BIOCHEMISTRY
Structural Studies of the Immunoglobulins. 11. Antigenic and Chemical Properties of yA Myeloma Globulins” George M. Bernier,t Kikuo Tominaga,: Caroline W. Easley, and Frank W. Putnams
The polypeptide chain structure of yA myeloma globulins of antigenic types I and I1 has been studied by physical and chemical techniques. After reductive dissociation and alkylation, two kinds of chains were separated by gel filtration, heavy and light. The light chains differ structurally for the two antigenic types but are virtually identical with the Bence-Jones protein of the same patient, both in amino ABSTRACT:
0
f the three classes of human serum immunoglobulins, least is known about the structure of the yA-globulins, partly because of the difficulty of purifying these proteins from normal human serum. yA myeloma globulins, on the other hand, can be obtained in relatively pure form and may serve as useful models for structural studies of normal yA-globulin (Ballieux, 1963),just as the yG myeloma globulins have facilitated structural analysis of normal yG-globulin (Putnam, 1962). In the latter case the yG-globulin molecule has been shown to contain two kinds of polypeptide chains: y chains which are the heavy chains characteristic of the yG-globulin class and light chains which are common moieties throughout the immunoglobulin family (Edelman and Poulik, 1961; Fleischman et ul., 1963; Fahey, 1963). Likewise, in the case of yG-globulin, limited enzymatic hydrolysis with papain has produced biologically active fragments (Fc and Fab fragments) which retain many of the properties of the yG-globulin molecule (Porter, 1959; Hsiao and Putnam, 1961; Franklin, 1960). In the case of the yA-globulins, however, papain cleavage results in poorly defined products that do not correspond to the Fc and Fab fragments of yG-globulin (Heremans, 1960; Deutsch, 1963). Reductive dissocia-
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* From the Department of Biochemistry, College of Medicine, University of Florida, Gainesville. Received Ma.v IO, 1965. Supported by a grant (CA-02803) from the National Cancer Institute, National Institutes of Health, Bethesda, Md. For the first paper in this series see Putnam and Easley (1965). t Present address: University Hospital, Cleveland, Ohio. Present address: Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan. $ Present address: Division of Biological Sciences, Indiana University, Bloomington, Indiana. The nomenclature used was proposed at the WHO Meeting on Nomenclature of Human Immunoglobulins o n May 29-30, 1964, at Prague. Synonyms may be found in the draft memorandum(Bull. Wld. Hlrh. 0%.(1964), 30, 447).
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E A Y L E Y , A N D F.
acid composition and in tryptic peptide maps. Many of the peptides of the light chains of antigenic type I are identifiable with peptides of known sequence that occur in Bence-Jones proteins of the same antigenic type and in normal yG-globulins. However, part of the light chain appears to be unique for each specimen. The heavy chains have antigenic determinants and peptide maps characteristic for yA-globulins, but the number and structure of these chains is not well defined.
tion of yA myeloma globulins has yielded fractions separable by gel filtration that are analogous to the heavy and light chains of $3-globulin (Cohen, 1963; Carbonara and Heremans, 1963; Fahey, 1963). The present study was designed to define the polypeptide chain structure of yA myeloma globulins by partial enzymatic cleavage, by reductive dissociation of polypeptide chains, and by comparison of the yA myeloma globulin and Bence-Jones protein of individual patients. In this study, the two antigenic types of yA myeloma globulins (type I and type 11) are shown to possess a common structural moiety that is representative of the a chain and differs from the y chain. The yA myeloma globulins also possess light chains which define the antigenic type (I or 11) and which are virtually identical with the Bence-Jones protein excreted by the patient.
