Controlled Deamidation of Peptides and Proteins: an Experimental Hazard and a Possible Biological Timer* Arthur B

Proceedings ofthe National Academy of Sciences Vol. 66, No. 3, pp. 753-757, July 1970

Controlled Deamidation of Peptides and Proteins: An Experimental Hazard and a Possible Biological Timer* Arthur B. Robinson,t James H. McKerrow, and Paul Cary

DEPARTMENT OF BIOLOGY, UNIVERSITY OF CALIFORNIA, SAN DIEGO (LA JOLLA) Communicated by Martin D. Kamen, April 13, 1970 Abstract. Experiments on model peptides show that the rate of deamidation of asparaginyl residues depends strongly on the nature of neighboring residues. The natural distribution of glutaminyl and asparaginyl residues is ordered with respect to the biological lifetime of the peptides and the functional groups of the residues neighboring to glutaminyl and asparaginyl residues. The rates of deamidation of such amide peptides under physiological conditions could serve as useful timers of development and aging.

The instability of the amino acids glutamine and asparagine with respect to hydrolytic deamidation of their functional side chains under relatively mild chemical conditions is well known. 1-4 The instability of glutaminyl and asparaginyl residues is a hazard during purification and primary structure de- terminations of peptides and proteins. In addition, now that Merrifield's solid- phase peptide synthesis5-726 procedure has made the synthesis of peptides easily available to all laboratories, it seems likely that the differential stabilities of amide side chains during synthetic procedures will provide an additional problem. The experiments of T. Flatmark8-10'27 on the properties of the deamidated forms of horse heart cytochrome c show that variations occur in the rate of deamidation of different amides in the same protein molecule and that the deamidated cytochromes exhibit altered biological activity and structure. Deamidation has also been observed in numerous other proteins.11 Why are these potentially unstable residues so widely distributed in nature? We suggest that controlled deamidation of glutaminyl and asparaginyl residues is a mechanism by which molecular and organismic development and aging are controlled. The rates of deamidation of glutaminyl and asparaginyl residues in proteins are probably determined by the primary and tertiary structures that govern distribution of amino acid residues surrounding the glutaminyl and asparaginyl residues in proteins. We have undertaken a study of the primary sequence dependence of deamidation of glutaminyl and asparaginyl residues in l)eptides. By comparing our results with the rates of deamidation of peptides of natural sequences and their corresponding proteins, we should also be able to make estimates of the tertiary structure dependence of deamidation. The results of our MAierrifield syntheses of the needed model peptides should aid in the predic- tion of difficulties during peptide synthesis and purification. 753 Downloaded by guest on September 25, 2021 754 BIOCHEMISTRY: ROBINSON ET AL. PROC. N. A. S.

