515
Commentary
Isoaspartate-dependent molecular switches for
integrin–ligand recognition
Angelo Corti* and Flavio Curnis
Division of Molecular Oncology and IIT Network Research Unit of Molecular Neuroscience, San Raffaele Scientific Institute, via Olgettina 58,
20132 Milan, Italy
*Author for correspondence ([email protected])
Journal of Cell Science
Journal of Cell Science 124, 515-522
© 2011. Published by The Company of Biologists Ltd
doi:10.1242/jcs.077172
Summary
Integrins are cell-adhesion receptors that mediate cell–extracellular-matrix (ECM) and cell–cell interactions by recognizing specific
ligands. Recent studies have shown that the formation of isoaspartyl residues (isoAsp) in integrin ligands by asparagine deamidation
or aspartate isomerization could represent a mechanism for the regulation of integrin–ligand recognition. This spontaneous posttranslational modification, which might occur in aged proteins of the ECM, changes the length of the peptide bond and, in the case of
asparagine, also of the charge. Although these changes typically have negative effects on protein function, recent studies suggested
that isoAsp formation at certain Asn-Gly-Arg (NGR) sites in ECM proteins have a gain-of-function effect, because the resulting
isoAsp-Gly-Arg (isoDGR) sequence can mimic Arg-Gly-Asp (RGD), a well-known integrin-binding motif. Substantial experimental
evidence suggests that the NGR-to-isoDGR transition can occur in vitro in natural proteins and in drugs containing this motif, thereby
promoting integrin recognition and cell adhesion. In this Commentary, we review these studies and discuss the potential effects that
isoAsp formation at NGR, DGR and RGD sites might have in the recognition of integrins by natural ligands and by drugs that contain
these motifs, as well as their potential biological and pharmacological implications.
Key words: Integrin, Extracellular matrix, Fibronectin, RGD, DGR, NGR, Asparagine deamidation, Aspartate isomerization, isoDGR, Tumor
targeting, Disintegrins, NGR-hTNF
Introduction
Integrins are  heterodimeric transmembrane receptors involved
in the regulation of many aspects of cell behavior, including
adhesion, growth, survival, proliferation, migration and invasion
(Barczyk et al., 2010). The integrin family comprises 24 members
made up of different combinations of  and  subunits. The specific
combination of subunits in each heterodimer determines the
function of the integrin and its ligand specificity. Individual
integrins may bind more than one ligand and individual ligands
can bind to more than one integrin (Humphries et al., 2006; Plow
et al., 2000). For example, the v3 heterodimer can recognize the
sequence Arg-Gly-Asp (RGD) that is present in fibronectin,
vitronectin, fibrinogen, osteopontin, vonWillebrand factor, tenascin
and thrombospondin, whereas a subset of integrins, such as 51-,
v1-, v3-, v5-, v6-, 81- and IIb3-integrins, can
recognize the sequence RGD in fibronectin (Pankov and Yamada,
2002). Other integrins recognize different sequences in the
extracellular matrix (ECM) or other proteins. For example, 41-,
47- and 91-integrins, can recognize the sequence LDV in
fibronectin and SVVYGLR in osteopontin, whereas 11-, 21-,
101- and 111-integrins recognize the GFOGER sequence in
collagen (Humphries et al., 2006). Besides these well-characterized
binding motifs, it has been proposed that the NGR and DGR
sequences also have a role in integrin recognition, although
controversial data have been reported with regard to the integrinbinding properties of these motifs (Corti et al., 2008; Hautanen et
al., 1989; Jullienne et al., 2009; Koivunen et al., 1999; Liu et al.,
1997; Rusnati et al., 1997; Soncin, 1992; Yamada and Kennedy,
1987). Interestingly, these motifs and many other integrin
recognition sequences (reviewed by Ruoslahti, 1996) contain
aspartate (Asp) or asparagine (Asn), i.e. two amino acid residues
that spontaneously can undergo post-translational modifications to
form isoaspartyl residues (isoAsp or isoD). There is substantial
evidence that formation of this non-standard -amino acid can
occur during tissue aging in the ECM and in other secreted proteins
with a slow turnover, e.g. in collagen, fibronectin and -crystallin
(Lanthier and Desrosiers, 2004; Lindner and Helliger, 2001; Weber
and McFadden, 1997b). Although this protein modification typically
causes a loss-of-function, recent data suggest that isoAsp formation
at NGR or DGR sites, by Asn deamidation or Asp isomerization,
might have a gain-of-function effect, because the resulting isoDGR
motif can mimic RGD and recognize the RGD-binding site of
integrins (Curnis et al., 2006).
In this Commentary, we discuss the potential role that isoAsp
formation in proteins of the ECM have as a molecular switch for
negative or positive regulation of integrin recognition by ligands,
particularly those containing RGD, NGR and DGR sequences.
