From the Department of Biological Sciences, National University of Singapore, Singapore 119260
Received for publication, May 23, 2002, and in revised form, October 30, 2002
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ABSTRACT |
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The vitellogenin receptor (VtgR) belongs to the
low density lipoprotein receptor (LDLR) gene family. It mediates the
uptake of vitellogenin (Vtg) in oocyte development of oviparous
animals. In this study, we cloned and characterized two forms of
Oreochromis aureus VtgR. Northern analysis showed that VtgR
was specifically expressed in ovarian tissues. However, reverse
transcription-PCR indicates that either there are trace levels
of expression of VtgR or a homolog of LDLR exists in nonovarian
tissues. The VtgR is highly homologous to the very low density
lipoprotein receptor. To better understand the mechanism by which
similar structural modules in the ligand-binding domain bind different
ligands, we used the yeast two-hybrid system to screen for the minimal
interaction motifs in Vtg and VtgR. The amino-terminal region of the
lipovitellin I domain of Vtg interacts with the ligand-binding domain
of VtgR. The first three ligand-binding repeats of the receptor were
found to be essential for ligand binding. Computational analysis of the
binding sequence indicates that Vtg has a similar receptor-binding region to apolipoprotein (apo) E and apoB. Site-directed mutagenesis of
this region indicates electrostatic interaction between Vtg and its
receptor. Sequence analysis suggests the coevolution of receptor-ligand pairs for the LDLR/apo superfamily and suggests that
the mode of binding of LDLR/very low density lipoprotein receptor to
apoB and apoE is inherited from the electrostatic attraction of VtgR
and Vtg.
During vitellogenesis, vitellogenin
(Vtg),1 a
lipophosphoglycoprotein, is synthesized in the liver and transported to
the ovary via blood circulation. This major yolk precursor protein
binds to vitellogenin receptor (VtgR) on the surface of oocytes and is
taken up by receptor-mediated endocytosis (1). A large amount of
Vtg accumulates in the oocytes within a relatively short time. Once in
the oocytes, Vtg is cleaved into yolk proteins, namely, lipovitellin
(LV) and phosvitin, which are stored as nutrients for the developing
embryo (2). Sequence analysis showed that the amino-terminal 700 amino
acids of Vtg and apolipoprotein (apo) B-100 are homologous, although
the similarity is limited (3). Coincidentally, Vtg also binds lipids
and transports them into the oocytes. The sequence and functional
relationship of these two proteins support the idea that they have a
common ancestor.
VtgR belongs to the low density lipoprotein receptor (LDLR) family (4).
The members of this family bind to various ligands and are involved in
lipid metabolism in both vertebrates and invertebrates. These receptors
have common structural features (5, 6) including (i) cysteine-rich
ligand-binding repeats (LBRs), (ii) cysteine-rich epidermal growth
factor precursor (EGFP)-like repeats spaced by cysteine-poor spacer
regions, (iii) a single transmembrane domain, and (iv) a short
carboxyl-terminal cytoplasmic tail. In addition, a short region highly
enriched in serine and threonine residues may exist in some receptors.
The number of LBRs varies among different receptors. LDLR contains
seven LBRs, whereas very low density lipoprotein receptor (VLDLR) and
VtgR in vertebrates have eight LBRs. Larger receptors such as
LDLR-related protein and megalin have more than 30 LBRs in several
clusters (7, 8). Each LBR consists of about 40 amino acids including 6 cysteine residues, participating in the formation of three disulfide
bonds, which are crucial for its proper folding (9). At the carboxyl
terminus of each LBR, there is a consensus acidic tripeptide,
Ser-Asp-Glu (SDE). Recent structural study by NMR and x-ray diffraction
analysis of LBRs 1, 2, 5, and 6 from LDLR have revealed that the side
chains of many of the aspartate and glutamate residues in the consensus peptides are involved in coordinating the calcium ion into a folded calcium cage (10-15).
The binding sites of Vtg for VtgR were presumed to be located on the
lipovitellin I domain, LV1 (16). Residue modification studies showed
that lysine and arginine residues were important for binding with the
acidic clusters in LBR of VtgR through ionic interactions (17).
However, new structural studies of LBR (10-15) indicate that those
acidic residues might not be accessible to Vtg. This necessitates a
reassessment of current models for the binding of VtgR to Vtg. Because
the sequences of LBRs in different receptors are highly homologous,
their backbone structures are very likely to be identical (9). The
distinct affinity to different ligands may result from differential
participation of individual LBRs, for example, repeat 5 is essential
for binding of apoE, and repeats 2-7 cooperatively bind apoB (18).
Thus, Vtg binding may require the involvement of different LBRs of
VtgR.
Given the relationship among VtgR, LDLR, and VLDLR and the relationship
between Vtg and apoB, the understanding of Vtg-VtgR recognition would
contribute insights into the mechanism of interaction between LDLR gene
family members and their ligands. Many Vtg and VtgR genes have been
cloned and characterized in recent years (19). However, knowledge on
Vtg-VtgR interaction remains limited. In our study, we have cloned
full-length and different domains of Vtg and VtgR from tilapia and
examined their interactions. Using yeast two-hybrid and GST pull-down
assays, we found that the minimal binding domain of VtgR was the first
three LBRs and that there might be more than one binding site. The
receptor recognizes the 84-amino acid fragment in the amino-terminal of
Vtg. Site-directed mutagenesis study indicates that lysine 185 in Vtg
is particularly important for electrostatic interaction with VtgR.
