In this article, similar experiments performed on the a-DG C-terminal domain pinpointed two residues, G563 and P565, as possible binding counterparts of the two b-DG phenylalanines.. In
Trang 1the dystroglycan precursor
Francesca Sciandra1,*, Manuela Bozzi2,*, Simona Morlacchi1,3, Antonio Galtieri4, Bruno Giardina1,2 and Andrea Brancaccio1
1 Istituto di Chimica del Riconoscimento Molecolare (CNR), c ⁄ o Istituto di Biochimica e Biochimica Clinica, Universita` Cattolica del
Sacro Cuore, Rome, Italy
2 Istituto di Biochimica e Biochimica Clinica, Universita` Cattolica del Sacro Cuore, Rome, Italy
3 Dipartimento di Biologia Animale ed Ecologia Marina, Universita` degli Studi di Messina, Italy
4 Dipartimento di Chimica Organica e Biologica, Universita` di Messina, Italy
Introduction
Dystroglycan (DG) is a ubiquitous
membrane-span-ning protein complex that was originally identified and
characterized in rabbit skeletal muscle [1–3] DG is expressed in skeletal and cardiac muscle, in the central
Keywords
alanine scanning; dystroglycan; dystroglycan
precursor; laminin binding; post-translational
processing
Correspondence
A Brancaccio, Istituto di Chimica del
Riconoscimento Molecolare (CNR), c ⁄ o
Istituto di Biochimica e Biochimica Clinica,
Universita` Cattolica del Sacro Cuore,
L.go F Vito 1, 00168 Rome, Italy
Fax: +39 6 3053598
Tel: +39 6 3057612
E-mail: andrea.brancaccio@icrm.cnr.it
*These two authors contributed equally to
this work
(Received 29 April 2009, revised 10 June
2009, accepted 3 July 2009)
doi:10.1111/j.1742-4658.2009.07196.x
The interaction between a-dystroglycan (a-DG) and b-dystroglycan (b-DG), the two constituent subunits of the adhesion complex dystroglycan, is crucial
in maintaining the integrity of the dystrophin–glycoprotein complex The importance of the a–b interface can be seen in the skeletal muscle of humans affected by severe conditions, such as Duchenne muscular dystrophy, where the a–b interaction can be secondarily weakened or completely lost, causing sarcolemmal instability and muscular necrosis The reciprocal binding epi-topes of the two subunits reside within the C-terminus of a-DG and the ectodomain of b-DG As no ultimate structural data are yet available on the a–b interface, site-directed mutagenesis was used to identify which specific amino acids are involved in the interaction A previous alanine-scanning analysis of the recombinant b-DG ectodomain allowed the identification of two phenylalanines important for a-DG binding, namely F692 and F718 In this article, similar experiments performed on the a-DG C-terminal domain pinpointed two residues, G563 and P565, as possible binding counterparts
of the two b-DG phenylalanines In 293-Ebna cells, the introduction of ala-nine residues instead of F692, F718, G563 and P565 prevented the cleavage
of the DG precursor that liberates a- and b-DG, generating a pre-DG of about 160 kDa This uncleaved pre-DG tetramutant is properly targeted at the cell membrane, is partially glycosylated and still binds laminin in pull-down assays These data reinforce the notion that DG processing and its membrane targeting are two independent processes, and shed new light on the molecular mechanism that drives the maturation of the DG precursor
Structured digital abstract
l MINT-7214494 : alpha DG (uniprotkb: Q62165 ) binds ( MI:0407 ) to beta DG (uni-protkb: Q62165 ) by solid phase assay ( MI:0892 )
l MINT-7214516 : laminin (uniprotkb: P19137 ) binds ( MI:0407 ) to beta DG (uniprotkb: Q62165 )
by pull down ( MI:0096 )
Abbreviations
DG, dystroglycan; DGC, dystrophin–glycoprotein complex; EGFP, enhanced green fluorescent protein; WGL, wheat germ lectin.
