Commentary 515

IntroductionIntegrins are heterodimeric transmembrane receptors involvedin the regulation of many aspects of cell behavior, includingadhesion, growth, survival, proliferation, migration and invasion(Barczyk et al., 2010). The integrin family comprises 24 membersmade up of different combinations of and subunits. The specificcombination of subunits in each heterodimer determines thefunction of the integrin and its ligand specificity. Individualintegrins may bind more than one ligand and individual ligandscan bind to more than one integrin (Humphries et al., 2006; Plowet al., 2000). For example, the v3 heterodimer can recognize thesequence Arg-Gly-Asp (RGD) that is present in fibronectin,vitronectin, fibrinogen, osteopontin, vonWillebrand factor, tenascinand thrombospondin, whereas a subset of integrins, such as 51-,v1-, v3-, v5-, v6-, 81- and IIb3-integrins, canrecognize the sequence RGD in fibronectin (Pankov and Yamada,2002). Other integrins recognize different sequences in theextracellular matrix (ECM) or other proteins. For example, 41-,47- and 91-integrins, can recognize the sequence LDV infibronectin and SVVYGLR in osteopontin, whereas 11-, 21-,101- and 111-integrins recognize the GFOGER sequence incollagen (Humphries et al., 2006). Besides these well-characterizedbinding motifs, it has been proposed that the NGR and DGRsequences also have a role in integrin recognition, althoughcontroversial data have been reported with regard to the integrin-binding properties of these motifs (Corti et al., 2008; Hautanen etal., 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 integrinrecognition sequences (reviewed by Ruoslahti, 1996) containaspartate (Asp) or asparagine (Asn), i.e. two amino acid residues

that spontaneously can undergo post-translational modifications toform isoaspartyl residues (isoAsp or isoD). There is substantialevidence that formation of this non-standard -amino acid canoccur during tissue aging in the ECM and in other secreted proteinswith a slow turnover, e.g. in collagen, fibronectin and -crystallin(Lanthier and Desrosiers, 2004; Lindner and Helliger, 2001; Weberand McFadden, 1997b). Although this protein modification typicallycauses a loss-of-function, recent data suggest that isoAsp formationat NGR or DGR sites, by Asn deamidation or Asp isomerization,might have a gain-of-function effect, because the resulting isoDGRmotif can mimic RGD and recognize the RGD-binding site ofintegrins (Curnis et al., 2006).

In this Commentary, we discuss the potential role that isoAspformation in proteins of the ECM have as a molecular switch fornegative or positive regulation of integrin recognition by ligands,particularly those containing RGD, NGR and DGR sequences.Furthermore, we discuss the structural basis of the molecularmimicry of RGD by isoDGR in integrin recognition, the regulatorymechanisms, and the potential functional implications of isoAspformation in natural ligands and in synthetic drugs that containthese motifs.

Formation of isoAsp in ECM proteins and itsfunctional effectsFormation of isoAsp and ECM protein damageThe formation of isoAsp residues at Asn or Asp residues in proteinsis generally considered a degradation reaction as a consequence ofprotein aging. IsoAsp formation has been shown to occur in ECMproteins, including collagen types I and III, in fibronectin, in -crystallin and in the ECM of the mammalian brain (Lanthier andDesrosiers, 2004; Lindner and Helliger, 2001; Reissner and Aswad,

SummaryIntegrins are cell-adhesion receptors that mediate cell–extracellular-matrix (ECM) and cell–cell interactions by recognizing specificligands. Recent studies have shown that the formation of isoaspartyl residues (isoAsp) in integrin ligands by asparagine deamidationor aspartate isomerization could represent a mechanism for the regulation of integrin–ligand recognition. This spontaneous post-translational modification, which might occur in aged proteins of the ECM, changes the length of the peptide bond and, in the case ofasparagine, also of the charge. Although these changes typically have negative effects on protein function, recent studies suggestedthat isoAsp formation at certain Asn-Gly-Arg (NGR) sites in ECM proteins have a gain-of-function effect, because the resultingisoAsp-Gly-Arg (isoDGR) sequence can mimic Arg-Gly-Asp (RGD), a well-known integrin-binding motif. Substantial experimentalevidence suggests that the NGR-to-isoDGR transition can occur in vitro in natural proteins and in drugs containing this motif, therebypromoting integrin recognition and cell adhesion. In this Commentary, we review these studies and discuss the potential effects thatisoAsp formation at NGR, DGR and RGD sites might have in the recognition of integrins by natural ligands and by drugs that containthese motifs, as well as their potential biological and pharmacological implications.

