Effects of amino acid sequence, buffers, and ionic strength on the rate and mechanism of deamidation of asparagine residues in small peptides

The nonenzymatic rates of deamidation of Asn residues in a series of pentapeptides with the sequences VSNXV and VXNSV, where X is one of 10 different amino acids, were determined at neutral, alkaline, and acid pH values. The results demonstrate that in neutral and alkaline solutions the amino acid residue on the amino side of the Asn had little or no effect on the rate of deamidation regardless of its charge or size. The group on the carboxyl side of Asn affected the rate of deamidation significantly. Increasing size and branching in the side chain of this residue decreased the rate of deamidation by as much as 70-fold compared to glycine in the N-G sequence, which had the greatest rate of deamidation. In acidic solution, the rate of deamidation of the Asn residue was not affected by the amino acid sequence of the peptide. The products for each deamidation reaction were tested for the formation of isoAsp residues. In neutral and alkaline solutions, all products showed that the isoAsp:Asp peptide products were formed in about a 3:1 ratio. In acidic solution, the Asp peptide was the only deamidation product formed. All peptides in which a Ser residue follows the Asn residue were found to undergo a peptide cleavage reaction in neutral and alkaline solutions, yielding a tripeptide and a dipeptide. The rate of the cleavage reaction was about 10% of the rate of the deamidation pathway at neutral and alkaline pH values. The rates of deamidation of Asn residues in the peptides studied were not affected by ionic strength, and were not specific base catalyzed. General base catalysis was observed for small bases like ammonia. A model for the deamidation reaction is proposed to account for the observed effects.     

Succinimide formation from aspartyl and asparaginyl peptides as a model for the spontaneous degradation of proteins

Nonenzymatic intramolecular reactions can result in the deamidation, isomerization, and racemization of protein and peptide asparaginyl and aspartyl residues via succinimide intermediates. To understand the sequence dependence of these reactions, we measured the rate of succinimide formation in a series of synthetic peptides at pH 7.4. These peptides (Val-Tyr-Pro-X-Y-Ala) contained an internal aspartyl, asparaginyl, aspartyl beta-methyl ester, or aspartyl alpha-methyl ester residue (X) followed by a glycyl, seryl, or alanyl residue (Y). The rates of succinimide formation of the asparaginyl peptides were found to be 13.1-35.6 times faster than those of the aspartyl peptides. The rates of succinimide formation for the glycyl peptides were 6.5-17.6 times faster than those of the alanyl peptides, while the rates for the seryl peptides were 1.6-4.5 times faster than those of the alanyl peptides. The overall 232-fold range in these reaction rates for aspartyl and asparaginyl residues suggests that sequence can be an important determinant in their stability in flexible peptides. In proteins, there may be a much larger range in the rates of succinimide formation because specific conformations may greatly enhance or inhibit this reaction.     

Structure-dependent nonenzymatic deamidation of glutaminyl and asparaginyl pentapeptides.

Nonenzymatic deamidation rates for 52 glutaminyl and 52 asparaginyl pentapeptides in pH 7.4, 37.0 degrees C. 0.15 m Tris-HCl buffer have been determined by direct injection mass spectrometry. These and the previously reported 306 asparginyl rates have been combined in a self-consistent model for peptide deamidation. This model depends quantitatively upon peptide structure and involves succinimide, glutarimide and hydrolysis mechanisms. The experimental values and suitable interpolated values have been combined to provide deamidation rate values in pH 7.4, 37.0 degrees C. 0.15 m Tris-HCl buffer for the entire set of 648 single-amide permutations of ordinary amino acid residues in GlyXxxAsnYyyGly and GlyXxxGlnYyyGly. Thus, knowledge about sequence-dependent deamidation in peptides is extended to include very long deamidation half-times in the range of 2-50 years.     