Cloning of VtgR cDNA--
mRNA was purified from
previtellogenic ovaries of Oreochromis aureus using Oligotex
Direct mRNA kit (Qiagen). Reverse transcription was performed with
oligo(dT) using the Thermoscript RT-PCR system (Invitrogen). Two
primers (A1, 5'-GTITGCAAGGCIGTIGG-3'; A2, 5'-TCCAIIAIACICGATCCTC-3'; I = inosine) designed from the consensus sequence of VtgR/VLDLR in
other species such as human, mouse, rabbit, rat chicken, and Xenopus were used for PCR using platinum Taq
polymerase (Invitrogen) to obtain fragment A in the EGFP-like domain.
Two other fragments, fragments B and C, adjacent to and overlapping the
5' and 3' ends of fragment A, were obtained by PCR amplification using
B1 (5'-GAGCAGTGIGGICGICAGCC-3') and B2 (5'-GCGTTTCTCTGCTGCTCC-3') and
C1 (5'-GTGCTCCAGTCTTCAGAG-3') and C2 (5'-ATTGCIGGITAIGTGTG-3'),
respectively, as primers. The 5'- and 3'-RACE system (Roche Molecular
Biochemicals) was used to obtain full-length VtgR cDNA. The primer
for 3'-RACE was C1. The primers for 5'-RACE were D
(5'-GCCAGTAGCAAGACGGGTAGTATG-3') and E (5'-GCAGTTCCCATCGTCACATTTG-3').
All PCR products were cloned into pGEM-T easy vector (Promega) and
sequenced (Fig. 1). Full-length VtgR cDNA was obtained by PCR using
high-fidelity platinum Pfx polymerase (Invitrogen).
Northern Analysis--
The total RNAs from ovary, liver, muscle,
brain, spleen, and intestine were purified using Trizol reagent
(Invitrogen). Aliquots of 20 µg of total RNAs were resolved in 1%
agarose formaldehyde gel. The separated RNAs were transferred onto a
nylon membrane (GeneScreenPlus; PerkinElmer Life Sciences) using
capillary transfer. The membrane was UV-cross-linked, prehybridized in
DIG Easy Hyb (Roche Molecular Biochemicals) for 4 h at
50 °C, and hybridized overnight at 50 °C in Easy Hyb with
DIG-labeled PCR fragment B as probe. After hybridization, the membrane
was washed twice in 2× SSC, 0.1% SDS for 15 min each at room
temperature and twice in 0.5× SSC, 0.1% SDS for 15 min each at
65 °C. The membrane was incubated for 1 h in blocking solution
followed by a 30-min incubation in anti-DIG-AP (Roche Molecular
Biochemicals) diluted 1:10,000 in blocking solution. After incubation,
the membrane was washed twice in washing buffer (100 mM
maleic acid, 150 mM NaCl, 0.3% Tween 20, pH 7.5), and the
hybridized band was detected by chemiluminescence (CSPD reagent;
Roche Molecular Biochemicals). The RT-PCR Analysis--
Approximately 500 ng each of mRNA from
ovary, liver, muscle, brain, spleen, and intestine, isolated using the
Oligotex Direct mRNA kit, was reverse transcribed with oligo(dT)
and Thermoscript reverse transcriptase. The cDNAs were amplified by
PCR using 0.2 µM concentrations of C1
(5'-GTGCTCCAGTCTTCAGAG-3') and G (5'-ATCGCGGGGTACGTGTG-3') primers that
are specific for VtgR, in a final volume of 50 µl, containing 2 units
of platinum Taq DNA polymerase, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, and 1.7 mM
MgCl2. The PCR reaction was carried out by incubating the
samples at 94 °C for 1 min, followed by 35 cycles of 94 °C for
30 s, 52 °C for 30 s, and 72 °C for 1 min.
Yeast Two-hybrid Constructs--
Unless otherwise stated, the
Vtg and VtgR insert cDNAs were generated by linker PCR using
platinum Pfx polymerase. The Vtg and VtgR cDNAs were then cloned
in-frame into pGADT7 and pGBKT7 yeast two-hybrid vectors
(Clontech), respectively. The template cDNAs
used for VtgR constructs were reverse transcription products of ovary
mRNA. The cDNAs for Vtg constructs were derived from pOAVtg1
(20). The full-length cDNA of VtgR flanked by
NdeI-XmaI was cloned into pGBKT7 to obtain
pGBK-VtgR. All sequential LBR deletion constructs were PCR-amplified
and cloned into pGBKT7 to obtain the respective pGBK-LBR constructs.
The VtgRs lacking the LBR domain were PCR-amplified and inserted into
pGBKT7 NdeI-XmaI sites to obtain pGBK-NLBR. The
full-length Vtg insert was PCR-amplified and cloned into
NdeI sites of pGADT7. The LV1 of Vtg was cloned into
NdeI-XmaI sites of pGADT7. The deletion
constructs of Vtg were obtained by digesting pGADVtg with the
corresponding enzymes and religating the plasmids. All constructs were
sequenced to confirm the inserts.