Key words: Integrin, Extracellular matrix, Fibronectin, RGD, DGR, NGR, Asparagine deamidation, Aspartate isomerization, isoDGR, Tumortargeting, Disintegrins, NGR-hTNF

Journal of Cell Science 124, 515-522 © 2011. Published by The Company of Biologists Ltddoi:10.1242/jcs.077172

Isoaspartate-dependent molecular switches forintegrin–ligand recognitionAngelo Corti* and Flavio CurnisDivision of Molecular Oncology and IIT Network Research Unit of Molecular Neuroscience, San Raffaele Scientific Institute, via Olgettina 58,20132 Milan, Italy*Author for correspondence (corti.angelo@hsr.it)

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2003; Weber and McFadden, 1997a). IsoAsp formation can occurnot only in vitro, e.g. during protein storage, but also in vivo, ashas been documented for the extracellular matrix of mammalianbrain or the vascular wall (Reissner and Aswad, 2003; Weber andMcFadden, 1997b). This post-translational modification mightcause changes in the charge or the length of the peptide bond (seeFig. 1A), typically causing loss of function. For example, incollagen-I or in peptides containing RGDSR and KDGEA (arecognition sequence for 21-integrin), the formation of isoAspreduces their capability to promote cell motility (Lanthier andDesrosiers, 2004). Treatment of aged collagen I with protein-L-isoAsp-O-methyltransferase (PIMT), an enzyme that can convertisoAsp into Asp, partially recovers cell migration. In addition,fibronectin, an adhesive protein that has important roles inhaemostasis, thrombosis, inflammation, wound repair, angiogenesisand embryogenesis (Humphries et al., 1989; Pankov and Yamada,2002), also accumulates isoAsp during in vitro aging (Lanthier andDesrosiers, 2004). Similarly, treatment of aged fibronectin orretronectin – a fibronectin fragment containing RGD – with PIMTincreases its pro-adhesive activity on endothelial cells (Curnis etal., 2006) (A.C. and F.C., unpublished data). Taken together, ittherefore appears that the biological properties of collagen,fibronectin and their fragments are impaired by Asp-to-isoAsptransitions that occur at certain sites in these proteins and aresubsequently restored by transforming isoAsp back into Asp.

Formation of isoAsp at NGR sites with gain-of-functionAlthough isoAsp formation in aged proteins is generally viewed asa deleterious event with regard to their function, a growing body

of evidence suggests that this reaction, in some instances, constitutesa mechanism for the intentional modification of a protein structurewith a useful function. For example, it has been proposed thatisoAsp formation, which might occur faster in proteins that havelost their structural integrity, can provide a type of molecularindicator of protein damage, which is capable to activate selectivedegradation mechanisms (Lanthier and Desrosiers, 2004; Pepperkoket al., 2000; Reissner and Aswad, 2003; Robinson and Robinson,2001; Weintraub and Deverman, 2007). Another striking exampleof this concept is the formation of isoAsp in fibronectin fragments.Fibronectin (FN) contains three types of repeating homologousmodules, in addition to alternatively spliced modules (Mohri, 1997;Pankov and Yamada, 2002). Notably, four FN modules, i.e. thefifth 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 NGRsequences. It has been suggested that isoAsp formation at the NGRsite of FN-I5 activates a latent v3-integrin-binding site (Curniset al., 2006; Takahashi et al., 2007). Here, deamidation of Asn263leads to a transformation of NGR into isoDGR and DGR. Structure–activity relationships studies have shown that isoDGR and notDGR is the integrin-binding motif.