Evidence for the in vivo deamidation and isomerization of an asparaginyl residue in cytosolic serine hydroxymethyltransferase

Rabbit liver cytosolic serine hydroxymethyltransferase exists in several subforms which have different isoelectric points. Incubation of the purified enzyme with chymotrypsin cleaves the enzyme at Trp14. The released amino-terminal 14-mer peptide was shown to exist in three forms of equal concentration. The peptides differ in structure only at the asparaginyl residue at position 5. In addition to asparagine at this position we found both aspartyl and isoaspartyl residues. The deamidation of Asn5 does not appear to occur during the purification of the enzyme. The in vitro rate of deamidation of Asn5 in the enzyme is more than 5-fold slower than the rate of deamidation of this residue in the free 14-mer peptide. The isoaspartyl residue at position 5 serves as a substrate for protein carboxyl methyltransferase both in the free 14-mer peptide and the native enzyme. The enzyme which has had the amino-terminal 14 residues removed by digestion with chymotrypsin still exists in several forms with different isoelectric points. Reaction of peptides from this enzyme with carboxyl methyltransferase suggests that there is at least one more asparaginyl residue in this enzyme other than Asn5 which has undergone deamidation with the formation of isoaspartyl bonds.     

Effect of protein conformation on rate of deamidation: ribonuclease A

The effect of the folded conformation of a protein on the rate of deamidation of a specific asparaginyl residue has been determined. Native and unfolded ribonuclease A (RNase A) could be compared under identical conditions, because stable unfolded protein was generated by breaking irreversibly the protein disulfide bonds. Deamidation of the labile Asn-67 residue of RNase A was followed electrophoretically and chromatographically. At 80 degrees C, similar rates of deamidation were observed for the disulfide-bonded form, which is thermally unfolded, and the reduced form. At 37 degrees C and pH 8, however, the rate of deamidation of native RNase A was negligible, and was more than 30-fold slower than that of reduced, unfolded RNase A. This demonstrates that the Asn-67 residue is located in a local conformation in the native protein that greatly inhibits deamidation. This conformation is the beta-turn of residues 66-68.     

Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. Succinimide-linked reactions that contribute to protein degradation

Aspartyl and asparaginyl deamidation, isomerization, and racemization reactions have been studied in synthetic peptides to model these spontaneous processes that alter protein structure and function. We show here that the peptide L-Val-L-Tyr-L-Pro-L-Asn-Gly-L-Ala undergoes a rapid deamidation reaction with a half-life of only 1.4 days at 37 degrees C, pH 7.4, to give an aspartyl succinimide product. Under these conditions, the succinimide product can further react by hydrolysis (half-time, 2.3h) and by racemization (half-time, 19.5 h). The net product of the deamidation reaction is a mixture of L- and D-normal aspartyl and beta-transpeptidation (isoaspartyl) hexapeptides. Replacement of the asparagine residue by an aspartic acid residue results in a 34-fold decrease in the rate of succinimide formation. Significant racemization was found to accompany the deamidation and isomerization reactions, and most of this could be accounted for by the rapid racemization of the succinimide intermediate. Replacement of the glycyl residue in the asparagine-containing peptide with a bulky leucyl or prolyl residue results in a 33-50-fold decrease in the rate of degradation. Peptide cleavage products are observed when these Asn-Leu and Asn-Pro-containing peptides are incubated. Our studies indicate that both aspartic acid and asparagine residues may be hot spots for the nonenzymatic degradation of proteins, especially in cells such as erythrocytes and eye lens, where these macromolecules must function for periods of about 120 days and 80 years, respectively.     

Mass spectrometric evaluation of synthetic peptides as primary structure models for peptide and protein deamidation.

Solid-phase peptide synthesis and deamidation measurements using a novel mass spectrometric technique were carried out for 94 model asparaginyl peptides from 3 to 13 residues in length. Deamidation rates of these peptides in pH 7.4, 37.0 degrees C, 0.15 M Tris-HCl buffer were measured and evaluated. It was found that they validate the use of pentapeptide models as surrogates for the primary sequence dependence of peptide and protein deamidation rates and the discovery by difference of secondary, tertiary and quaternary structure effects. Deamidation of the pentapeptide models, compared with that of longer peptides of more intricate structure, is discussed, and the application of this technique to deamidation measurement of intact proteins is demonstrated.     

