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Deamidation in Proteins and Peptides
Author: Glen Teshima

Published 21 November 2000

Introduction

Deamidation of asparagine residues is one of the most common post-translational modifications occurring in therapeutic proteins produced using recombinant DNA technology.  A reduction/loss of in vitro or in vivo biological activity has been reported for a variety of proteins including recombinant human DNAse (Cacia et al., J. Chromatogr. 1993, 634:229-239) and recombinant soluble CD4 (Teshima et al., Biochemistry 1991, 30:3916-3922) while others appear unaffected (recombinant human growth hormone; hGH); Becker et al., Biotech. Appl. Biochem, 1988, 10:326-337).  It is therefore important to establish methods for characterizing the sites of deamidation as well as for evaluating the effect on biological activity and antigenicity.

 

What is deamidation?

Deamidation is a common post-translational modification resulting in the conversion of an asparagine residue to a mixture of isoaspartate and aspartate.  Deamidation of glutamine residues can occur but does so at a much lower rate.

 

What is the role of deamidation?

Not known with certainty.  It has been postulated that deamidation may provide a signal for protein degradation thereby regulating intracellular levels.

 

What is the mechanism of deamidation?

Neutral pH (Beta-aspartyl shift mechanism)

The non-enzymatic modification of asparagine and aspartate residues at physiological pH occurs primarily through intramolecular rearrangement as shown in Figure 1.

Step 1

The peptide bond nitrogen (reactive anion) of the N + 1 amino acid attacks the carbonyl carbon of the asparagine or aspartate side chain forming a five-membered ring structure referred to as a succinimide or cyclic imide.

Step 2

The succinimide is then rapidly hydrolyzed at either the alpha or beta carbonyl group to yield iso-aspartate (beta-aspartate) and aspartate in a ratio of approximately 3:1.

Acidic pH

At low pH (<2), direct hydrolysis of the side chain amide generates aspartate as the sole product.

 

What conditions favor deamidation?

Sequence considerations

Effect of following residue (N + 1)

Highest frequency in proteins with Asn-Gly sequences

For proteins in which deamidation has been established and the site characterized, glycine has far and away been the most common (N + 1) neighboring residue.

Intermediate frequency in Asn residues followed by a polar amino acid with a relatively small side chain (i.e. Ser, Thr, Asp).  Low frequency in Asn residues followed by a hydrophobic amino acid with a bulky side chain.

Explanation for different rates of reactivity

Steric hindrance by the bulky side chain of the N + 1 amino acid may limit accessibility of the reactive nitrogen anion to the asparagine side chain amide thereby reducing the rate of reactivity.

Conformational effects

The asparagine and flanking regions must be solvent accessible and reside within a conformationally flexible region of the molecule.  Crystal structure data, if available, allows the most complete assessment of local conformational flexibility and exposure of amino acid residues and used in conjunction with primary sequence information should provide the most accurate prediction of deamidation.

Exposure to alkaline pH

Exposure to alkaline pH results in an increased rate of succinimide formation due to greater deprotonation of the peptide bond nitrogen at higher pH values.

 

How does one identify the sites of deamidation?

Isolation of intact protein variant by chromatography

Hint: If rate of deamidation is very low and therefore difficult to detect, incubation of protein samples in slightly alkaline buffer (i.e. 20mM sodium phosphate, pH ~ 8) or at elevated temperature (i.e. 37C) can accelerate the rate of reactivity at potential deamidation sites.

Ion exchange

Ion-exchange chromatography is the most widely applied chromatographic technique for the isolation of deamidated proteins.

Summary of IEC approach

Advantages

Selective for changes in net charge therefore optimal for the resolution of Asn and deamidated products (Asp and IsoAsp) (ref 1,2), as well as for distinguishing Asp from succinimide. (ref 3)

Higher loading capacity compared to reversed phase LC facilitating the isolation of sufficient quantities for characterization studies (i.e. identity, in vitro/in vivo bioassay).  Separation performed under non-denaturing conditions therefore native conformation preserved; a requirement for the evaluation of bioactivity and antigenicity properties.