Experimental Section Proteins. yA myeloma globulins were prepared by ammonium sulfate precipitation, column or batch chromatography using DEAE-cellulose or carboxymethyl-cellulose, and in the case of l e 1 4 S yA-globulins gel filtration on Sephadex G-200. Bence-Jones proteins were prepared by ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration (Bernier and Putnam, 1964). The proteins are designated by abbreviations referring to the patient’s name (e.g., Ha, Ln, and Mo). Antisera. The following rabbit antisera were employed in this study: antiserum to Ha yA myeloma globulin, antiserum to Ln yA myeloma globulin, antisera to Bence-Jones proteins of type I (Ag) and type II ( B ~ ) antiserum , to the Franklin protein ( c r ) which Possesses antigenic determinants of yG-globulin heavy chain only (Franklin et al., 1964), and antiserum
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to reduced and alkylated heavy chains of y A myeloma globulin Ln and y A myeloma globulin Ha. Anu1)'iicuI Techniqurs. Two-dimensional mapping o f tryptic peptides was performed a t p H 3.7 its described by Putnam and Easley (1965). The maps were stained with ninhydrin. Pauly, Sakaguchi, tyrosine. and chloroplatinic acid reagents both singly and in comhinalion, as described by Easley (1965). Proteins wcre prepared for tryptic digestion by performic acid oxidation or reduction-alkylation. Amino acid analysis was performed in duplicate on the Beckman Model 1211 automatic amino acid analyzer. Ultracentrifugation was done in the Spinco Model E at 59,780 rpm, and Tiselius electrophoresis was performed in a Spinco Model H apparatus. Thermosolubilities were tested in capillary tubes by a previously described method (Bernier and Putnam, 1964). Antigenic analysis was performed by double dilrusion in agar and by immuiioelectrophoresis (Migita and Putnam. 1963). Starch ureii gel electrophoresis was performed in the discontinuous lormate bulrer system hased on the method of Srnithic\
(IY62). R e ~ u r f i o n ~ A l ~ ? l u r i oProteins n. were reduced and
'IAHLI: I: Physical Properties and Antigenic Clilssitication of y A Myeloma Globulins ( y A ) and Bence-Joncs Proteins (BJ).
Antigenic Symbol Class Type
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Mo Ha Ln Mo
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/ I ( , ( iti ~. i I I I . I ~ ~ ~ I I ~ ~ u ( . :. ~' i i l ~ i ' ~r ci ii i i i i ' . j i i i i c , \ I ,c originiil 7 4 iii)eliiiiw g l d w l i n (Ha) WIII lI%i !lie prcjiaration iiftcr rcduc!ion and ;ilkyl;ition in detergent (SDS),; ~ n d( C ) aiter a secund reduction and alkylation in detergent. Note that tlie twice-reduced material shows onI> 2 S and 2.1 S components. The \,erticiil &sIi line indicates the ;ipproximatc sedimentation cuetiicient i t t the time indicated. ~
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* Expressed in Svedberg units. '' Exprehsed in units o f IO-: cm'sec~ v at 0" in pH 8.6 Verona1 bulrer. 0.1 ionic strength.
/'u,xun ( l w r o ~ < ,Limited . proteolytic cluiivage of t w o y A niyeloma globulins (Hi1 and L n ) was pcrforniod with crystiilline papain (Hsiao and I'utnani. I Y h l ) . The digesi wah fractionated hy gcl filtration w i t h Scphiidex G-200 with tlie aid of the Beckiiian Model 130 Spectrochrom, 'The eluted iractians were iissayed for Ihcxosc content hy thc mcthod o f Duhois ul. (lY5hl and fur I-cactivity with ninhydrin (Moore and Stcin.
lY541. alkylated by the method of Fleischman ''I ul. (lYh3) and by a modification of the method of Jaquct ei id (lY64). I n the latter method two sequential reduction alkylations were performed. Gel tiltration was performed on 250 mg o f reduced-alkylated protein by passage through G-200 Sephadex, 0.5 Lt sodium phosphate, pH 6.8, 100 X 3.8 cm. The Sephadex was not equilibrated with detergent. The only detergent present was that which could n o t he removed by dialysib of tlie reduced-alkylated protein. Fractions obtained by gel tiltration were pressure dialyzed. and the detergent wiis removed by hatch chromatogntphy using Dowex I (acetate).