Effect of protein structure on deamidation rate in the Fc fragment of an IgG1 monoclonal antibody

The effects of secondary structure on asparagine (N) deamidation in a 22 amino acid sequence (369‐GFYPSDIAVEWESNGQPENNYK‐390) of the crystallizable (Fc) fragment of a human monoclonal antibody (Fc IgG1) were investigated using high‐resolution ultra performance liquid chromatography with tandem mass spectrometry (UPLC/MS). Samples containing either the intact Fc IgG (～50 kD) (“intact protein”), or corresponding synthetic peptides (“peptide”) were stored in Tris buffer at 37°C and pH 7.5 for up to forty days, then subjected to UPLC/MS analysis with high energy MS1 fragmentation. The peptide deamidated only at N₃₈₂ to form the isoaspartate (isoD₃₈₂) and aspartate (D₃₈₂) products in the ratio of ～4:1, with a half‐life of ～3.4 days. The succinimide intermediate (Su₃₈₂) was also detected; deamidation was not observed for the other two sites (N₃₈₇ and N₃₈₈) in peptide samples. The intact protein showed a 30‐fold slower overall deamidation half‐life of ～108 days to produce the isoD₃₈₂ and D₃₈₇ products, together with minor amounts of D₃₈₂. Surprisingly, the D₃₈₂ and isoD₃₈₇ products were not detected in intact protein samples and, as in the peptide samples, deamidation was not detected at N₃₈₈. The results indicate that higher order structure influences both the rate of N‐deamidation and the product distribution.     

Effect of lysine residues on the deamidation reaction of asparagine side chains

The effect of lysine residues on the deamidation reaction of the asparagine side chain has been studied on the peptide and on its lysine-acetylated derivative in a wide range of pH values. The amino acid sequence of these peptides is similar to the local sequence flanking the labile Asn-67 in RNAse A. The experimental data show that Lys influences both the deamidation rate and the relative yield of the two reaction products, i.e., the aspartic acid and beta-aspartic acid containing peptide. These effects are pH dependent and can be rationalized based on the mechanism previously proposed for the deamidation reaction via succinimide derivative.     

Enzymatic methyl esterification of a deamidated form of mouse epidermal growth factor

The enzyme S-adenosylmethionine:protein carboxyl-O-methyl-transferase, type II (EC 2.1.1.77; PCMT) from eukaryotes methyl esterifies peptides containing isoAsp residues, which can arise from spontaneous deamidation of labile Asn residues. We report here a study on in vitro methyl esterification of mouse EGF by bovine brain PCMT. This peptide contains two Asn in the sequences Asn1-Ser2 and Asn16-Gly17. It is known from the literature that the presence of a small residue on the carboxyl side of asparaginyl makes this residue susceptible to deamidation through the spontaneous formation of a succinimide intermediate. Therefore EGF was incubated under deamidating conditions (pH 9.0, 37 degrees for 48 h) and the extent of deamidation monitored by enzymatically measuring the NH3 produced during the alkali treatment: a release of 0.80 mol NH3/mol EGF was calculated. The alkali-treated EGF, analyzed by anion-exchange chromatography, shows two major components identified as native EGF (nEGF) and its deamidated form (dEGF). When incubated in the presence of purified PCMT neither nEGF nor dEGF showed any methyl accepting capability. Since it is known that the three-dimensional structure of a protein may hinder the methyl esterification of a potential ethyl accepting site, dEGF was unfolded by reducing and alkylating the intrachain disulfide bridges. Only a slight increase in the methyl accepting capability could be observed. Conversely, when EGF was deamidated after its unfolding, the resulting protein was stoichiometrically methylated by PCMT, presumably at level of isoAsp16. Our findings strongly suggest that the three-dimensional structure of a protein is a major specificity determinant for both deamidation and methyl esterification processes.     