Ref 1: Teshima et.al. Deamidation of Soluble CD4 at Asparagine-52 Results in Reduced Binding Capacity for the HIV-1 Envelope Glycoprotein gp120.  A deamidated variant at Asn52-Asp53 of desialylated recombinant soluble CD4 was isolated by cation exchange chromatography using a sulfopropyl column.

Ref 2: Cacia et. al. J. Chromatogr. 1993, 634:229-239  A separation of native and deamidated forms of recombinant human DNAse was achieved using a LiChrosphere tentacle cation exchange column.

Ref 3: Teshima et. al. Isolation and Characterization of a Succinimide Variant of Methionyl Human Growth Hormone  In a study using an aged sample of recombinant human growth hormone, a succinimide variant at Asp130-Gly131 was isolated by anion exchange chromatography using a DEAE column. The succinimide was unstable in solution and precautions had to be taken to minimize its hydrolysis.

Key disadvantage

Formation of either a succinimide from an Asn or an IsoAsp from Asp will not result in a change in net charge and therefore resolution of the intact and variant forms by IEC would not be expected.

 

Reversed phase

Summary of RPLC approach

The potential exists for distinguishing Asn from succinimide as well as Asp from IsoAsp by exploiting subtle differences in hydrophobicity and hence retention behavior associated with the structural modifications.

Ref: Bischoff, Biochemistry 1993, 32:725-734  Stable succinimides of recombinant hirudin at Asn33-Gly34 and Asn53-Gly54 were resolved as later eluting peaks by RPLC. Separation of the Asn and succinimide forms has not been achieved by IEC.

The "contact region", i.e. the residues interacting with the stationary phase bed, can be manipulated by varying the mobile phase conditions (buffer pH and type of organic modifier). However, typical RPLC conditions can result in partial to complete protein denaturation and therefore interpretation of in vitro/in vivo bioactivity results must be made with caution.

 

Hydrophobic interaction chromatography (HIC)

Summary of HIC approach

The selectivity mechanism is based on hydrophobicity, as in RPLC, however the conditions are much less denaturing due to the lack of organic modifier in the mobile phase and the use of lower density, less non-polar stationary phases. Sample loading capacity is relatively high.

Ref: Di Donato et.al. J.Biol.Chem. 1993, 268:4745-4751  The resolution of Asp and IsoAsp forms at Asn67-Gly68 of Ribonuclease A was achieved by HIC. Deamidated (Asp, IsoAsp) and native forms (Asn) were resolved by cation-exchange chromatography using a Mono-S column. The deamidated fraction was then analyzed on a Spherogel HIC-CAA column. Two peaks were resolved and characterized as IsoAsp (earlier eluting) and Asp.

 

Peptide mapping of proteolytic digests (LC-MS)

Introduction

It is often not possible to directly characterize variants resulting from single site amino acid modifications in large, glycosylated proteins (>20-30kD) due to the limits in mass resolution as well as the heterogeneity introduced by the carbohydrate structures.

In general, cleavage of the deamidated protein variant into smaller fragments using specific proteases, i.e. trypsin which cleaves at the C-terminal end of lysing and arginine residues, is necessary for the identification of the site of modification.

The peptide fragments of varying hydrophobicity are separated by reversed-phase HPLC resulting in a pattern of peaks or "fingerprint" diagnostic for a particular protein and used to monitor product identity and purity.

The effluent from the HPLC is directed into the electrospray mass spectrometer (quadrapole or ion-trap), in a technique referred to as LC-MS, allowing the identification of the various theoretical peptides as well as "new" peptides arising from post-translational modifications such as deamidation and oxidation.

 

Mass changes resulting from deamidation 

Deamidation of an Asn to Asp/IsoAsp results in an increase in mass of a single dalton.  Conversion of Asp to succinimide results in a decrease in mass of 18 daltons due to loss of water.  Conversion of Asn to succinimide results in a decrease in mass of 17 daltons due to release of ammonia.  Asp and IsoAsp have identical masses.  For future reference refer to the deamidation mass table on the reaction page.