Furthermore, we discuss the structural basis of the molecular
mimicry of RGD by isoDGR in integrin recognition, the regulatory
mechanisms, and the potential functional implications of isoAsp
formation in natural ligands and in synthetic drugs that contain
these motifs.
Formation of isoAsp in ECM proteins and its
functional effects
Formation of isoAsp and ECM protein damage
The formation of isoAsp residues at Asn or Asp residues in proteins
is generally considered a degradation reaction as a consequence of
protein aging. IsoAsp formation has been shown to occur in ECM
proteins, including collagen types I and III, in fibronectin, in crystallin and in the ECM of the mammalian brain (Lanthier and
Desrosiers, 2004; Lindner and Helliger, 2001; Reissner and Aswad,
Journal of Cell Science 124 (4)
2003; Weber and McFadden, 1997a). IsoAsp formation can occur
not only in vitro, e.g. during protein storage, but also in vivo, as
has been documented for the extracellular matrix of mammalian
brain or the vascular wall (Reissner and Aswad, 2003; Weber and
McFadden, 1997b). This post-translational modification might
cause changes in the charge or the length of the peptide bond (see
Fig. 1A), typically causing loss of function. For example, in
collagen-I or in peptides containing RGDSR and KDGEA (a
recognition sequence for 21-integrin), the formation of isoAsp
reduces their capability to promote cell motility (Lanthier and
Desrosiers, 2004). Treatment of aged collagen I with protein-LisoAsp-O-methyltransferase (PIMT), an enzyme that can convert
isoAsp into Asp, partially recovers cell migration. In addition,
fibronectin, an adhesive protein that has important roles in
haemostasis, thrombosis, inflammation, wound repair, angiogenesis
and embryogenesis (Humphries et al., 1989; Pankov and Yamada,
2002), also accumulates isoAsp during in vitro aging (Lanthier and
Desrosiers, 2004). Similarly, treatment of aged fibronectin or
retronectin – a fibronectin fragment containing RGD – with PIMT
increases its pro-adhesive activity on endothelial cells (Curnis et
al., 2006) (A.C. and F.C., unpublished data). Taken together, it
therefore appears that the biological properties of collagen,
fibronectin and their fragments are impaired by Asp-to-isoAsp
transitions that occur at certain sites in these proteins and are
subsequently restored by transforming isoAsp back into Asp.
of evidence suggests that this reaction, in some instances, constitutes
a mechanism for the intentional modification of a protein structure
with a useful function. For example, it has been proposed that
isoAsp formation, which might occur faster in proteins that have
lost their structural integrity, can provide a type of molecular
indicator of protein damage, which is capable to activate selective
degradation mechanisms (Lanthier and Desrosiers, 2004; Pepperkok
et al., 2000; Reissner and Aswad, 2003; Robinson and Robinson,
2001; Weintraub and Deverman, 2007). Another striking example
of this concept is the formation of isoAsp in fibronectin fragments.
Fibronectin (FN) contains three types of repeating homologous
modules, in addition to alternatively spliced modules (Mohri, 1997;
Pankov and Yamada, 2002). Notably, four FN modules, i.e. the
fifth type I (FN-I5), the first type II (FN-II1), the seventh type I
(FN-I7) and the ninth type III (FN-III9) repeats, contain NGR
sequences. It has been suggested that isoAsp formation at the NGR
site of FN-I5 activates a latent v3-integrin-binding site (Curnis
et al., 2006; Takahashi et al., 2007). Here, deamidation of Asn263
leads to a transformation of NGR into isoDGR and DGR. Structure–
activity relationships studies have shown that isoDGR and not
DGR is the integrin-binding motif.
Mechanisms of isoAsp formation
The formation of isoAsp in proteins can occur by non-enzymatic
Asn deamidation or Asp isomerization reactions (Geiger and Clarke,
1987; Weintraub and Deverman, 2007). Asn deamidation occurs
by a nucleophilic attack of the backbone NH center at the carbonyl
group of Asn side chain, which leads to formation of a succinimide
ring (Fig. 1B). The formation of a succinimide ring is also the first
Formation of isoAsp at NGR sites with gain-of-function
Although isoAsp formation in aged proteins is generally viewed as
a deleterious event with regard to their function, a growing body
A
N
α
C
C
O
H
C
N
N
O
C
C
O
C
NH2
H
O
N
C
H β CH2
H
H β CH2
α
N
C
H
H
O
C
O
H
O
β
O
α
C
C
CH2 C
N
O
Journal of Cell Science
516
H
O
O
Asn
Asp
isoAsp
B
L/D-isoAsp
(isoDGR)
O
O
H
N
α
α
N
H
NH2
β
N
H
Deamidation
NH3
O
L-Asn
β
H
N
PIMT
H2O
α
O
CH3OH
H2O
β
N
H
N
H
OCH3
β
O
Methyl ester
O
N
H
O
O
α
N
Succinimide
Dehydration
SAH
only for
L-isoAsp
O
H
N
SAM
O
H2O
O
(NGR)
H2O
N
H
O
H2O
H
N
α
N
H
O
β
O
O
L-Asp
L/D-Asp
(DGR)
(DGR)
Fig. 1. Mechanisms of spontaneous formation of isoDGR by
deamidation of Asn or isomerization of Asp residues.
