Solid-phase binding assays, performed with recombinant fragments corresponding to the C-terminal domain of a-DG harboring progressive deletions, have shown that the b-DG-binding epitope
Trang 1to the b-dystroglycan ectodomain strongly inhibits the
interaction with a-dystroglycan in vitro
Manuela Bozzi1,*, Francesca Sciandra2,*, Lorenzo Ferri2, Paola Torreri3, Ernesto Pavoni2,
Tamara C Petrucci3, Bruno Giardina2and Andrea Brancaccio2
1 Istituto di Biochimica e Biochimica Clinica, Universita` Cattolica del Sacro Cuore, Rome, Italy
2 CNR, Istituto di Chimica del Riconoscimento Molecolare c ⁄ o Istituto di Biochimica e Biochimica Clinica, Universita` Cattolica del Sacro Cuore, Rome, Italy
3 Dipartimento di Biologia Cellulare e Neuroscienze, Istituto Superiore di Sanita`, Rome, Italy
Dystroglycan (DG) is an adhesion molecule composed
of two subunits, a-DG and b-DG [1], encoded by a
single gene, dag1, which produces a unique polypeptide
precursor consisting of 895 amino acids A post-trans-lational cleavage, performed by a still unidentified pro-tease at the Gly653-Ser654 site, produces two subunits,
Keywords
alanine scanning; cell transfection;
dystroglycan; protein–protein interaction;
site-directed mutagenesis
Correspondence
A Brancaccio, CNR, Istituto di Chimica del
Riconoscimento Molecolare c ⁄ o Istituto di
Biochimica e Biochimica Clinica, Universita`
Cattolica del Sacro Cuore, Largo Francesco
Vito 1, 00168 Rome, Italy
Fax: +39 6 3053598
Tel: +39 6 3057612
E-mail: andrea.brancaccio@icrm.cnr.it
*These authors contributed equally to this
work
(Received 10 July 2006, revised 1 September
2006, accepted 6 September 2006)
doi:10.1111/j.1742-4658.2006.05492.x
The dystroglycan adhesion complex consists of two noncovalently interact-ing proteins: a-dystroglycan, a peripheral extracellular subunit that is extensively glycosylated, and the transmembrane b-dystroglycan, whose cytosolic tail interacts with dystrophin, thus linking the F-actin cytoskele-ton to the extracellular matrix Dystroglycan is thought to play a crucial role in the stability of the plasmalemma, and forms strong contacts between the extracellular matrix and the cytoskeleton in a wide variety of tissues Abnormal membrane targeting of dystroglycan subunits and⁄ or their aberrant post-translational modification are often associated with several pathologic conditions, ranging from neuromuscular disorders to carcinomas A putative functional hotspot of dystroglycan is represented
by its intersubunit surface, which is contributed by two amino acid stret-ches: approximately 30 amino acids of b-dystroglycan (691–719), and approximately 15 amino acids of a-dystroglycan (550–565) Exploiting alanine scanning, we have produced a panel of site-directed mutants of our two consolidated recombinant peptides b-dystroglycan (654–750), corresponding to the ectodomain of b-dystroglycan, and a-dystroglycan (485–630), spanning the C-terminal domain of a-dystroglycan By solid-phase binding assays and surface plasmon resonance, we have determined the binding affinities of mutated peptides in comparison to those of wild-type a-dystroglycan and b-dystroglycan, and shown the crucial role of two b-dystroglycan phenylalanines, namely Phe692 and Phe718, for the a–b interaction Substitution of the a-dystroglycan residues Trp551, Phe554 and Asn555 by Ala does not affect the interaction between dystroglycan subunits in vitro As a preliminary analysis of the possible effects of the aforementioned mutations in vivo, detection through immunofluorescence and western blot of the two dystroglycan subunits was pursued in dystro-glycan-transfected 293-Ebna cells
Abbreviations
DG, dystroglycan; DGC, dystroglycan–glycoprotein complex; EGFP, enhanced green fluorescent protein; SPR, surface plasmon
resonance.