Propensity for spontaneous succinimide formation from aspartyl and asparaginyl residues in cellular proteins

One mechanism for the spontaneous degradation of polypeptides is the intramolecular attack of the peptide bond nitrogen on the side chain carbonyl carbon atom of aspartic acid and asparagine residues. This reaction results in the formation of succinimide derivatives and has been shown to be largely responsible for the racemization, isomerization, and deamidation of these residues in several peptides under physiological conditions (Geiger, T. & Clarke, S. J. Biol. Chem. 262, 785-794 (1987]. To determine if similar reactions might occur in proteins, I examined the sequence and conformation about aspartic acid and asparagine residues in a sample of stable, well-characterized proteins. There did not appear to be any large bias against dipeptide sequences that readily form succinimides in small peptides. However, it was found that aspartyl and asparaginyl residues generally exist in native proteins in conformations where the peptide bond nitrogen atom cannot approach the side chain carbonyl carbon to form a succinimide ring. These orientations also represent energy minimum states, and it appears that this factor may account for a low rate of spontaneous damage to proteins by succinimide-linked reactions. The presence of aspartic acid and asparagine residues in other conformations, such as those in partially denatured, conformationally flexible regions, may lead to more rapid succinimide formation and contribute to the degradation of the molecule. The possible role of isoimide intermediates, formed by the attack of the peptide oxygen atom on the side chain carboxyl group, in protein racemization, isomerization, and deamidation is also considered.     

Composition variation of papain-catalyzed esterification of a fibroin peptide mixture

Composition variation of a complex peptide mixture under enzymatic transformation can be tracked by mass spectrometry (MS). In this report, papain-catalyzed esterification of fibroin peptides was investigated at the individual peptide level using liquid chromatography-mass spectrometry with selected ion monitoring. Optimal conditions for maximizing ester formation were obtained using a water-to-pentanol ratio of 1:9 at pH 2.8 and 40°C; however, the optimum conditions varied for individual peptides. The optimum pH levels were 2.5 and 2.8 for the tetrapeptides with a tyrosine or a valine residue and those with alanine or serine residues, respectively. The optimum pH shifted to 3.4 for dipeptide esters with a tyrosine residue. Tetrapeptides had a relatively higher rate of esterification above 50°C. Alhough, the profiles of peptides and their esters in the esterification reaction were significantly affected by the reaction conditions, alanyl-glycine ester represented the largest fraction in the mixture under most reaction conditions. As demonstrated here, MS analysis of peptide mixtures can be used to elucidate specific reaction conditions for the enrichment of particular peptide products.     

Deamidation of glutaminyl residues: dependence on pH, temperature, and ionic strength

We have shown the dependence of the deamidation half-times of the peptides, GlyLeuGlnAlaGly and GlyArgGlnAlaGly upon pH, temperature, and ionic strength. Increase in temperature or ionic strength, variation of pH to pH′s higher or lower than pH 6, and the use of phosphate buffer rather than Tris buffer at high pH all decrease the half-time of dcamidation. Temperature increase of 20°C or pH change of 2 pH units decreases the half-time about fivefold, while increase of one ionic strength unit decreases the half-time about twofold. In pH 7.4, I = 0.2, 37.0°C phosphate buffer, the deamidation half-times are 663 ± 74 and 389 ± 56 days respectively for the two peptides, GlyLeuGlnAlaGly and GlyArgGlnAlaGly.These experiments should serve as a warning to peptide and protein experimenters that even the more stable glutaminyl residues are unstable with respect to deamidation in certain solvent conditions. These experiments also provide, along with previously reported experiments on asparaginyl peptides (7), some quantitative data to help with the extrapolation of in vitro deamidation experiments to in vivo deamidation conditions.     

