric assembly of the
monomers(3) . TTR binds the thyroid hormones, T
and
T
, directly in this central
channel(1, 4, 5) , and it transports retinol
(vitamin A) indirectly by interacting with serum retinol-binding
protein (RBP)(6, 7) .
Both thyroid hormones and metabolites of retinol, acting through their respective nuclear receptors, have been shown to be important in the embryonic development of many vertebrates including oviparous (egg laying) species, such as the chicken(8, 9, 10, 11, 12) . But despite these effects, it is not yet known how the oocytes acquire the necessary thyroid hormones and retinoids. Stated differently, many questions remain about the fate of circulatory TTR. Which cell types can interact with TTR? Do the ligands of TTR play any role in its cellular interactions? Does such interaction lead to endocytosis of TTR and/or RBP?
Uptake of TTR has thus far been shown to occur in
cultured human HepG2 hepatoma cells and has been proposed to occur in
other human and rat cells (e.g. adenocarcinoma, primary
hepatocytes, neuroblastoma) by a receptor-mediated
mechanism(13) . However, there are also some reports that free
T and T
can be taken up directly by primary
hepatocytes (14) and erythrocytes (15) via cell
membrane transporters. In a previous study, we detected TTR in
clathrin-coated vesicles of chicken oocytes and proposed that its
uptake occurs by receptor-mediated endocytosis(16) . Indeed,
the very rapid growth phase of the oocyte has been shown to involve
receptor-mediated endocytosis of certain other circulatory proteins and
lipoproteins(17, 18, 19, 20, 21) .
In this report, we provide the first direct evidence for the uptake of
serum transthyretin into growing chicken oocytes, and we identify a
specific TTR-binding oocyte membrane protein that may act as a receptor
in this process. We show further that TTR differs from the previously
characterized chicken proteins that are transported from the serum into
the yolk in that its serum levels are not significantly influenced by
estrogen action.
Chicken ovarian granulosa cells and embryonic fibroblasts were cultured, and membrane extracts were prepared from them as described in (25) .
The precipitates were mixed with 150 µl of SDS-PAGE sample buffer (non-reducing), heated at 95 °C for 2 min to release the bound IgG and ligand complexes, and briefly microcentrifuged at maximum speed. Maximum volumes of supernatants (60,000 cpm from the immune and 11,000 cpm from the non-immune reactions) were then loaded on a 4.5-18% SDS-polyacrylamide gel and subjected to electrophoresis under non-reducing conditions. The gels were subsequently stained, destained, dried, and subjected to autoradiography(22) .
Figure 1: Detection of intravenously administered biotinylated chicken serum TTR in yolk. Whole laying hen serum was biotinylated and then reinjected into a laying hen as described under ``Experimental Procedures.'' Subsequently, oocytic yolk from the injected hen (lanes1 and 3) and a control hen (lanes2 and 4) was subjected to anti-TTR immunoaffinity chromatography. The column eluates were analyzed for biotinylated proteins with streptavidin-horseradish peroxidase (A) and for TTR with anti-TTR IgG (B); detection was performed by the ECL method. The positions of migration of the two expected TTR species (monomer, 13 kDa; dimer, 33 kDa) are indicated by arrowheads. The two larger size protein bands observed in lane1 likely represent biotinylated proteins that interact nonspecifically with the IgG-Sepharose immunoaffinity matrix (see ``Results'').
The anti-chicken TTR IgG-Sepharose
immunoaffinity column for isolation of TTR has been previously
described(16) . Control and biotin yolk (15 ml each) were mixed
with 1.5 ml of 10 PBS and subjected to TTR immunoaffinity
chromatography as described(16) , except that the final low pH
column eluate was collected in 1-ml aliquots into tubes containing 100
µl of 2 M Tris-HCl, pH 8. The contents of the tubes
containing the eluted protein peak (A
> 0.1)
were pooled and concentrated using Centricon 10 microconcentrators
(Amicon). The TTR component of the eluted biotin-TTR was detected by
immunoblotting with anti-TTR IgG followed by protein A-horseradish
peroxidase, and its biotin moiety was detected by probing the blots
with streptavidin-horseradish peroxidase. For both blots, the final
step involved the horseradish peroxidase-dependent generation and
detection of ECL according to the instructions supplied with the ECL
kit (Amersham Corp.).