Trang 2and peripheral nervous system and in several epithelial
tissues [3,4] Homozygous null mice for the DG gene
dag-1die early during embryogenesis, at day E6.5, as a
result of defects in Reichert’s membrane, the first
extra-embryonic basement membrane deposited during
murine development [5]
Indeed, DG plays a crucial role in the assembly of
several basement membranes, promoting the
recruit-ment of laminins and other extracellular matrix
mole-cules during morphogenesis, tissue remodelling, cell
polarization and wound healing [6–10] DG is also
implicated in the myelinization of nerves and in the
sta-bilization of the neuromuscular junction [11,12]
More-over, in skeletal muscle, together with sarcoglycans,
dystrobrevins, syntrophins and sarcospan, DG forms
the dystrophin–glycoprotein complex (DGC), which
connects the extracellular matrix to the actin
cytoskele-ton, and is thought to offer stabilization to the muscle
fibres during the contraction–relaxation cycle [13]
Although no primary genetic alterations of DG have
been linked to human diseases to date, mutations in
other components of the DGC are associated with
dis-tinct forms of muscular dystrophy Primary mutations
in dystrophin, laminin-2 and any of the sarcoglycans
cause Duchenne muscular dystrophy, congenital
mus-cular dystrophy and limb-girdle musmus-cular dystrophy,
respectively [2] In these forms of muscular dystrophy,
DG membrane targeting and stability can be strongly
perturbed
DG is composed of two interacting subunits, a and
b, which are translated from a single mRNA molecule,
generating a precursor protein of 895 residues that is
post-translationally cleaved into the two noncovalently
associated subunits [1] The cleavage site is highly
con-served among vertebrates and lies between residues
G653 and S654 [14,15] The detailed mechanism and
functional significance of the post-translational
pro-cessing of the DG precursor are still largely unknown,
but experimental evidence has demonstrated its
impor-tance for the correct function of DG Indeed, a
trans-genic mouse overexpressing the uncleaved precursor
developed muscular dystrophy, and the expression of
the noncleavable DG protein in neuroepithelial cells
reduced their proliferation and differentiation in
neurons [16,17]
b-DG is a transmembrane protein whose
cytoplas-mic domain binds actin via the interaction with
dystro-phin, and may act as a scaffold platform for signalling
proteins interacting with the adaptor protein Grb2,
but also with ezrin and extracellular signal-regulated
kinase [18] a-DG, in turn, is a peripheral protein
char-acterized by a dumbbell-like structure with two
globu-lar domains at the N- and C-termini, separated by an
elongated central and highly glycosylated mucin-like domain [19] a-DG binds with high affinity a variety
of extracellular matrix molecules, such as laminin, agrin and perlecan The reduction of the glycosylated shell of DG is thought to perturb its binding affinity towards extracellular matrix molecules [20] Indeed, several forms of congenital muscular dystrophy are caused by mutations in a number of known or putative glycosyltransferases, leading to hypoglycosylation of a-DG in both skeletal muscle and brain [21]
However, a-DG retains contact with the plasma membrane through binding with b-DG, and the inter-action is independent of glycosylation [22,23] The interaction between the two subunits involves the C-terminal domain of a-DG and the extracellular domain of b-DG, which belongs to the increasingly populated family of natively unfolded proteins, charac-terized by high conformational plasticity [22,24] The reciprocal binding epitopes have been mapped between amino acids 550 and 565 of the C-terminal domain of a-DG and in the region located between the amino acid positions 691 and 719 of b-DG [24,25] Recently, detailed mutagenesis analysis of the interaction between the two DG subunits identified two phenylala-nine residues (F692 and F718), belonging to the b-DG ectodomain, that are essential for the binding to a-DG
in vitro [26] In this study, extending the molecular analysis to the C-terminal portion of the b-DG binding epitope of a-DG [25], we identified some new residues that are important for the stability of the a–b inter-face
Results
Alanine scanning of the b-DG binding epitope within the C-terminal domain of a-DG
We have previously demonstrated that a linear amino acid sequence of 15 residues between positions 550 and
565 of a-DG is sufficient to interact with b-DG in experiments carried out with recombinant proteins [25] Following these preliminary data, alanine scan-ning was performed on three amino acid positions belonging to the N-terminal portion of this linear sequence, namely W551, F554 and N555, in order to evaluate the contribution of each amino acid side-chain to the stability of the a–b interface [26] As none
of these three mutations seem to significantly affect the interaction with b-DG, we extended our alanine-scan-ning approach to the C-terminus of the 550–565 linear sequence
We expressed and purified a series of recombinant proteins spanning the C-terminal domain of a-DG,
Trang 3a-DG(485–630), carrying the following point
muta-tions: S556A, Q559A, M561A, Y562A, G563A, L564A
and P565A (Fig 1) The affinity of each mutant
towards the soluble recombinant biotinylated b-DG
ectodomain, b-DG(654–750), was measured by
solid-phase binding assays Although solid-solid-phase binding
assays were carried out in nonequilibrium conditions,
they provide apparent dissociation constants that are
fully comparable with those measured with more
accu-rate techniques, such as surface plasmon resonance
[26] a-DG(485–630) and its mutants a-DG(485–630)
S556A, a-DG(485–630)Q559A, a-DG(485–630)M561A,
a-DG(485–630)Y562A, a-DG(485–630)G563A, a-DG
(485–630)L564A and a–DG(485–630)P565A were coated
onto a microtitre plate, whereas biotinylated b-DG(654–
750) was used as a soluble ligand at increasing
concentra-tions (up to 20 lm)
The mutants a-DG(485–630)S556A, a-DG(485–630)
Y562A and a-DG(485–630)L564A bind b-DG(654–
750) with the same affinity as the wild-type (see
Fig 2A), whereas a-DG(485–630)Q559A, a-DG(485–
630)M561A, a-DG(485–630)G563A and a-DG(485–
630)P565A show a slightly reduced affinity for
b-DG(654–750), suggesting that these latter mutations
might destabilize the a–b interface (see Fig 2B) In
Table1, it can be seen that the lowest affinities
(corre-sponding to the highest apparent dissociation
constants) refer to the mutants a-DG(485–630)G563A and a-DG(485–630)P565A In order to further validate these results, we have produced the double mutant a-DG(485–630)G563A-P565A and measured its affin-ity towards b-DG(654–750) The double substitution
Fig 1 Panel of mutants of murine DG fused to GFP The a- and
b-subunits of mammalian DG contain several well-conserved
domains: (a) the N-terminal domain, the mucin-like region and the
C-terminal region of a-DG, the latter containing the b-DG binding
epi-tope (amino acids 550–565); (b) the ectodomain, the transmembrane
region (TM) and the cytosolic domain of b-DG The b-DG binding
epi-tope was mutated by alanine scanning to produce the following
mutants: S556A, Q559A, M561A, Y562A, G563A, L564A and
P565A A mutant deleted of the whole b-DG binding epitope
(DGD550–565) was also generated All the mutations were
intro-duced into the wild-type murine DG cDNA sequence and cloned into
a pEGFP vector for cell transfection experiments, or introduced into
a plasmid, allowing quantitative expression of recombinant
C-termi-nal a-DG peptides in E coli cells (see Experimental procedures).