Mechanisms of isoAsp formationThe formation of isoAsp in proteins can occur by non-enzymaticAsn deamidation or Asp isomerization reactions (Geiger and Clarke,1987; Weintraub and Deverman, 2007). Asn deamidation occursby a nucleophilic attack of the backbone NH center at the carbonylgroup of Asn side chain, which leads to formation of a succinimidering (Fig. 1B). The formation of a succinimide ring is also the first

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Fig. 1. Mechanisms of spontaneous formation of isoDGR bydeamidation 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 aconsequence of Asn deamidation or Asp isomerization changes thelength of the peptide bond and, in the case of Asn, also the charge ofthe side chain. (B)Schematic representation of L-Asn deamidationand L-Asp isomerization in proteins and peptides. This reaction canoccur by nucleophilic attack of the adjacent peptide bond nitrogenatom on the side chain carbonyl group of L-Asn (indicated by thecurved arrow), leading to formation of a five-membered succinimidering. Racemization of the succinimide intermediate and its rapidhydrolysis at either the - or the -carbonyl group then generatesmixtures of L-Asp, D-Asp, L-isoAsp and D-isoAsp, with L-isoAspbeing the most abundant. L-isoAsp can be converted into L-Asp bythe enzyme protein-L-isoAsp-O-methyltransferase (PIMT) throughmethyl-esterification in the presence of S-adenosylmethyonine(SAM). L-Asp isomerization can occur by a similar mechanism thatinvolves L-Asp dehydration and formation of the succinimideintermediate.

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step for Asp isomerization (Fig. 1B). Hydrolysis can then occur atboth carbonyl groups of the cyclic imide, leading to the formationof a mixture of Asp and isoAsp residues, typically with a ratio ofapproximately 1:3. Racemization and hydrolysis of the succinimideintermediate can also lead to the formation of Asp and isoAsp inD-configuration, but this typically occurs with much lowerefficiency (Geiger and Clarke, 1987; Stephenson and Clarke, 1989;Tyler-Cross and Schirch, 1991). Thus, the resulting Asp and isoAspin L-configuration are more relevant. The above deamidation andisomerization reactions can take hours, days or even years –depending on several factors. For example, secondary, tertiary andquaternary structural elements in a protein can place Asn or Aspresidues in proximity to functional groups that either inhibit oraccelerate these reactions (Robinson, 2002; Robinson andRobinson, 2001; Robinson et al., 2004). In particular, the presenceof a Gly residue following Asn or Asp generally accelerates thesereactions owing to its small size and flexibility (Robinson andRobinson, 2001). Other crucial factors are the pH, the ionic strengthand the temperature (Robinson and Robinson, 2001; Tyler-Crossand Schirch, 1991).

Kinetics of the NGR-to-isoDGR transitionApproximately 17% of proteins that are classified by using thekeyword ‘adhesion’ in vertebrate protein databanks contain NGRsites (Corti et al., 2008). Considering the known effects of 3D-structural protein elements on the deamidation rate of Asn residuesdiscussed above, only certain NGR sites with suitable accessibility,flexibility and side chain orientation are likely to undergodeamidation 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 cellculture medium of only ~4 hours (Curnis et al., 2006). Thesurprising velocity of this reaction is probably owing to the factthat here Asn is followed by a Gly residue. Furthermore, the Asnand Gly residues are part of the GNGRG loop that is well exposedon the surface of this fibronectin fragment, thereby possiblyfavoring the correct orientation of the Asn side chain and peptidebackbone for a nucleophilic attack (Di Matteo et al., 2006).Similarly, fast deamidation rates have been observed in peptidesthat contain the disulfide-bonded CNGRC loop (Curnis et al.,2010; Curnis et al., 2006).

By contrast, full-length plasma fibronectin was shown to beconsiderably more resistant to Asn deamidation than short FNfragments 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 isoDGRformation in a 70 kDa N-terminal fragment of fibronectin (Xu etal., 2010). It therefore appears that rapid deamidation of NGR infibronectin requires proteolytic processing and/or conformationalchanges. Nevertheless, the in vivo formation of isoDGR sites infibronectin, e.g. after deposition in tissues, still remains to bedemonstrated.