In previous biochemical analyses of the distribution of TTR by immunoaffinity chromatography and Western blotting, we have shown its presence in yolk(16) . In fact, TTR and RBP could be demonstrated in oocytic coated vesicles(16) , indicating a specific uptake pathway. Moreover, analysis of chicken yolk by immuno-electron microscopy has revealed that other serum components, for which specific receptors have been identified, are found in the electron-lucent phase of yolk(19, 20) , which represents a compartment derived from endocytic transport vesicles(16, 18, 20) .
Thus, we first wanted to determine directly whether circulatory TTR is the precursor of TTR found in the yolk of growing oocytes. To this end, whole laying hen serum was biotinylated and reinjected into the hen, and biotin-TTR was recovered from yolk. Biotinylation was selected instead of radioiodination because of greater sensitivity in combination with ECL detection and to avoid in vivo and reisolation experiments with large amounts of radioactivity. The biotinylation reaction was highly efficient as determined by probing control and biotinylated serum proteins, after SDS-PAGE and transfer to nitrocellulose, with streptavidin-horseradish peroxidase (data not shown). Chromatography of yolk from oocytes of the hen injected with biotinylated serum proteins on an anti-TTR IgG-Sepharose column and blotting of the immunoaffinity column eluate with streptavidin-horseradish peroxidase revealed the presence of serum biotin-TTR in the oocyte (Fig. 1A, lane1 and B, lane3). The control yolk from an uninjected hen, as expected, was only positive for the TTR analysis with the antibody (Fig. 1B, lane4) but not for biotin-TTR (Fig. 1A, lane2).
With evidence from other studies (17, 18, 19, 20, 21) that
oocytic uptake of certain serum components in oviparous organisms is a
specific, receptor-mediated process, we tested oocyte membrane proteins
for the presence of TTR-binding components by ligand blotting ( Fig. 2and Fig. 3) and chemical cross-linking (Fig. 4). The ligand blots revealed a I-TTR-binding component with a relative molecular mass of
approximately 115 kDa (Fig. 2, lane1). The
binding of
I-TTR to the 115-kDa protein could be competed
for by unlabeled TTR (Fig. 2, lane2) but not
by the serum albumin present in the blotting buffer. This component
clearly was distinct from the abundant and well characterized 95- and
380-kDa oocytic lipoprotein receptors (19, 26) (Fig. 2, lane3).
Membranes from cultured chicken granulosa cells (somatic cells that
enclose the oocyte in the ovarian follicle) (Fig. 2, lane4) and chicken embryo fibroblasts (lane5) were also tested. However, no TTR-binding component
could be detected, even though much higher amounts of somatic cell
membrane proteins were analyzed than used for oocytes. Recognition of
TTR and/or the TTR
RBP complex by the 115-kDa oocyte membrane
protein was confirmed by ligand blotting using the isolated unlabeled
ligand complex, followed by detection with anti-TTR IgG (Fig. 3).
Figure 2:
Ligand blotting analysis of cellular
membrane proteins for the presence of TTR-binding components. The blots
were performed as described under ``Experimental
Procedures.'' Membrane proteins from chicken oocytes (lanes1-3, 75 µg each), granulosa cells (lane4, 200 µg), and embryo fibroblasts (lane5, 200 µg) were transferred to nitrocellulose and
probed with I-TTR (4 µg/ml, 600 cpm/ng) in the
absence (lanes1, 4, and5) or presence (lane2) of a 30-fold
molar excess of unlabeled TTR. Lane3 was incubated
with
I-vitellogenin (20 µg/ml, 50 cpm/ng). Molecular
mass standards (kDa) are indicated to the left of lane1.