Trang 2a-DG and b-DG a-DG is a highly glycosylated
per-ipheral membrane protein that interacts with several
extracellular matrix proteins such as laminin, perlecan
and agrin [2] b-DG spans the membrane and binds
a-DG in a noncovalent way, providing a connection
between the extracellular matrix and the cytoskeleton
inside the cells, where it interacts with dystrophin,
utrophin and other cytosolic proteins, such as rapsyn,
caveolin-3 and Grb2 [3–5] Together with sarcoglycans,
dystrobrevins, syntrophins, and sarcospan, DG forms
the dystrophin–glycoprotein complex (DCG), which
plays an essential role as a scaffold for cells in muscle
and in a wide variety of nonmuscle tissues [6,7],
inclu-ding the central and peripheral nervous systems, and
several epithelial tissues [8]
The importance of the DCG is dramatically
appar-ent in several forms of muscular dystrophy, where
mutations in DCG proteins lead to instability and
pro-gressive weakness of the muscle fibers [9] Although no
natural mutations have been detected in DG, it is
sub-stantially altered or absent in muscular dystrophies
For this reason, detailed molecular characterization of
the subunit interface should be considered of primary
importance for our understanding of the overall
stabil-ity of the DGG, and perhaps in the future for surgical
modulation of its function with the purpose of
allevi-ating severe human diseases [10]
Primary structure analysis and electron microscopy
have shown that a-DG has a dumbbell-like structure
organized in two globular domains, the N-terminal and
C-terminal domains, connected by an elongated central
mucin-like region that contains highly glycosylated
sequences, rich in prolines, serines and threonines [11]
A structural characterization of the N-terminal domain
was recently obtained by a crystallographic analysis
carried out on a murine a-DG N-terminal fragment,
and revealed the presence of two autonomous modules
connected by a long and flexible linker The N-terminal
module shows Ig-like folding, whereas the C-terminal
module appears to be very similar to the ribosomal
RNA-binding proteins [12] The only structural hints
concerning the C-terminal domain of a-DG come from
a sequence alignment approach, which has shown some
similarities with cadherin domains [13]
Previous studies, carried out employing a series of
independent techniques such as IR, CD [14] and NMR
spectroscopy [15], have revealed the absence of any
classic secondary structural element in the recombinant
b-DG ectodomain, which shows high conformational
plasticity, typical of a natively unfolded protein
The noncovalent interaction between the two DG
subunits occurs between the C-terminal region of
a-DG and the N-terminal ectodomain of b-DG, and is
apparently independent of glycosylation [16] Solid-phase binding assays, performed with recombinant fragments corresponding to the C-terminal domain of a-DG harboring progressive deletions, have shown that the b-DG-binding epitope resides between amino acids 550 and 585 [17], and further NMR analysis has narrowed this location to amino acids 550–565 [18] In addition, extensive NMR structural characterization
of our 15N⁄13C b-DG(654–750) recombinant fragment, spanning the b-DG ectodomain, suggested that the a-DG-binding epitope corresponds to an amino acid stretch located between positions 691 and 719 [15]
In order to identify the specific amino acids within the linear interacting epitopes involved in the complex between a-DG and b-DG, alanine scanning of some of the residues that were mainly influenced in NMR titra-tions [15] was performed on recombinant fragments a-DG(485–630) and b-DG(654–750) The reciprocal affinities of wild-type a-DG and b-DG peptides vs the panel of mutated recombinant fragments were meas-ured using two independent techniques: solid-phase binding assays, exploiting biotinylated recombinant lig-ands, and surface plasmon resonance (SPR), in which one peptide is covalently immobilized on a sensor chip while the other is used in soluble phase without labels Such mutations were also imported into full-length
DG constructs cloned into an appropriate vector to transfect eukaryotic cells, in order to set up a suitable cellular system to study the effect of site-directed mut-agenesis on the processing and targeting of DG in vivo
Results
Mutations within the b-DG(654–750) recombinant fragment
The phenylalanine in position 718, belonging to the putative a-DG-binding epitope (691–719) within the ectodomain of b-DG, was found to be one of the most influenced residues during the titration of [15N]b-DG(654–750) with thioredoxin-a-DG(485–620) [15] Therefore, we decided to mutate it to an alanine, together with two other phenylalanines, Phe692 and Phe700, the only other aromatic residues located within the a-DG-binding epitope that are highly con-served in all the species so far analyzed A more dras-tic alteration of the protein primary structure was produced by deleting six amino acids, located within the a-DG-binding epitope, between positions