Rapid NGR-to-isoDGR transformation also occurs inrecombinant proteins that contain CNGRC fused to the N-terminus 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 notspecific for the GNGRG site in FN-I5. Thus, it is possible thatother proteins also use this mechanism to activate latent integrinrecognition sites, and this should be taken into account when

analyzing potential integrin-binding sites of proteins that containthe NGR sequence.

Recognition of RGD-dependent integrins by isoDGR andits functional rolePeptides that contain the isoDGR motif can recognize members ofthe RGD-dependent integrin family, such as v3-, v5-, v6-,v8- and 51-integrins, but not other, such as 11-, 31-,47-, 64- or 91-integrins (Curnis et al., 2010). The affinityand specificity of the interaction between isoDGR and integrindepend on the molecular scaffold of isoDGR (Curnis et al., 2010).For instance, cyclic peptides that contain isoDGR flanked by Cysresidues can bind v3-integrin with an affinity that is 10–100-fold higher than that for other members of the RGD-dependentintegrin family (for example, Kd is ~9 nM for v3-integrin).Replacement of the Cys by two Gly residues leads to a marked lossof affinity for all integrins and a change of specificity (Curnis etal., 2010). It appears, therefore, that isoDGR, like RGD, candifferentially recognize integrins depending on its molecularscaffold.

These findings and the results of a series of in vitro experimentsperformed with fibronectin and fibronectin fragments suggest thatNGR deamidation has a functional role in the regulation of celladhesion. For example, a deamidated FN-I4-5 fragment enhancedthe adhesion of endothelial cells to microtiter plates, but not whenits NGR sequence was replaced with SGS (Curnis et al., 2006).Similarly, replacement of NGR in the FN-I5 and FN-I7 modules ofa 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 infibronectin modules (Xu et al., 2010).

Although isoDGR-containing compounds can promote celladhesion when they are adsorbed onto microtiterplates, they canexert inhibitory effects in other assays. For example, deamidatedFN-I5 and the peptide CisoDGRC inhibited endothelial cellproliferation and adhesion to vitronectin when these peptides wereadded as soluble ligands in the cell culture medium (Curnis et al.,2006). These peptides also recognized v3-integrin-positiveendothelial cells in tumor vessels, and inhibited tumor growthwhen they were systemically administered to tumor-bearing mice(Curnis et al., 2010; Curnis et al., 2006). Given the growing bodyof evidence that implicates v3-integrin in angiogenesis (Brookset al., 1995; Eliceiri and Cheresh, 2000), it is possible that theinteraction of the isoDGR site in fibronectin or fibronectinfragments with v3-integrin has an important role in cancer andin other diseases that involve angiogenesis.

Tissue fibronectin is organized in fibrils, which mediate a varietyof cellular interactions with the extracellular matrix (Humphries etal., 1989; Pankov and Yamada, 2002). The formation of fibronectinfibrils in the ECM is a cell-mediated process that involves therearrangement of the cytoskeleton and integrins in order to transmitthe forces necessary to unfold fibronectin and assemble it intofibrils (Geiger, 2001; Ohashi et al., 2002; Pankov et al., 2000;Zamir et al., 2000). Mice that were genetically engineered toexpress a fibronectin variant with a non-functional RGE in placeof RGD and that are, thus, unable to bind integrins through thissite, can still assemble fibrils in vivo (Takahashi et al., 2007). Thesame authors also showed that a cyclic peptide containing isoDGRinhibits fibril formation of a RGE-fibronectin mutant in vitro andthey proposed that the GNGRG of FN-I5 can function in fibril

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assembly. However, other investigators showed that fibronectinmutants, in which NGR is replaced with QGR in the FN-I5 andFN-I7 modules, can still form fibrils, thus arguing against a crucialrole of these sites in fibril formation (Xu et al., 2010). As the rolein fibril formation of those NGR sites located in FN-II2 and FN-III9 has not yet been investigated, further studies are necessary toclarify the function of different NGR sites in the formation offibronectin fibrils.

