Journal ofhnmunokJgical Methods, 153 (1992) 223-227
223
© 1992 ElsevierSciencePublishers B.V. All rights reserved 111122.1759/92/$05.00
JIM06406
13C-NMR spectral analysis of the structures of mouse immunoglobulin G1 carrying allotypes a and j H a H y u n g Kim a, Koichi Kato ", A t s u k o Higuchi b, Noriko N o m u r a h, Hiroshi Noguchi b and Yoji A r a t a ~ '* Factdty of Pharmaceutical Seiences, Unit'ersity of Tokyo, Hongo, Tokyo 113. Japan, and b Biotechnology Laboratory, Takarazuka Research Center, Sumitomo Chemical Co., Ltd., Takarazuka, Hyogo 665. Japan
(Received27 February 1992.accepted 15 April 1992)
A novel ~3C nuclear magnetic resonance (NMR) method is described for the detection of subtle structural differences between mouse immunoglobulins carrying different allotypes. Fc fragments of mouse lgG1 antibodies carrying allotypes a and j have been selectively labeled with [I-13C]methionine. t3C-NMR spectra have shown that the microenvironment around Met-398 is significantly different for the two kinds of allotypes. Peptide mapping and amino acid sequence analyses have revealed that Val-406 of IgG1 carrying allotype a is substituted for lie in the case of allotype j. X-ray crystallographic data indicate that Met-398 is in close spatial proximity to Val (!1e)-406. We therefore conclude tha +. the t3C-NMR method can provide us with a novel spectroscopic probe for the structural characterization of allotypic markers. Key words: 13C-NMR;AIIotype;Immunoglobulin
Introduction
Allotypes of mouse immunoglobulins have been studied extensively (for reviews, see Lieber-. man, 1978; Parsons et al., 1986). Mouse allotypes have traditionally been characterized by physical or serological properties, especially by using anti-ailotype antibodies obtained from other
Correspondence to: Yoji Arata, Faculty of Pharmaceutical Sciences, Universityof Tokyo, Hongo,Tokyo 113,Japan. Tel.: 3-3815-65401 Fax: 3-3813-5099. " Abbreviations: NMR, nuclear magnetic resonance; TrisHCI, tris-(hydroxymethyl)aminomethanehydrochloride; DTT, dithiothreitol;EDTA, ethylenediaminetetraaceticacid; HPLC, high-performance liquid chromatography; TFA, trifluoroacetic acid.
mouse strains. In the case of mouse IgG2a, lgG2b, lgM and IgE, complete nucleotide sequences of the heavy chain constant regions for both alleles a and b were determined and gene conversion and polymorphism have been discussed on the basis of the comparative data of nucleotide sequences (Schreier et al., 1981, 1986; O11o and Rouge~a, 1982, 1983; Shinkai et al., 1988). However, individual allotypic determinants have not always been successfully correlated to specific amino acid substitutions. Thus, the tertiary and quaternary structure of the immunoglobulin molecules was suggested to be important for presenting allotypic determinants (Parsons et al., 1983). In a previous work, we have shown that the t H nuclear magnetic resonance (NMR) peak originating from His-435 can be a useful probe for the
224 identification of human lgG3 carrying G3m(st) allotypes (Shimizu et al., 1983). We have recently established a laC-NMR method for the molecular structural analysis of immunoglobulins (Kato et al., 1989, 1991a,b). In this method the earbonyl carbons of the main chain are specifically labeled using a [1-13C]amino acid. In the present communication, we report a novel ~3C-NMR approach to the detection of subtle structural differences at atomic resolution between mouse monoclonal antibodies that are defined to different allotypes.