(A)Schematic representation of the backbone of peptides with Asn,
Asp and isoAsp residues (shaded area). The formation of isoAsp as a
consequence of Asn deamidation or Asp isomerization changes the
length of the peptide bond and, in the case of Asn, also the charge of
the side chain. (B)Schematic representation of L-Asn deamidation
and L-Asp isomerization in proteins and peptides. This reaction can
occur by nucleophilic attack of the adjacent peptide bond nitrogen
atom on the side chain carbonyl group of L-Asn (indicated by the
curved arrow), leading to formation of a five-membered succinimide
ring. Racemization of the succinimide intermediate and its rapid
hydrolysis at either the - or the -carbonyl group then generates
mixtures of L-Asp, D-Asp, L-isoAsp and D-isoAsp, with L-isoAsp
being the most abundant. L-isoAsp can be converted into L-Asp by
the enzyme protein-L-isoAsp-O-methyltransferase (PIMT) through
methyl-esterification in the presence of S-adenosylmethyonine
(SAM). L-Asp isomerization can occur by a similar mechanism that
involves L-Asp dehydration and formation of the succinimide
intermediate.
IsoAsp and integrin–ligand recognition
step for Asp isomerization (Fig. 1B). Hydrolysis can then occur at
both carbonyl groups of the cyclic imide, leading to the formation
of a mixture of Asp and isoAsp residues, typically with a ratio of
approximately 1:3. Racemization and hydrolysis of the succinimide
intermediate can also lead to the formation of Asp and isoAsp in
D-configuration, but this typically occurs with much lower
efficiency (Geiger and Clarke, 1987; Stephenson and Clarke, 1989;
Tyler-Cross and Schirch, 1991). Thus, the resulting Asp and isoAsp
in L-configuration are more relevant. The above deamidation and
isomerization reactions can take hours, days or even years –
depending on several factors. For example, secondary, tertiary and
quaternary structural elements in a protein can place Asn or Asp
residues in proximity to functional groups that either inhibit or
accelerate these reactions (Robinson, 2002; Robinson and
Robinson, 2001; Robinson et al., 2004). In particular, the presence
of a Gly residue following Asn or Asp generally accelerates these
reactions owing to its small size and flexibility (Robinson and
Robinson, 2001). Other crucial factors are the pH, the ionic strength
and the temperature (Robinson and Robinson, 2001; Tyler-Cross
and Schirch, 1991).
Journal of Cell Science
Kinetics of the NGR-to-isoDGR transition
Approximately 17% of proteins that are classified by using the
keyword ‘adhesion’ in vertebrate protein databanks contain NGR
sites (Corti et al., 2008). Considering the known effects of 3Dstructural protein elements on the deamidation rate of Asn residues
discussed above, only certain NGR sites with suitable accessibility,
flexibility and side chain orientation are likely to undergo
deamidation under physiological conditions and, subsequently,
bind integrins. Remarkably, NGR deamidation in the FN-I5
fragment is very rapid, with a half-life of the NGR site in cell
culture medium of only ~4 hours (Curnis et al., 2006). The
surprising velocity of this reaction is probably owing to the fact
that here Asn is followed by a Gly residue. Furthermore, the Asn
and Gly residues are part of the GNGRG loop that is well exposed
on the surface of this fibronectin fragment, thereby possibly
favoring the correct orientation of the Asn side chain and peptide
backbone for a nucleophilic attack (Di Matteo et al., 2006).
Similarly, fast deamidation rates have been observed in peptides
that contain the disulfide-bonded CNGRC loop (Curnis et al.,
2010; Curnis et al., 2006).
By contrast, full-length plasma fibronectin was shown to be
considerably more resistant to Asn deamidation than short FN
fragments or peptides (Lanthier and Desrosiers, 2004) and,
accordingly, freshly isolated plasma fibronectin contains only 0.03–
0.05 pmol of isoAsp per pmol of protein (Curnis et al., 2006).