The DGR-to-isoDGR transition and integrin recognitionThe frequency of the DGR sequence element in proteins classifiedas having a role in cell adhesion is higher than that of other xGRsequences (where x represents any amino acid residue other thanAsn 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 inintegrin 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 offibroblasts to laminin and collagen type I when added to the cellculture medium (Yamada and Kennedy, 1987). Furthermore,fibroblast growth factor 2 (FGF2), a pro-angiogenic heparin-bindingcytokine that contains two DGR sites, can bind to v3-integrinand promote endothelial cell adhesion (Rusnati et al., 1997).Mapping of the cell-adhesive domains has shown that the regionsfrom amino acids 38 to 61 and 82 to 101 in FGF2, which containthe DGR sequences, are responsible for this activity (Rusnati et al.,1997). In addition, it was also shown that the GVCTDGR sequenceof connective tissue growth factor can bind 51-integrin (Gaoand Brigstock, 2006). However, other studies found SDGR andother DGR peptides to be inactive in cell attachment assays, whichargues 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 theisomerization of Asp, one explanation for these apparentlycontroversial data may be that DGR, like NGR, can function as alatent integrin-binding site that is differentially activated dependenton its molecular context and the conditions. In agreement with this

hypothesis, we have observed that the interaction of FGF2 withv3-integrin is inhibited by treatment with PIMT (A.C. and F.C.,unpublished data), supporting the idea that isoAsp residues inFGF2 are crucial for this interaction.

Considering that a number of proteins contain DGR it is possiblethat this mechanisms is more general. However, as discussed abovefor NGR, it should be born in mind that the residues flanking DGRand its microenvironment can crucially affect the kinetics of Aspisomerization and its transformation into isoDGR. Furthermore,the rate of Asp isomerization is generally slower than that of Asndeamidation in similar peptides (Geiger and Clarke, 1987). Thisreaction is, therefore, likely to have a significant role in only asmall subset of proteins.

Structural basis of the molecular mimicry ofRGD by isoDGR in integrin recognitionThe structural basis of ligand recognition by RGD-dependentintegrins has been studied intensively (Luo et al., 2007; Takagi,2004). Biochemical studies have shown that both - and -integrinsubunits span the plasma membrane and have short cytoplasmicdomains. Outside the plasma membrane, the - and -subunits lieclose together and form the RGD-binding pocket (Xiong et al.,2001). Substantial experimental evidence suggests that isoDGRbinds to the RGD-binding site of integrins. First, the cyclicCisoDGRCGVRY peptide (called isoDGR-2C) might inhibit thebinding of a cyclic RGD peptide to v3-integrin in a competitivemanner (Curnis et al., 2006). Notably, the affinities of cyclicisoDGR-2C and RGD-2C peptides for v3-integrin were similar(Curnis et al., 2006). Replacement of isoAsp with Asp (as in DGR-2C) or with its enantiomer in D-configuration (D-isoAsp) causes amarked loss of binding affinity, pointing to highly selective andstereospecific 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 v3-integrin dockingexperiments showed that isoDGR, but not DGR and NGR, fit intothe RGD-binding pocket and that it favorably interacts with thisintegrin (Curnis et al., 2006; Spitaleri et al., 2008) (Fig. 2). Notably,

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isoDGR RGD

Fig. 2. Model for the interaction between isoDGR and the RGD-binding site of v3-integrin. Representation of the v3-integrin-binding pocket in complexwith either CisoDGRC or CRGDC (courtesy of Giovanna Musco, San Raffaele Institute, Italy) (Spitaleri et al., 2008). - and -integrin subunits are represented inpink 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 labeledwith the three- and one-letter code, respectively. Dotted lines denote hydrogen bonds between ligands and integrin. For details on peptide structure analysis anddocking experiments see Spitaleri et al. 2008 (Spitaleri et al., 2008). IsoDGR favorably interacts with the RGD-binding site of this integrin, thereby recapitulatingRGD-binding contacts and establishing additional polar interactions.

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isoDGR docks onto v3-integrin in an inverted orientationcompared with RGD docking to v3-integrin. This orientationallows isoDGR to bind the integrin - and -subunits through theisoAsp and Arg residues. The acidic and basic side chains of theseresidues are at the correct distance and orientation to engagestabilizing interactions with the polar regions of the integrin, thusreproducing the canonical interactions between RGD and v3-integrin (Spitaleri et al., 2008). These data are in line with theresults of binding studies showing that isoDGR, but not DGR andNGR, can efficiently interact with v3-integrin. Therefore,isoDGR, unlike DGR and NGR, is a natural fit for the RGD-binding pocket of v3-integrin and supports the hypothesis thattransforming DGR or NGR into isoDGR can result in a molecularswitch able to activate integrin recognition.