Materials and methods
We compared two kinds of mouse monoclonal IgGl antibodies, which are produced by Ex-3C7 and 27-44 hybridoma cells. The Ex-3C7 cells from B A L B / c mice (haplotype a) produce an antiPseudomonas aen,ginosa exotoxin A IgGI(K) (Ohtsuka et al., 1992). The 27-44 cells from C 3 H / S W mice (haplotype j) produce an antidansyl IgGI(K) (Reidler et al., 1982; Oi et al., 1984). Hereafter, the Ex-3C7 and 27-44 antibodies will simply be referred to as I g G P and lgG1 i, respectively. Fc fragments of l g G P and IgG1 i antibodies were prepared as described previously (Kato et al., t991a). Hybridoma ceils were grown in a serum-free medium (Nissui NYSF-404) that contains an appropriate [1-~3C]amino acid. For example, IgG selectively labeled with ~3C at the methionyi carbonyl carbon was prepared by using the medium where unlabeled methionine had been replaced by an equal amount of L-[1-~3C]methionine (ISOTEC, Inc.; the isotope enrichment is higher than 99%). After cell growth, the supernatant was concentrated with an ultrafiltration system (Miilipore Minitan) and applied to a protein A affinity column (Bio-Rad). Typically 20 mg of purified lgG1 proteins were obtained per liter of the cell culture. The purified antibodies were digested by papain (Sigma) at pH 7.0, 37°C, in 75 mM sodit:m phosphate buffer which contains 75 mM NaCI, 2 mM ethylenediaminetetraacetic acid (EDTA), and 5 mM NaN 3. The protein concentration and the enzyme:substrate ratio (w/w) were 5 m g / m l and 1:50, respectively. After incubation for 2 h in the presence of
2 m g / m l of L-Cysteine. HCi. H20, the reaction was terminated by addition of 30 mM of N-ethylmaleimide. The reaction mixture was loaded onto a Pharmacia Mono Q FPLC column equilibrated with 20 mM Tris-HCI, pH 8.0, and the Fe fragment was eluted by increasing NaCI concentration up to 400 mM. Purity of the Fc preparations were checked by sodium dodecyi sulfate-polyacrylamide gel electrophoresis. For NMR measurements, the Fc solutions were concentrated to a final volume of 2 ml in 5 mM phosphate buffer, pH 7.4, containing 200 mM NaCI and 3 mM NaN 3 in D20. The final concentration of the Fc was 0.2 mM. NMR measurements were made on a Bruker AM 400 spectrometer. laC-NMR spectra were recorded at 100 MHz by using a Waltz 16 composite pulse decoupling sequence. The free induction decay was recorded with 32 K data points and a spectral width of 24,000 Hz. For resolution enhancement, the free induction decay was multiplied by a Gaussian window function prior to Fourier transformation. Chemical shifts are given in ppm from internal dioxane. The probe temperature was 30°C.
Results and discussion
Fig. 1 compares the ~3C-NMR spectra of the Fc fragments of I g G P and IgG1 j, both of which were selectively labeled with [1-~3C]methionine. The Fc fragment labeled with [1-13C]methionine will hereafter be designated as [M]Fc. Each of these lgG1 antibodies possesses four Met residues in the Fc portion at positions 309, 358, 368 and 398 *. The results of spectral assignments for [M]Fc of IgGP have already been reported in a previous paper (Kato et al., 1991a). As Fig. 1 shows, the resonances originating from Met-398 are significantly different in chemical shift for I g G P and IgG1 j, indicating the microenvironment surrounding Met-398 is different for these two kinds of proteins. We therefore conclude that there is (are) amino acid substitution(s) in the C u 3 domain.
* The numbering system used in this paper is based on protein Eu for the 'yl chain (Edelman et al., 1969).
In order to locate the position(s) of the amino acid substitution(s), we made peptide maps of the two kinds of Fc fragments. The Fc fragment was reduced by 10 mM dithiothreitol ( D T r ) at room temperature for 1 h in 1.5 M Tris-HC1, pH 8.5, containing 2 mM E D T A and then alkylated by adding 22 mM iodoaeetie acid. The reaction mixture was incubated at room temperature for 20 min in the dark. The reaction was terminated by adding 10 mM DTT and dialyzed against 10 mM phosphate buffer, pH 7.2, containing 150 mM NaCI. The reduced and alkylated Fc fragment was digested by lysyl endopeptidase (Wako) in 50 mM Tris-HCI buffer, pH 8.5, containing 4 M urea. The enzyme:substrate molar ratio was 1 : 100 and the incubation.time was 4 h at 37°C. The reaction mixture was loaded onto a YMC ODS A-312 reverse-phase column (Yamamura Chemical Laboratories) connected into a Shimadzu LC-6A pump system equilibrated with an aqueous solution containing 5% acetonitrile and
1I
0z
0~ 0.
A A' 'L
!
i 'l !
;
~ "/
~ 0,51 ~l°A 0,5 o~ o
!Bi o
1o
~
, ~i
ao 4o r n ( n~nl
5o
6o
Fig. 2. Elution profiles of the lysyl endopeptidase digestion products of the Fc fragments of (upper trace) IgG,~" and (lower trace) lgGI i on a YMC ODS A-312 revere-phase HPLC column. Eluted at a flow rate of 1.0 ml/min with a linear gradient of 5-75% acetonitrile in 0.1% TFA for 811rain
at 40°C. Absorbance detected at 230 nm. Peaks A, A'. B and B' were fractionated and subjected to the amino acid sequence analyses.
A
, ::,', .,..
