Estimation of the Deamidation Rates of Major Deamidation Sites in a Fab Fragment of Mouse IgG1‑κ by Capillary Isoelectric Focusing of Mutated Fab Fragments Kiyohito Shimura,*,† Makoto Hoshino,‡ Keiichiro Kamiya,‡ Manabu Enomoto,‡ Sunao Hisada,§ Hiroyuki Matsumoto,∇ Mark Novotny,⊥ and Ken-ichi Kasai‡ †
Laboratory of Chemistry, School of Medicine, Fukushima Medical University, Fukushima, Fukushima 960-1295, Japan Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamihara, Kanagawa 252-5195, Japan § Systems Division, Hamamatsu Photonics K.K., Hamamatsu, Shizuoka 431-3196, Japan ∇ Central Research Laboratory, Hamamatsu Photonics K.K., Hamakita, Shizuoka 434-8601, Japan ⊥ J. Craig Venter Institute, 10355 Science Center Drive, La Jolla, California 92121, United States ‡
ABSTRACT: The deamidation of asparagine (Asn or N) residues in proteins is a common post-translational chemical modiﬁcation. The identiﬁcation of deamidation sites and determination of the degree of deamidation have been carried out by the combination of peptide mapping and mass spectrometry. However, when a peptide fragment contains multiple amides, such analysis becomes diﬃcult and sometimes impossible. In this report, a quantitative method for estimating the deamidation rate of a speciﬁc amide in a protein is presented without using peptide mapping. Five Asn residues of a recombinant fragment antigen binding (rFab) (mouse IgG1, κ) were mutated to a serine (Ser) residue, one by one, through site-directed mutagenesis, and the single-residue deamidation rates of the original rFab and the mutants were determined using capillary isoelectric focusing. The diﬀerence of the rate between the original rFab and the mutant was assumed to be equal to the deamidation rate of the speciﬁc Asn residue, which had been mutated. Among ﬁve mutants established, three major deamidation sitesH chain Asn135, L chain Asn157, and L chain Asn161, using the Kabat numbering systemwere identiﬁed, accounting for 66%, 29%, and 7% of the single-residue deamidation of the original rFab, respectively. Although the former two have been known by peptide mapping, the last one, which resides on the same tryptic peptide that carries one of the former two, previously has not been identiﬁed. For the ﬁrst time, the deamidation rate constants of the three sites were estimated to be 10.5 × 10−3 h−1, 4.6 × 10−3 h−1, and 1.1 × 10−3 h−1 in 0.1 M phosphate buﬀer, pH 7.5 at 37 °C, respectively, with corresponding half-life of 2.8 days, 6.3 days, and 27 days. The method should be applicable to any recombinant proteins.
identiﬁcation of the deamidation site could become impossible. Therefore, the currently available method to identify deamidation sites and quantitatively estimate the contribution of each site to the deamidation of a whole protein molecule is not satisfactory. With isoelectric focusing (IEF), we observed some heterogeneity in recombinant fragments antigen binding (rFabs) derived from an IgG1-κ-type monoclonal antibody, and the heterogeneity seemed to be attributable to deamidation. Some deamidation-related isoforms of mouse IgG1-κ type immunoglobulins have been known to exist.4 By using peptide mapping and mass spectrometry, Asn135 of H chains of IgG15 and Asn157 of κ-type L chains,5,6 using the
eamidation of asparagine (Asn) residue in proteins or peptides results in the change to either aspartic acid (Asp) or iso-aspartic acid (iso-Asp) residues.1,2 Either change adds one negative charge to a protein in a neutral to basic pH range. The deamidation rate of Asn residues depends on the size of the side chain of the nearest carboxyl neighboring amino acid residues. Amino acid residues having small side chains such as glycine (Gly) and serine (Ser) facilitate deamidation of Asn. Flexibility of the surrounding structure also increases the rate.1 The combination of peptide mapping and mass spectrometry is powerful for identifying the sites of deamidation, and additionally quantifying the degree of deamidation.3 In principle, this technique is applicable to the determination of the deamidation rate of each amide in a protein. However, the number of such applications is limited, probably because of technical diﬃculties involved with the analysis. Furthermore, when a peptide fragment contains multiple amides, even © 2013 American Chemical Society
Received: October 13, 2012 Accepted: January 1, 2013 Published: January 1, 2013 1705
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Kabat numbering system, were identiﬁed as major deamidation sites, both in the constant domain of Fab. In this report, a quantitative method for estimating the deamidation rate of a speciﬁc amide is presented. We chose ﬁve Asn residues and one glutamine (Gln) residue with Gly or Ser at its nearest neighboring carboxyl side, and made six mutants by replacing each amino acid residue with a Ser residue, using site-directed mutagenesis. Each mutant was labeled at its hinge region with a ﬂuorescent dye. The change of each mutant to more-acidic species during incubation was measured by capillary IEF (CIEF), and the single-residue deamidation rate of each Fab molecule was determined from the decrease of the original peak. The deamidation rate of a speciﬁc amide was estimated by subtracting the rate of the speciﬁc mutant from that of the wild type. We conﬁrmed that the known two sites are certainly the major sites of deamidation. Another Asn residue that deamidates slower than the two was identiﬁed in the constant domain of κ-chain by the present method. This site locates in the same tryptic peptide that contains one of the formerly known deamidation sites. In addition, the deamidation rate of each site was estimated for the ﬁrst time. A double mutant at the two major deamidation sites exhibited a high stability against deamidation, showing less than a tenth of the deamidation of the original Fab.