TABLE 1. Comparative deamidation rates of asparaginyl residues. Ionic strength Ionic strength Deamidated 1.0 and 370C 0.15 and 370C during (sodium carbonate (Sodium phosphate preparation buffer) buffer) Peptide (%) k X 106, pH 10.0 k X 106, pH 7.0 Gly* Ala Asn Ala Gly 0 50 0. 20 (half-life 40 days) Gly* Thr Asn Thr Gly 0 18 ... Gly* LysAsn Lys Gly 13t 10 0.09 (half-life 3 months) Gly* Arg Asn Arg Gly 20 9 0.73 (half-life 11 days) Gly* Glu Asn Glu Gly 0 7 Gly* Thr Asn Glu Gly 0 2.0§ 0. 37§ (half-life 22 days) Gly* Gly Thr Asn Glu 0 2.2§ 0.41§ (half-life 20 days) Asnt ... 0.18 (ionic strength ... 0.1) (ethanola- mine-HCl buffer) Cyt I -- Cyt II T. Flatmark8 (assayed 2. 33§ 0. 62§ (half-life by evolution of am- 2.85§ 13 days) monia) 1. 61§ (half-life Cyt II -) Cyt III ... 5 days) Asn 0.18 (ionic strength ... 0.1) * 4C-Glycine. t Deamidation of this peptide during preparation was first observed by T. Flatmark and A. B. Robinson (cf. Ph.D. thesis, A. B. Robinson, University of California (San Diego) 1967). t 3H-Asparagine. § Values are for pH 6.8 and pH 10.4. The results of initial experiments in this study are shown in Table 1. The variation of the first-order rate constant k with variation in sequence near the asparaginyl residue is evident. In addition, it is seen that if an asparaginyl residue is next to a lysyl or arginyl residue, it undergoes some deamidation during synthesis. This deamidation probably occurs during the exposure of the peptide to strongly acidic solution for a short time after the hydrogen fluoride cleavage of the blocking groups. We have chosen five-residue peptides for our models, in order to minimize the effects of the charged-end amino acids. Some experiments on the deamidation at 100°C of dipeptides and tripeptides that contain glutaminyl residues have been reported. 12 In these experiments the complicating effect of the end residues is evident. We have also begun some experiments on similar models that contain glutaminyl residues, and we also are studying the rates of deamidation of the glutaminyl and asparaginyl residue portions of cytochrome c, subtilisin, lysozyme, A-1 EAE brain protein, and histone IV."3 If the rate of deamidation of glutaminyl and asparaginyl residues is of biological importance in proteins and if this deamidation is primarily controlled by the amino acid residues next to the asparaginyl and glutaminyl residues, then we might expect some correlation between the rates of deamidation of our models and the frequency with which difftrent amino acid residues occur next to glutaminyl and asparaginyl residues in proteins. We might also expect some cor- Downloaded by guest on September 25, 2021 VOL. 66, 1970 BIOCHEMISTRY: ROBINSON ET AL. 755

relation between the number of glutaminyl and asparaginyl residues and the lifetime of a protein under physiological conditions. Table 2 shows the frequency of occurrence of some amino acid di-residues in 43 proteins and peptides selected without regard to biological function.14 They contained 290 asparaginyl residues, 222 glutaminyl residues, and 5698 amino acid residues. The number of times that a given amino acid residue would be expected to occur at random on either side of an asparaginyl or glutaminyl residue was calculated. Standard deviations were calculated as the square root of each such number. A comparison between the expected number and the observed number was made in units of the standard deviation, a. For example, an alanyl residue was expected next to a glutaminyl residue 40 times and this pair- ing occurs 60 times. Hence, A = (60 - 40)/V'i = 3.2 a. This is designated ++++ in the table where (+) means 0 < A < 0.5, + means 0.5 < A < 1.0, ++ means 1.0 < A < 2.0, +++ means 2.0 < A < 3.0, and ++++ means 3.0 < A. + Values indicate more-often-than-expected occurrence, and - values indicate less-often-than-expected occurrence. Table 2 can be extended to any of the other 18 amino acid residues in proteins,

TABLE 2. Frequency distribution of residues near asparaginyl and glutaminyl residues. Neighboring Neighboring residue* GIN Asn residue* GIN Asn Ala ++++ - Arg ++ ++ Gly +++- Lys -- (-) Ser - ++ Phe - (+) Thr - + Tyr (+ ) Cys + + + His -- - Met + + + Trp + (-) Asp -- (+) Leu - (+) Glu (+) -- lieu -- (+) Asn (-) + Val (-) Gin - (-) Pro -- + End (+) + * There is also some indication that some residues are preferred on the amino side of asparaginyl residues and glutaminyl residues, whereas others are preferred on the carboxyl side. A larger or more selective sampling of sequences should be used to establish these "side" preferences. although it is interesting that a greater variation from the expected random distribution occurs for the glutaminyl residue than for any of the other 19 amino acid residues. In addition, Table 3 shows the correlation between percentage glutaminyl and asparaginyl residues and the biological lifetime of a protein. The lifetime of a protein seems to be a function of the number of its glutaminyl and asparaginyl residues. We suggest that a biological function of glutaminyl and asparaginyl residues in proteins is to effect a timed alteration of chemical structure through deamidation. Materials and Methods. Our experimental procedure involved synthesis of the peptides by the usual methods of Merrifield solid-phase peptide synthesis-7'26 fol- lowed by HF-anisole removal of the resin and side-chain blocking groups.'5'8 One residue in each peptide was 40-glycine. After one step of purification on G-10 Sephadex in pyri- dine acetate-pH7 buffer and freeze drying, buffered solutions of the peptides were sealed Downloaded by guest on September 25, 2021 756 BIOCHEMISTRY: ROBINSON ET AL. PROC. N. A. S.