701 and 706 A previous NMR characterization of the b-DG ectodomain [15] revealed that the amino acids between positions 701 and 704 are so flexible as to be undetectable under the experimental conditions used
Trang 3for NMR analysis We believed that it would be
inter-esting to verify whether such a flexible amino acid
stretch, located within the a-DG-binding epitope,
might play a role in the interaction between the a-DG
and b-DG subunits Three additional mutations were
introduced outside the putative a-DG-binding epitope
to check whether perturbing the b-DG ectodomain
elsewhere might also influence its interaction with
a-DG We produced two mutations upstream of the
a-DG-binding epitope, such as Trp659fi Ala, because
Trp659 is the only aromatic residue in this portion of
the protein, and Glu667fi Ala; only one mutation,
Val736fi Ala, was generated within the C-terminal
region of the b-DG ectodomain, downstream of
the a-DG-binding epitope A map of all the mutations
produced within the recombinant fragments
b-DG(654–750) and a-DG(485–620) is given in Fig 1
In order to measure the affinity of such mutants
for a-DG(485–630), a series of solid-phase binding
assays was carried out Typically, in solid-phase binding
assays, a-DG(485–630) was coated onto
micro-titer plates, whereas b-DG(654–750) and its
mutants, b-DG(654–750)Trp659fi Ala, b-DG(654–
750)Glu667fi Ala, b-DG(654–750)Phe692fi Ala, b-DG(654–
750)Phe700fi Ala, b-DG(654–750)Phe718fi Ala, b-DG(654–
750)Val736fi Ala, b-DG(654–750)Phe692fi Ala ⁄ Phe718 fi Ala,
b-DG(654–750)Phe692fi Ala ⁄ Phe700 fi Ala ⁄ Phe718 fi Ala and
b-DG(654–750)D(701)706), were biotinylated and used as
soluble ligand at increasing concentrations (up to 20 lm)
The apparent affinity for a-DG(485–630), exhibited
by the mutants b-DG(654–750)Glu667fi Ala and b-DG(654–750)Val736fi Alaand evaluated by solid-phase binding assays, was very similar to that displayed by b-DG(654–750) (Fig 2A, Table 1), whereas all the other single mutants, namely b-DG(654–750)Trp659fi Ala, b-DG(654–750)Phe692 fi Ala, b-DG(654–750)Phe700 fi Ala and b-DG(654–750)Phe718fi Ala, showed reduced affinity for a-DG(485–630) (Fig 2B) Also, the deletion mutant b-DG(654–750)D(701)706) was able to bind a-DG(485– 630) with the same affinity as the wild type, demonstra-ting that the highly flexible stretch corresponding to positions 701–706 is not involved in the interaction with a-DG(485–630) and does not alter significantly the b-DG ectodomain conformation (Fig 2A, Table 1) On the other hand, double and triple mutations, such as Phe692fi Ala ⁄ Phe718 fi Ala and Phe692fi Ala ⁄ Phe700fi Ala ⁄ Phe718 fi Ala, completely abolished the binding between b-DG(654–750) and a-DG(485– 630), at least in the ligand concentration range explored (Fig 2C) To rule out the possibility that the lower affinity for a-DG(485–630) exhibited by some b-DG mutants might be due to some major proteolytic event, all the samples used to perform solid-phase binding assays were checked by Tricine⁄ SDS ⁄ PAGE before and after biotinylation, and showed the same mobility as wild-type b-DG(654–750), indicating that no degrada-tion occurred within mutated recombinant fragments; similarly, we did not observe any evident aggregation behavior when analyzing the various mutated b-DG peptides by native gel electrophoresis (data not shown) The solid-phase binding assay data were confirmed
by SPR experiments, in which a-DG(485–630) was immobilized on a sensor chip, and b-DG(654–750) and its mutants were used in soluble phase as analytes First, the dissociation equilibrium constant KD was measured for the interaction between the two wild-type recombinant fragments a-DG(485–630) and b-DG(654–750) It should be noted that the affinity constant value for the b-DG(654–750)–a-DG(485–630) interaction, measured by immobilizing b-DG(654–750) and using a-DG(485–630) as analyte (KD2.73 lm) (Table 1, Supplementary Fig S1), was fully compar-able to the value obtained when a-DG(485–630) was immobilized and b-DG(654–750) was used as analyte (KD2.66 lm) (Table 1) The thermodynamic constant
KDwas also measured for the interaction between the wild-type recombinant fragment a-DG(485–630) and the mutant b-DG(654–750)Phe700fi Ala, and confirmed its reduced affinity for a-DG(485–630) with respect to the wild type (KD7.00 lm; Table 1)
The kinetic SPR profiles obtained for all the single mutants b-DG(654–750)Phe692fi Ala, b-DG(654–
Fig 1 A panel of mutations hitting the reciprocal a-DG–b-DG
bind-ing epitopes was generated In the C-terminal region of a-DG,
between amino acids 550 and 565, Trp551, Phe554 and Asn555
were mutated to alanine In the a-DG-binding epitope comprising
residues 691–719 of the b-DG ectodomain, the mutations
Phe692 fi Ala, Phe700 fi Ala and Phe718 fi Ala were generated
while the residues from 701 to 706 were knocked-in Three
addi-tional mutations were introduced: Glu667 fi Ala and Trp659 fi Ala
upstream, and Val736 fi Ala downstream, of the a-DG-binding
epi-tope.