Empirical rules predicting conformation of cyclic tetrapeptides from primary structure

A useful set of empirical rules is put forward to predict the conformations of cyclic tetrapeptides and cyclic tetradepsipeptides on the basis of primary structure, briefly presented as follows: A conformation allowing an intramolecular hydrogen bond (IMHB) of gamma-turn is preferred, and an ester bond always adopts a trans form. On a right-handed peptide ring, the carbonyl group acylating a D residue is oriented to the upper side of the main ring. The carbonyl group acylating a D proline or an N-methyl-D-amino acid residue is oriented to the lower side of the ring, forming a cis bond. The LDDL configurational sequence adopts a cis-trans-cis-trans backbone with Ci symmetry. A glycine residue behaves as a D residue in an L-peptide. Conformations of cyclotetrapeptides containing two glycine residues at diametric positions or containing an N-methyl-dehydroamino acid residue are predicted by use of appendices of rule 5. Almost all conformations of cyclic tetrapeptides are predicted by these rules. Energetical rationalization of the rules and prediction of possible new conformations are described. Conformations of cyclo(-L-Pro-L-Leu-D-Tyr(Me)-L-Ile-)(1) and cyclo (-L-Pro-D-Leu-D-Tyr(Me)-L-Ile)(2) are compared. Results of n.m.r. experiments showed that compound 1 adopts a unique cis-trans-trans-trans backbone with a gamma-turn IMHB, and 2 has a cis-trans-cis-trans backbone with Ci symmetry. These observations confirmed the rules described above. Peptides 1 and 2 are the first diastereomeric peptides with trans (LD) and cis (DD) secondary amide bonds.     

Inactivation of mammalian fructose diphosphate aldolases by COOH terminus autophosphorylation

Rabbit skeletal muscle and liver fructose 1,6-diphosphate aldolases autophosphorylate in the presence of inorganic phosphate at physiological and alkaline pH. ATP as well as nonhydrolyzable ATP analogues inhibits autophosphorylation. Autophosphorylation of aldolases abolishes catalytic activity, which is restored upon treatment with alkaline phosphatase. Limited proteolysis of aldolase preferentially hydrolyzes the COOH terminus and liberates a phosphorylated peptide. Treatment of rabbit aldolases with carboxypeptidase, which liberates the COOH terminal residue Tyr 363, although modifying catalytic activity does not affect autophosphorylation. Amino acid analyses are consistent with results of autophosphorylation of the COOH terminus showing residue His 361 in muscle aldolase and Tyr 361 in liver aldolase. Phosphate lability in acid pH by phosphorylated muscle aldolase but not by phosphorylated liver aldolase corroborates the amino acid assignment. Autophosphorylation of the aldolases in the crystalline state is consistent with an intramolecular mechanism. The pH dependence of autophosphorylation being dependent on the enzyme's physical state (soluble or crystalline) is not inconsistent with crystallization stabilizing a conformer having different amino acid pka values and/or reactivities than those of the soluble state.     

Use of Merrifield solid phase peptide synthesis in investigations of biological deamidation of peptides and proteins

Merrifield solid phase peptide synthesis has been the principle research procedure used in the study of the chemistry and biological use of deamidation of asparaginyl and glutaminyl residues in peptides and proteins during the past 40 years. During the initial years of investigation, it permitted the qualitative demonstration that primary, secondary, and tertiary structure-determined deamidation half-times vary over a wide range under biological conditions and the discovery of two biological systems in which deamidations serve as molecular clocks. More recently, it has made possible such a thorough quantitative understanding of the structural dependence of deamidation that the deamidation rates of asparaginyl residues in proteins can be predicted from protein three-dimensional structures with a high degree of reliability. This, in turn, has led to the discovery that amide residues serve as molecular clocks in many biological systems and the demonstration of additional examples. In these investigations, Professor R. B. Merrifield contributed his techniques, time, and laboratory resources, both in personally teaching his methods to the principle investigators and in making available his laboratory in which more than 900 peptides were synthesized for this work.     