Table 1. Mutants of Fab mutant γN135S γN162S γQ179S κN157S κN161S κN190S γN135S, κN157S a
Codon changes for mutation
AQTNSMV VTWNSGS AVLQSDL ERQNGVL GVLNSWT ERHNSYT same as above
AQTSSMV VTWSSGS AVLSSDL ERQSGVL GVLSSWT ERHSSYT same as above
AAC > AGC AAC > AGC CAG > TCG AAT > AGT AAC > AGC AAC > AGC same as above
Replaced amino acid residues with a Ser residue are underscored.
× 10−6 M in 50 mM sodium acetate buﬀer (pH 5.0) were diluted 100-fold with 0.1 M sodium phosphate buﬀer (pH 7.5) containing 0.01% bovine serum albumin (BSA) and incubated at 37 °C for 15 and 30 h. The incubation was stopped by transferring the samples to an ice bath, and they were analyzed within 1 h as follows. The incubated sample was diluted 100fold with 2.5% (v/v) Pharmalyte 3−10 containing 0.01% BSA and 10−11 M each of pI markers, when they were included, and was subjected to CIEF analysis in a polydimethylacrylamidecoated fused silica capillary10 (50 μm i.d., 375 μm o.d., 180 mm long), as previously described.7 Focusing was carried out at 500 V/cm for 10 min at room temperature with 20 mM phosphoric acid as an anode solution and 20 mM NaOH as a cathode solution. Labeled mutants were detected using a scanning laserinduced ﬂuorescence detector at 590 nm with a 543.5 nm He− Ne laser for excitation at a scanning speed of 3 mm/s. After incubation in the pH 7.5 buﬀer, one or two peaks appeared on the acidic side of the original peak. Triply deamidated peaks were too small to be detected. The area of each peak was obtained by integrating the ﬂuorescence signal using a data processor for chromatography (ChromatoPack 4, Shimadzu), and the ratio of the peak area of the original peak to the sum of the three peaks was calculated. The reduction of the ratio along the incubation was analyzed according to the ﬁrst-order kinetics.
MATERIALS AND METHODS Chemicals and Materials. The following were obtained from commercial sources: Pharmalyte, Sephadex G25 (ﬁne), Agarose IEF, anti-E tag antibody immobilized column (Pharmacia Biotech AB, Uppsala, Sweden); tetramethylrhodamine 5-iodoacetamide (5-TMRIA) “single isomer” (Molecular Probes, Eugene, OR, USA); α1-antitrypsin (from human plasma, Calbiochem-Novabiochem Corp., La Jolla, CA, USA); N,N-dimethylacrylamide (Wako Pure Chemical Industries, Osaka, Japan); puriﬁed Tween 20 solution (Surfact-Amps 20) (Pierce, Rockford, IL, USA); and fused silica capillaries (GL Sciences, Tokyo, Japan). Fluorescence-Labeled Recombinant Fab. Fluorescencelabeled recombinant Fab for α1-antitrypsin was prepared as previously reported.7 Fab DNA harbored in the pAK400E vector, in which the 6His tag of the pAK400 vector8 was replaced with a pentadeca peptide tag, E tag, being attached at the C-terminus of the L chain, was expressed in the periplasmic space of Escherichia coli. After puriﬁcation by aﬃnity chromatography on an anti-E tag antibody-immobilized column, the rFab was labeled with 5-TMRIA at the Cys residue locating at the C-terminal hinge region of the Fd chain derived from the H chain and puriﬁed by passing a column of Sephadex G25. The colored pass through fraction was further puriﬁed by IEF on a slab of Agarose IEF with Pharmalyte pH 4−6.5. The major cathodic band was excised, and the labeled rFab was eluted to 50 mM sodium acetate buﬀer (pH 5.0) by allowing it to diﬀuse at 4 °C. Mutants of Recombinant Fab. A rFab, 12C-L3, for α1antitrypsin was used as the original Fab.7 It was composed of γ1-class Fd chain, i.e., VH and CH1 domains, and κ-type L chain. Site-directed mutagenesis by overlap extension using polymerase chain reaction (PCR) was used for the induction of the mutations.9 The mutants were summarized in Table 1, and the mutations at the speciﬁc sites were conﬁrmed by sequencing of the entire length of the rFab DNAs. Determination of Deamidation Rates of Mutants. Labeled and IEF-puriﬁed mutant Fabs at a concentration of ∼5
RESULTS AND DISCUSSION Selection of Mutations. The ﬂuorescent-dye-labeled rFab preparation showed two major bands on separation by CIEF. After puriﬁcation of the basic band by IEF on a slab gel, regeneration of the acidic band was observed during storage under conditions of a physiological pH. We suspected that deamidation of Asn or Gln residues of the rFab might have occurred. Deamidation of Asn in peptides and proteins is considered to occur through the formation of a succinimide intermediate by the attack of the imide nitrogen of the carboxyl-side peptide bond to the carbonyl carbon of the amide.1,2 From the measurement of deamidation rate of Asn in 425 pentapeptides with the sequence Gly-Xxx-Asn-Yyy-Gly, it was found that Gly at position Yyy was the most favorable for the deamidation of Ans, followed by Ser and His.1,11 The numbers of Asn residues in the recombinant Fab are 3, 1, 3, and 8 in the VH, VL, CH1, and CL domains, respectively. Among the total of 15 Asn residues, 1 has a Gly residue at the nearest carboxyl neighbor and 4 have Ser residues at the same relative position. These 5 Asn residues, which are expected to be labile for deamidation, are all located in the constant domain of the rFab. The 5 Asn 1706