Trang 4750)Phe700fi Ala and b-DG(654–750)Phe718fi Ala were in line with the reduction of affinity measured by solid-phase binding assays, indicating a lower association rate and a higher dissociation rate with respect to wild-type a-DG The only exception was the mutant b-DG(654–750)Phe700fi Ala, which showed a higher association rate but also a higher dissociation rate in comparison to the wild type; the double mutant b-DG(654–750)Phe692fi Ala ⁄ Phe718 fi Ala did not bind a-DG(485–630) at all (Fig 3)
Mutations within the a-DG(485–630) recombinant fragment
Our previous NMR analysis using a synthetic peptide corresponding to a-DG(550–585) and the recombinant fragment b-DG(654–750) indicated that the residues between positions 550 and 565 of a-DG belong to an amino acid stretch that is likely to be involved in the
A
B
C
Fig 2 Solid-phase binding assays a-DG(485–630) was immobilized
on plates, whereas b-DG(654–750) (black) and its mutants
b-DG(654–750) Glu667 fi Ala (blue), b-DG(654–750) Val736 fi Ala (red),
b-DG(654–750)D(701)706) (green) (A), b-DG(654–750) Trp659 fi Ala
(magenta), b-DG(654–750)Phe692fi Ala (green), b-DG(654–
750) Phe700 fi Ala (blue), b-DG(654–750) Phe718 fi Ala (yellow) (B),
b-DG(654–750) Phe692 fi Ala ⁄ Phe718 fi Ala
(cyan), and b-DG(654–
750)Phe692fi Ala ⁄ Phe700 fi Ala ⁄ Phe718 fi Ala(red) (C), were used as
biot-inylated ligands Every point is an average of three or more
inde-pendent experiments The continuous line represents fitting of
experimental data using a single class of equivalent binding sites
equation The maximal binding of control b-DG(654–750),
extrapola-ted by fitting experimental data, was set as 100% (see
Experimen-tal procedures).
Table 1 (A) Equilibrium dissociation constants (K D ) calculated by solid-phase binding assays and SPR Mean apparent K D values and relative standard deviations, calculated for the interaction between wild-type and mutated recombinant fragments, b-DG(654–750) and a-DG(485–630), by solid-phase binding assays The values are averaged over a number of independent experiments, indicated in parentheses For the b-DG mutants showing reduced affinity for a-DG(485–630), K D values cannot be calculated (ND, not determined; see Experimental procedures) (B) KD values for the interaction between wild-type recombinant fragments, b-DG(654–750) and a-DG(485–630), and b-DG(654–750) Phe700 fi Ala
and a-DG(485–630),
as measured by SPR.
(A) Solid-phase binding assays Immobilized protein ⁄ biotinylated protein Apparent KD(l M ) a-DGwt⁄ b-DG wt
2.8 ± 0.9 (9) a-DG wt ⁄ b-DG Glu667 fi Ala 3.5 ± 0.9 (3)
a-DGwt⁄ b-DG Val736 fi Ala
2.9 ± 2 (4)
a-DG wt ⁄ b-DG Phe700 fi Ala
ND (3) a-DGwt⁄ b-DG Phe718 fi Ala
ND (8) a-DG wt ⁄ b-DG Phe692 fi Ala ⁄ Phe718 fi Ala ND (3) a-DG wt ⁄ b-DG Phe692 fi Ala ⁄ Phe700 fi Ala ⁄ Phe718 fi Ala
ND (3) a-DGTrp551fi Ala⁄ b-DG wt
1.3 ± 0.8 (4) a-DG Phe554–Ala ⁄ b-DG wt 2.3 ± 0.9 (5) a-DG Asn555 fi Ala
a-DGTrp551fi Ala ⁄ Phe554 fi Ala⁄ b-DG wt
2.4 ± 0.7 (3) (B) SPR
a-DGwt⁄ b-DG Phe700 fi Ala
7.00
Trang 5interaction with b-DG(654–750), based on data
collec-ted at the level of their NH and CHa [18]
In order to test the role of individual amino acid
side chains at the a-DG–b-DG interface, alanine
scan-ning of positions Trp551, Phe554 and Asn555 was
carried out Solid-phase binding assays were carried
out, in which a-DG(485–630) and its mutants,
a-DG(485–630)Trp551fi Ala, a-DG(485–630)Phe554fi Ala,
a-DG(485–630)Asn555fi Ala and a-DG(485–
630)Trp551fi Ala ⁄ Phe554 fi Ala were coated onto the
microtiter plate and biotinylated b-DG(654–750) was
used as soluble ligand All the mutants showed the
same affinity for b-DG(654–750) as the wild type,
indi-cating that such mutations are not likely to have major
effects on the interaction between a-DG and b-DG
(Supplementary Fig S2) Accordingly, the apparent
