Spontaneous Formation of L-Isoaspartate and Gain of Function in Fibronectin

Abstract

Isoaspartate formation in extracellular matrix proteins, by aspartate isomerization or asparagine deamidation, is generally viewed as a degradation reaction occurring in vivo during tissue aging. For instance, non-enzymatic isoaspartate formation at RGD-integrin binding sites causes loss of cell adhesion sites, which in turn can be enzymatically "repaired" to RGD by protein-L-isoAsp-O-methyltransferase. We show here that isoaspartate formation is also a mechanism for extracellular matrix activation. In particular, we show that deamidation of Asn 263 at the Asn-Gly-Arg (NGR) site in fibronectin N-terminal region generates an ␣ v ␤ 3 -integrin binding site containing the L-isoDGR sequence, which is enzymatically "deactivated" to DGR by protein-L-isoAsp-O-methyltransferase. Furthermore, rapid NGRto-isoDGR sequence transition in fibronectin fragments generates ␣ v ␤ 3 antagonists (named "isonectins") that competitively bind RGD binding sites and inhibit endothelial cell adhesion, proliferation, and tumor growth. Time-dependent generation of isoDGR may represent a sort of molecular clock for activating latent integrin binding sites in proteins.Fibronectins are adhesive proteins that mediate a variety of cellular interactions with extracellular matrix and play important roles in hemostasis, thrombosis, inflammation, wound repair, angiogenesis, and embryogenesis (1, 2). About 20 isoforms of human fibronectin can be generated as a result of alternative splicing of the primary transcript (1, 3). Fibronectins are large glycoproteins (ϳ450 kDa) composed of two nearly identical disulfide-bonded subunits present in most body fluids and extracellular matrix of many tissues. Each subunit consists of three types of repeating homologous modules termed FN-I, FN-II, and FN-III repeats. Alternatively spliced modules, called EDA, EDB, and IIICS, can also be present (1, 3). Single modules or groups of modules may contain binding sites for different molecules, including sulfated glycosaminoglycans, DNA, gelatin, heparin, and fibrin (1, 3, 4). Furthermore, fibronectins contain binding sites for about half of the known cell surface integrin receptors (5, 6). In particular, the FN-III 10 repeat contains an RGD site that can bind, and ␣II b ␤ 3 integrins, while the FN-III 9 repeat contains the so-called "synergy site" PHSRN that cooperates with RGD in the binding of ␣ 5 ␤ 1 and ␣II b ␤ 3 (1, 7).Primary and tertiary structure analysis of human fibronectin showed that this protein contains two GNGRG loops, located in FN-I 5 and FN-I 7 modules, that are conserved in bovine, murine, rat, amphibian, and fish (8). Two additional NGR sites, less conserved, are also present in human FN-II 1 and FN-III 9 (see Fig. 1). Recent experimental work showed that peptides containing the NGR motif can inhibit ␣ 5 ␤ 1 -and ␣ v ␤ 1 -mediated cell adhesion to fibronectin (9).These notions prompted us to investigate the functional role of NGR in fibronectin. We observed that the NGR sequence of FN-I 5 (residues 263-265) promotes endothelial cell adhesion via an...

Oxidation-induced Structural Changes of Ceruloplasmin Foster NGR Motif Deamidation That Promotes Integrin Binding and Signaling

“…Thus, for each protein it is important to assess whether specific Asn residues can undergo or not this post-translational modification. For example, we previously reported that the deamidation of an NGR site of fibronectin does not occur in the intact protein, whereas it can rapidly occur in protein fragments (10). The results reported here show that deamidation of the 568 NGR site, which is exposed on the protein surface, does not require structural changes.…”

“…To assess whether cell adhesion was mediated by isoDGR, we incubated ox/AmBic-treated Cp with PIMT, an enzyme that converts L-isoAsp residues to L-Asp (10,30). PIMT treatment almost completely inhibited the pro-adhesive activity of ox/AmBic-treated Cp and of isoDGR peptide, the latter serving as a control (Student's t test, p Ͻ 0.0001) (Fig.…”

“…Deamidated Cp was prepared as follows: purified Cp was diluted 1 mg/ml in 100 mM ammonium bicarbonate (AmBic) buffer, pH 8.5, incubated for 16 h at 37°C, and stored at Ϫ20°C until analysis. This condition is known to favor Asn deamidation at the NGR site (10). Before use in the assays, Cp was dialyzed against PBS using Slide-A-lyzer dialysis cassette 10,000 molecular weight cutoff (Pierce).…”

Substrates of the Arabidopsis thaliana Protein Isoaspartyl Methyltransferase 1 Identified Using Phage Display and Biopanning

“…This conversion can be detrimental to protein function and/or solubility (2)(3)(4)(5) or in rare instances necessary (6) or as an intermediate step that effects an Asn 3 isoAsp 3 Asp conversion that optimizes protein function post-translationally (7,8). Protein L-isoaspartyl methyltransferase (PIMT) 4 is an intracellular enzyme present in most life forms.…”

Chronoregulation by Asparagine Deamidation

“…Fibronectin contains two highly conserved Gly-Asn-GlyArg-Gly (NGR) loops, which had been suggested to have a role in integrin-mediated cell-fibronectin interactions (38). Curnis and colleagues found that fibronectin aging increases cellfibronectin interactions because of the deamidation of the asparagine in an NGR loop (39). Generation of an isoaspartate at the deamidation site in the NGR loop is necessary to promote the interaction, and consequently, treatment with PIMT, the enzyme that converts isoaspartate to aspartate, decreases adhesiveness.…”