Evidence that intracellular and secreted light chains of a mouse IgG2a plasmacytoma are monomers carrying a free thiol group

lmmunochemistry, 1977, Vol. 14, pp. 62%631. Pergamon Press. Printed in Great Britain

EVIDENCE THAT INTRACELLULAR A N D SECRETED LIGHT CHAINS OF A MOUSE IgG2,~ PLASMACYTOMA ARE M O N O M E R S CARRYING A FREE THIOL G R O U P ANNE COOKE* and ARNOLD FEINSTEIN School of Biological Sciences, University of Sussex, Brighton, Sussex; A.R.C. Institute of Animal Physiology, Babraham, Cambridge, Cambs, U.K. (First received 3 November 1976; in revised form 7 March 1977)

Abstract--We have studied the intracellular light chains of PI, a mouse plasmacytoma derived from ADJ PC5, which secreted IgG2, and excess light chains, and its mutant X2P1 which secreted only light chains. In both cases the intracellular light chains were not covalently dimerised, carrying, as did the freshly secreted light chains, one free thiol group per polypeptide chain. The presence of a free thiol group intracellularly indicates that temporary blocking cannot play a significant role in the control of assembly of the L chain into IgG2~. The light chains excreted in the urine of mice carrying these plasmacytomas have blocked thiol groups, and these results show that such blocking must occur extracellularly after secretion.

INTRODUCTION In various studies of murine plasmacytomas an intracellular pool of monomer light chains has been detected (Baumal et al., 1971; Baumal & Scharff, 1973; Laskov et al., 1971; Schubert & Cohn, 1968; Namba & Hanaoka, 1969; Askonas & Williamson, 1966). The tumour P1, derived from M. Potters' original ADJ PC-5, synthesises ~2a heavy chain and x light chains (Schubert et al., 1968), and secretes immunoglubulin (H2L2) and, unlike the parent ADJ PC-5 some excess light chain. When the cells were lysed in a dissociating medium light chain monomers were found (Baumal et al., 1971 ; Schubert & Cohn, 1968), and the data can be interpreted as showing that these L chains were dissociated from non-covalently assembled complexes with heavy chains, with little or no free L chain pool. This would be consistent with the analysis of intermediates presented by Baumal et al. (Baumal et al., 1971; Baumal & Scharff, 1973). The evidence suggests that little or no L chain dimer is present (Baumal et al., 1971; Schubert & Cohn, 1968). In a mutant, XP1 (Schubert & Cohn, 1968; Schubert et al., 1968) which had lost the ability to secrete IgG but continued to secrete L chain, none of the intracellular L chain pool was found to be associated non-covalently with the H chain also present. However, here it is difficult to determine from the polyacrylamide-gel patterns to what extent L2 dimers may be present in the large H chain pool (Schubert & Cohn, 1968). When L chain monomers are found as Bence Jones protein in urine, they are stabilized by having their

C-terminal SH group bridged to a half cystine 0Viiistein, 1966). We considered the possibility that the intracellular L chain monomers were blocked in some such manner, and have examined the P1 tumour and a mutant, X2P1, which resembles XP1 in secreting light chains but no heavy chains. Stott and Feinstein (1973) have demonstrated that MOPC 104E, a plasma cell tumour which secretes IgM and free light chain, contains unblocked light chain monomers. We wished to look for free monomer L chains in P1 and X2P1, and to see whether these L chains carry free or blocked sulphydryl groups. For the latter pur4 pose, carboxymethylation was employed, since suet] modification of the SH group alters the electro~ phoretic mobility of an L chain band by changing the net charge (Stott & Feinstein, 1973; Feinstein, 1968, 1969; Feinstein & Stott, 1972). MATERIALS AND METHODS Preparation of mouse and human Bence Jones proteins

These were isolated by ammonium sulphate precipitation from the urine of a human patient (Pet) or from the urine of X2P1 tumour bearing mice. All preparation s moved as essentially single components in the analytical centrifuge and in agar gel electrophoresis. Turnout lines The light chain-secreting tumour mutant X2P1 arose between passages of line P1, which in turn derived from tumour ADJ PC5 obtained from Dr. M. Potter.

Labelling of intracellular protein (a) For columnfractionation. Mice were killed by cervical dislocation and the tumour washed and rinsed in Hanks *Present address: Department of Immunology, The BSS.t The cells were released by gentle homogenisation, Middlesex Hospital Medical School, London WlP9PG, washed three times in Hanks BSS, and resuspended at a U.K. cell density of 2.5 x 107/ml in 10 ml of Eagles medium t Abbreviations used: BSS, balanced salt solution; PBS, lacking lencine but containing the normal amounts of the phosphate buffered saline; TKM, Tris/KC1/MgC12 other amino acids. The cells were pre-incubated at 37°C medium; SDS, sodium dodecyl sulphate. for 15 min and then for 1 hr in the presence of 14C leucine

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ANNE COOKE and ARNOLD FEINSTEIN

(10#Ci/ml 40--60mCi/m-mole). The incorporation of radioactivity was terminated by chilling on ice. (b} For isoelectrofocusing. This was performed as described previously (Stott & Feinstein, 1973). Both the intracellular and secreted immunoglobulin produced by the PI tumour were examined. The tumour, although not of the same generation as that used for the column fractionation, also secreted immunoglobulin and excess light chain.