dissociation constant values obtained by fitting the
experimental data are very similar to the values
calcu-lated for the interaction between the wild-type peptides
a-DG(485–630) and b-DG(654–750) (Table 1) To
fur-ther confirm that residues Trp551, Phe554 and Asn555
are not involved in the interaction with b-DG, the
affinities of some b-DG mutants for the mutants
a-DG(485–630)Trp551fi Ala, a-DG(485–630)Phe554fi Ala
and a-DG(485–630)Asn555fi Ala were also estimated
by solid-phase binding assays The affinities of
b-DG(654–750)Trp659fi Ala, b-DG(654–750)Phe692fi Ala,
b-DG(654–750)Phe700fi Ala, b-DG(654–750)Phe718fi Ala
and b-DG(654–750)Phe692fi Ala ⁄ Phe700 fi Ala ⁄ Phe718 fi Ala
for immobilized a-DG mutants were very similar to
the reduced affinity exhibited for wild-type a-DG(485–
630), indicating that the effect measured can be ascribed to the mutations within the b-DG ectodomain (data not shown) Interestingly, a western blot experi-ment showed that Trp551, Phe554 and Asn555 are also not likely to be key residues for the interaction with the mAb sx⁄ 3 ⁄ 50 ⁄ 25 directed against the b-DG-bind-ing epitope (residues 549–567 of a-DG) [19], as the antibody is able to recognize the bands relative to a-DG recombinant peptides in western blot experi-ments (Fig 4A) Moreover, the antibody is also able
to bind a-DG mutated peptides in solid-phase binding assays, as it inhibits the interaction between b-DG(654–750) and the mutant a-DG(485– 630)Phe554fi Ala (Fig 4B), as previously shown for wild-type a-DG(485–600) [19]
A
B
Fig 4 (A) Western blot on 12% SDS ⁄ PAGE of wild-type and mutated recombinant fragments of a-DG Recombinant fragments were detected using mAb sx ⁄ 3 ⁄ 50 ⁄ 25 Lane 1: a-DG(485– 630) Trp551–Ala ⁄ Phe554 fi Ala Lane 2: a-DG(485–630) Asn555 fi Ala Lane 3: a-DG(485–630) Phe554 fi Ala
Lane 4: a-DG(485–630) Trp551 fi Ala
Lane 5 (control): a-DG(485–630) (B) Solid-phase binding assays were performed by immobilizing a-DG(485–630) (d) and a-DG(485– 630) Phe554 fi Ala
(h) and using biotinylated b-DG(654–750) as soluble ligand, in the presence (empty symbols) and in the absence (full symbols) of mAb sx ⁄ 3 ⁄ 50 ⁄ 25.
Fig 3 SPR kinetic profiles of the interaction between immobilized
a-DG(485–630) and b-DG(654–750) (black) and its mutants,
b-DG(654–750) Phe692 fi Ala (green), b-DG(654–750) Phe700 fi Ala (blue),
b-DG(654–750)Phe718fi Ala(yellow), b-DG(654–750)Phe692–Ala⁄ Phe718 fi Ala
(cyan), used as analytes at a fixed concentration of 10 l M
Trang 6Transfection of 293-Ebna cells with wild-type and
mutated DG constructs
In order to verify the correct membrane targeting
of mutated DG, DNA constructs spanning the entire
DG gene, including its signal peptide, and carrying
the mutations analysed in vitro, such as Trp551fi
Ala, Phe554fi Ala, Asn555 fi Ala, Glu667 fi Ala,
Phe692fi Ala, Phe700fi Ala, Phe718fi Ala, Phe692fi Ala ⁄ Phe718 fi Ala and Val736fi Ala, were included in an appropriate mammalian expression vector and then transfected into human 293-Ebna cells The cytomegalovirus promoter drives the efficient tran-scription of the DG exogenous gene, which was strongly expressed in the transfected cells (Fig 5) None of the mutations seemed to significantly alter the
A
Fig 5 Immunostaining of wild-type DG and its mutants in transiently transfected 293-Ebna cells (A) The a-subunits were stained with a polyclonal antibody directed against the C-terminal domain of a-DG on intact 293-Ebna cells (B) Detection of b-DG was carried out using b-DG antibody in permeabilized 293-Ebna cells Both subunits of wild-type and mutated DG were clearly overexpressed with respect to non-transfected cells displaying a much lower and diffuse staining due to endogenous DG; the double mutation Phe692 fi Ala ⁄ Phe718 fi Ala does not alter the membrane targeting of a-DG.