Figure 3:
Ligand blot analysis using isolated
TTRRBP complex. Oocyte membrane proteins (75 µg/lane) were
separated by SDS-PAGE, transferred to nitrocellulose, and incubated
with chicken TTR
RBP complex (lane1, none; lane2, 4 µg/ml) for 3 h at 24 °C. Bound TTR
was detected by incubation with anti-chicken TTR IgG followed by
I-labeled protein A and autoradiography. The position of
migration of molecular mass standards are indicated on the right (from top to bottom, in kDa: 200, 116, 97, 68,
and 45).
Figure 4:
Cross-linking analysis for TTR-binding
components on chicken oocyte membranes. Detergent-solubilized membranes
were incubated with chicken I-TTR in the absence (lanes1 and 3) or presence (lanes2 and 4) of 100-fold excess of unlabeled human
TTR, cross-linked, and immunoprecipitated with rabbit anti-chicken TTR
IgG (lanes1 and 2) or non-immune IgG (lanes3 and 4) as described under
``Experimental Procedures.'' Electrophoresis was performed
under nonreducing conditions. Molecular mass standards (kDa) and the
top of the separating gel are indicated to the left of lane1.
Finally, we addressed the characterization of the
putative TTR receptor by an alternative method. With oocyte membrane
proteins in the soluble phase, we performed cross-linking reactions
with I-TTR followed by immunoprecipitation with anti-TTR
IgG (Fig. 4). The cross-linking reaction between TTR and oocyte
membrane proteins resulted in a ligand-membrane protein complex of
175-180 kDa and a very minor, less defined component of
approximately 160 kDa. Possibly, this minor component is a breakdown
product of the major reactive protein or represents a different
TTR-binding species. Other minor bands seen in the immunoprecipitates (lanes1 and 2) but not in the nonimmune
controls (lanes3 and 4) may represent
higher order TTR structures (e.g. tetramers and/or products of
the oxidative action of Iodo-Gen). Since under these conditions TTR can
be expected to exist as a tetramer of 60 kDa, the apparent size of the
cross-linked complex is compatible with the binding of a TTR tetramer
to the 115-kDa protein identified in ligand blots. The major bands (13
and 33 kDa) seen in Fig. 4, lane1, represent
the TTR monomer and dimer, respectively, which are stable in SDS under
nonreducing conditions (described in (23) and (24) ).
Because the anti-chicken TTR antibody does not recognize human TTR,
these bands were undiminished when excess unlabeled human TTR was
included in the incubations (Fig. 4, lane2).
Thus, by a variety of methods using human or chicken TTR, the 115-kDa
protein was consistently identified as a specific TTR-binding oocyte
membrane protein.
At the onset of egg laying, the sudden rise in
circulatory estrogen levels causes a dramatic induction of hepatic
synthesis and serum levels of egg yolk precursor proteins and
lipoproteins(28, 29, 30, 31, 32, 33) .
The effect of estrogen is readily observed and typically studied in
roosters by injecting them with
17-estradiol(28, 29, 30, 31, 32) .
Previously characterized proteins and lipoproteins, e.g. VTG (Fig. 5), which are induced in the hen, taken up by the oocyte,
and deposited into yolk, are also induced rapidly (within 24-48
h) (cf. (31) ) in the rooster
model(28, 29, 30, 32, 33) .
In contrast, Fig. 5shows that estrogen treatment does not
significantly change serum TTR levels relative to those in control
roosters over a period of at least 125 h (Fig. 5, upperpanel). As expected, serum vitellogenin is strongly
induced, with peak values reached approximately 60 h following hormone
administration (Fig. 5, lowerpanel).
Figure 5: Effects of estrogen treatment on serum TTR (upper panel) and VTG (lower panel) levels in roosters. Each lane of the Western blots contains the proteins of 2 µl of rooster serum (prepared as described under ``Experimental Procedures'') treated with 50 mM dithiothreitol. Polyclonal antibodies against TTR and VTG were used to detect the two respective proteins at the following times for post-estrogen treatment: lane1, 12 h; lane2, 26 h; lane3, 33 h; lane4, 57 h; lane5, 81 h; lane6, 125 h. Lane7 represents serum of the untreated control. Molecular mass standards are indicated to the left of the respective panels.