A
B
C
Fig 2 Solid-phase binding assays a-DG(485–630) (black) and its mutants, a-DG(485–630)S556A (red), a-DG(485–630)Y562A (green), a-DG(485–630)L564A (blue) (A), a-DG(485–630)Q559A (red), a-DG (485–630)M561A (green), a-DG(485–630)G563A (blue), a-DG(485– 630)P565A (magenta) (B) and a-DG(485–630)G563A-P565A (red) (C), were coated onto a microtitre plate, whereas biotinylated b-DG(654–750) was used as a soluble ligand at increasing concen-trations Each continuous line corresponds to a representative experiment (from a set of at least three experiments with similar results), and was obtained by fitting experimental data to a single class of equivalent binding sites equation (see Experimental procedures).
Trang 4of both G563 and P565 with alanine completely
inhib-ited the interaction between a-DG(485–630) and
b-DG(654–750), at least in the ligand concentration
range explored (Fig 2C) Interestingly, G563 and P565
are also fully conserved in DGs from phylogenetically
distant species (Fig S1, see Supporting information)
These results indicate that, together, G563 and P565
might significantly contribute to the a–b interface and
to the stability of the whole DG complex
Transfection of 293-Ebna cells with mutated DGs:
western blot and fluorescence microscopy
In order to analyse in eukaryotic cells the effects of the
point mutations that impair the interaction between
a-and b-DG, the same mutations tested in solid-phase
binding assays were introduced within the entire
mur-ine DG cDNA, which was cloned into the pEGFP
vec-tor and used to transiently transfect 293-Ebna cells
Enhanced green fluorescent protein (EGFP) was fused
at the C-terminal region of b-DG to increase its
molec-ular mass by 25 kDa; the presence of GFP allows
endogenous b-DG to be distinguished unambiguously
from exogenous b-DG-EGFP in western blot analysis
Western blot of total protein extracts of cells
overex-pressing DG-EGFP constructs carrying the single
point mutations S556A, Q559A, M561A, G563A,
L564A and P565A confirmed the presence of the
expected 68 kDa band corresponding to exogenous
b-DG-EGFP when the samples were probed with both
anti-b-DG and anti-EGFP IgG (Fig 3A,B)
However, G563A displayed an additional faint band
of about 100⁄ 200 kDa (Fig 3A,B) Interestingly, the
same band was also detectable in the two double mutants, G563A⁄ P565A and F692A ⁄ F718A (Fig 3C– E) The latter mutant hits the two phenylalanines belonging to the b-DG ectodomain, F692A and F718A, that have been shown previously to be key residues for binding with a-DG in vitro [26] This higher band is likely to correspond to the unprocessed DG precursor (hereafter pre-DG), as the mutation S654A, located at the physiological a⁄ b maturation cleavage site G653– S654, produces a single band with a molecular weight estimated at 160 kDa that has the same electrophoretic mobility as displayed by the double mutants G563A⁄ P565A and F692A⁄ F718A (Fig 3C,D) [16,27,28]
On the basis of these results, we hypothesized that perturbation of the network of interactions that is likely to stabilize the a–b interface within the DG com-plex may interfere with the cleavage of the DG precur-sor To further validate this hypothesis, we generated two additional constructs, one carrying the four mutations G563A, P565A, F692A and F718A, DGG563A_P565A_F692A_F718A, and the second with deletion of the whole b-DG binding epitope between amino acids 550 and 565 within the C-termi-nal domain of a-DG, DGD550–565 [25] As expected, the products of both constructs appeared on SDS-PAGE as a single 160 kDa band, albeit less intense than that observed for the mutant S654A, indicating
an instability and a major susceptibility to degradation
of the former mutant pre-DGs (Fig 3C–E) A possible scale in the amounts of pre-DG is as fol-lows: DGS654A > DGG563A_P565A_F692A_F718A
> DG(D550–565) > DGF692AF718A ‡ DGG563A P565A > DGG563A (Fig 3E)
Fluorescence microscopy analysis showed that the
