From the The assembly of triglyceride-rich lipoproteins
requires the formation in the endoplasmic reticulum of a complex
between apolipoprotein B (apoB), a microsomal triglyceride transfer
protein (MTP), and protein disulfide isomerase (PDI). In the MTP
complex, the amino-terminal region of MTP (residues 22-303) interacts
with the amino-terminal region of apoB (residues 1-264). Here, we
report the identification and characterization of a site on apoB
between residues 512 and 721, which interacts with residues 517-603 of
MTP. PDI binds in close proximity to this apoB binding site on MTP. The
proximity of these binding sites on MTP for PDI and amino acids
512-721 of apoB was evident from studies carried out in a yeast
two-hybrid system and by co-immunoprecipitation. The expression of PDI
with MTP and apoB16 (residues 1-721) in the baculovirus expression system reduced the amount of MTP co-immunoprecipitated with apoB by
73%. The interaction of residues 512-721 of apoB with MTP facilitates lipoprotein production. Mutations of apoB that markedly reduced this
interaction also reduced the level of apoB-containing lipoprotein secretion.
Apolipoprotein B (apoB)1
is an obligatory component of chylomicrons, very low density
lipoproteins and low density lipoproteins, which transport lipid to all
body tissues. The assembly of apoB-containing lipoproteins has an
absolute requirement for a microsomal triglyceride transfer protein
(MTP) complexed to an endoplasmic reticulum-resident chaperone, protein
disulfide isomerase (PDI) (1-3). The MTP complex facilitates the
loading of apoB with lipid. ApoB that does not acquire sufficient lipid
to form a lipoprotein is rapidly degraded by the proteosome (4-7).
ApoB and MTP, along with the major lipid transport proteins of
arthropods, share a common ancestry with the vitellogenins (VTGs) of
nematodes (8). The VTGs transport lipid to the yolk sac (9). We have
used the crystal structure of lamprey lipovitellin (LV) (10), the
mature product of VTG, to derive molecular models of the amino termini
of MTP and apoB (8). In the modeled structures, the amino-terminal
~300 residues of each protein form a MTP and apoB interact during lipoprotein assembly (15, 16). We have
studied this interaction with a series of carboxyl-terminally truncated
forms of apoB (8). The results established that residues 1-264 of apoB
(apoB5.8) interact with the predicted Yeast Two-hybrid--
Vectors pSB202 and pJG4-5, the reporter
plasmid pSH18-34, and EGY48 were gifts from Professor Brent (Harvard
Medical School, Boston, MA). MTP cDNAs were fused to the amino
terminus of LexA. ApoB and PDI were fused to the B42 transcription
activation domain. The PDI-aeb construct encodes amino acids 18-274 of
PDI, representing the first three domains of PDI (designated "aeb"
by Freedman et al. (17)), and was constructed from a cDNA
clone provided by Professor Kivirikko (Collagen Research Unit,
University of Oulu, Finland). Transformations were undertaken as
described (18).
Sequence Alignment--
A WU-BLASTP search of the Protein Data
Bank, SwissProt+SPubdate+PIR, and the nonredundant GenBank Coding
Sequences translation data bases was performed with residues 1-1000 of
lamprey LV, using blosum 62 as the scoring matrix. The most significant
matches were tobacco hornworm apolipophorin, human (Homo
sapiens) apoB, Drosophila melanogaster retinoid/fatty
acid binding protein, and H. sapiens MTP (p
values of 3.4 × 10 Expression Constructs--
Details of oligonucleotides are
available on request (from C. C. S.). Polymerase chain reaction and
restriction enzymes were used to manipulate the MTP, apoB, and PDI
sequences. Epitope tags were fused in-frame to the carboxyl termini of
cDNAs and juxtaposed to a terminator codon. The baculovirus vectors
were pVL1392 and 1393 (Invitrogen BV, Leek, The Netherlands). All
constructs were sequenced.
Baculovirus Expression--
Cells were maintained in Grace's
medium (Life Technologies, Inc.), supplemented with 10%
insect-qualified fetal calf serum (Life Technologies, Inc.).