Kesult, Thc three yA myelom;i globulins bludicd (Ln. Mo, and Ha) manifestcd polymer-type heterogeneity i n die ultr;icentriluge and in starch-gel electrophoresis, illthough by Tiselius clcctl-ophoresis they appeared nithcr homogeneous. Some 0 1 thc physical properties of these plottins itre summarired i n Table 1. Two prepiiration~ (Ln and Mo) contained large iimounts o f 10 S y A ; these componcnts w'erc isoltiied hy gel liltratiun using Sephadex G-200 cqnilihrated i n I hi NaCI, 0.1 11 Tris HCI. pH Y.1) hull'err. In I h c x two preparations. tlic y A ~nye~onui globulin wiis isolalccl virtually free O S c o w
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FIGURE 2: Gel-filtration elution pattern of a yA myeloma globulin of antigenic type I (Ln), reduced and alkylated by the method of Fleischman et a / . (1963). The first peak contained only y A specific antigenic determinants; the second peak contained only type I determinants.
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I V M I N A G Ar. , w. E A S L E Y ,
A N D
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FIGURE 3 : Starch urea gel electrophoretic pattern comparing proteins and separated chains from patient Ha. (1) Untreated yA myeloma globulin; (2) yA myeloma reduced and alkylated twice in detergent; (3) heavy chain; (4) intermediate fraction containing heavy and light chains; ( 5 ) light cliain; ( 6 ) Bence-Jones protein, reduced and alkylated twice in detergent; and (7) untreated Bence-Jones protein. H and L represent the position of the heavy and light chains, respectively.
lamination with yG-globulin. The Ha yA myeloma globulin contained trace amounts of yG-globulin, detectable by immunoelectrophoresis. The three purified yA myeloma globulin preparations were studied by the methods of reductive dissociation, papain cleavage, and comparative peptide mapping. Reducrive Dissociuriiin ofPdypeptide Clmins. Figure 1 shows the change in ultracentrifugal pattern on progressive reduction by the detergent method of yA myeloma globulin Ha. Although fair separation of heavy and light chains of detergent-treated proteins was achieved on Sephadex G-200, the separation of these chains was generally more complete using the method of Fleischman et rrl. (1963) which involves reductive dissociation in aqueous buffers and separation of the heavy and light chains on Sephadex G-I00 equilibrated in acetic acid (Figure 2). By various physical and chemical criteria the reducedalkylated light chains of the yA myeloma globulin appeared similar to or identical with the Bence-Jones protein from the same patient. The reduced-alkylated light chain of the yA myeloma globulin H a (prepared by the detergent method) was similar to the Bence-Jones protein Ha in electrophoretic mobility in starch urea gel, while the heavy chain had a markedly slower mobility (Figure 3). In thermal coagulability the Ha light chain and the Bence-Jones protein were virtually identical; both proteins were precipitated by heating a t 54" and underwent thermal dissolution a t 1 I O " . Antigenic Identity ofthe Light Chuins und Bence-Jones Proteins. Immunodiffusion analysis confirmed the identity of the light chains of a y A myeloma globulin and the Bence-Jones protein of the same patient, which was suggested by their similarity in chemical and physical properties. In both c a m tested (Ha and Ln), the light chain and the Bence-Jones protein were antigenically identical when treated with antiserum to
BEKNIEK,
2
the original yA myeloma globulin (Figures 4A and 4B). In Figure 4A the intact yA myeloma globulin spurs with the precipitin lines of both proteins, reflecting additional antigenic determinants due to the heavy chain. In Figure 4 8 the nonidentity of the heavy and light chains of the yA myeloma globulin is illustrated. In this case, the original yA myeloma globulin spurs with the heavy chain reflecting the presence of light chain determinants in the intact globulin. Pupuin Digesli