DG precursors are likely to be properly targeted at the plasma membrane, as cells expressing the un-cleavable DG mutants are indistinguishable from those expressing wild-type DG (Fig 4) In addition, the quadruple mutation G563A⁄ P565A ⁄ -F692A⁄ F718A and the deletion of the 550–565 region did not significantly affect the trafficking or membrane targeting of pre-DG (Fig 4) The diffused and punctuated label throughout the cytoplasm and around the plasma membrane, featured by cells transfected with both wild-type and mutated DG, was probably a result of overexpression of exogenous EGFP-tagged proteins
Wheat germ lectin (WGL)-driven enrichment of mutant pre-DGs
The DG gene encodes a unique polypeptide precur-sor consisting of 895 amino acids with a calculated
Table 1 Apparent equilibrium dissociation constants (K D )
calcu-lated by solid-phase binding assays Mean apparent K D values and
relative standard deviations, calculated for the interaction between
a-DG(485–630) and its mutants and b-DG(654–750) in solid-phase
binding assays The values were averaged over a number of
inde-pendent experiments indicated in parentheses For the a-DG(485–
630) double mutant, showing a strongly reduced affinity towards
b-DG(654–750), the K D value could not be calculated (n.d.; see
Experimental procedures).
Immobilized protein ⁄ biotinylated protein KD,app(l M )
a-DG(S556A) ⁄ b-DG wt
3.2 ± 1.2 (4)
a-DG(Y562A) ⁄ b-DG wt
3.1 ± 0.8 (5)
a-DG(P565A) ⁄ b-DG wt
5.5 ± 1.2 (4)
Trang 5molecular mass of about 98 kDa Based on their
apparent mobility on SDS-PAGE, pre-DGG
563A_P565A, pre-DGG563A_P565A_F692A_F718A,
pre-DG(D550–565) and pre-DGS654A should be
highly, or at least partially, glycosylated In order to
further clarify this aspect, total protein extracts
obtained from 293-Ebna cells transfected with the
uncleavable DG mutants were incubated with
aga-rose-immobilized WGL that specifically binds
N-acet-ylglucosamine residues (Fig 5A) All the pre-DG
mutants were pulled down and enriched by this
pro-cedure, suggesting the presence of
N-acetylglucos-amine moieties within the uncleaved precursors
(Fig 5B) Densitometric analysis confirmed the minor
stability of preDGG563A_P565A, preDGG563A_
P565A_F692A_F718A and preDG(D550–565) when
compared with pre-DGS654A (Fig 5C)
Laminin binding properties of mutant pre-DGs
DG serves as a receptor for a variety of extracellular ligands, such as laminin, agrin and perlecan Full chemi-cal deglycosylation of a-DG in vitro is known to disrupt its ability to bind other extracellular matrix proteins [29,30] Therefore, laminin conjugated to Sepharose beads was used to test the capacity of the mutant DGs (DGG563A_P565A_F692A_F718A,
pre-DGD550–565 and pre-DGS654A) to interact with com-mercial mouse laminin-1 (Fig 6A) Mutant pre-DGs remained bound to laminin even after several washing steps (Fig 6B) This interaction was inhibited using EDTA, suggesting that the binding between laminin and the mutant pre-DGs is reversible and dependent on divalent calcium cations, as expected for the laminin–
DG interaction (data not shown) [29,31]
C D
E
Fig 3 Western blot of total protein
extracts 293-Ebna cells were transfected
with DG mutants and their protein extracts
were probed with anti-b-DG (anti-43-DAG)
(A and C) or anti-GFP (B and D) The DG
mutants carrying the point mutations
S556A, Q559A, M561A, G563A, L564,
P565A display a single band corresponding
to the cleaved b-DG-GFP (A and B) The
double DG mutants, G563A ⁄ P565A and
F692A ⁄ F718A, show the presence of an
additional higher band at 160 kDa that is
likely to correspond to the unprocessed
pre-DG (C and D) Pre-DG is also expressed
in the presence of the mutation S654A,
the quadruple mutation G563A ⁄ P565A ⁄
F692A ⁄ F718A and the deletion of the entire
b-DG binding epitope between amino acids
550–565 (C and D) A lower band, at about
50 kDa, probably originates from further
pro-teolysis of b-DG-GFP The black boxes
indi-cate pre-DG-GFP and b-DG-GFP The
amounts of cleaved DG-GFP (open bars) and
unprocessed pre-DG (filled bars) were
quan-tified by densitometry, averaging the values
of the band intensities obtained from five
independent experiments (E) Such a
quanti-tative analysis shows how the differences in
band intensities between some of the
mutants are not significant.