Transfections were with liposomes, linearized BacPAK 6 viral DNA
(CLONTECH), and the appropriate baculovirus transfer plasmid. The
apoA-I virus was a gift from Dr. David Booth (Imperial College School
of Medicine, London). Infections were at a multiplicity of 2.5. Cells
were harvested 42-46 h postinfection. Cells for labeling were washed
and resuspended in 7 ml of methionine-free Grace's medium and gently
agitated at 27 °C for 45 min. Labeling was for 75 min with 0.43 mCi
of L-[35S]methionine (Pro-Mix, Amersham
International PLC). Analysis of expression was undertaken on microsomal
fractions at 4 °C. Cells were washed in phosphate-buffered saline,
pH 7.4, homogenized in 0.25 M sucrose containing 20 mM imidazole, pH 7.4, and protease inhibitors, layered onto
a gradient of 1.8 M sucrose in 20 mM imidazole
and 0.5 M sucrose in 20 mM imidazole, and
centrifuged at 100,000 × g for 60 min. The pellicle of
microsomes at the 0.5-1.8 M sucrose interface was
resuspended in buffer A (10 mM Tris, pH 7.4, containing 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, and
protease inhibitors). ApoB, apoA-I, and MTP-FLAG were
immunoprecipitated with saturating quantities of sheep anti-human apoB
(Boehringer Mannheim from Roche Diagnostics Ltd.), anti-apoA-I (Genzyme
Diagnostics), rabbit anti-human MTP antibodies, or anti-FLAG M2
affinity gel (Sigma), respectively. Immobilized proteins were washed
with buffer A and recovered by boiling in SDS sample buffer.
Expression of ApoB and MTP in COS-1 Cells--
The details of
the coordinate expression of MTP and apoB have been described, as have
the L-[35S]methionine labeling of cells and
the immunoprecipitation of apoB (2). The chase was for 3 h.
To screen for sites of interaction between apoB and MTP, distal to
residues 1-264 of apoB, we used a yeast two-hybrid system. Short,
overlapping cDNAs encoding residues 247-1147 of apoB (apoB5-25) (Fig. 1A) were assayed for
interaction with the MTP amino-terminal (residues 22-304), predicted
To fine map the region on MTP that interacts with apoB, predicted
helices 1-8 (residues 297-442), 9-13 (residues 447-529), and 13-17
(residues 517-603) of MTP were expressed with residues 349-583
(apoB8-13) and 512-721 (apoB11-16) of apoB (Fig. 1C). The
interactions with predicted helices 13-17 of MTP were ~10- and
5-fold higher than with predicted helices 1-8 and 9-13, respectively. These results indicate that residues 512-721 (apoB11-16) of apoB interact with helices 13-17 of MTP, which also form a major binding site for PDI (8). The PDI binding site on MTP was also mapped in a
yeast two-hybrid system and corroborated by the characterization of
mutant forms of full-length MTP expressed in COS-1 and Sf9 cells.
To fine map the region on apoB that interacts with MTP, we examined the
interaction of subfragments of apoB11-16 (residues 512-721) with
predicted helices 13-17 of MTP (Fig. 1D). The removal of
residues 512-524 from the amino terminus of residues 512-721 of apoB
(apoB11-16) abolished the interaction. The deletion of residues
701-721 and 664-721 from the carboxyl terminus of residues 512-721
of apoB (apoB11-16) reduced the interaction by 29 and 49%,
respectively. These data indicate that helices 13-17 of MTP (residues
517-603) interact with a site encompassing predicted helices 13-17
(residues 512-592) of apoB and the carboxyl-terminal portion (residues
593-721) of apoB16. The homologous interacting regions of apoB and MTP
(Fig. 2A) are encoded by exons
with conserved intron demarcations (Fig. 2, A and
B).
Medical Research Council Molecular Medicine
Group,
Gene and Genome Evolution Group,
ABSTRACT
Top
Abstract
Introduction
References
INTRODUCTION
Top
Abstract
Introduction
References
-barrel. The next 300 residues form a double-layered
-helical structure containing 17 helices. The carboxyl-terminal portion of this structure is stabilized
by a conserved buried salt bridge, which in lamprey LV underlies the
homodimerization interface (10). In MTP, the surfaces of outer helices
15 and 17 form the major PDI binding site (8). This interaction is
required for the production of soluble and active MTP (11, 12) and may,
in addition, anchor MTP at the site of apoB translocation, as PDI alone
contains the "KDEL" endoplasmic reticulum retention sequence (13,
14).
-barrel of MTP (8). In the
present study, we have identified a second region of apoB that
interacts with a binding site on MTP in close proximity to the major
PDI binding site.
MATERIALS AND METHODS
16, 2.2 × 10
14, 1.6 × 10
12, and 2.7 × 10
8, respectively). GenBank accession numbers are X75500
(H. sapiens MTP), Y00354 (Xenopus laevis VTG),
M88749 (Ichthyomyzon unicuspus (lamprey) VTG), and X03044
(Caenorhabditis elegans VTG 5). Sequences were aligned with
the Clustal version W11.6 program (19) using default values and minimal
manual adjustments, without reference to the position of intron/exon boundaries.