Interestingly, cystine reduction and alkylation enhanced isoDGR
formation in a 70 kDa N-terminal fragment of fibronectin (Xu et
al., 2010). It therefore appears that rapid deamidation of NGR in
fibronectin requires proteolytic processing and/or conformational
changes. Nevertheless, the in vivo formation of isoDGR sites in
fibronectin, e.g. after deposition in tissues, still remains to be
demonstrated.
Rapid NGR-to-isoDGR transformation also occurs in
recombinant proteins that contain CNGRC fused to the Nterminus of tumor necrosis factor  (TNF-) (Curnis et al., 2006;
Curnis et al., 2008) or to the C-terminus of interferon  (IFN)
(Curnis et al., 2005), suggesting that this phenomenon is not
specific for the GNGRG site in FN-I5. Thus, it is possible that
other proteins also use this mechanism to activate latent integrin
recognition sites, and this should be taken into account when
517
analyzing potential integrin-binding sites of proteins that contain
the NGR sequence.
Recognition of RGD-dependent integrins by isoDGR and
its functional role
Peptides that contain the isoDGR motif can recognize members of
the RGD-dependent integrin family, such as v3-, v5-, v6-,
v8- and 51-integrins, but not other, such as 11-, 31-,
47-, 64- or 91-integrins (Curnis et al., 2010). The affinity
and specificity of the interaction between isoDGR and integrin
depend on the molecular scaffold of isoDGR (Curnis et al., 2010).
For instance, cyclic peptides that contain isoDGR flanked by Cys
residues can bind v3-integrin with an affinity that is 10–100fold higher than that for other members of the RGD-dependent
integrin family (for example, Kd is ~9 nM for v3-integrin).
Replacement of the Cys by two Gly residues leads to a marked loss
of affinity for all integrins and a change of specificity (Curnis et
al., 2010). It appears, therefore, that isoDGR, like RGD, can
differentially recognize integrins depending on its molecular
scaffold.
These findings and the results of a series of in vitro experiments
performed with fibronectin and fibronectin fragments suggest that
NGR deamidation has a functional role in the regulation of cell
adhesion. For example, a deamidated FN-I4-5 fragment enhanced
the adhesion of endothelial cells to microtiter plates, but not when
its NGR sequence was replaced with SGS (Curnis et al., 2006).
Similarly, replacement of NGR in the FN-I5 and FN-I7 modules of
a large N-terminal fragment of fibronectin (FN-70 kDa) by QGR,
a sequence that can deamidate but with a much slower rate
(Robinson and Robinson, 2001), reduced its cell adhesion activity,
further supporting a functional role for the NGR sequence in
fibronectin modules (Xu et al., 2010).
Although isoDGR-containing compounds can promote cell
adhesion when they are adsorbed onto microtiterplates, they can
exert inhibitory effects in other assays. For example, deamidated
FN-I5 and the peptide CisoDGRC inhibited endothelial cell
proliferation and adhesion to vitronectin when these peptides were
added as soluble ligands in the cell culture medium (Curnis et al.,
2006). These peptides also recognized v3-integrin-positive
endothelial cells in tumor vessels, and inhibited tumor growth
when they were systemically administered to tumor-bearing mice
(Curnis et al., 2010; Curnis et al., 2006). Given the growing body
of evidence that implicates v3-integrin in angiogenesis (Brooks
et al., 1995; Eliceiri and Cheresh, 2000), it is possible that the
interaction of the isoDGR site in fibronectin or fibronectin
fragments with v3-integrin has an important role in cancer and
in other diseases that involve angiogenesis.
Tissue fibronectin is organized in fibrils, which mediate a variety
of cellular interactions with the extracellular matrix (Humphries et
al., 1989; Pankov and Yamada, 2002). The formation of fibronectin
fibrils in the ECM is a cell-mediated process that involves the
rearrangement of the cytoskeleton and integrins in order to transmit
the forces necessary to unfold fibronectin and assemble it into
fibrils (Geiger, 2001; Ohashi et al., 2002; Pankov et al., 2000;
Zamir et al., 2000). Mice that were genetically engineered to
express a fibronectin variant with a non-functional RGE in place
of RGD and that are, thus, unable to bind integrins through this
site, can still assemble fibrils in vivo (Takahashi et al., 2007). The
same authors also showed that a cyclic peptide containing isoDGR
inhibits fibril formation of a RGE-fibronectin mutant in vitro and
they proposed that the GNGRG of FN-I5 can function in fibril
518
Journal of Cell Science 124 (4)
assembly. However, other investigators showed that fibronectin
mutants, in which NGR is replaced with QGR in the FN-I5 and
FN-I7 modules, can still form fibrils, thus arguing against a crucial
role of these sites in fibril formation (Xu et al., 2010). As the role
in fibril formation of those NGR sites located in FN-II2 and FNIII9 has not yet been investigated, further studies are necessary to
clarify the function of different NGR sites in the formation of
fibronectin fibrils.