Trang 6Towards the identification of single amino acids
within the C-terminal region of a-DG that are
crucial for the interaction with b-DG
In this study, we focused alanine scanning on the
a-DG amino acid linear sequence 550–565 in an
attempt to identify which residues were responsible for
binding with b-DG A series of point mutations,
S556A, Q559A, M561A, Y562A, G563A, L564A and
P565A, was introduced into the recombinant protein
a-DG(485–630), and their affinities towards
recombi-nant biotinylated b-DG(654–750) were measured The
KDvalues reported in Table 1 show that only the point
mutations hitting odd positions (i.e G563A and
P565A) elicit some slight effect on the interaction with
b-DG, whereas those at even positions (i.e S556A,
Y562A and L564A) do not produce any effect (see
Fig 2A,B and Table 1) Moreover, the double
muta-tion G563A⁄ P565A completely inhibits the interaction
between a- and b-DG (Fig 2C)
The interaction between a- and b-DG seems to
induce some local secondary structures Indeed, our
results may suggest that the a-DG linear sequence
QLMYGLP assumes a b-strand conformation, with
the amino acids Q559, M561, G563 and P565 pointing
towards the b-DG ectodomain and interacting with it
Retrospectively, our previous NMR experiments, car-ried out by exploiting the synthetic peptide a-DG(550– 585) in free and b-DG-bound fashion, suggested a greater involvement of Q559, M561 and G563 than of S558, L560, Y562 and L564 in binding the recombi-nant b-DG ectodomain; the alternate fashion of these side-chain contributions could indeed be reminiscent of
a b-strand conformation (see Fig 4 of [25]) Further-more, the hypothesis that the QLMYGLP amino acid stretch would assume a b-strand conformation is corroborated by a model of the a-DG C-terminal domain based on sequence homology with a member
of the cadherin family [32] However, further experi-ments are needed to validate this hypothesis
Interestingly, the crucial importance of G563 and P565 could be deduced from the analysis of a multiple alignment of DG sequences from species phylogeneti-cally distant from humans or mouse, including lower vertebrate and several invertebrate species (Fig S1, see Supporting information), where these amino acids are always conserved despite a very low overall sequence homology Our new data on G563 and P565, together with the results of our previous study, in which two phenylalanines belonging to the b-DG ectodomain, F692 and F718, were recognized as key residues for the interaction with a-DG, point towards the identifi-cation of the major molecular cornerstones of the a–b interface
Fig 4 Immunofluorescence of 293-Ebna cells transfected with the pEGFP vector, empty or carrying wild-type or mutated DGs All the uncleavable mutants are expressed and targeted to the plasma membrane (open arrowheads), showing a fluorescence pattern similar to that
of wild-type DG (WT) GFP was expressed throughout the cytoplasm.
Trang 7The a–b interface is essential for the correct cleavage of the DG precursor
A heterologous cell expression system was used to verify whether the mutations analysed in vitro might also influence the expression and stability of DG in cells 293-Ebna cells were transfected with the entire
DG gene carrying the single mutations, S556A, Q559A, M561A, G563A, L564A and P565A, and cloned into a pEGFP vector As demonstrated by western blot of total cell extracts, the single point mutations do not drastically alter the stability of DG, which is correctly processed into the two subunits (Fig 3A,B) Only the mutant G563A showed an addi-tional faint band at about 160 kDa, probably caused
by a small amount of the uncleaved DG precursor, pre-DG, which spans both the a- and b-subunits
of DG (Fig 3A,B,E) Interestingly, the two DG
A
B
C
Fig 5 WGL enrichment of total protein extracts of untransfected
cells (NT) and cells transfected with wild-type or mutated DGs (A)
Assay rationale: WGL specifically binds to the N-acetylglucosamine
moieties covalently linked to the core protein of a-DG Therefore,
b-DG-GFP, which is noncovalently associated with a-DG (or directly
pre-DG), can be retained from the immobilized WGL molecules.
(B) Western blot carried out with the anti-b-DG IgG clearly shows
that both wild-type (b-DG-GFP) and mutant (b-DG-GFP and mainly
pre-DG) DG proteins can be specifically eluted by WGL beads.
Only the eluted fractions, collected upon extensive washing, were
loaded onto the gel; the wash fractions did not contain any
rele-vant signal (data not shown) (C) The amounts of cleaved DG-GFP
and unprocessed pre-DG were quantified by densitometry,
averag-ing the values of the band intensities obtained from three
indepen-dent experiments.
A
B
Fig 6 Laminin-Sepharose pull-down of mutant pre-DGs (A) Assay rationale: laminin, covalently bound to CNBr-Sepharose, interacts with a-DG b-DG-GFP is retained by laminin-Sepharose beads through the interaction with the a-subunit (B) Pull-down of wild-type (WT) DG or DG carrying the mutations indicated on the spe-cific panels Western blot carried out with the anti-b-DG IgG clearly shows that both wild-type and mutant DG proteins specifically bind laminin (lane E: elution fraction) after extensive washing (lanes W1 and W5); FT (flow-through) The black boxes indicate b-DG-GFP and pre-DG-GFP.