RESULTS
-helical (residues 298-603), and carboxyl-terminal domains
(residues 604-894). ApoB residues 349-583 (apoB8-13) and 512-721
(apoB11-16) produced an interaction with the predicted
-helical
domain of MTP (Fig. 1B). The strongest interaction was with
apoB residues 512-721 (apoB11-16). No other site of interaction was
detected (data not shown).
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Fig. 1.
Interaction of apoB with MTP in a yeast
two-hybrid system. A, ApoB subfragments used to map the
interaction of apoB with MTP. The domain organization of apoB is based
on the primary and predicted secondary structural homology with lamprey
LV (8). B, residues 349-583 (apoB8-13) and 512-721 of
apoB (apoB11-16) interact with the predicted -helical domain of
MTP. C, interaction of helices 13-17 of MTP with apoB8-13
and apoB11-16. The control plasmid encodes residues 918-1147 of apoB.
D, interaction of subfragments of apoB11-16 (residues
512-721) with predicted helices 13-17 of MTP. In B, C, and
D,
-galactosidase activity was assayed in duplicate as
described (18). Values (n
10) are mean ± S.D.
aa, amino acids.
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Fig. 2.
Structural relationships between the VTGs,
apoB, and MTP. A, sequence alignment of helices 13-17
of lamprey LV (I.u) with H. sapiens
(H.s) apoB, H.s MTP, X. laevis
(X.l) VTG, and C. elegans (C.e) VTG 5. The percentage identities and similarities of the helical domain
(residues 298-607) of lamprey LV with the predicted -helical
domains (22) of H.s apoB and MTP are 24 and 44.3 and 18.2 and 37.9, respectively. The arrowheads indicate salt bridge
residues. The bracketed arrowheads indicate the use of an
alternative glutamate in the MTPs. With the exception of apoB, numbers
include the signal peptide. Residues on a black background
are identical in all five sequences. Residues in gray boxes
are identical in four sequences. Residues in white boxes
have similarities of >0.5 (23) and are conserved in
4 sequences. For
apoB, MTP, and X.l VTG, the first and last residues of this
exon are underlined. Cylinders indicate
-helices. B, the interacting regions of apoB and MTP are
encoded by conserved exons. Black and gray
triangles denote conserved boundaries (within 12 nucleotides). The
codon sequence at each conserved boundary is interrupted at the same
position. White triangles denote remaining boundaries
(24-26). The narrow black box highlights the conserved
intron demarcations of exon sequences encoding residues 520-663 of MTP
and residues 514-663 of apoB. SP, signal peptide.
C, molecular model of helices 13-17 of MTP and of the
buried salt bridge formed between Arg-540, Glu-570, and Asn-531.
Tyr-554, Met-555, Lys-558, Ile-592, Arg-594, and Arg-595 perturb the
binding of apoB or PDI to MTP in the yeast two-hybrid system. Val-520,
Lys-521, and Arg-526 (shaded) make no significant
contribution to either apoB or PDI binding. D, molecular
model of helices 13-17 of apoB and of the buried salt bridge. In
C and D, side chains are depicted as van der
Waals spheres.
In view of the co-localization of the binding sites for PDI and
residues 512-721 of apoB (apoB11-16) to helices 13-17 of MTP, we
examined whether mutations of MTP that disrupted the interaction of MTP
with PDI (8) might also impair apoB binding. MTP R540A and R540H, which
disrupt the predicted buried salt bridge, and a series of mutant MTPs
that had been used to map the PDI binding site (8) were expressed with
either PDI (residues 18-274) or apoB (residues 512-721). The
predicted positions of the salt bridge residue (Arg-540) and of mutated
residues Val-520, Lys-521, Arg-526, Tyr-554, Met-555, Lys-558, Ile-592,
Arg-594, and Arg-595 at the surface of helices 13-17 of MTP are shown
in Fig. 2C. MTP R540H and double mutant R594A/R595A had a
more marked effect on the interaction of MTP with apoB than on the
interaction of MTP with PDI (Fig. 3).
Mutants R540A, Y554A/M555A, Y554A/M555A/K558A, and Y554A/M555A/I592A
were more deleterious for the interaction of MTP with PDI. The
disruption for mutants V520A, K521A, R526A, and I592A was similar for
both proteins (Fig. 3). These results suggest that the apoB and PDI
binding sites on the -helical region of MTP are in close
proximity.