Journal of Cell Science
The DGR-to-isoDGR transition and integrin recognition
The frequency of the DGR sequence element in proteins classified
as having a role in cell adhesion is higher than that of other xGR
sequences (where x represents any amino acid residue other than
Asn or Asp) and is similar to that of NGR and RGD (Corti et al.,
2008). Interestingly, it has been shown that DGR has also a role in
integrin recognition and cell adhesion (Gao and Brigstock, 2006;
Rusnati et al., 1997; Yamada and Kennedy, 1987). For example,
SDGR-containing peptides were found to inhibit the attachment of
fibroblasts to laminin and collagen type I when added to the cell
culture medium (Yamada and Kennedy, 1987). Furthermore,
fibroblast growth factor 2 (FGF2), a pro-angiogenic heparin-binding
cytokine that contains two DGR sites, can bind to v3-integrin
and promote endothelial cell adhesion (Rusnati et al., 1997).
Mapping of the cell-adhesive domains has shown that the regions
from amino acids 38 to 61 and 82 to 101 in FGF2, which contain
the DGR sequences, are responsible for this activity (Rusnati et al.,
1997). In addition, it was also shown that the GVCTDGR sequence
of connective tissue growth factor can bind 51-integrin (Gao
and Brigstock, 2006). However, other studies found SDGR and
other DGR peptides to be inactive in cell attachment assays, which
argues against a functional role of this motif in integrin recognition
(Hautanen et al., 1989; Liu et al., 1997; Soncin, 1992).
Given that DGR can be converted into isoDGR by the
isomerization of Asp, one explanation for these apparently
controversial data may be that DGR, like NGR, can function as a
latent integrin-binding site that is differentially activated dependent
on its molecular context and the conditions. In agreement with this
isoDGR
hypothesis, we have observed that the interaction of FGF2 with
v3-integrin is inhibited by treatment with PIMT (A.C. and F.C.,
unpublished data), supporting the idea that isoAsp residues in
FGF2 are crucial for this interaction.
Considering that a number of proteins contain DGR it is possible
that this mechanisms is more general. However, as discussed above
for NGR, it should be born in mind that the residues flanking DGR
and its microenvironment can crucially affect the kinetics of Asp
isomerization and its transformation into isoDGR. Furthermore,
the rate of Asp isomerization is generally slower than that of Asn
deamidation in similar peptides (Geiger and Clarke, 1987). This
reaction is, therefore, likely to have a significant role in only a
small subset of proteins.
Structural basis of the molecular mimicry of
RGD by isoDGR in integrin recognition
The structural basis of ligand recognition by RGD-dependent
integrins has been studied intensively (Luo et al., 2007; Takagi,
2004). Biochemical studies have shown that both - and -integrin
subunits span the plasma membrane and have short cytoplasmic
domains. Outside the plasma membrane, the - and -subunits lie
close together and form the RGD-binding pocket (Xiong et al.,
2001). Substantial experimental evidence suggests that isoDGR
binds to the RGD-binding site of integrins. First, the cyclic
CisoDGRCGVRY peptide (called isoDGR-2C) might inhibit the
binding of a cyclic RGD peptide to v3-integrin in a competitive
manner (Curnis et al., 2006). Notably, the affinities of cyclic
isoDGR-2C and RGD-2C peptides for v3-integrin were similar
(Curnis et al., 2006). Replacement of isoAsp with Asp (as in DGR2C) or with its enantiomer in D-configuration (D-isoAsp) causes a
marked loss of binding affinity, pointing to highly selective and
stereospecific interactions (Curnis et al., 2010; Curnis et al., 2006).
Second, NMR structure analysis of cyclic isoDGR-2C, RGD-2C,
DGR-2C and NGR-2C peptides, and v3-integrin docking
experiments showed that isoDGR, but not DGR and NGR, fit into
the RGD-binding pocket and that it favorably interacts with this
integrin (Curnis et al., 2006; Spitaleri et al., 2008) (Fig. 2). Notably,
RGD
Fig. 2. Model for the interaction between isoDGR and the RGD-binding site of v3-integrin. Representation of the v3-integrin-binding pocket in complex
with either CisoDGRC or CRGDC (courtesy of Giovanna Musco, San Raffaele Institute, Italy) (Spitaleri et al., 2008). - and -integrin subunits are represented in
pink and pale cyan, respectively. The RGD and isoDGR residues are shown in green, cystine in gray, and nitrogen, oxygen and sulfur atoms in blue, red and yellow,
respectively. Ca2+ bound to the adhesion site dependent on metal ions is represented by a red sphere. Integrin and ligand residues involved in binding are labeled
with the three- and one-letter code, respectively. Dotted lines denote hydrogen bonds between ligands and integrin. For details on peptide structure analysis and
docking experiments see Spitaleri et al. 2008 (Spitaleri et al., 2008). IsoDGR favorably interacts with the RGD-binding site of this integrin, thereby recapitulating
RGD-binding contacts and establishing additional polar interactions.