Our experiments provide direct evidence for the endocytosis of circulatory transthyretin by growing oocytes in an oviparous animal, the chicken. We show, by the detection of biotinylated TTR in the yolk of growing oocytes following intravenous administration of biotinylated serum proteins, that efficient TTR uptake occurs in vivo. This represents the first such demonstration for a non-lipoprotein precursor and suggests a general procedure to study the formation of yolk from serum components amenable to biotin tagging.
Previous studies have
indicated that most of the yolk precursors present in serum are taken
up by the oocyte via specific, receptor-mediated
processes(17, 18, 19, 20, 21, 26) .
We have shown that there are two (95 and 380 kDa) receptors on the
chicken oocyte membrane that interact with the major yolk components
very low density lipoprotein, VTG, and possibly
-macroglobulin(19, 21, 22, 26) .
Here, we show in two differently designed ligand-blotting experiments ( Fig. 2and Fig. 3) that free as well as RBP-complexed TTR
interacts with a different oocyte component, i.e. a
115-kDa membrane protein. In addition, chemical cross-linking (Fig. 4) confirmed the binding of a similarly sized membrane
protein to the 60-kDa TTR tetramer. In this context, it is noted that a
considerable portion of circulatory TTR in vertebrates is present
complexed to RBP ( (16) and Refs. therein), and in addition to
TTR, TTR
RBP complexes can be isolated from yolk(16) . It
would be of interest to address TTR
RBP transport by the
biotinylation technique described here for TTR; unfortunately, however,
it cannot be applied to complexed RBP, as it is not readily labeled by
biotinylation (nor by iodination). (
)Future studies are
necessary to determine if the TTR receptor we have identified is
involved in the oocyte-directed uptake of free TTR as well as of
TTR
RBP complexes.
Estrogen treatment of roosters mimics the
laying hen situation in that it dramatically increases the levels of
serum components that, in the laying hen, are known to be massively
endocytosed by the oocyte (28, 29, 30, 31, 32, 33) .
Our present results, however, do not show an increase in the serum
levels of TTR after estrogen administration. Furthermore, in Western
blots of serum, laying hen TTR levels do not appear higher than in
roosters (data not shown). Although we have not ruled out that estrogen
affects both the rate of synthesis and catabolism (or
clearance) of serum TTR, it is clear that TTR belongs to a class of
proteins whose serum levels are not significantly changed by estrogen
but which nevertheless are efficiently transported to the oocyte. One
possible explanation for this difference between TTR and most major
yolk proteins is that large fluctuations in the levels of TTR could
adversely affect the controlled delivery of the important regulatory
ligands, thyroid hormones and retinol, to peripheral cells. Consistent
with this idea, preliminary results suggest that serum levels of RBP
are similarly refractory to estrogen. ()Thus, cell-specific
receptors such as the 115-kDa protein identified here are likely
mediating the accumulation of regulatory molecules in growing oocytes.
It is not known whether the TTR protein itself plays any role in
oogenesis and embryogenesis. However, the hormones that TTR transports
from the serum into the oocyte have been shown to have important
functions in embryogenesis in a wide variety of species. In the
chicken, thyroid hormones have been reported to accumulate in the yolk
during oogenesis(34) . Although the role of TTR was not
examined in that study, it was noted that the highest accumulation of
the hormones per unit mass occurs in the smallest oocytes(34) .
We have previously reported that TTR levels relative to total yolk
protein are also highest in the smaller oocytes(16) . The
thyroid hormone, T, as well as various derivatives of
retinol (e.g. all-trans retinoic acid and 9-cis retinoic acid)
affect expression of important regulatory genes through their
respective nuclear
receptors(8, 10, 12, 35, 36, 37) .
Furthermore, the nuclear receptors for T
and retinoids can
affect each others' activities through their common
heterodimerization partner, retinoid X receptor, another nuclear
retinoid-binding receptor (38, 39, 40, 41) . In this respect,
it is interesting to speculate that the uptake of both retinol and
T
into the oocyte may be co-regulated as a result of their
common interaction with TTR and the subsequent interaction of the
complex with the oocytic TTR receptor.