Trang 7correct membrane localization of a-DG and b-DG, at
least 24 h after transfection, as all the mutants could
be stained with a polyclonal antibody directed against
the C-terminal region of a-DG [20] and with a
com-mercial antibody directed against the cytoplasmic tail
of b-DG (Fig 5A,B) Also, the double mutation
Phe692fi Ala ⁄ Phe718 fi Ala, which greatly reduces
the affinity of b-DG for the a-subunit in vitro, did not
influence the localization of the two DG subunits
(Fig 5A,B) In order to detect any effect of the double
mutation Phe692fi Ala ⁄ Phe718 fi Ala, which may
have evaded the immunostaining analysis [21], the entire DG carrying this mutation was cloned into the pEGFP vector, which codes for the enhanced green fluorescence protein (EGFP) fused at the C-terminus
of b-DG This vector was used to transiently transfect 293-Ebna cells Other two pEGFP vectors were produced, carrying wild-type DG and its mutant
DGPhe554fi Ala, respectively, to be used as a control (Fig 6)
EGFP increases the molecular mass of b-DG
by 25 kDa, allowing us to distinguish between the
B
Fig 5 (Continued).
Trang 8exogenous b-DG and the endogenous b-DG in western
blot experiments Western blot analysis carried out on
total protein extracts from 293-Ebna cells transiently
transfected with wild-type and mutated (Phe554fi Ala
or Phe692fi Ala ⁄ Phe718 fi Ala) DG genes did not
show any aberrant processing or glycosylation patterns
of DG Although lower expression of a-DG was
detected in all transfected cells (including those
transfected with empty pEGFP or wild-type pDG–
EGFP; Fig 7A), the amount of a-DG in the cells
transfected with the double-mutated (Phe692fi Ala ⁄
Phe718fi Ala) DG construct was similar to that
measured in wild-type DG-transfected cells (Fig 7)
Discussion
In vitro inhibition of the a-DG–b-DG interaction
via Phe to Ala mutations within the ectodomain
of b-DG
We investigated the interaction between a-DG and
b-DG recombinant peptides carrying a series of
site-directed mutations We performed amino acid
substitu-tions using alanine, because this residue does not show
any propensity for a specific secondary structure, and
therefore does not perturb the overall protein
confor-mation while highlighting the role of the side chain
functional group that it replaces [22,23] This
charac-teristic makes alanine the amino acid of choice for site-directed mutagenesis [22–26] For the first time, we have measured the affinity between recombinant pep-tides spanning the C-terminal domain of a-DG and the b-DG ectodomain by solid-phase binding assays and SPR, demonstrating that despite the intrinsic pit-falls of solid-phase binding techniques, extensively reviewed by Tangemann & Engel [27], the apparent
KDvalues measured with our ‘two-step’ biotin enzyme-linked streptavidin approach are in full agreement with those measured with an independent and accurate technique such as SPR, whose reliability has been dem-onstrated in recent years for protein–protein interac-tions displaying very low affinity [28] Our SPR measurements simply demonstrate that the apparent constants that we have estimated with the solid-phase binding approach are reliable In this case, it seems that even a complex displaying a relatively low affinity (KDabout 3 lm; Table 1) can be populated, when rap-idly washed without major perturbations and then titrated with streptavidin [17,29]
In previous studies, we had already estimated that the a-DG-binding epitope of b-DG might involve a relatively extended region of approximately 30 amino acids, between positions 691 and 719 [15] In order to obtain a deeper insight into the a–b interface, we have introduced a series of mutations within the b-DG ecto-domain located in three different protein regions:
D C
Fig 6 293-Ebna cells transiently transfected with the vector pEGFP, empty (A) or containing DNA constructs corresponding to wild-type DG (B) and its mutants DG Phe554 fi Ala (C) and
DGPhe692fi Ala ⁄ Phe718 fi Ala(D), where enhanced green fluorescent protein (EGFP) was fused at the C-terminus of b-DG All the images are magnified 10· EGFP alone was uniformly distributed throughout the cytoplasm, whereas DG–EGFP and its two mutants were mostly localized around the cellular periphery In order to better visualize the DG complex location at the cellular periphery, a 40· magnified image was obtained referring to wild-type DG–EGFP, which clearly shows the membrane targeting of the chimeric wild-type DG–EGFP construct [inset in (B), red arrowheads].