Journal of Cell Science
IsoAsp and integrin–ligand recognition
isoDGR docks onto v3-integrin in an inverted orientation
compared with RGD docking to v3-integrin. This orientation
allows isoDGR to bind the integrin - and -subunits through the
isoAsp and Arg residues. The acidic and basic side chains of these
residues are at the correct distance and orientation to engage
stabilizing interactions with the polar regions of the integrin, thus
reproducing the canonical interactions between RGD and v3integrin (Spitaleri et al., 2008). These data are in line with the
results of binding studies showing that isoDGR, but not DGR and
NGR, can efficiently interact with v3-integrin. Therefore,
isoDGR, unlike DGR and NGR, is a natural fit for the RGDbinding pocket of v3-integrin and supports the hypothesis that
transforming DGR or NGR into isoDGR can result in a molecular
switch able to activate integrin recognition.
Such a model might also explain the controversial data reported
in the literature for the interaction of integrin with compounds that
contain DGR – as discussed above – or NGR. The suggestion that
NGR and integrin interact originated from several observations.
For example, in vitro panning of phage-display libraries against
v3- and 51-integrins selected peptides that contain NGR
(Healy et al., 1995; Koivunen et al., 1993; Koivunen et al., 1994).
Furthermore, adeno-associated virus type 2 contains an NGR site
that is crucial for 51-integrin binding and viral cell entry (Asokan
et al., 2006), and adenoviruses that have been genetically engineered
to express the linear NGR sequence on their surface bind to v3integrin-expressing cells (Majhen et al., 2006). However, in vitro
binding studies with purified v3-integrin and peptides that
contain the CNGRC or GNGRG sequences show only a modest
interaction, if any at all (Curnis et al., 2010). Although we cannot
totally exclude that NGR sites that are embedded in viral particles
behave in a different manner, we hypothesize that integrin binding
observed with certain NGR-containing ligands can be explained
by binding of isoDGR generated from NGR. For example, phages
that have been selected as carrying the NGR sequence might
instead have displayed isoDGR on their surface owing to
deamidation reaction during library preparation and in vitro
selection. Thus, we think it is important that any isoAsp formation
must be ruled out before integrin recognition of NGR-containing
compounds is proposed.
Although isoDGR- and RGD-containing ligands can share the
same binding site on integrins, their effects on integrin function are
not necessarily identical for the following reasons. It is well
established that the interaction between integrins and ligands is
regulated by conformational changes (Arnaout et al., 2005; Luo et
al., 2007). Conformational changes and integrin activation can be
transmitted bidirectionally across the membrane through
interactions with intracellular molecules (inside-out signaling) and
with extracellular ligands (outside-in signaling) (reviewed in Askari
et al., 2009). It has been suggested that different RGD-containing
ligands induce different signaling pathways (Askari et al., 2009).
Thus, it is possible that RGD and isoDGR ligands induce
differential conformational changes and signaling in vivo.
Furthermore, although peptides containing CRGDC or CisoDGRC
sequences recognize purified v3-integrin with similar affinities
in vitro (Curnis et al., 2006), their kon and koff, which have not yet
been measured, might not necessarily be similar. Considering the
importance of these parameters for their biological properties,
kinetic analysis of binding data and characterization of the signaling
mechanisms induced by isoDGR-containing ligands are needed to
define the extent to which isoDGR mimics – or differs – from
RGD.
519
Regulation of isoAsp formation in tissues
As discussed above, Asp isomerization and Asn deamidation are
thermodynamically spontaneous reactions that do not require
enzymatic catalysis. It is currently unknown whether specific Asn
deamidases or Asp isomerases exist that could further accelerate
this process. However, the observation that NGR deamidates faster
in short fibronectin fragments compared with soluble plasma
fibronectin (discussed above) and the finding that isoDGR can be
converted into DGR by PIMT (Curnis et al., 2006) point to possible
enzymatic regulatory mechanisms.