In Vitro Pull-Down Assays--
The ligand-binding domain of VtgR
was amplified by PCR and cloned into the Escherichia coli
expression vector, pGEX4T-3 (Amersham Biosciences), in-frame with GST.
The cDNA fragment corresponding to amino acids 162-246 of Vtg was
amplified using PCR and cloned in-frame into the
NdeI-EcoRI site of expression vector pET22-b (Novagen). To facilitate cloning, these restriction sites were added to
the primers in both cases. The recombinant plasmids were transformed
into E. coli strain BL21. The bacterial culture was grown at
25 °C to A600 nm = 0.6-0.8 and then
induced with 0.1 mM
isopropyl-1-thio- Site-directed Mutagenesis--
The QuikChange XL mutagenesis kit
(Stratagene) was used to mutate 3 residues in Vtg and 1 residue in
VtgR. These are sites that were anticipated to contribute crucially to
their interaction. The primers were designed according to the manual.
The mutation reaction was carried out by incubating samples at 95 °C
for 1 min, followed by 18 cycles of 94 °C for 50 s, 60 °C
for 50 s, and 68 °C for 28 min. After PCR, the sample was
incubated with 1 µl of DpnI at 37 °C for 1 h to
digest the template DNA. Five µl of reaction was used to transform
the XL10-Gold competent cells. The mutation was confirmed by
sequencing. The mutated constructs were used to transform the yeast,
and the resulting interaction was analyzed by Two Forms of VtgR cDNA Were Identified--
To clone VtgR
cDNA, RT-PCR was performed based on primers designed to obtain a
688-bp fragment A in the EGFP-like domain (Fig. 1). The sequence information was used to
design another two pairs of primers. By PCR, two fragments (fragments B
and C) overlapping fragment A were cloned and sequenced. Fragment B was
734 bp, encompassing LBRs 5-8. Two forms of fragment C, of 580 and 520 bp, were observed in the PCR product. The corresponding amino acid
sequence shows that the 580-bp cDNA has the O-linked
sugar domain, which is a threonine- and serine-rich region. The 520-bp
fragment lacks this region. The primers for 5'- and 3'-RACE were used
to obtain the full-length cDNA sequence (GenBankTM
accession number AF514281). The analysis of cDNA sequences revealed two open reading frames of 2500 and 2560 bp, encoding 800 and
820 residues, respectively. The alignment of the amino acid sequences
of VtgR to the VLDLR and VtgR from other species showed high
homology.
Differential Expression of VtgR in Various Tissues--
To examine
the transcription of the VtgR gene, RT-PCR was carried out with C1 and
G primers flanking the O-linked sugar domain. All the
tissues consistently exhibited two forms of VtgR mRNA of 420 and
360 nucleotides (Fig. 2A). The
size difference between these two forms was probably attributable to
the differential splicing of a short region, as observed in the
comparison of the sequences of these two PCR products. By aligning with
VtgR and VLDLR genes of other species, this region was found to be
located in the O-linked sugar domain. Interestingly, when
Northern analysis was performed using fragment B (containing LBRs 5-8)
as the probe, only the ovary exhibited one transcript of VtgR mRNA
of 3.3-kb nucleotides (Fig. 2B). These data indicate that
the VtgR gene was transcribed in both ovarian and nonovarian tissues.
However, only the ovarian VtgR mRNA is sufficiently abundant to be
detectable by Northern analysis. In our study, all the RT-PCR products
from different tissues share the same sequence. This result excludes the possibility that the RT-PCR amplified the LDLR that is homologous to VtgR. When fragment C was obtained from ovarian mRNA by RT-PCR, the dominant product was the one lacking the O-linked sugar
domain. Additionally, in 3'-RACE, the same product was obtained (data not shown). These data indicate that the major form of VtgR in the
ovary lacks the O-linked sugar domain. In chicken, the VtgR was reported to function as VLDLR/VtgR in different tissues (4). The
existence of VtgR mRNA in nonovarian tissues suggests that this
receptor may function as VLDLR or VtgR in different piscine tissues.
The LBR of VtgR Is Sufficient for Vtg Binding--
In an effort to
locate the important interactive domains between VtgR and Vtg, we first
examined the interactions between full-length Vtg and different domains
of VtgR using yeast two-hybrid assays to confirm that the yeast
two-hybrid assay is suitable for the study of interaction between Vtg
and VtgR. Although two forms of VtgR were identified in the ovary, the
results indicate that the major form lacks the O-linked
sugar domain. Therefore, the VtgR constructs used in the interaction
studies were from the VtgR lacking O-linked sugar domain.