Trang 9upstream, downstream and inside the putative
a-DG-binding epitope By measuring the affinity of these
mutants for the recombinant fragment a-DG(485–630),
corresponding to the C-terminal domain of a-DG, via
solid-phase binding assays and SPR experiments, we
have found three different behaviors: some mutants,
namely b-DG(654–750)Glu667fi Ala, b-DG(654–
750)Val736fi Ala and b-DG(654–750)D(701)706), show the
same affinity for a-DG(485–630) as the wild-type
recombinant fragment; a second intermediate group,
comprising b-DG(654–750)Trp659fi Ala, b-DG(654–
750)Phe692fi Ala, b-DG(654–750)Phe700fi Ala and
b-DG(654–750)Phe718fi Ala, shows a reduced affinity
for a-DG(485–630); the double and triple mutants,
such as b-DG(654–750)Phe692fi Ala ⁄ Phe718 fi Ala and
b-DG(654–750)Phe692fi Ala ⁄ Phe700 fi Ala ⁄ Phe718 fi Ala are
completely unable to bind a-DG(485–630)
The behavior of b-DG(654–750)Glu667fi Ala and
b-DG(654–750)Val736fi Ala is not surprising, as these
point mutations are located upstream and downstream
of the a-DG-binding epitope, respectively, in por-tions of the protein that are not involved in the forma-tion of the a–b interface [15] However, this simple argument cannot be applied to explain the reduced affinity for a-DG(485–630) shown by the mutant b-DG(654–750)Trp659fi Ala, whose amino acid substitu-tion is located upstream of the a-DG-binding epitope
A possible interpretation of this result can be sugges-ted on the basis of previous studies showing that the recombinant fragment b-DG(654–750) is organized into an N-terminal region, consisting of approximately
70 amino acids, which is characterized by restricted conformational mobility, and a highly flexible C-ter-minal region of approximately 30 residues [15] In this context, an amino acid substitution introduced within the region of restricted mobility, such as Trp659fi Ala, although located outside the putative a-DG-binding epitope, may perturb the b-DG(654– 750) conformational equilibrium, driving it to adopt non-native conformations unable to efficiently bind a-DG(485–630)
All the point mutations located inside the puta-tive a-DG-binding epitope, namely b-DG(654– 750)Phe692fi Ala, b-DG(654–750)Phe700fi Ala and b-DG(654–750)Phe718fi Ala, result in reduced affinity between a-DG and b-DG recombinant peptides, which completely lose their ability to interact when two phenylalanines, Phe692 and Phe718, are simultaneously substituted with alanine Surprisingly, the deletion of six residues corresponding to amino acids 701–706, located between the two important phenylalanines Phe692 and Phe718, does not perturb the interaction between a-DG and b-DG recombinant peptides,
as can be deduced by comparing the values of the apparent KD for the b-DG(654–750)wt–a-DG(485–630) interaction and the b-DG(654–750)D(701)706) –a-DG(485–630) interaction, as measured by solid-phase binding assays (Table 1) It should be noted that the choice to delete these specific amino acids (701–706) is based on the observation that the residues between positions 701 and 704, although belonging to the region of restricted mobility, are so flexible to be unde-tectable under the experimental conditions used for NMR experiments [15]
The stretch 701–706 may be part of a flexible linker that could bring two separate regions of the a-DG-binding epitope (carrying Phe692 and Phe718, respect-ively) closer to each other when they bind a-DG Apparently, deleting the amino acid stretch 701–706 has no effect on the a–b interaction, so it is likely that the knock-in does not significantly alter the spatial distance between the two important phenyl-alanines
A
B
Fig 7 Immunoblot of total protein extracts from 293-Ebna cells
nontransfected (lane 1) or transfected with the empty pEGFP
vector (lane 2), or containing DG–EGFP (lane 3) and its mutants
DG Phe554 fi Ala
–EGFP (lane 4) and DG Phe692 fi Ala ⁄ Phe718 fi Ala
–EGFP (lane 5) (A) a-DG bound to the plasma membrane was probed
with the commercial antibody VIA4-1 (Upstate, Charlottesville, VA,
USA) (B) The amount of b-DG–EGFP (68 kDa) compared with that
of endogenous b-DG (43 kDa) was detected with the commercial
antibody directed against the cytoplasmatic tail of b-DG (upper
panel) and with antisera against EGFP (middle panel) Anti-actin
serum was used as loading control (lower panel).