, ....
.
ii~i:~,~
223
© 1992 ElsevierSciencePublishers B.V. All rights reserved 111122.1759/92/$05.00
JIM06406
13C-NMR spectral analysis of the structures of mouse immunoglobulin G1 carrying allotypes a and j H a H y u n g Kim a, Koichi Kato ", A t s u k o Higuchi b, Noriko N o m u r a h, Hiroshi Noguchi b and Yoji A r a t a ~ '* Factdty of Pharmaceutical Seiences, Unit'ersity of Tokyo, Hongo, Tokyo 113. Japan, and b Biotechnology Laboratory, Takarazuka Research Center, Sumitomo Chemical Co., Ltd., Takarazuka, Hyogo 665. Japan
(Received27 February 1992.accepted 15 April 1992)
A novel ~3C nuclear magnetic resonance (NMR) method is described for the detection of subtle structural differences between mouse immunoglobulins carrying different allotypes. Fc fragments of mouse lgG1 antibodies carrying allotypes a and j have been selectively labeled with [I-13C]methionine. t3C-NMR spectra have shown that the microenvironment around Met-398 is significantly different for the two kinds of allotypes. Peptide mapping and amino acid sequence analyses have revealed that Val-406 of IgG1 carrying allotype a is substituted for lie in the case of allotype j. X-ray crystallographic data indicate that Met-398 is in close spatial proximity to Val (!1e)-406. We therefore conclude tha +. the t3C-NMR method can provide us with a novel spectroscopic probe for the structural characterization of allotypic markers. Key words: 13C-NMR;AIIotype;Immunoglobulin
Introduction
Allotypes of mouse immunoglobulins have been studied extensively (for reviews, see Lieber-. man, 1978; Parsons et al., 1986). Mouse allotypes have traditionally been characterized by physical or serological properties, especially by using anti-ailotype antibodies obtained from other
Correspondence to: Yoji Arata, Faculty of Pharmaceutical Sciences, Universityof Tokyo, Hongo,Tokyo 113,Japan. Tel.: 3-3815-65401 Fax: 3-3813-5099. " Abbreviations: NMR, nuclear magnetic resonance; TrisHCI, tris-(hydroxymethyl)aminomethanehydrochloride; DTT, dithiothreitol;EDTA, ethylenediaminetetraaceticacid; HPLC, high-performance liquid chromatography; TFA, trifluoroacetic acid.
mouse strains. In the case of mouse IgG2a, lgG2b, lgM and IgE, complete nucleotide sequences of the heavy chain constant regions for both alleles a and b were determined and gene conversion and polymorphism have been discussed on the basis of the comparative data of nucleotide sequences (Schreier et al., 1981, 1986; O11o and Rouge~a, 1982, 1983; Shinkai et al., 1988). However, individual allotypic determinants have not always been successfully correlated to specific amino acid substitutions. Thus, the tertiary and quaternary structure of the immunoglobulin molecules was suggested to be important for presenting allotypic determinants (Parsons et al., 1983). In a previous work, we have shown that the t H nuclear magnetic resonance (NMR) peak originating from His-435 can be a useful probe for the
224 identification of human lgG3 carrying G3m(st) allotypes (Shimizu et al., 1983). We have recently established a laC-NMR method for the molecular structural analysis of immunoglobulins (Kato et al., 1989, 1991a,b). In this method the earbonyl carbons of the main chain are specifically labeled using a [1-13C]amino acid. In the present communication, we report a novel ~3C-NMR approach to the detection of subtle structural differences at atomic resolution between mouse monoclonal antibodies that are defined to different allotypes.