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The proximity of binding sites on MTP for apoB and PDI was further evaluated by studies performed in a baculovirus expression system (Fig. 4). ApoB was co-immunoprecipitated with MTP using anti-MTP antibodies, whereas MTP was co-immunoprecipitated with anti-apoB antibodies. The coordinate expression of PDI reduced the amount of apoB17 (residues 1-781) co-immunoprecipitated with MTP as detected by Western blotting (Fig. 4A). This reduction was also visually evident with apoB16 (residues 1-721) but not with apoB13 (residues 1-590) or apoB11 (residues 1-499) (Fig. 4B). Rather, PDI expression increased the amount of apoB11 and apoB13 co-immunoprecipitated with MTP by around 25%. This increase corresponds to a 3-fold higher level of MTP in these cells, presumably because of increased solubilization of MTP by PDI (Fig. 4B). Thus, on a mole to mole basis, PDI substantially reduced the amount of apoB11 and apoB13 co-immunoprecipitated with MTP. Similar results were obtained in an independent experiment in which MTP was co-immunoprecipitated with anti-apoB antibodies. PDI reduced the amount of MTP co-immunoprecipitated with apoB16 (residues 1-721), apoB13 (residues 1-590), and apoB11 (residues 1-499) by 73, 52, and 29%, respectively (Fig. 4C). No reduction was observed with apoB5.8 (residues 1-264). These results support the close proximity of the binding sites on MTP for residues 512-721 of apoB and 18-274 of PDI and, in addition, indicate that the amino-terminal limit of this apoB binding site may extend further upstream than residue 499 of apoB.
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Next, we evaluated whether mutations in predicted helices 13-17
(residues 512-592) of apoB (Fig. 2A) might disrupt the
binding of amino acids 512-721 of apoB to MTP. The predicted buried
salt bridge residues Arg-531 and Glu-557 in apoB (Fig. 2D)
were individually replaced with alanine. R531H was also created,
because the replacement of the homologous residue (Arg-540) in MTP with
histidine causes abetalipoproteinemia (20). In the yeast two-hybrid
system, R531A, R531H, and E557A reduced the interaction of helices
13-17 of MTP with amino acids 512-721 of apoB to 20.4 ± 6, 9.2 ± 3.2, and 56.1 ± 26% of wild type, respectively (Fig.
5A). To examine the
consequence of this loss of binding between apoB and MTP for
lipoprotein assembly, R531A, R531H, and E557A were introduced into
apoB36, and their impact on the secretion of apoB-containing
lipoproteins from COS-1 cells was examined. Mutants R531H and R531A,
which had markedly reduced the interaction of amino acids 512-721 of
apoB with MTP in the yeast two-hybrid system, reduced apoB secretion,
whereas mutant E557A did not (Fig. 5B). Secretion levels
were 20, 53, and 95% of wild type, respectively. Thus, the loss of the
interaction between amino acids 512-721 (apoB11-16) of apoB and the
carboxyl terminus of the predicted -helical domain of MTP is
deleterious for the production of apoB-containing lipoproteins.
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DISCUSSION |
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In this study, we have identified a region on MTP that interacts with both apoB and PDI. The delineation of the apoB and MTP binding sites has been facilitated by the use of well characterized molecular models of residues 294-592 and 304-598 of apoB and MTP, respectively. The models were generated from the crystal structure of lamprey LV (10). The structural relationship between MTP, apoB, and VTG is supported by conservation of gene structure, primary sequence, predicted secondary structure, and site-directed mutagenesis (8).
We have previously shown that the predicted amino-terminal -barrels
of apoB and MTP (first ~300 residues) interact (8). Here, we describe
the interaction between residues 512-721 of apoB and helices 13-17
(residues 517-603) of MTP. This interaction was identified and
characterized in a yeast two-hybrid system. The binding site on MTP for
apoB is in close proximity to the major PDI binding site. Mutation of
the buried salt bridge residue Arg-540 of MTP and of residues at the
surface of predicted helices 15 (Tyr-554, Met-555, Lys-558) and 17 (Ile-592, Arg-594, Arg-595) impair the interaction of MTP with apoB and
PDI, whereas the equivalent mutation of residues in helix 13 (Val-520,
Lys-521, and Arg-526) of MTP do not. Accordingly, we propose that apoB
and PDI have binding sites centered on predicted helices 15-17
(residues 556-598) of MTP. The fact that 4 of the 10 MTP mutations
were more deleterious for the interaction of MTP with PDI than with
residues 512-721 of apoB, whereas 2 were more deleterious for the
interaction between MTP and apoB, suggests that apoB and PDI do not
bind to the same site on MTP. However, the mutation data do not exclude
the possibility that the critical residues on MTP for binding to apoB
and PDI may differ and that these critical residues form part of a
common binding site.