Trang 8constructs carrying the double mutations,
G563A⁄ P565A and F692A ⁄ F718A, which, in
solid-phase assays completely inhibit the binding between
a- and b-DG (Fig 2C) [26], display significant
amounts of pre-DG, with respect to the correctly
cleaved b-DG-EGFP (Fig 3C–E)
The correct cleavage is completely inhibited in the
DG construct carrying the four mutations G563A,
P565A, F692A and F718A, (Fig 3C–E), suggesting
that interaction between the reciprocal binding
epi-topes of the two subunits forming the mature DG
complex is necessary for correct processing of the DG
precursor Consistent with this hypothesis, the entire
deletion (knockin) of the b-DG binding epitope within
the a-DG subunit (positions 550–565) abolishes the
cleavage, producing the 160 kDa pre-DG (Fig 3C–E)
The mutants pre-DGG563A_P565A_F692A_F718A,
pre-DGD550–565 and pre-DGS654A specifically bind
WGL, which indicates that they are at least partially
glycosylated (Fig 5B); furthermore, laminin pull-down
experiments show that mutated DG precursors
har-bour some laminin binding epitopes (Fig 6B), clearly
indicating a residual functionality of hypoglycosylated
and unprocessed pre-DGs
Depicting a possible model for DG precursor
processing
The mechanism and functional significance of DG
pre-cursor processing still remain largely elusive In several
human and murine cell lines and tissues, DG was
always detected as a heteromeric complex, suggesting
that precursor cleavage is a very early
post-transla-tional event along the route of DG maturation
Muta-tions in the amino acid sites crucial for the interaction
between a- and b-DG, namely G563 and P565 within
the C-terminal domain of a-DG and their counterparts
F692 and F718 within the b-DG ectodomain, ‘freeze’
the DG precursor as a relatively stable and partially
glycosylated monomeric intermediate Our results
strongly suggest that the network of interactions
important for the build up of the a–b interface on
pre-cursor cleavage is already established within the
unc-leaved DG precursor and is strictly necessary for
processing into the two subunits The impairment of
the correct formation of the a–b interface may
destabi-lize pre-DGs; indeed, both pre-DGG563A_P565A_
F692A_F718A and pre-DG(D550–565) display lower
expression levels compared with pre-DGS654A, in
which most of the interactions underlying the a–b
interface are still likely to take place (Fig 3E) Such a
network of interactions may also influence the
glyco-sylation pattern of the DG precursor This could be
inferred from the different electrophoretic behaviour displayed by the uncleavable DGs Indeed,
pre-DGS654A displays in western blot as a broader band (which could imply the presence of more carbohydrate groups) with respect to pre-DGG563A_P565A_ F692A_F718A and pre-DG(D550–565), where most of the a–b interactions cannot be established (Fig 3C,D) The correct folding of the DG precursor may there-fore be important for the recognition by glyco-syltransferases, which should primarily take place at the level of the N-terminal portion of a-DG [33,34] In particular, O-glycosyltransferases are thought to be crucial, especially for the extensive sugar decoration of the DG central mucin-like domain [19] Apart from the correct folding of what could be defined as the
‘pre-a–b interface’, a few other factors have been proposed to play an important role in DG precursor processing: for example, the disulfide bridge between C669 and C713, within the b-DG ectodomain [35], and N-glycosylation [27] The formation of this disulfide bridge may also contribute to the stabilization of the correct folding of the DG precursor necessary for spe-cific cleavage As far as N-glycosylation is concerned,
it has been shown by others that alanine substitution
of N662, a putative N-glycosylation site in the b-DG ectodomain, prevents the cleavage of the precursor and strongly reduces its expression [27] However, whether N-glycosylation really influences DG precursor cleav-age is still a matter of debate: other studies have shown that blocking N-glycosylation does not prevent cleavage [36]
On the basis of our data and other evidence from the literature, we propose the following scenario for
DG maturation (shown in Fig 7): immediately after translation, the DG core protein is translocated into the endoplasmic reticulum, where it is likely to adopt a stable three-dimensional conformation prior