Three constructs of VtgR, full-length VtgR, the extracellular part of
VtgR, and the LBR of VtgR, were tested for binding to Vtg. The
quantitative LBRs 1-3 of VtgR Are Involved in the Interaction with Vtg--
To
determine which subdomains of the receptor are important to Vtg
binding, 25 different fragments of the VtgR ligand-binding domain were
cloned into pGBKT7. The interaction between the pGBKT7-VtgR deletion
constructs and pGADVtg was tested by the yeast two-hybrid assay. All
the conditions used in the transformation of yeast and the enzymatic
assays for all the constructs were the same. This made the yeast
two-hybrid assay semiquantitative. The difference in the
Five carboxyl-terminal deletion constructs of LBD, including LBR1-7,
LBR1-6, LBR1-5, LBR1-4, and LBR1-3, did not disrupt the binding to
Vtg. The two carboxyl-terminal deletion constructs of LBD, namely,
LBR1-2 and LBR1, lost binding to Vtg (Fig.
4). When the first LBR was deleted in
constructs LBR2-8, LBR 2-7, LBR 2-6, and LBR2-3, dramatic drops in
the The LBR of VtgR Interacts with the Amino-terminal Fragment of Vtg
between Ala162 and Ile246--
To define the
actual binding sites in Vtg for VtgR, seven 3' deletion constructs of
Vtg together with full-length Vtg were tested for interaction with the
VtgR LBRs (Fig. 5). The LBRs interacted with full-length Vtg and interacted to different extents with the
following deletion constructs: pGADVtg-ClaI (1584 amino
acids), pGADVtg-XmaI (1505 amino acids), pGADLV1 (1089 amino
acids), pGADVtg-BamHI (286 amino acids), and
pGADVtg-EcoRI (246 amino acids). No interaction was observed
with pGADVtg-SacI (162 amino acids) and
pGADVtg-XhoI (52 amino acids). Deletion of Vtg upstream of
246 amino acids completely abolished its binding to VtgR. Thus, the Vtg
binding site is either between amino acids 162 and 246 or between amino acids 1 and 162. The deletion from 162 to the carboxyl terminus may cause a drastic change in the conformation of the upstream region
that is necessary for binding with VtgR. Therefore, to delineate the
actual binding site, the amino-terminal 84-amino acid fragment (flanked
by SacI and EcoRI), VtgSE, containing amino acids
162-246 was subcloned into the pGAD vector to examine its interaction
with LBR. The Direct Binding of LBR and Vtg in Vitro--
To test whether the
interaction between LBD and the short fragment VtgSE is direct and to
confirm the results of the yeast two-hybrid assay, the LBD and VtgSE
were tested by in vitro pull-down assay. LBR and VtgSE were
expressed as GST fusion protein and His fusion protein, respectively,
in E. coli. The GST and GST-LBR proteins were immobilized on
the glutathione-Sepharose beads and incubated with VtgSE cell lysate.
After extensive washing, the elution of GST-LBR contained VtgSE,
whereas the elution of GST did not (Fig. 6B). Thus, it is
clear that the LBR and VtgSE interact directly in vitro. In
the presence of DDT, the GST-LBR did not bind VtgSE. This suggests that
the interaction was dependent not only on the charged residues in LBRs
but also on the integrity of the disulfide bonds, which are crucial for
the three-dimensional structure to confer the functional LBD.
Site-directed Mutagenesis of Vtg Reveals the Importance of
Lys185 for Interaction with VtgR--
By aligning
the sequence of the binding site in Vtg with the apoB
major binding site and the apoE binding site for LDLR (23, 24), a short
motif in Vtg with basic residues was observed (Fig. 7A). This suggests that
vitellogenin may utilize the same mechanism as apoB and apoE to bind
the VtgR. To test this hypothesis, we mutated the positively charged
amino acids at His182, Lys185, and
Lys187 in VtgSE into alanine. These residues are
correspondingly important in apoB and apoE for binding to their
respective receptors. We examined the effect on binding of VtgR by
yeast two-hybrid assay. The mutation constructs VtgSE(H182A) and
VtgSE(K187A) showed similar binding of LBD and LBR1-3. However, the
binding between the mutation construct VtgSE(K185A) and either LBD or
LBR1-3 is attenuated (Fig. 8). This
result highlights the importance of the residue Lys185 in
the interaction between Vtg and VtgR.
When the sequences of different VtgR LBRs were aligned (Fig.
7C), the signature sequences of the LDLR superfamily emerged as well conserved. In LBR3, the three conserved acidic residues are
EDE, whereas in all other LBRs, the sequence is SDE. Bajari et
al. (25) have searched for the minimal binding site in chicken VtgR for receptor-associated protein (RAP) and proposed that the EDE
region in LBR3 might be important for ligand binding because it has the
highest negative charge density. Consistently, our study has
empirically confirmed that LBR3 is critical to the interaction with
Vtg. To demonstrate a potential relationship between this subtle
difference in the acidic residues in LBR3 and its affinity for Vtg, we
mutated the E144S in both the LBD and LBR1-3 and tested their
interactions with Vtg and VtgSE. However, this mutation did not affect
the binding between LBD/LBR1-3 and Vtg/VtgSE. Furthermore, the
mutation of SDE to EDE in LBR6 of LBR4-7 did not show any gain of
function for binding Vtg (data not shown). Hence, contrary to chicken
VtgR, the increased negative charge density in EDE of piscine VtgR is
not related to ligand binding. Instead, we propose that the EDE region
in LBR3 is more likely to play a role in the formation of the calcium
cage, which is also found in other LBRs of LDLR (9).