Such a model might also explain the controversial data reportedin the literature for the interaction of integrin with compounds thatcontain DGR – as discussed above – or NGR. The suggestion thatNGR and integrin interact originated from several observations.For example, in vitro panning of phage-display libraries againstv3- and 51-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 sitethat is crucial for 51-integrin binding and viral cell entry (Asokanet al., 2006), and adenoviruses that have been genetically engineeredto express the linear NGR sequence on their surface bind to v3-integrin-expressing cells (Majhen et al., 2006). However, in vitrobinding studies with purified v3-integrin and peptides thatcontain the CNGRC or GNGRG sequences show only a modestinteraction, if any at all (Curnis et al., 2010). Although we cannottotally exclude that NGR sites that are embedded in viral particlesbehave in a different manner, we hypothesize that integrin bindingobserved with certain NGR-containing ligands can be explainedby binding of isoDGR generated from NGR. For example, phagesthat have been selected as carrying the NGR sequence mightinstead have displayed isoDGR on their surface owing todeamidation reaction during library preparation and in vitroselection. Thus, we think it is important that any isoAsp formationmust be ruled out before integrin recognition of NGR-containingcompounds is proposed.

Although isoDGR- and RGD-containing ligands can share thesame binding site on integrins, their effects on integrin function arenot necessarily identical for the following reasons. It is wellestablished that the interaction between integrins and ligands isregulated by conformational changes (Arnaout et al., 2005; Luo etal., 2007). Conformational changes and integrin activation can betransmitted bidirectionally across the membrane throughinteractions with intracellular molecules (inside-out signaling) andwith extracellular ligands (outside-in signaling) (reviewed in Askariet al., 2009). It has been suggested that different RGD-containingligands induce different signaling pathways (Askari et al., 2009).Thus, it is possible that RGD and isoDGR ligands inducedifferential conformational changes and signaling in vivo.Furthermore, although peptides containing CRGDC or CisoDGRCsequences recognize purified v3-integrin with similar affinitiesin vitro (Curnis et al., 2006), their kon and koff, which have not yetbeen measured, might not necessarily be similar. Considering theimportance of these parameters for their biological properties,kinetic analysis of binding data and characterization of the signalingmechanisms induced by isoDGR-containing ligands are needed todefine the extent to which isoDGR mimics – or differs – fromRGD.

Regulation of isoAsp formation in tissuesAs discussed above, Asp isomerization and Asn deamidation arethermodynamically spontaneous reactions that do not requireenzymatic catalysis. It is currently unknown whether specific Asndeamidases or Asp isomerases exist that could further acceleratethis process. However, the observation that NGR deamidates fasterin short fibronectin fragments compared with soluble plasmafibronectin (discussed above) and the finding that isoDGR can beconverted into DGR by PIMT (Curnis et al., 2006) point to possibleenzymatic regulatory mechanisms.

