|
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.
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
- Digest protein with trypsin (lowered pH and temp.)
- Methylate with PIMT.
- Analyze by RPLC; compare elution profile with unlabeled
(control) digest.
- 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.
|