Age-dependent deamidation of asparagine residues in proteins

Lindner, Herbert; Helliger, Wilfried

doi:10.1016/s0531-5565(01)00140-1

Cited by 109 publications

(87 citation statements)

References 35 publications

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“…It is well known that proteins may contain isoAsp residues due to post-translational Asn deamidation or Asp isomerization reactions (21,31,(37)(38)(39). These reactions can occur in vivo, e.g.…”

Section: Discussionmentioning

confidence: 99%

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

Curnis

Longhi

Crippa

et al. 2006

Journal of Biological Chemistry

181

230

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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...

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Section: Discussionmentioning

confidence: 99%

“…These reactions can occur in vivo, e.g. in extracellular matrix proteins with slow turnover (30,31,37), and in vitro during protein isolation and storage (22,40,41). In general these phenomena are associated with protein "loss of function" and, for this reason, are generally viewed as deleterious events associated with protein aging.…”

Section: Discussionmentioning

confidence: 99%

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

Curnis

Longhi

Crippa

et al. 2006

Journal of Biological Chemistry

181

230

View full text Add to dashboard Cite

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“…This can arise for a number of reasons that reflect both biological and nonbiological processes [3][4][5][6]. In both cases, characterization of the heterogeneity is crucial.…”

Section: Discussionmentioning

confidence: 99%

“…Similarly, proteins coexpressed by a multi-gene family can vary by a single amino acid, e.g., lysine to glutamate, with an associated mass shift of less than 1 Da [5]. Nonbiological LMH can arise as a result of experimental manipulation and storage [6]. Whilst such subtle heterogeneity is difficult to resolve, it has the potential to indicate important biological processes and sample integrity.…”

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confidence: 99%

Observation of heterogeneous gene products by FT-ICR MS

Robertson

Wong

Beynon

et al. 2008

J. Am. Soc. Mass Spectrom.

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The ability of FT-ICR MS to resolve isotopic variants of intact proteins for each of the charge states formed by electrospray ionization offers a sensitive, rapid method for detecting "low mass" heterogeneity, where this is defined as the presence of structural variants differing in mass by 2 Da or less. Such heterogeneity may reflect biological or chemical modifications of structure or may result from the coexpression of related proteins from a multi-gene family. In the analytical approach described here, comparisons are made between observed isotopic distributions and those expected for predicted protein sequences. Close agreement is demonstrated for a homogeneous model protein, and the utility of the method has been evaluated in the study of mouse major urinary proteins (MUPs), a group of closely related sequences. Divergence of the experimental isotopic distribution from distributions predicted for known MUP sequences can be explained, in quantitative terms, by the coexpression of closely related sequences. This approach provides a facile method for the assessment of protein homogeneity and for the detection of structural variants, without recourse to proteolytic digestion and analysis of the resulting products. (J

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“…An important age-dependent hydrolytic modification is the deamidation of protein asparagine to aspartic and isoaspartic acid, and racemization reactions interconverting L-and D-aspartic acid as well as L-and D-isoaspartic acid (Lindner & Helliger, 2001;Clarke, 2003). Deamidation can have profound biological consequences such as the initiation of amyloid formation (Nilsson, Driscoll, & Raleigh, 2002;Shimizu et al, 2002) or the loss of the anti-apoptotic activity of Bcl-x L (Deverman et al, 2002(Deverman et al, , 2003.…”

Section: Biological Aging and Post-translational Protein Modificmentioning

confidence: 99%

Mass spectrometry in aging research

Schöneich

2004

Mass Spectrometry Reviews

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This review covers the application of mass spectrometric techniques to aging research. Modern proteomic strategies will be discussed as well as the targeted analysis of specific proteins for the correlation of post-translational modifications with protein function. Selected examples will show both the power and also current limitations of the respective techniques. Experimental results and strategies are discussed in view of current theories of the aging process. # 2004 Wiley Periodicals, Inc., Mass Spec Rev 24: [701][702][703][704][705][706][707][708][709][710][711][712][713][714][715][716][717][718] 2005

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Age-dependent deamidation of asparagine residues in proteins

Cited by 109 publications

References 35 publications

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

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

Observation of heterogeneous gene products by FT-ICR MS

Mass spectrometry in aging research

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