The deamidated products can be distinguished by;

Blockage of Edman sequencing at IsoAsp
The additional methylene group in the peptide backbone of the IsoAsp residue prevents cyclization of the phenylthiocarbamyl peptide to form the anilinothiazolone derivative.

Differences in their MS/MS side chain cleavage product ions using a high energy FAB-MS magnetic sector instrument (Electrospray is a "soft" ionization technique and does not generate product ions resulting from side chain cleavage).
Ref: Carr et al. Anal. Chem, 1991 63:2802-2824.

Selective methylation of isoaspartyl sites with protein carboxyl methyl transferase (PIMT).  PIMT catalyzes the transfer of the methyl group from S-adenosyl-L-methinonine (SAM) onto the free alpha-carboxyl of the isoaspartyl residue.  The additional methyl group in the labeled IsoAsp peptide should increase the overall hydrophobicity and hence retention time relative to the unlabeled Asp-containing peptide.  

 

Protein carboxyl methyltransferase (PIMT): A useful tool for isoaspartyl analysis

Introduction

PIMT catalyzes the transfer of the methyl group from S-adenosyl-L-methinonine (SAM) onto the free alpha-carboxyl of the isoaspartyl residue.  It is found in most cells and is thought to play a major role in the removal of isoaspartyl residues.

Ref: Aswad, Deamidation and Isoaspartate Formation in Peptides and Proteins, CRC Series in Analytical Biotechnology, 1999.  

Determination of the sites of modification

Potential sites were identified in "thermally stressed" samples of recombinant human growth hormone (Johnson et al., J. Biol. Chem., 1989, 264:14262-14271) and recombinant tissue plasminogen activator (Paranandi, et al., J. Biol. Chem. 1994, 269:243-253).

General procedure

  1. Digest protein with trypsin (lowered pH and temp.)
  2. Methylate with PIMT.
  3. Analyze by RPLC; compare elution profile with unlabeled (control) digest.
  4. Methylated (IsoAsp) peptide should elute later due to increased hydrophobicity.

Care must be taken not to introduce artifactual deamidation during the trypsin digestion procedure.  Normally digests are performed at elevated pH and temperature which could cause further deamidation of the protein.  Method development may be in order to lower the pH and temperature parameters of the digestion.

How does deamidation affect biological activity?

Introduction

The published results to date indicates a lack of any definitive trend; i.e. a significant number of proteins deamidate without any apparent effect on their biological activity while others are adversely affected.

References:

T. Wright, Amino Acid Abundance and Sequence Data: Clues to the Biological Significance of Nonenzymatic Asparagine and Glutamine Deamidation in Proteins, in; Deamidation and Isoaspartate Formation in Peptides and Proteins, D. Aswad Ed., CRC Press, 1995.  A list of proteins that deamidate non-enzymatically and the effect on their in vitro/in vivo biological activity has been compiled.

Teshima et al. In; Deamidation and Isoaspartate Formation in Peptides and Proteins, D. Aswad Ed., CRC Press, 1995.  A review on the effect of deamidation and isoaspartate formation on the activity of proteins.

 

Correlation with location relative to binding site

There appears to be a relationship between the location of the deamidation site relative to the receptor binding region as it affects biological activity for hGH and CD4.  Deamidation at Asn52-Asp53 of CD4 reduced the in vitro biological activity while deamidation at Asn149-Ser150 of hGH had no apparent effect.  These results are consistent with the location of the sites of deamidation in relation to the binding region(s).  In the case of CD4, Asn52 is located in a region of the molecule involved in binding to the human immunodeficiency virus (HIV), while for hGH, Asn149 resides outside the two receptor binding regions.

 

Need for further clinical testing

While knowledge of the binding sites can be useful in predicting the effect of deamidation on biological activity at a particular site, the final determination must be made from the results of human clinical trials.

 

 

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