In humans, PIMT is encoded by PCMT1, which is localized on
chromosome 6 (6q24–q25). This enzyme (EC 2.1.1.77) catalyzes
the transfer of a methyl group from S-adenosyl-L-methionine to
the free carboxyl groups of D-Asp and L-isoAsp residues (Reissner
and Aswad, 2003). The resulting methyl ester can then form a
cyclic succinimide that, after hydrolysis, can generate Asp and
isoAsp (Fig. 1B). In peptide substrates, there appears to be a
preference for isoAsp residues that are flanked by bulky
hydrophobic group at the position preceeding isoAsp and by neutral
or positive groups at the positions that follow isoAsp (Lowenson
and Clarke, 1991). PIMT is present in all vertebrate tissues and
represents the major protein methyltransferase in the brain, testis,
erythrocytes and eye (Yamamoto et al., 1998). PIMT is normally
an intracellular enzyme that exists in two isoforms due to
differential mRNA splicing (Takeda et al., 1995). The C-terminus
of one isoenzyme ends with the sequence RWK, whereas the other
isoform ends with RDEL, a known endoplasmic reticulum retention
sequence (MacLaren et al., 1992). It has been shown that
calmodulin, tubulin, histone H2B and synapsin I are natural
substrates of PIMT action in vivo (Furuchi et al., 2010). However,
experimental evidence suggests that this enzyme also targets
extracellular proteins, such as collagen type I and type II (Weber
and McFadden, 1997b). Interestingly, it has been demonstrated
that PIMT is released into the extracellular environment by
damaged vessels and by injured tissues, and that it becomes
entrapped in the extracellular space (Weber and McFadden, 1997a;
Weber and McFadden, 1997b). In uninjured blood vessels, up to
90% of total isoAsp residues are inaccessible to endogenous
intracellular PIMT (Weber and McFadden, 1997b). However, after
vessel injury PIMT is released into the extracellular environment
and methylation of extracellular proteins, such as aged collagen
type I and type II, can then ensue (Weber and McFadden, 1997b).
The biological importance of PIMT is also supported by the
observation that PIMT-deficient mice have a smaller body and
spleen, a larger brain, an abnormal neuronal organization and
atypical patterns of motor activity, and that they die from
progressive epilepsy at 4–10 weeks after birth (Kim et al., 1997;
Yamamoto et al., 1998).
An important point to keep in mind is that PIMT can restore the
original sequence in the case of Asp isomerization, but not in the
case of Asn deamidation, because isoAsp is converted into Asp in
both cases. Thus, whereas PIMT can potentially repair RGD or other
damaged functional sites that undergo Asp isomerization, it can lead
to inactivation of isoDGR sites derived from NGR or DGR.
Pharmacological implications and therapeutic
opportunities for peptides that target RGDdependent integrins
Integrins are involved in many physiological and pathological
processes, such as inflammation, thrombosis, osteoporosis,
angiogenesis and cancer. The role of integrins in cancer biology
Journal of Cell Science
520
Journal of Cell Science 124 (4)
and angiogenesis is one of the most studied functions of these cell
adhesion receptors. For example, v3-integrin, an integrin
overexpressed in the tumor vascular endothelium, has an important
role in angiogenesis and tumor growth (Brooks et al., 1995; Eliceiri
and Cheresh, 2000; Friedlander et al., 1996; Kumar, 2003). RGDcontaining peptides with a variable degree of affinity and selectivity
for this integrin have been developed and used for delivering a
variety of drugs and nanoparticles to tumor vessels (Desgrosellier
and Cheresh, 2009; Liu et al., 2008; Ruoslahti et al., 2010). Given
the structural and functional similarities between RGD and isoDGR
it is conceivable that peptides containing the isoDGR motif could
also be exploited as specific ligands for the targeted delivery of
drugs, imaging agents or other compounds to tumors. Consistent
with this, a cyclic CisoDGRC peptide that was coupled to
fluorescent nanoparticles (quantum dots) was shown to bind v3integrin and colocalize with antibodies against v3-integrin in
vessels of human renal cell carcinoma (Curnis et al., 2008).
Furthermore, extremely low doses (1–10 pg) of a recombinant
protein made up of CisoDGRC fused to TNF-, a cytokine capable
of inflicting damages to tumor vessels, induced anti-tumor effects
in tumor-bearing mice through specifically targeting TNF- to
tumor sites (Curnis et al., 2008).
The isoDGR motif might also be exploited, similar to RGD, for
the generation of integrin antagonists, which are currently being
investigated for the treatment of cancer, osteoporosis and
coagulation disorders (Desgrosellier and Cheresh, 2009; Liu et al.,
2008). For example, c(RGDf[NMe]V) (also known as Cilengitide)
is a cyclic RGD-peptide that binds v3- and v5-integrins and
inhibits angiogenesis. This drug is currently tested in a phase III
trial in patients with glioblastoma (Desgrosellier and Cheresh,
2009). In principle, new integrin antagonists could be developed
by using isoDGR instead of RGD. However, it should be born in
mind that simply replacing RGD with isoDGR within the context
of an existing compound is unlikely to work, because isoDGR has
to interact with the RGD-binding pocket in an inverted orientation
(see Fig. 3 for a schematic representation) and, moreover, the
flanking residues also contribute to integrin-binding affinity and
specificity as discussed above. The potential use of small molecules
that contain isoDGR is supported by the results of our recent study,
which shows that a cyclic CisoDGRC peptide alone inhibits the
growth of tumors in mouse models (Curnis et al., 2010). Further
work is necessary to assess the potential use of isoDGR compared
with RGD, regarding stability (also to proteases), integrin-binding
properties (Kd, kon and koff), signaling and pharmacological
properties.