In humans, PIMT is encoded by PCMT1, which is localized onchromosome 6 (6q24–q25). This enzyme (EC 2.1.1.77) catalyzesthe transfer of a methyl group from S-adenosyl-L-methionine tothe free carboxyl groups of D-Asp and L-isoAsp residues (Reissnerand Aswad, 2003). The resulting methyl ester can then form acyclic succinimide that, after hydrolysis, can generate Asp andisoAsp (Fig. 1B). In peptide substrates, there appears to be apreference for isoAsp residues that are flanked by bulkyhydrophobic group at the position preceeding isoAsp and by neutralor positive groups at the positions that follow isoAsp (Lowensonand Clarke, 1991). PIMT is present in all vertebrate tissues andrepresents the major protein methyltransferase in the brain, testis,erythrocytes and eye (Yamamoto et al., 1998). PIMT is normallyan intracellular enzyme that exists in two isoforms due todifferential mRNA splicing (Takeda et al., 1995). The C-terminusof one isoenzyme ends with the sequence RWK, whereas the otherisoform ends with RDEL, a known endoplasmic reticulum retentionsequence (MacLaren et al., 1992). It has been shown thatcalmodulin, tubulin, histone H2B and synapsin I are naturalsubstrates of PIMT action in vivo (Furuchi et al., 2010). However,experimental evidence suggests that this enzyme also targetsextracellular proteins, such as collagen type I and type II (Weberand McFadden, 1997b). Interestingly, it has been demonstratedthat PIMT is released into the extracellular environment bydamaged vessels and by injured tissues, and that it becomesentrapped in the extracellular space (Weber and McFadden, 1997a;Weber and McFadden, 1997b). In uninjured blood vessels, up to90% of total isoAsp residues are inaccessible to endogenousintracellular PIMT (Weber and McFadden, 1997b). However, aftervessel injury PIMT is released into the extracellular environmentand methylation of extracellular proteins, such as aged collagentype I and type II, can then ensue (Weber and McFadden, 1997b).The biological importance of PIMT is also supported by theobservation that PIMT-deficient mice have a smaller body andspleen, a larger brain, an abnormal neuronal organization andatypical patterns of motor activity, and that they die fromprogressive 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 theoriginal sequence in the case of Asp isomerization, but not in thecase of Asn deamidation, because isoAsp is converted into Asp inboth cases. Thus, whereas PIMT can potentially repair RGD or otherdamaged functional sites that undergo Asp isomerization, it can leadto inactivation of isoDGR sites derived from NGR or DGR.

Pharmacological implications and therapeuticopportunities for peptides that target RGD-dependent integrinsIntegrins are involved in many physiological and pathologicalprocesses, such as inflammation, thrombosis, osteoporosis,angiogenesis and cancer. The role of integrins in cancer biology

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and angiogenesis is one of the most studied functions of these celladhesion receptors. For example, v3-integrin, an integrinoverexpressed in the tumor vascular endothelium, has an importantrole in angiogenesis and tumor growth (Brooks et al., 1995; Eliceiriand Cheresh, 2000; Friedlander et al., 1996; Kumar, 2003). RGD-containing peptides with a variable degree of affinity and selectivityfor this integrin have been developed and used for delivering avariety of drugs and nanoparticles to tumor vessels (Desgrosellierand Cheresh, 2009; Liu et al., 2008; Ruoslahti et al., 2010). Giventhe structural and functional similarities between RGD and isoDGRit is conceivable that peptides containing the isoDGR motif couldalso be exploited as specific ligands for the targeted delivery ofdrugs, imaging agents or other compounds to tumors. Consistentwith this, a cyclic CisoDGRC peptide that was coupled tofluorescent nanoparticles (quantum dots) was shown to bind v3-integrin and colocalize with antibodies against v3-integrin invessels of human renal cell carcinoma (Curnis et al., 2008).Furthermore, extremely low doses (1–10 pg) of a recombinantprotein made up of CisoDGRC fused to TNF-, a cytokine capableof inflicting damages to tumor vessels, induced anti-tumor effectsin tumor-bearing mice through specifically targeting TNF- totumor sites (Curnis et al., 2008).

The isoDGR motif might also be exploited, similar to RGD, forthe generation of integrin antagonists, which are currently beinginvestigated for the treatment of cancer, osteoporosis andcoagulation 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 v3- and v5-integrins andinhibits angiogenesis. This drug is currently tested in a phase IIItrial in patients with glioblastoma (Desgrosellier and Cheresh,2009). In principle, new integrin antagonists could be developedby using isoDGR instead of RGD. However, it should be born inmind that simply replacing RGD with isoDGR within the contextof an existing compound is unlikely to work, because isoDGR hasto interact with the RGD-binding pocket in an inverted orientation(see Fig. 3 for a schematic representation) and, moreover, theflanking residues also contribute to integrin-binding affinity andspecificity as discussed above. The potential use of small moleculesthat contain isoDGR is supported by the results of our recent study,which shows that a cyclic CisoDGRC peptide alone inhibits thegrowth of tumors in mouse models (Curnis et al., 2010). Furtherwork is necessary to assess the potential use of isoDGR comparedwith RGD, regarding stability (also to proteases), integrin-bindingproperties (Kd, kon and koff), signaling and pharmacologicalproperties.