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residues were chosen to induce mutations, each being substituted with a Ser residue (see Table 1). Gln as an amino acid or an N-terminal amino acid residue is known to release ammonia more rapidly than Asn as an amino acid through the formation of pyrrolidone-ring structure by the attack of the α-amino group to the carbonyl carbon of the amide. Gln residues in peptides and proteins except at their Ntermini, generally deamidate several hundred times slower than Asn residues.1 The numbers of Gln residues in the rFab are 6, 2, 6, and 3 in the VH, VL, CH1, and CL domains, respectively. One Gln residue, with a Ser residue at the nearest carboxyl neighbor, was also selected to mutate to a Ser residue, for the sake of comparison. Preparation of Labeled rFab Mutants. The original recombinant Fab and its mutants were expressed in E. coli and puriﬁed from periplasmic extracts. They were site-speciﬁcally labeled at the single cysteine residue at the hinge region near the C-terminus of the Fd chain, VH + CH1, with a thiolreactive ﬂuorescent dye. When the labeled rFabs were subjected to the separation by IEF on an agarose gel slab, most Fabs appeared as two bands. The cathodic major bands were recovered from the gel as the intact rFabs, although there is a possibility that the protein has an extremely rapid deamidation site(s) that cannot be noticed by the IEF separation (i.e., the presence of completed deamidation site(s)). Such rapid deamidation is out of the scope of the present method. When analyzed by the CIEF, the major peak of each puriﬁed labeled mutant accounted for more than 98% of the detected peaks nearby the major peak, although this purity is not relevant to the determination of the single-residue deamidation rate of a protein (i.e., the rate for the ﬁrst deamidation of a protein).1 With the ﬂuorescence-labeled peptide pI markers as standards, the pI values of the mutants were determined.12 All the mutants focused at the same pI of the original rFab of 5.60, being consistent with the substitution of a nonionic amino acid residue, Asn or Gln, with a nonionic amino acid residue, Ser. Determination of Deamidation Rates. Incubation of the original Fab, 12C-L3, in 0.1 M sodium phosphate buﬀer (pH 7.5) at 37 °C for 15 or 30 h caused the formation of additional two peaks, at pH 5.42 and 5.26, that were attributable to deamidation (Figure 1). Each mutant presented a similar change of the CIEF pattern during incubation but at a diﬀerent rate (Figure 2). Because of the error in the determination of the concentration of the labeled Fabs, there is some variations in the total peak area of each chart, in comparison with those of the marker peaks. The deamidation was evaluated by the decrease of the relative peak area of the original peak; thus, the variation of the total peak area of the labeled Fabs does not aﬀect the following estimation of deamidation rates. The variation could also be attributable to the diﬀerence in the recovery of the Fabs in the CIEF separation. Even under such conditions, if we can assume the same recovery for the deamidated rFabs as that for the nondeamidated rFab, the evaluation of the deamidation should be successful. Although a specially made CIEF instrument with a scanning laser-induced ﬂuorescence detector was used in this report, when protein samples can be prepared at a higher concentration, commercially available capillary electrophoresis instruments with UV detection also should be able to perform the analysis.13 The single-residue deamidation rate (ks) for each Fab molecule was determined from the decrease of the relative area (Ar) of the original peak according to the ﬁrst-order
Figure 1. Changes of IEF patterns during incubation at pH 7.5 at 37 °C. A ﬂuorescence-labeled recombinant Fab with the original sequence was incubated at 37 °C in 0.1 M sodium phosphate buﬀer (pH 7.5) for 15 and 30 h. The samples were analyzed by CIEF using Pharmalyte 3− 10 in a polydimethylacrylamide-coated fused silica capillary, 50 μm i.d. and 180 mm long, with scanning laser-induced ﬂuorescence detection over a distance of 135 mm. Peak numbering: 0, the original peak; 1, singly deamidated product; and 2, doubly deamidated product.