Reaction of peptide 89-169 of bovine myelin basic protein with 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine.

The C-terminal half of the bovine myelin basic protein, peptide 89-169, was treated with BNPS-skatole [2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine], and the products were isolated by repeated gel filtration through Sephadex G-50. They consisted of uncleaved peptide 89-169 in which approximately 30% of the tyrosine had been monobrominated and the tryptophan converted to oxindolealanine, peptide 116-169 modified by partial bromination (30%) of the tyrosine, and two chromatographic forms of peptide 89-115. The major form contained the lactone of dioxindolealanine at the C terminus; the minor form contained the uncyclized oxidation product. Each form of peptide 89-115 was resolved into several components by electrophoresis in polyacrylamide gels (10%, w/w) containing 1 M acetic acid and 8 M urea. The presence of three of these components could be explained by partial deamidation of Asn-91 and Gln-102. Studies on the oxidation of tryptophan-containing model peptides by BNPS-skatole indicated that the reaction can also include partial bromination of the dioxindole and its lactone and partial cleavage at the amino peptide bond of the tryptophan.     

Isolation and structure of the principal products of preproglucagon processing, including an amidated glucagon-like peptide

The principal products derived from in vivo processing of anglerfish preproglucagon II were isolated and their structures determined. The structures were confirmed by a combination of automated Edman degradation, amino acid analysis, and fast atom bombardment mass spectrometry. The peptide corresponding to anglerfish preproglucagon II-(22-49) (numbering from the amino terminus of preproglucagon) was isolated intact and defines the site of signal cleavage to be between Gln-21 and Met-22. Glucagon from the anglerfish preproglucagon gene II was found to correspond to preproglucagon II-(52-80) (numbering from the amino terminus). Three forms of a glucagon-like peptide derived from preproglucagon II were also isolated. The structure of the longest form was consistent with the sequence of preproglucagon II-(89-122) deduced from the cDNA, His-Ala-Asp-Gly-Thr-Tyr-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Gln-Asp-Gln-Ala- Ala-Lys-Asp-Phe-Val-Ser-Trp-Leu-Lys-Ala-Gly-Arg-Gly-Arg-Arg-Glu. The carboxyl-terminal portion deduced from the cDNA remains intact in this form. A second form, preproglucagon II-(89-119) appears to result from proteolytic processing of the major form at the two adjacent arginine residues occurring at the carboxyl terminus. This second form has a glycine residue at its carboxyl terminus and is processed to the third form (preproglucagon II-(89-118)) which contains a carboxyl-terminal arginineamide. Radiolabeling studies in primary tissue culture support the observation that glucagon (preproglucagon II-(52-80], preproglucagon II-(89-122), and preproglucagon II-(89-119) are products of proglucagon processing in vivo.     

Competitive inhibitors of renin. Inhibitors effective at physiological pH.

Previously we reported the development of competitive inhibitors of renin effective at pH 5.5 (Poulsen, K., Burton, J., and Haber, E. (1973), Biochemistry 12, 3877). At physiologic pH (7.5), the inhibitory constants (Ki) increased and solubility decreased to the point that inhibition could not be demonstrated with these peptides. Modification of the octapeptide sequence, His-Pro-Phe-His-Leu-Leu-Val-Tyr, either by addition of serinol to the carboxyl terminus or by replacement of valine-7 with an isosteric threonyl residue failed to yield peptides active at pH 7.5. Attachment of polyproline sequences to the amino terminus increased solubility from threefold to tenfold and decreased Ki so that competitive inhibition was demonstrable at physiologic pH. In addition, if leucine-6 was replaced in these peptides with a phenylalanyl or tyrosyl residue, Ki decreased (3-12 muM) to give effective competitive inhibitors at physiologic pH in both buffer and in plasma.