The capability of isoDGR peptides to interact with integrins
might also have important implications for another class of drugs,
i.e. drugs containing the NGR motif. NGR peptides have been
used by different investigators to deliver cytokines,
chemotherapeutic drugs, liposomes, anti-angiogenic compounds,
viruses, imaging agents and DNA complexes to tumor
neovasculature, in order to improve their therapeutic or imaging
properties (reviewed by Corti and Curnis, 2011). For example, the
CNGRCG peptide, fused to the N-terminus of TNF- (NGRTNF), and the GNGRAHA-peptide, fused to the C-terminus of
tissue factor, are currently tested in several phase I, II and III
clinical studies (Bieker et al., 2009; Gregorc et al., 2010a; Gregorc
et al., 2010b; van Laarhoven et al., 2010; Corti et al., 2011). The
rationale for using these peptides is the observation that NGR can
recognize a membrane-bound form of aminopeptidase N (CD13)
that is expressed by angiogenic vessels (Curnis et al., 2002;
RGD
NGR
isoDGR
α
β
PIMT
DGR
Fig. 3. Schematic representation of an isoDGR-dependent molecular
switch for integrin–ligand recognition. As highlighted in this schematic
representation, isoDGR can derive from NGR by deamidation of Asn, but it
has to assume an inverted orientation (compared with RGD) to be able to
interact with v3-integrin. isoDGR can also be converted into DGR by PIMT
that, as the orginal NGR sequence element, cannot efficiently interact with this
integrin.
Pasqualini et al., 2000). Remarkably, in these compounds the NGRto-isoDGR transition, which may occur during purification, storage
or even in vivo for molecules that are circulating a long while,
might simultaneously switch off CD13 and switch on integrin
binding (Curnis et al., 2010) – with potentially important
pharmacological and toxicological implications.
Conclusions
The formation of isoAsp in proteins of the ECM might represent
a new mechanism for the regulation of integrin recognition by
their ligands. Although, this protein modification typically impairs
the functional properties of ECM proteins, it might however – in
certain cases – generate new integrin-binding sites. The spontaneous
transformation from NGR to isoDGR in fibronectin fragments,
through Asn deamidation, and the consequent activation of integrinbinding sites is an example of how isoAsp formation can confer
new functional properties to proteins. Spontaneous formation of
isoAsp in proteins might occur also at Asp residues through
isomerization reactions, suggesting that also the DGR motif can
work, in principle, as an inactive precursor of isoDGR. Whereas
the formation of isoDGR may represent a molecular switch for
‘turning on’ integrin binding, the release of PIMT from damaged
cells could represent an enzyme-dependent mechanism for ‘turning
off’ the switch by promoting conversion of isoDGR to DGR.
However, to be physiologically relevant, the kinetics of these on–
off changes need to be faster than protein turnover and the resulting
isoDGR site accessible to integrins. Thus, although substantial
experimental evidence suggests that proteins of the extracellular
matrix can accumulate isoAsp in vivo, further studies are necessary
to demonstrate that this reaction can, indeed, occur at NGR sites
in vivo, and that it generates isoDGR in an amount that is able to
substantially affect cell adhesion. As isoAsp formation can take
hours, days or even years, depending on the molecular
microenvironment, this mechanism is likely to have a significant
role only in certain proteins. Further work is necessary to identify
those proteins with sequence motifs that are transformed into
isoDGR, to quantify its formation in normal and pathological
tissues, and to assess their physiological roles.
The observation that isoDGR is generated in purified proteins
and in drugs that contain the NGR motif suggests that this reaction
IsoAsp and integrin–ligand recognition
also has important implications for their pharmacological and
toxicological properties. Furthermore, the ability of synthetic
isoDGR-containing peptides to mimic RGD and to bind to tumor
vessels suggests that these compounds can be exploited, similar to
RGD, as tools for targeted delivery of drugs and diagnostic
nanoparticles to tumor vessels or for the development of new
integrin antagonists.
This work was supported by Associazione Italiana per la Ricerca sul
Cancro (AIRC) and Alleanza Contro il Cancro (ACC) of Italy, and
FIRB.
Journal of Cell Science
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