Trang 10Our previous results indicate that although the
ecto-domain of b-DG is a natively unfolded protein, its
conformation is still maintained by a delicate network
of long-range reciprocal interactions that govern major
structural–functional events [30], to which the two
phe-nylalanine residues, which are quite distant within the
b-DG ectodomain linear sequence, may make an
important contribution
When the binding experiments were performed with
a-DG(485–630) and its mutants, we found that
Trp551, Phe554 and Asn555 are not key residues
for the interaction with b-DG(654–750), because no
reduction of the affinity of the mutants a-DG
(485–630)Trp551fi Ala, a-DG(485–630)Phe554fi Ala,
a-DG(485–630)Asn555fi Ala and a-DG(485–
630)Trp551fi Ala ⁄ Phe554 fi Ala for b-DG(654–750),
com-pared to wild-type a-DG(485–630), was measured by
solid-phase binding assays (Supplementary Fig S2) It
should be pointed out that our previous NMR
experi-ments showed that a-DG residues Trp551, Phe554 and
Asn555, among others, were significantly influenced by
the presence of b-DG(654–750) at the level of the
pro-tein backbone (i.e at their NH and CHa), although at
that time no data were collected on their side chains,
which therefore could be substantially unaffected by
b-DG binding [18], as is now strongly suggested by the
results herein presented
A possible implication of our results is that the
specificity of a-DG(485–630) binding to b-DG(654–
750) could depend mainly on its local conformation
and only to a lesser extent on the chemical nature
of the amino acid side chains involved in the
bind-ing To further analyze this hypothesis, it will be
necessary to introduce, within residues 550–565 of
a-DG, amino acids that require stringent steric
con-straints, such as proline or isoleucine, which may
significantly perturb the local structural
characteris-tics of the b-DG-binding epitope Interestingly, the
amino acid substitutions that we analyzed do not
impair binding to a monoclonal antibody, mAb
sx⁄ 3 ⁄ 50 ⁄ 25, as suggested by the western blot in
Fig 4A Moreover, mAb sx⁄ 3 ⁄ 50 ⁄ 25 is able to
effi-ciently inhibit the interaction between wild-type
a-DG and b-DG peptides [19] and also between
mutated a-DG and b-DG (Fig 4B), suggesting that
other a-DG residues, belonging to the approximately
550–565 residues epitope, but located in the
C-ter-minal region downstream of those so far analyzed,
may be involved in the interaction both with mAb
sx⁄ 3 ⁄ 50 ⁄ 25 and b-DG(654–750) Therefore, additional
amino acid substitutions, such as Ser556fi Ala,
Gly563fi Ala and Pro565 fi Ala, will be tested in
the future [18]
Analysis of DG subunit targeting in vivo:
detection of DG subunits in 293-Ebna cells Few studies are currently available that focus on the identification and analysis of polymorphisms and⁄ or point mutations within the DG gene in human popula-tions [31,32]; nevertheless, it has been shown in a cellu-lar system that single mutations may strongly influence the processing of the DG precursor and targeting at the cell surface of its subunits [33] Mutations affecting some putative glycosylation sites lead to impaired membrane targeting of DG and also to defective clea-vage of the precursor into the two subunits [33] Fur-thermore, mutation at the precursor maturation cleavage site (Gly653-Ser654) induces muscular dystro-phy in mice [34]
To investigate whether the amino acid substitutions tested in vitro influence the stability and the localization
of DG at the plasma membrane, we transfected 293-Ebna cells with the full-length murine DG gene harboring the mutations Trp551fi Ala, Phe554 fi Ala, Asn555fi Ala, Glu667fi Ala, Phe692fi Ala, Phe700fi Ala, Phe718fi Ala, Val736fi Ala and Phe692fi Ala ⁄ Phe718 fi Ala Cell-staining experi-ments showed that none of these mutations significantly affects the subcellular trafficking and plasmalemmal tar-geting of exogenous murine DG in transfected human 293-Ebna cells (Fig 5) In fact, all the mutants were overexpressed, and a strong fluorescent signal was detec-ted at the plasmalemma, exploiting a monoclonal anti-body directed against the cytoplasmic tail of b-DG Apparently, even the double mutation Phe692fi Ala ⁄ Phe718fi Ala, which greatly impairs binding between the two DG subunits in vitro, has no evident effect on the plasmalemmal targeting of b-DG (Fig 5B) The cor-rect membrane targeting of DG was also confirmed by the immunodetection of the a-subunit in human cells transfected with all the mutants (Fig 5A) In order to further analyze possible effects of the double mutation Phe692fi Ala ⁄ Phe718 fi Ala in vivo, we also cloned the mutant DGPhe692fi Ala ⁄ Phe718 fi Alawithin the
pEG-FP vector, together with wild-type DG and the mutant
DGPhe554fi Alaas controls (Fig 6) Western blot analy-sis of total protein extracts from 293-Ebna cells, transi-ently transfected with these constructs, confirmed the results of cell-staining experiments (Fig 7) The amount
of a-DG in cells transfected with the mutant
DGPhe692fi Ala ⁄ Phe718 fi Ala is in fact fully comparable with that observed in cells transfected with the empty pEGFP vector or containing wild-type DG or
DGPhe554fi Ala(Fig 7A) The reduced amount of a-DG detected by VIA4-1 in all the 293-Ebna transfected cell lines probably results from reduction and⁄ or