to any post-translational modifications At this stage,
an essential contribution for achieving a confor-mation that will allow subsequent cleavage is pro-vided by a network of interactions (in which G563, P565, F692 and F718 play a crucial role) that are likely to stabilize the mature a–b interface also on cleavage
It is still unclear whether cleavage is carried out by
an unidentified protease or whether it occurs via an autocatalytic mechanism [28] However, our data clearly show that precursor cleavage is dispensable for correct trafficking and membrane targeting of DG, as all our novel uncleavable mutants can be detected at the plasma membrane, and their localization is indis-tinguishable from that characterizing wild-type DG (Fig 4); furthermore, they are still capable of binding
Trang 9laminin, fulfilling one of the most important functions
of DG (Fig 6)
Conclusions
Based on the available evidence, during evolution,
there was a ‘free choice’ for the liberation, or not, of
the two DG subunits For example, in Caenorhabditis
elegans, in which not only DG but a whole DGC
orthologue has been identified and functionally
charac-terized [37], the maturation of the DG complex into
two subunits has not been observed and, accordingly,
the motif Gly-Ser (653–654) at the cleavage site has
not been conserved [38] Clearly, further work is
needed in order to fully understand the biological
sig-nificance of why the two DG subunits are liberated
[39]
How an abnormal a–b interface would affect human
DG function is not yet known, as no primary
muta-tions of the dag1 gene have been identified so far
However, in principle, it should be possible to find
spe-cific mutations, or more likely polymorphisms, which,
in mammals, would interfere with DG processing
with-out grossly impairing DG function and displaying very
mild phenotypic signs in virtually asymptomatic
carri-ers This is suggested by recent papers from other
laboratories showing that DG does not take part in
the later stages of embryonic development, or that
hy-poglycosylated DG can be partially functional [40–42]
However, it will also be important to rule out the
pos-sibility that the presence of an uncleaved DG precur-sor may, instead, be linked to severe neuromuscular pathologies
A concerted effort of biochemical, genetic and clini-cal studies is needed in order to finally address these points At the present stage, our identification of mul-tiple point mutations that inhibit or affect the DG maturation pathway may provide a useful tool to investigate and shed light on the molecular details of such an important and mysterious process
Experimental procedures
DNA manipulation The full-length cDNA encoding for murine DG was used
as a template to generate, by PCR, two DNA constructs, one corresponding to the N-terminal region of b-DG, b-DG(654–750), and the other to the C-terminal region of a-DG, a-DG(485–630) [22] Appropriate primers were used
to amplify the DNA sequences of interest For b-DG(654– 750): forward, 5¢-CCCGGATCCTCTATCGTGGTGG AATGGACCAACA-3¢; reverse, 5¢-CCCGAATTCTTAG TAAACATCGTCCTCACTGCTCTCTTC-3¢ (BamHI and EcoRI restriction sites are given in italic type) For a-DG(485–630): forward, 5¢-CCCGTCGACAGTGGAGTG CCCCGTGGGGGAGAAC-3¢; reverse, 5¢-CCCGAATTC TTATACCAAAGCAATTTTTCTTGTGAATG-3¢ (SalI and EcoRI restriction sites are given in italic type) Single point mutations were introduced into the murine DG gene, cloned into the pEGFP vector, using the QuikChange
site-PreDG
PreDG
Fig 7 Schematic model showing the
influ-ence of the a–b interface on pre-DG
cleav-age (A) In the wild-type pre-DG, the correct
interaction between the a- and b-domains
stabilizes pre-DG in a conformation that can
be proteolytically processed at its G ⁄ S
cleavage site, liberating the a- and
b-subun-its The black double-headed arrow indicates
the pre-a–b interface (B) When S654, part
of the cleavage site, is mutated, pre-DG is
not proteolytically processed It is possible
that the interactions within the a–b interface
are formed even in the uncleaved precursor,
ensuring a certain stability of pre-DG The
black double-headed arrow indicates the
pre-a–b interface (C) When the a–b
inter-face is impaired by specific mutations hitting
the amino acids G563, P565 (within a-DG),
F692 and F718 (within b-DG), pre-DG does
not reach a conformation suitable for
proteo-lytic cleavage.