An interaction between MTP and residues 270-570 of apoB has recently
been reported by Hussain et al. (21) in a third experimental system. The present study provides strong evidence that this apoB binding site encompasses helices 13-17 (residues 514-592) of the predicted -helical domain of apoB, as well as structural motifs formed by residues 640-721 at the carboxyl-terminal end of apoB16. The
site may also include residues 430-511 of apoB, which are predicted to
form helices 9-12 of its
-helical structure (8). In the
experimental system of Hussain et al. (21), an expression construct encoding residues 270-509 of apoB produced an interaction with MTP that was 58% of that observed with residues 270-570 of apoB,
whereas a corresponding construct that comprised residues 270-430 of
apoB produced virtually no interaction. These results, combined with
our yeast two-hybrid data, indicate that the interaction of apoB with
helices 13-17 of the predicted
-helical domain of MTP is centered
on residues 512-592 (apoB11-apoB13) of apoB and includes flanking
residues 430-511 (apoB9-apoB11) and 640-721 (apoB14-apoB16).
Our baculovirus expression studies also indicate that PDI and amino
acids 512-721 of apoB bind to the same helical region of MTP. From our
previous study (8) and the present yeast two-hybrid data we made two
predictions. First, we predicted that the coordinate expression of PDI
with MTP would not impair the interaction of residues 1-264 of apoB
(apoB5.8) with the predicted -barrel of MTP, which is confirmed in
this study. Second, we predicted that PDI would reduce the interaction
of MTP with apoB16 (residues 1-721), apoB13 (residues 1-590), and
apoB11 (residues 1-499) and that the maximum reduction would be with
apoB13 and apoB16. Thus, we show that PDI expression successively
reduced the interaction of MTP with apoB11 (residues 1-499), apoB13
(residues 1-590), and apoB16 (residues 1-721).
The present study demonstrates that disruption of predicted helices 13-17 (residues 512-592) at the center of the distal binding site on apoB impairs the secretion of apoB-containing lipoproteins. Thus, we show that the mutation of the apoB buried salt bridge residue, Arg-531, which virtually abolished the interaction of MTP with apoB in the yeast two-hybrid system, had a marked impact on lipoprotein secretion. These results indicate that the assembly of apoB-containing lipoproteins is perturbed by the loss of interaction of the distal apoB binding site (residues 512-721) with MTP. An alternative explanation would be that the apoB Arg-531 mutation supports lipoprotein assembly but impairs the secretion of apoB-containing lipoproteins through aberrant misfolding. The present study does not directly address this issue. However, our data and those of Hussain et al. (21) are consistent with a loss of interaction between residues 512-721 of apoB and MTP as being the primary cause of reduced secretion. Incubation of cells with a pharmacological agent that specifically blocks this interaction represents an attractive strategy for determining its importance for lipoprotein production.
The location of the two binding sites on apoB and MTP would serve to
align nascent apoB in a head to head orientation with MTP. This would
position the predicted lipid binding domain of MTP with the lipid
binding structures of apoB and presumably facilitate the transfer of
lipid from MTP to apoB. The proximity of the distal apoB binding site
and of the PDI binding site on MTP raises the question as to how this
arrangement might facilitate lipoprotein assembly. It will be important
to establish whether apoB displaces PDI from MTP only to be replaced
again by PDI as lipidation progresses in the distal parts of the
secretory pathway.
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ACKNOWLEDGEMENTS |
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We are grateful to the British Medical Research Council and the British Heart Foundation and to Tamsin Grantham for technical assistance.
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FOOTNOTES |
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* This work was supported by the British Medical Research Council and by Grants PG/97011 and PG/98032 from the British Heart Foundation.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.
Recipient of a research award from the Bristol-Myers Squibb Corporation.
§§ To whom correspondence should be addressed: MRC Molecular Medicine Group, Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Rd., London, W12 0NN, U. K. Tel: 44-181-383-8308; Fax: 44-181-383-2028; E-mail: cshoulde{at}rpms.ac.uk.
The abbreviations used are: apoB, apolipoprotein B; LV, lipovitellin; MTP, microsomal triglyceride transfer protein; PDI, protein disulfide isomerase; VTG, vitellogenin.
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REFERENCES |
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