In this study, using RT-PCR and 5'- and 3'-RACE, we cloned a
piscine VtgR, a member of the LDLR family. The VtgR contains eight low
density lipoprotein complement type A ligand-binding repeats. Northern
analysis indicates that this receptor is expressed mainly in ovarian
tissues. Using the yeast two-hybrid system, we show that the
ligand-binding domain is sufficient to bind Vtg. The deletions of
different LBRs provide evidence that LBRs 1, 2, and 3 constitute the
major ligand-binding subdomain in VtgR for binding of Vtg. The binding
sites within Vtg are localized to 84 amino acids at the amino-terminal
of LV1, between amino acids 162 and 248. Sequence analysis of the
binding sites in Vtg and VtgR together with other members of LDLR and
apolipoprotein suggests that VtgR may interact with Vtg via
electrostatic attraction.
The piscine VtgR is highly homologous to the VtgR of amphibians, birds,
and the LDLR/VLDLR of mammals. Both VtgR and VLDLR contain eight type A
repeats in the ligand-binding domain. It was reported that the receptor
in chicken functions as both VtgR and VLDLR in different tissues (26).
In mammals, members of the LDLR family function not only in
receptor-mediated endocytosis but also in transducing signals that are
important during embryonic development (27). Given the fact that
VLDLR/VtgR plays diverse roles in different tissues, it was proposed
that they could be uniformly named LR8, according to the number of
repeats in ligand-binding domain (28). The existence of two forms of
VtgR, one with and one without O-linked sugar domain, was
also reported in other species and in other tissues (28-30). The
function of this O-linked sugar domain is still unclear. It
may be responsible for controlling receptor recycling and degradation
(31, 32). In the present study, Northern analysis showed an apparent
single transcript. This may be attributable to the difference between
the two forms of mRNA, which is subtle and very short (60 bp),
compared with full-length VtgR mRNA (~3.5 kb). Previous studies
indicated that lack of this 60-bp region would not affect the binding
of the receptors to their ligands. The presence of VtgR transcript in the nonovarian tissues suggests that there are piscine versions of
VLDLR/LDLR, which have distinct functions from VtgR in the ovarian
tissues. However, the mRNA levels of both forms of VtgR, with or
without O-linked sugar domain, in nonovarian tissues are very low. It is still not clear whether this trace amount of
transcription has any distinct biological significance.
The ligand-binding domains in members of the LDLR family have been
studied extensively. It is interesting that the highly conserved
ligand-binding domains can form promiscuous interactions with various
ligands. Analysis of the naturally occurring and engineered mutations
in LDLRs has shown that LBRs 2-6 are required for the binding of
apoB, whereas the binding of apoE depends critically on LBR5.
Previous efforts to search for the minimal binding domain in chicken
VtgR indicated that LBR3 is important for binding of RAP (25). It was
reported that the minimal binding unit in LDLR for RAP binding was any
two adjacent repeats (33). There is, however, no report on the
participation of LBRs in the interaction with Vtg. Using the yeast
two-hybrid system, we show that binding to Vtg requires LBRs 1, 2, and
3. Savonen et al. (34) reported that RAP interacts equally
well with repeats 1-3 and 1-5, but not with repeats 6-8 in VLDLR. An
analysis by Mikhailenko et al. (35) suggested that the RAP
binding site of VLDLR is located within the amino-terminal four repeats
and also suggested that the first three repeats are especially
important for RAP binding. Consistent with these reports, we also
observed the importance of the first three LBRs to binding Vtg. LBR3 is
the only repeat that contains EDE instead of the consensus sequence,
SDE. In chicken VtgR/VLDLR, this acidic region has been proposed to
bind the basic residues on RAP because it has the highest negative
charge density (25). However, according to the known structures of LBRs
1, 2, 5, and 6, many residues in these regions are involved in the formation of calcium cage and are most likely not accessible to the
ligand. In our study, site-directed mutagenesis in LBR3 affirmed that
the change of EDE sequence to SDE did not affect the binding of Vtg. In
addition, mutagenesis of SDE to EDE in LBR6 failed to gain the function
of binding Vtg. This also indicates that EDE sequence alone is
insufficient for Vtg binding. The structural determinants in different
LBRs, viz., three disulfide bonds and calcium cage-forming
amino acids, are highly conserved. The backbone structures of those
LBRs are expected to be similar. Thus the specificity of the LBRs to
different ligands may be attributable to the nonconserved acidic
groups, which are still available for interaction. The GST pull-down
assay in the presence of dithiothreitol confirms the importance of
disulfide bonds toward an appropriate architecture of LBRs for
effective binding of Vtg (Fig. 6B). This clearly
demonstrates that the primary sequence of LBD is not sufficient for
binding Vtg. The three-dimensional structure of LBD must constitute the
correct surface patch, which can recognize Vtg.
Both Vtg and apolipoprotein belong to the large lipid transfer protein,
and they were found to be evolutionarily related not only in function
but also in sequence (36). In our study, we found that the binding
region, VtgSE, showed a pattern similar to the binding sites of apoB
and apoE. The site-directed mutagenesis of the basic residues in VtgSE
further confirmed that the positively charged residue,
Lys185, plays a crucial role in receptor binding.