Trang 10directed mutagenesis kit (Stratagene, Cedar Creek, TX,
USA); all constructs were verified by automated
sequenc-ing The primers used for mutagenesis are reported below
with the mutated codons in italic:
S556A forward: 5¢-TGGGTTCAGTTTAACGCCAACA
GCCAGCTCATG-3¢
S556A reverse: 5¢-CATGAGCTGGCTGTTGGCGTTA
AACTGAACCCA-3¢
Q559A forward: 5¢-TTTAACAGCAACAGCGCGCTC
ATGTATGGCCTG-3¢
Q559A reverse: 5¢-CAGGCCATACATGAGCGCGCT
GTTGCTGTTAAA-3¢
M561A forward: 5¢-AGCAACAGCCAGCTCGCGTAT
GGCCTGCCTGAC-3¢
M561A reverse: 5¢-GTCAGGCAGGCCATACGCGAG
CTGGCTGTTGCT-3¢
Y562A forward: 5¢-AACAGCCAGCTCATGGCT
GGCCTGCCTGACAGC-3¢
Y562A reverse: 5¢-GCTGTCAGGCAGGCCAGCCAT
GAGCTGGCTGTT-3¢
G563A forward: 5¢-AGCCAGCTCATGTATGCCCTG
CCTGACAGCAGC-3¢
G653A reverse: 5¢-GCTGCTGTCAGGCAGGGCATA
CATGAGCTGGCT-3¢
L564A forward: 5¢-CAGCTCATGTATGGCGCGCCTG
ACAGCAGCCAT-3¢
L564A reverse: 5¢-ATGGCTGCTGTCAGGCGCGCC
ATACATGAGCTG-3¢
P565A forward: 5¢-CTCATGTATGGCCTGGCTGAC
AGCAGCCATGTG-3¢
P565A reverse: 5¢-CACATGGCTGCTGTCAGCCAG
GCCATACATGAG-3¢
S654A forward: 5¢-CAGAACATCACTCGGGGCGC
TATCGTGGTGGAATGGACC-3¢
S654A reverse: 5¢-GGTCCATTCCACCACGATAGCGC
CCCGAGTGATGTTCTG-3¢
G563AP565A forward: 5¢-AGCCAGCTCATGTATG
CCCTGGCTGACAGCAGC-3¢
G563AP565A reverse: 5¢-GCTGCTGTCAGCCAGGG
CATACATGAGCTGGCT-3¢
The full-length DNA constructs carrying the point
muta-tions were also used as templates to generate, by PCR, the
DNA constructs for the expression of the a-DG(485–630)
mutants in the Escherichia coli recombinant system (see
below), employing the same primers as used to amplify the
wild-type a-DG(485–630) sequence
For the production of the DG(D550–565) deletion
mutant, the knocked-in DNA construct was generated by
the overlap extension method [43] using 5¢-CCCGAAT
TCATGTCTGTGGACAACTGGCTACTG-3¢ and
5¢-TTTCTCACCTACTAACTGCTGCTCT-3¢ as forward and
reverse primers, respectively, for the first PCR, and
5¢-CAGTTAGTAGGTGAGAAAGACAGCAGCCATGTG-3¢
and 5¢-CCCGAATTCGGCTAGGGGGAACATACGGAG
GGGG-3¢ for the second PCR
Protein expression, purification and biotinylation The DNA constructs were cloned into a bacterial vector that was appropriate for the expression of the protein as a thioredoxin fusion product, also containing an N-terminal 6His tag and a thrombin cleavage site [44] The recombi-nant fusion proteins were expressed in E coli BL21(DE3) Codon Plus RIL strain and purified using nickel affinity chromatography The fragments of interest were obtained
on thrombin cleavage Tricine⁄ SDS-PAGE was used to check the purity of the recombinant proteins under analy-sis For solid-phase binding assays, recombinant b-DG(654–750) was biotinylated in 5 mm sodium phos-phate buffer at pH 7.4, with 0.5 mgÆmL)1 sulfo-N-hydroxyl-succinimido-biotin (S-NHS-biotin, Pierce, Rockford, IL, USA) The reaction was carried out for 30 min on ice and
in the dark, and dialysed overnight against 10 mm Tris⁄ HCl, 150 mm NaCl, pH 7.4 The optimal dilution for signal detection was determined by dot blot analysis and revealed by enhanced chemiluminescence (Pierce)
Solid-phase binding assays
To assess the binding properties of recombinant a-DG(485– 630) and its mutants with respect to biotinylated recombi-nant b-DG(654–750), solid-phase assays were performed as follows: approximately 0.5 lg of a-DG(485–630), its mutants and BSA were immobilized on microtitre plates in coating buffer (50 mm NaHCO3, pH 9.6) overnight at 4C After washing with NaCl⁄ Pi buffer (2.5 mm KCl, 2 mm
KH2PO4, 2 mm Na2HPO4, 140 mm NaCl, pH 7.4) contain-ing 0.05% (v⁄ v) Tween-20, 1.25 mm CaCl2 and 1 mm MgCl2, wells were incubated with decreasing concentrations
of recombinant biotinylated b-DG(654–750) in NaCl⁄ Pi containing 0.05% (v⁄ v) Tween-20, 3% (w ⁄ v) BSA, 1.25 mm CaCl2and 1 mm MgCl2for 3 h at room temperature After washing, the biotinylated b-DG(654–750) bound fraction was detected with alkaline phosphatase Vectastain AB Complex (Vector Laboratories, Burlingame, CA, USA) Five milligrams of p-nitrophenyl phosphate dissolved in
10 mL of 10 mm diethanolaminine and 0.5 m MgCl2 were added to every well containing 100 lL of this solution, and used as a substrate for the reaction with alkaline phospha-tase; the absorbance values were recorded at 405 nm For each b-DG(654–750) concentration, the absorbance value (Ai) originating from coated BSA was subtracted from the values obtained with the coated wild-type or mutated a-DG samples under analysis The data were fitted using a single class of equivalent binding sites equation, Ai= A-sat[c⁄ (KD+ c) + A0], where Ai represents the absorbance measured at increasing concentrations of ligand, KD is the dissociation constant, c is the concentration of ligand, bioti-nylated b-DG(654–750), and Asat and A0 are the absor-bances at saturation and in the absence of ligand, respectively Data were normalized and reported as the