The capability of isoDGR peptides to interact with integrinsmight also have important implications for another class of drugs,i.e. drugs containing the NGR motif. NGR peptides have beenused by different investigators to deliver cytokines,chemotherapeutic drugs, liposomes, anti-angiogenic compounds,viruses, imaging agents and DNA complexes to tumorneovasculature, in order to improve their therapeutic or imagingproperties (reviewed by Corti and Curnis, 2011). For example, theCNGRCG peptide, fused to the N-terminus of TNF- (NGR-TNF), and the GNGRAHA-peptide, fused to the C-terminus oftissue factor, are currently tested in several phase I, II and IIIclinical studies (Bieker et al., 2009; Gregorc et al., 2010a; Gregorcet al., 2010b; van Laarhoven et al., 2010; Corti et al., 2011). Therationale for using these peptides is the observation that NGR canrecognize a membrane-bound form of aminopeptidase N (CD13)that is expressed by angiogenic vessels (Curnis et al., 2002;

Pasqualini et al., 2000). Remarkably, in these compounds the NGR-to-isoDGR transition, which may occur during purification, storageor even in vivo for molecules that are circulating a long while,might simultaneously switch off CD13 and switch on integrinbinding (Curnis et al., 2010) – with potentially importantpharmacological and toxicological implications.

ConclusionsThe formation of isoAsp in proteins of the ECM might representa new mechanism for the regulation of integrin recognition bytheir ligands. Although, this protein modification typically impairsthe functional properties of ECM proteins, it might however – incertain cases – generate new integrin-binding sites. The spontaneoustransformation from NGR to isoDGR in fibronectin fragments,through Asn deamidation, and the consequent activation of integrin-binding sites is an example of how isoAsp formation can confernew functional properties to proteins. Spontaneous formation ofisoAsp in proteins might occur also at Asp residues throughisomerization reactions, suggesting that also the DGR motif canwork, in principle, as an inactive precursor of isoDGR. Whereasthe formation of isoDGR may represent a molecular switch for‘turning on’ integrin binding, the release of PIMT from damagedcells could represent an enzyme-dependent mechanism for ‘turningoff’ 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 resultingisoDGR site accessible to integrins. Thus, although substantialexperimental evidence suggests that proteins of the extracellularmatrix can accumulate isoAsp in vivo, further studies are necessaryto demonstrate that this reaction can, indeed, occur at NGR sitesin vivo, and that it generates isoDGR in an amount that is able tosubstantially affect cell adhesion. As isoAsp formation can takehours, days or even years, depending on the molecularmicroenvironment, this mechanism is likely to have a significantrole only in certain proteins. Further work is necessary to identifythose proteins with sequence motifs that are transformed intoisoDGR, to quantify its formation in normal and pathologicaltissues, and to assess their physiological roles.

The observation that isoDGR is generated in purified proteinsand in drugs that contain the NGR motif suggests that this reaction

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RGD

isoDGR α

β

DGR

NGR

PIMT

Fig. 3. Schematic representation of an isoDGR-dependent molecularswitch for integrin–ligand recognition. As highlighted in this schematicrepresentation, isoDGR can derive from NGR by deamidation of Asn, but ithas to assume an inverted orientation (compared with RGD) to be able tointeract with v3-integrin. isoDGR can also be converted into DGR by PIMTthat, as the orginal NGR sequence element, cannot efficiently interact with thisintegrin.

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also has important implications for their pharmacological andtoxicological properties. Furthermore, the ability of syntheticisoDGR-containing peptides to mimic RGD and to bind to tumorvessels suggests that these compounds can be exploited, similar toRGD, as tools for targeted delivery of drugs and diagnosticnanoparticles to tumor vessels or for the development of newintegrin antagonists.

This work was supported by Associazione Italiana per la Ricerca sulCancro (AIRC) and Alleanza Contro il Cancro (ACC) of Italy, andFIRB.

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