Lys185 is highly conserved in Vtgs. In the single deviant
case in chicken Vtg, the lysine residue is also substituted with a
basic residue, arginine (Fig. 7B). These observations
strongly suggest that electrostatic interaction is involved. The
existence of common basic residues in the receptor-binding region of
Vtg, apoB and apoE on one hand, and the inaccessibility of the
conserved acidic triplets in LBRs on the other, is apparently
contradictory to expectations for electrostatic interactions.
Nevertheless, it is still possible that electrostatic interactions
between receptors and ligands exist via other negatively charged
residues present within LBRs. This assumption is supported by reports
that all the LBRs in LDLR have negatively charged surface patch
(37).
The crystal structure of lamprey Vtg is already known (38, 39). By
alignment of the amino acid sequence, the receptor-binding region,
VtgSE, was located to the LV1n part of lamprey Vtg. LV1n contains 4 In nature, Vtg exists as a dimer containing symmetric binding sites
(40). In our study, the receptor-binding site was found to be located
in the VtgSE region. Comparable activities in the yeast two-hybrid
assays observed with VtgSE and full-length Vtg indicate that the VtgSE
region might be the only binding site in Vtg. The early study indicated
that Vtg dimer and VtgR interact in a 1:1 stoichiometry (41).
Therefore, to bind the Vtg dimer, VtgR must contain two Vtg-binding
sites. This was supported by the results of our studies in deletion
constructs of VtgR. LBRs 1-3 may contain more than one binding site
for Vtg. Fig. 9 illustrates our proposed
model of Vtg-VtgR interaction. Two molecules of Vtg dimerize through
the dimerization domain in LV1 (39). The symmetric receptor binding
sites in Vtg bind to two sites in the VtgR LBRs 1-3. The
carboxyl-terminal of Vtg will form the lipid-binding cavity to
transport lipid into the oocytes. Thus transportation of 2 Vtgs/VtgR
molecule into the oocytes presents an efficient mechanism to meet the
temporal demands of oogenesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin cDNA from zebrafish
was also labeled with DIG and used as a normalization control.
-Galactosidase Reporter Assay--
The yeast strain Y187
(Clontech) was cotransformed by the lithium acetate
method with pGAD and pGBK plasmids encoding the DNA binding domain and
activator fusion protein, respectively. As controls, the pGAD
constructs and pGBK constructs were also cotransformed with either
pGBKT7 or pGADT7 vector. The transformed yeast cells were plated on SD
minimal medium (Clontech) agar plates supplemented with -Trp-Leu DO supplement
(Clontech). The cultures were grown for 4 days at
30 °C, until colonies reached 1-2 mm in diameter. Three individual
colonies were inoculated into SD liquid cultures for
-galactosidase
assays. The
-galactosidase activities were quantified using
-galactosidase assay kits (Pierce). The absorbance of the cultures
at 600 nm was recorded. Aliquots of 350 µl of each mid-log phase
liquid culture were mixed with 350 µl of Y-PER reagent (Pierce),
followed by 300 µl of a 1 M Na2CO3 stop solution when the yellow color
appeared. As a control, 350 µl of SD medium was assayed as blank. The
absorbance of each sample at 420 nm was recorded against the blank.
-D-galactopyranoside for 4 h.
Cells harvested from 10 ml of culture were resuspended in 0.5 ml of
phosphate-buffered saline and sonicated. Triton X-100 was added to 1%,
and the cell debris was removed by centrifugation at 13,000 × g for 30 min. Glutathione-Sepharose 4B (Amersham
Biosciences) was incubated overnight at 4 °C with GST or GST-LBR
lysate, either with or without 2 mM DDT. The beads
were washed three times with 1× phosphate-buffered saline and 1%
Triton X-100 and then incubated with the second lysate of pET22-b or
pET22b-Vtg construct. The beads were washed three times with 1×
phosphate-buffered saline and 1% Triton X-100, and the eluates were
analyzed on 15% SDS-PAGE. Before using cell lysates in binding assays,
aliquots of each protein sample were analyzed on SDS-PAGE gels and
stained with Coomassie Blue to estimate protein concentrations, and
sample volumes were adjusted to equalize the amounts of fusion proteins
used in each assay.
-galactosidase assay.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The schematic representation of the structure
of VLDLR and VtgR genes. The organization of Vtg in different
domains is indicated. The positions of primers designed to clone VtgR
cDNA are annotated accordingly. Primers A1 and A2 were used to
amplify fragment A (688 bp) in the EGFP-like domain. Primers B1 and B2
were designed to replicate the conserved region in the ligand-binding
domain and EGFP-like domain, giving a product of 734 bp. Primers C1 and
C2 were used to obtain two fragments flanking the O-linked
sugar domain. Two different products, C big (656 bp) and C small (596 bp), were amplified. Primer D was used to obtain the 5' end fragment D
(872 bp) by 5'-RACE, and primer C1 was used to derive the 3' end
fragment E (1792 bp) by 3'-RACE. Primer G was used to study the
expression of two forms of VtgR by RT-PCR analysis.
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Fig. 2.
Tissue-specific expression of VtgR.