TABLE 3. Correlation between number of glutaminyl and asparaginyl residues and lifetimes of protein. Percentage of glutaminyl and asparaginyl Protein Half-life7-26 residuesl4,17,18,25 Ribonuclease (rat) Less than 1 day 13.7 Lysozyme (chicken) Less than 1 day 12.4 Transferrin (human) 7-8 days 7.7 yG immunoglobulin 9-11 days 9.4 Cytochrome c (rat liver mitochondria) 10 days 7.7 A1-EAE (rat brain) 141/2 days 6.9 -yM immunoglobulin 15-26 days 9.0 Hemoglobin (rabbit) 25-35 days 6.3 Hemoglobin (human) 60 days 4.9 Glyceraldehyde-3-phosphate dehydrogenase (pig) 100 days 5.4 Histone IV (rat) (100-150)? days 3.9 Collagen (human bone) Years 3.4 in glass tubes and placed at a constant temperature of 370C. Peptide concentrations were approximately 10-2 M. Periodically a tube was opened and its contents applied to a strip electrophoresis apparatus. The electrophoresis separated the starting peptide from the deamidated product and the resulting bands were eluted and assayed in a liquid scintillation counter. Discussion. It is necessary to understand deamidation of glutaminyl and asparaginyl residues because of its importance in experimental protein chemistry. Also, it may be that this conveniently variable timed change in protein structure plays an important role in the development, function, and aging of living systems. First, during the development of an organism, large numbers of different chemical processes must be timed to occur in the right sequence and for the right duration. We can see that, simply by varying the sequence of residues around asparaginyl and glutaminyl residues, one can cause timed changes which would be useful for development. Second, a simple method by which a living system could keep its protein pool at optimal metabolic activity would be to incorporate a "planned obsolescence" into each protein molecule by way of glutaminyl and asparaginyl residues that are timed to deamidate. Flatmark's experiments show that the deamidated forms of cytochrome c have more open structures that are more readily susceptible to degradation. The timed period would be selected as a compromise between the expected useful lifetime of the protein in its physiological chemical environment and the energy required to replace the protein. Third, a simple method by which a species could keep its organism pool at an optimal activity would be to incorporate a "planned obsolescence" into each organism by way of glutaminyl and asparaginyl residues that are timed to deamidate. This aging might be controlled by deamidation in proteins that the organism is not able to resynthesize. Tertiary structure effects on deamidation should be especially important for control of long time-interval deamidation, as can be seen by comparing the rates of deamidation of our model peptides with the fact that several amides in cytochrome c are so stable that Flatmark was un- able to measure rates of deamidation for them. Downloaded by guest on September 25, 2021 VOL. 66, 1970 BIOCHEMISTRY: ROBINSON ET AL. 757