Materials and methods
We compared two kinds of mouse monoclonal IgGl antibodies, which are produced by Ex-3C7 and 27-44 hybridoma cells. The Ex-3C7 cells from B A L B / c mice (haplotype a) produce an antiPseudomonas aen,ginosa exotoxin A IgGI(K) (Ohtsuka et al., 1992). The 27-44 cells from C 3 H / S W mice (haplotype j) produce an antidansyl IgGI(K) (Reidler et al., 1982; Oi et al., 1984). Hereafter, the Ex-3C7 and 27-44 antibodies will simply be referred to as I g G P and lgG1 i, respectively. Fc fragments of l g G P and IgG1 i antibodies were prepared as described previously (Kato et al., t991a). Hybridoma ceils were grown in a serum-free medium (Nissui NYSF-404) that contains an appropriate [1-~3C]amino acid. For example, IgG selectively labeled with ~3C at the methionyi carbonyl carbon was prepared by using the medium where unlabeled methionine had been replaced by an equal amount of L-[1-~3C]methionine (ISOTEC, Inc.; the isotope enrichment is higher than 99%). After cell growth, the supernatant was concentrated with an ultrafiltration system (Miilipore Minitan) and applied to a protein A affinity column (Bio-Rad). Typically 20 mg of purified lgG1 proteins were obtained per liter of the cell culture. The purified antibodies were digested by papain (Sigma) at pH 7.0, 37°C, in 75 mM sodit:m phosphate buffer which contains 75 mM NaCI, 2 mM ethylenediaminetetraacetic acid (EDTA), and 5 mM NaN 3. The protein concentration and the enzyme:substrate ratio (w/w) were 5 m g / m l and 1:50, respectively. After incubation for 2 h in the presence of
2 m g / m l of L-Cysteine. HCi. H20, the reaction was terminated by addition of 30 mM of N-ethylmaleimide. The reaction mixture was loaded onto a Pharmacia Mono Q FPLC column equilibrated with 20 mM Tris-HCI, pH 8.0, and the Fe fragment was eluted by increasing NaCI concentration up to 400 mM. Purity of the Fc preparations were checked by sodium dodecyi sulfate-polyacrylamide gel electrophoresis. For NMR measurements, the Fc solutions were concentrated to a final volume of 2 ml in 5 mM phosphate buffer, pH 7.4, containing 200 mM NaCI and 3 mM NaN 3 in D20. The final concentration of the Fc was 0.2 mM. NMR measurements were made on a Bruker AM 400 spectrometer. laC-NMR spectra were recorded at 100 MHz by using a Waltz 16 composite pulse decoupling sequence. The free induction decay was recorded with 32 K data points and a spectral width of 24,000 Hz. For resolution enhancement, the free induction decay was multiplied by a Gaussian window function prior to Fourier transformation. Chemical shifts are given in ppm from internal dioxane. The probe temperature was 30°C.
Results and discussion
Fig. 1 compares the ~3C-NMR spectra of the Fc fragments of I g G P and IgG1 j, both of which were selectively labeled with [1-~3C]methionine. The Fc fragment labeled with [1-13C]methionine will hereafter be designated as [M]Fc. Each of these lgG1 antibodies possesses four Met residues in the Fc portion at positions 309, 358, 368 and 398 *. The results of spectral assignments for [M]Fc of IgGP have already been reported in a previous paper (Kato et al., 1991a). As Fig. 1 shows, the resonances originating from Met-398 are significantly different in chemical shift for I g G P and IgG1 j, indicating the microenvironment surrounding Met-398 is different for these two kinds of proteins. We therefore conclude that there is (are) amino acid substitution(s) in the C u 3 domain.
* The numbering system used in this paper is based on protein Eu for the 'yl chain (Edelman et al., 1969).
In order to locate the position(s) of the amino acid substitution(s), we made peptide maps of the two kinds of Fc fragments. The Fc fragment was reduced by 10 mM dithiothreitol ( D T r ) at room temperature for 1 h in 1.5 M Tris-HC1, pH 8.5, containing 2 mM E D T A and then alkylated by adding 22 mM iodoaeetie acid. The reaction mixture was incubated at room temperature for 20 min in the dark. The reaction was terminated by adding 10 mM DTT and dialyzed against 10 mM phosphate buffer, pH 7.2, containing 150 mM NaCI. The reduced and alkylated Fc fragment was digested by lysyl endopeptidase (Wako) in 50 mM Tris-HCI buffer, pH 8.5, containing 4 M urea. The enzyme:substrate molar ratio was 1 : 100 and the incubation.time was 4 h at 37°C. The reaction mixture was loaded onto a YMC ODS A-312 reverse-phase column (Yamamura Chemical Laboratories) connected into a Shimadzu LC-6A pump system equilibrated with an aqueous solution containing 5% acetonitrile and
1I
0z
0~ 0.
A A' 'L
!
i 'l !
;
~ "/
~ 0,51 ~l°A 0,5 o~ o
!Bi o
1o
~
, ~i
ao 4o r n ( n~nl
5o
6o
Fig. 2. Elution profiles of the lysyl endopeptidase digestion products of the Fc fragments of (upper trace) IgG,~" and (lower trace) lgGI i on a YMC ODS A-312 revere-phase HPLC column. Eluted at a flow rate of 1.0 ml/min with a linear gradient of 5-75% acetonitrile in 0.1% TFA for 811rain
at 40°C. Absorbance detected at 230 nm. Peaks A, A'. B and B' were fractionated and subjected to the amino acid sequence analyses.
A
, ::,', .,..
, ....
.
ii~i:~,~