A, detection of VtgR mRNA in different tissues by
RT-PCR. mRNA at 200 ng each from ovary, liver, spleen, muscle,
intestine, brain, and heart was used as template for reverse
transcription, followed by amplification with two primers, C1 and G
flanking O-linked sugar domain. Aliquots of 10 µl from
each reaction were loaded onto the agarose gel together with 100-bp DNA
marker (M). B, Northern analysis of VtgR mRNA
in different tissues. mRNA at 200 ng each from liver, spleen,
muscle, intestine, brain, heart, and ovary was loaded onto the agarose
gel, and RNA was transferred to the membrane. The membrane was probed
with VtgR fragment B and normalized with zebrafish -actin.
-galactosidase assay confirms the binding of the
ligand-binding domain of VtgR or extracellular part of VtgR to
full-length Vtg in vivo (Fig. 3). However, full-length VtgR did not
show binding with full-length Vtg. This may be due to hindrance
by the transmembrane domain on the transportation of VtgR fusion
protein into the nucleus, where the interaction-dependent
activation of reporter gene transcription occurs in the yeast
two-hybrid assay. Although some earlier studies have indicated that the
EGFP-like domain might be required for ligand binding (21, 22), our
results clearly confirm that deletion of other parts of VtgR did not
disrupt the binding between LBRs and Vtg. The controls showed that the
interactions were not within the fusion proteins and the activation
domain or DNA-binding domain encoded by the pGADT7 or pGBKT7 vector.
These data suggest that the LBR itself is sufficient for binding Vtg.
The EGFP homology domain is not necessary for the receptor-ligand
interaction. Therefore, further studies on the receptor focused on the
LBD.
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Fig. 3.
The interaction between Vtg and different
domains of VtgR. The constructs tested for interactions are
schematically shown on the left, and the relative
-galactosidase activities quantifying the interactions are shown on
the right. The eight LBRs are numbered 1-8 in
circles. For each construct, the result was the mean of three
individual transformed colonies assayed. The
A420 nm of the
-galactosidase assay
reactions and A600 nm of the culture were
recorded. The relative
-galactosidase activities were obtained by
calculating the ratio of A420 nm to
A600 nm. Full-length Vtg was fused with the
activation domain (AD), and the different constructs of VtgR
were fused with the DNA-binding domain (BD). To rule out
nonspecific binding between the Vtg/VtgR and AD/BD backbones, the
constructs containing Vtg or VtgR domains were also assayed for
potential interactions with the vector backbones, pGADT7 or
pGBKT7.
-galactosidase activities most probably arose from the difference in
the interaction.
-galactosidase activities were observed. LBR2 alone did not show
significant binding to Vtg. When both the first and second LBRs were
deleted, all 11 constructs lost binding of Vtg. It is therefore clear
that the first three LBRs are most important for binding Vtg. Without
LBR3, the construct LBR1-2 exhibited weaker interaction with Vtg. On the other hand, constructs containing only LBR3 did not bind Vtg. Similarly, the deletion of LBR1 from various combinations of LBD (viz., constructs LBR2-8, LBR2-7, LBR2-6, LBR2-4, and
LBR2-3) attenuated but did not abolish the interaction with Vtg. These data indicate that the interaction between LBD and Vtg utilizes more
than one LBR in the LBD. There may be individual binding sites in the
first three LBRs. However, LBRs 1-3 are not mutually exclusive
in their interaction with Vtg. It is possible that one single binding
site is insufficient to stabilize the interaction. Thus, the minimum
ligand-binding region is a combination between either LBR1 and LBR2 or
LBR2 and LBR3 or LBR1 and LBR3. Thus, the region required for maximal
binding of Vtg is LBRs 1, 2, and 3.
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Fig. 4.
The interactions between LBD of VtgR
and different deletion constructs of Vtg. Seven carboxyl-terminal
deletion constructs of Vtg and full-length Vtg were fused with
activation domain (AD) and assayed for their interaction
with the LBD of VtgR. The first three LBRs are most critical for
VtgR-Vtg interaction. Individually, LBR1, LBR2, or LBR3 is
nonfunctional, thus suggesting the global effects of LBRs 1-3 on
binding Vtg.
-galactosidase assay shows that this VtgSE fragment
has a binding capacity similar to that of full-length Vtg (Fig.
6A), thus indicating that this
region contains the crucial binding site for VtgR and that deletion of
other parts of Vtg does not seem to affect its binding to receptor.
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Fig. 5.
The LBD binds to the amino-terminal region of
Vtg. The LBD of VtgR was fused with DNA-binding domain
(BD). The Vtg deletion constructs are demarcated by the
restriction sites, and the lengths are indicated by the amino acid
positions on the Vtg molecule. This result indicates that the
amino-terminal Vtg region upstream of EcoRI contains the
binding site for VtgR.
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Fig. 6.
Direct binding between VtgSE and VtgR is
confirmed by GST pull-down assay. A, yeast two-hybrid
assay of the interaction between VtgSE and the LBD of VtgR. VtgSE was
fused with the activation domain (AD) and tested for
interaction with the LBD of VtgR. As the positive control, the
interaction between full-length Vtg and the LBD of VtgR was also
assayed. B, in vitro direct binding between VtgSE
and LBD shown by GST pull-down. GST-LBD was expressed in E. coli and coupled to glutathione beads with or without 2 mM dithiothreitol. After washing, the expressed VtgSE was
incubated with the beads. The unbound proteins were washed away, and
the bound proteins were analyzed on SDS-PAGE. The protein bands were
visualized by Coomassie Blue staining. Interaction between GST (without
LBD) and pET22-b vector backbone was used as a negative control.