We are grateful to Professor T. Flatmark for many helpful discussions and instruction re- garding the problem of deamidation in proteins. We wish to thank Professor L. Pauling for his suggestions, Professors S. J. Singer and R. Doolittle for the loan of the electrophoresis apparatus used in these experiments, and Professor M. D. Kamen for his encouragement and financial support. * This work was supported by grants HD-01262 (National Institutes of Health) and GB- 7033X (National Science Foundation) to Professor M. D. Kamen. t Requests for reprints may be addressed to A. B. Robinson, Department of Biology, University of California, San Diego, La Jolla, Calif. 92037. 1 Chibnall, A. C., and R. W. Westall, Biochem. J., 26, 122 (1932). 2Vickery, H. B., G. W. Pucher, H. E. Clark, A. C. Chibnall, and R. G. Westall, Biochem. J., 29, 2710 (1935). 3 Gilbert, J. B., V. E. Price, and J. P. Greenstein, J. Biol. Chem., 180, 209 (1949). 4Schoffeniels, E., Traite Biochim. Gen., 3, 354-376 (1967). 6 Merrifield, R. B., J. Amer. Chem. Soc., 86, 304 (1964). 6 Marshall, G. R., and R. B. Merrifield, Biochemistry, 4, 2394 (1965). 7Marglin, A., and R. B. Merrifield, J. Amer. Chem. Soc., 88, 5051 (1966). 8 Flatmark, T., Acta Chem. Scand., 18, 1517 and 1656 (1964). 9 Flatmark, T., Acta Chem. Scand., 20, 1487 (1966). 10 Flatmark, T., J. Biol. Chem., 242, 2454 (1967). 11 Hartfenist, E. J., J. Amer. Chem. Soc., 75, 5528 (1953); R. G. Shephard, et al., J. Amer. Chem. Soc., 78, 5051 (1956); and others. 12 Fukawa, H., J. Chem. Soc. Jap., 88, 459 (1967). 13 Sharp, J., F. Westall, J. Strathern, and M. Legaz, experiments in progress. 14Selection is from Atlas of Protein Sequence and Structure, M. 0. Dayhoff and R. V. Eck (1969). Proteins used were horse heart cytochrome c, baker's yeast cytochrome c, Pseudo- monas fluorescens cytochrome c, bovine cytochrome B,, Pseudomona fluorescens azurin, Micro- coccus aerogenes rubredoxin, Clostridium pasteurianum ferredoxin, spinach ferredoxin, human adult alpha hemoglobin, sperm whale myoglobin, human AG-Bence-Jones kappa, bovine tryp- sinogen, Bacillus subtilis Carlsberg subtilisin, Escherichia coli tryptophan synthetase alpha, chicken lysozyme, bovine ribonuclease, Staphylococcus aureus V8 nuclease, bovine trypsin inhibitor, sheep lipotropin and human growth hormone, bovine posterior pituitary peptide, Rhodospirillum rubrum cytochrome c2, Desulfovibrio vulgaris cytochrome ca, Sipunculid worm hemerythrin, Escherichia cQli B thioredoxin, human hemoglobin beta chain, human hemoglobin gamma chain, human SH-Bence-Jones lambda chain, pig elastase, papain from papaya, bacterio- phages T4 and T2 lysozyme, pig glyceraldehyde-3-phosphate dehydrogenase, Staphylococcus aureus PC-i penicillinase, bovine basic trypsin inhibitor and kallikrein inactivator, bovine corticotropin (ACTH), pig secretin, pig glucagon, tobacco mosaic virus vulgare and OM coat protein, bacteriophage FR coat protein, human F and S haptoglobin alpha i, bovine and pea histone IV, E. coli E-26 acyl carrier protein, and pig proinsulin. 15 Sakakibara, S., Y. Shimonishi, M. Okada, and Y. Koshida, 8th European Peptide Sym- posium (1966). 16 Lenard, J., and A. B. Robinson, J. Amer. Chem. Soc., 89, 181 (1967). 17Hall, D. A., The Chemistry of Connective Tissue (Springfield, Ill.: Charles C Thomas, 1961). 18 Peeters, H., Protides of the Biological Fluids (Amsterdam: Elsevier 1967). 19 Velick, S. F., Biochim. Biophys. Acta, 20, 228 (1956). 20Munro, H. N. and J. B. Allison, Mammalian Protein Metabolism (New York: Academic Press, 1964), vol. I. 21 Phia, M. Cuenod, and H. Waelsch, J. Biol. Chem., 241, 2397 (1966). 22Keller, P. J., E. Cohen, and H. Neurath, J. Biol. Chem., 234, 311 (1959). 23 Morris, A. J., and S. R. Dickman, J. Biol. Chem., 235, 1404 (1960). 24 Fletcher, M. J., and D. R. Sanadi, Biochim. Biophys. Acta, 51, 356 (1961). 26 Shultz, H. E., and J. F. Hermans, Molecular Biology of Human Proteins (Amsterdam: Elsevier, 1966). 26 Stewart, J. Al., and J. D. Young, Solid Phase Peptide Synthesis (San Francisco: W. H. Freeman 1969). 27Flatmark, T., and K. Sletten, J. Biol. Chem., 243, 1623 (1968). Downloaded by guest on September 25, 2021