kinetics, i.e., −dAr/dt = ksAr (see Table 2 and Figure 3). The rate constant (ks) consists of the sum of the rate constants of deamidation of all amides in a particular Fab molecule as ks = k1 + k2 + k3 + ..., where k1, k2, k3, ... are the deamidation rate of individual amides. When the amino acid residue bearing amide 1 is mutated to a serine residue, the single-residue deamidation rate constant of the mutant, ks1, should be represented as ks1 = k2 + k3 + .... Thus, the rate constant of amide 1 can be estimated from the decrease of the single-residue deamidation rate constant of the mutant Fab, in which amide 1 has been removed, i.e., k1 = ks − ks1 (see Table 3). The most labile amide of the six was that of Asn135 of the γ heavy chain, accounting for 66% of the single-residue deamidation of the original rFab. The next labile one was that of Asn157 of the κ light chain, accounting for 29% of the single-residue deamidation of the original Fab (Table 2). These results are in good agreement with the previous reports about the major deamidation sites of IgG1-κ by using peptide mapping and mass spectrometry (i.e., γAsn135 5 and κAsn1575,6). Deamidation at κAsn161 was also detected with a rate of ∼1/10 of γAsn135 by the present method. The residue, κAsn161, resides in the same tryptic peptide carrying κAsn157, which has been known to be deamidated. Quite unexpectedly, an example of the incompetence of peptide mapping with MS analysis in the identiﬁcation of an additional deamidation site in a single peptide was demonstrated by the above results. In contrast to the three, deamidation was not detected at γAsn162 and γGln179 with the present method. The deamidation of κAsn190 seemed to occur, but the observed change fell in the range of the experimental error. The data of the deamidation rates of six model peptides, which are relevant to the ﬂanking sequence of the amidecontaining residues of the original rFab, were also collected in Table 3 from the Robinson and Robinson book,14 determined at 37 °C in 0.15 M Tris-HCl buﬀer (pH 7.4), which is known to be several times more suppressive for deamidation than the phosphate buﬀer used in this report.15 The deamidation rate of 1707
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Figure 2. (A) Changes of CIEF patterns of the labeled recombinant Fabs (original rFab, γN162S, γQ179S, and κN157S) during incubation at pH 7.5 at 37 °C. Experimental conditions were the same as those described in Figure 1. The small peaks at pI 6.18, 5.53, and 4.99 are those of ﬂuorescence pI markers at a concentration of 10−11 M each. Calculations of the peak area were carried out on the electropherograms without pI markers. Peak numbering: “0”, the original peak; “1”, single deamidation product; and “2”, double deamidation product. The scale of the ordinate was slightly changed from chart to chart to ﬁt the peaks. (B) Changes of CIEF patterns of the labeled recombinant Fabs (κN161S, κN190S, γN135S, and the double mutant (γN135S, κN157S)) during incubation at pH 7.5 at 37 °C. 1708
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Table 2. Rate Constants for Single-Residue Deamidation of Whole Fab Molecules ksa (× 1000 h−1) mutant
original Fab γN135S γN162S γQ179S κN157S κN161S κN190S γN135S, κN157S
15.7 4.5 c 15.3 10.7 13.6 14.3
16.1 5.0 c 15.1 12.1 14.8 15.3
15.8 5.4 15.8 17.3 11.9 15.8 16.5 1.0
16.6 6.9 17.0 17.3 11.4 15.6 16.9 1.6
15.9 5.5 16.0 16.0 11.4 14.9 15.6 1.3
2 16 4 7 5 5 6 23
100 34 101 100 71 93 98 8
1.8 5.3 1.8 1.8 2.5 1.9 1.9 22
15.3 14.9 10.7 14.5 14.8
a The ﬁrst-order rate constants for the decrease of the original peak, i.e., a single-residue deamidation rate of a whole rFab molecule. For mutants, it represents ksi to be strict. bCoeﬃcient of variation or relative standard variation. cDetermined ks values with r2 values of