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Fig. 7.
Sequence analysis of the binding region in
Vtg and VtgR. A, the binding region of Vtg. A short
fragment of Vtg was aligned with the binding sequences of apoB and
apoE. The positively charged residues His182,
Lys185, and Lys187 are labeled with
arrows. B, the alignment of the 8-amino acid
binding region of 27 Vtg sequences in different species. The highly
conserved Lys185 is highlighted. C,
the eight ligand-binding repeats in the LBD of O. aureus
VtgR are compared with each other. The conserved amino acids are
highlighted according to the different homology level.
Glu144 in LBR3 was selected for subsequent
mutagenesis.
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Fig. 8.
The effect of mutation in Vtg on its
interaction with VtgR. Different mutation constructs were fused
with the activation domain (AD), and the interactions
between the mutation constructs and LBD containing LBR1-8 or LBR1-3
were examined. The mutation K185A attenuated the interaction. This
indicates that the positively charged Lys185 is important
for the Vtg-VtgR interaction.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
helices and 12
strands, 11 of which form a barrel-like conformation. It is not clear whether this VtgSE receptor site is in
the
strands or the
helices. Using the secondary structure prediction program, we predict that VtgSE is located in the
helices
(data not shown). In both apoB and apoE, the receptor-binding region is
in the
helices conformation, thus suggesting that they may utilize
a similar mechanism for binding.
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Fig. 9.
The model of Vtg-VtgR interaction. The
LBRs are labeled 1-8. The EGFP-like repeats A, B, and C are
annotated in VtgR. In a stoichiometric ratio of interaction between
2Vtgs:1VtgR, the Vtg dimer facilitates the binding of the LBRs 1-3 via
the SE fragment (amino acids 162-248) in each amino-terminal of the
Vtgs. There is more than one ligand-binding site within LBRs
1-3, and binding of VtgR to Vtg requires the combination of these
three LBRs, which make electrostatic contacts with the VtgSE regions of
the Vtg dimer.
The receptor-ligand pairs of VtgR-Vtg and LDLR/VLDLR-apolipoprotein
have existed together in fish, amphibians, reptiles, and birds for
millions of years (42, 43). In the lower species, including insects and
nematode, VtgR was the predominant form of receptor. The existence of
VtgR in more ancient species such as Caenorhabditis elegans
indicates that the LDLR and VLDLR might have evolved from VtgR by
mutation and gene shuffling, and Vtg is the ancestor of apolipoprotein.
It is accepted that apoB and Vtg share a common ancestor. However, the
amino-terminal location of the receptor-binding site in Vtg is in
contrast to apoB, which contains receptor-binding site in the
carboxyl-terminal. This difference may arise from the structural change
for better adaptation to the function. The mode of binding of
LDLR/VLDLR to apolipoprotein is inherited from the electrostatic
attraction of VtgR-Vtg. However, the sequence changes in apoB,
especially in the region for lipid binding, probably facilitate the
specific function of lipid transportation (39, 44). Responsively, the
mutation in LDLR/VLDLR in different LBRs accumulates for the improved
specific binding to apoB, thus LDLR and VLDLR utilize different LBRs
for apoB binding. This coevolution of receptor-ligand pairs
probably creates the current functionally and structurally distinct
receptors and ligands. The receptor-binding sites in many ligands of
LDLR family members are still unknown. We predict that they also
contain domains rich in basic residues for binding electrostatically
with their cognate receptors.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Ge Ruowen for generously providing zebrafish actin cDNA clones. We also thank Subha, Lim Eng Hwa, Li Haifeng, and Jaspal Kumar for excellent technical assistance.
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FOOTNOTES |
---|
* This work was supported in part by National University of Singapore Grant RP399900/A.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF514281.
Recipient of a postgraduate research scholarship from the National
University of Singapore.
§ To whom correspondence should be addressed: Dept. of Biological Sciences, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260. Tel.: 65-68742776; Fax: 65-67792486; E-mail: dbsdjl@nus.edu.sg.
Published, JBC Papers in Press, November 11, 2002, DOI 10.1074/jbc.M205067200
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ABBREVIATIONS |
---|
The abbreviations used are: Vtg, vitellogenin; VtgR, vitellogenin receptor; apo, apolipoprotein; DIG, digoxigenin; EGFP, epidermal growth factor precursur; LBD, ligand-binding domain; LBR, ligand-binding repeat; LDLR, low density lipoprotein receptor; RAP, receptor-associated protein; RT-PCR, reverse transcription-PCR; DDT, dithiothreitol; SD, selective dextrose; VLDLR, very low density lipoprotein receptor; LV, lipovitellin; GST, glutathione S-transferase; RACE, rapid amplification of cDNA ends; CSPD, disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2'-(5'-chloro) tricyclo[3,3,1,13,T]decan}-4-yl)phenyl phosphate.
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