Cell lysis (a) For column fractionation. The cell suspension was split into two equal fractions and the cells washed three times in Hank's BSS, and resuspended in I ml of 0.2 M Tris buffer, at pH 8.2 containing, 0.5% Triton X100, 1 mg human Bence Jones (Pet) protein as carrier, and in the one case 0.05 M iodoacetate and the other 0.05 M iodoacetamide. The lysates were left for 1 hr for alkylation to occur and then centrifuged at 12,500g for 15rain. The supernatants were then dialysed in two changes of PBS, pH 7.5. (b) For focusing #el analysis. This was performed as described previously (Stott & Feinstein, 1973). Ceils were divided into two parts and lysed under nitrogen with 10 strokes of a Dounce homogeniser having a clearance of 25 n m in an equal volume of TKM containing 1% Nonidet P40 and either 0.1 M iodoacetate or 0.1 M iodoacetamide. A 10-min spin at 10,000 g to remove the nuclei and cell debris was followed by a 45-min spin at 200,000 g (Spinco SW50L rotor) to remove the ribosomes. The extracts were dialysed against 0.01 M sodium phosphate buffer pH 7.4 containing L-leucine (1 mg/ml) prior to electrofocusing.

Isolation of intracellular light chains The intracellular light chains were purified by gel filtration on a 100ml Sephadex G100 column equilibrated in phosphate buffered saline, pH7.5. More protein Pet (2 mg/ml) was added as a carrier and to mark the point of elution of light chains, 0.05 ml samples (X2P1) or 0.1 ml samples (P1) of each fraction were dried on planchettes and counted on a Nuclear-Chicago gas-flow counter. The light chain-containing fractions were pooled according to O.D. at 280 nm, dialysed against H20, then split into two fractions, for chromatography or for electrophoretic analysis. Both fractions were then lyophilised.

Separation of monomer light chain from dimer The lyophilised light chain sample was dissolved in 1 ml PBS, pH 7.5, dialysed against 1 M propionic acid and chromatographed on a 200 ml Sephadex G100 column equilibrated with 1 M propionic acid. The protein Pet (15 rag) was added in 0.5 ml 1 M propionic acid prior to gel filtration. Under identical conditions the O.D. elution profile Pet showed 80% dimer light chain and 20% monomer. This interpretation of the profile was confirmed by the fact that after reduction and carboxymethylation of Pet, the dimer was completely converted to the monomer. The added Pet thus served to identify the positions of emergence of dimer and monomer mouse light chains. In the case of X2P1 0.3 mi samples were counted directly by liqui d scintillation, but in the case of P1, fractions were first precipitated with 10%TCA in the presene of 100 p.g/ml carrier BSA. The precipitates were collected on Millipore filters and counted by liquid scintillation.

Agar gel electrophoresis The lyophilised intracellular light chain preparations were dissolved in 1 ml 0.1 M barbitone buffer and 5 mg each/ml of iodoacetate and iodoacetamide carboxymethylated mouse Bence Jones protein added as carrier. Three microlitre samples were electrophoresed on agar plates in 0.025 M barbitone buffer, pH 8.2, at 25 V/cm and 4°C for 40 min according to the method of Wieme (1959). The protein was fixed after electrophoresis and the agar gel was dried to a thin film, and autroadiographed using Kodirex X-ray film.

Polyacrylamide gel electrophoresis Mouse Bence Jones protein samples before and after mild reduction and carboxymethylation were examined in 4.25% gets containing SDS (Summers et al., 1965). A predominantly dimerised and a predominantly monomeric human Bence Jones protein were used as markers.

Electrofocusing in gels Samples of protein and cell extracts in 0.01 M sodium phosphate buffer pH 7.4 were electrofocused in pH graclients in thin layers of polyacrylamide gel, prepared as described by Awdeh et al. (1968) and Salaman and Williamson (1971). The pH gradient was measured directly on the gel after warming to ambient temperature using a Pye Ingold fiat membrane electrode. The gels were fixed, stained, dried in an oven at 37°C and autoradiographed using Kodirex X-ray film. RESULTS Of the four separations by gel filtration of the alkylated intracellular light chains, Fig. 1 shows one from X2P1 a n d one from P1. 0'6

6O0

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2o0

ILl

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Fraction no.

06

800

0,6

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Preparation of carrier mouse light chains Ten milligrams of mouse Bence Jones X2PI light chains were dissolved in 0.6 ml Tris buffer, pH 8.2. 0.1 ml 1 M mercaptoethanol was added, and after reduction for 1 hr the sample was divided into two batches. Iodoacetate 0.1 ml 1 M was added to one and 0.1 ml 1 M iodoacetamide was added to the other, and after I hr of alkylation the samples were dialysed against 0.9% NaC1 to remove excess reagents.

ol

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Fraction no.

Fig. 1. Sephadex GI00 gel filtration in phosphate buffered saline of cell lysate supernatants. (a) X2P1 (amidomethylated); (b) P1 (carboxymethylated). (O ..... O) Absorbanee 280nm; (O 0) counts/min. L chain fractions between the arrows were pooled for analysis.

Intracellular and Secreted Mouse Plasmacytoma Light Chains

629

addition of carrier Pet light chain, by gel filtration in molar propionic acid, and the elution patterns corresponding to (a) and (b) are shown in Figs. 2a and ,soo b. These patterns were interpreted by correlation with 0.4t~ fractionation of protein Pet under similar conditions 1000 ~' separating covalently bridged dimers from monomer IJJ light chains and establishing their O.D. profile. 0.2 Figure 2a shows that 87% (by planimetry) of the radioactivity in the light chain pool from tumour X2P1 is eluted in the monomer position, and only J I I 89/0 in the dimer position. The latter may be due to 20 ~0 60 80 monomer H chains which were found to be present Fraction no. in a similar L chain secreting mutant, XP1 (Schubert & Cohn, 1968). In the corresponding pattern for PI, 0-6 600 Fig. 2b, again the major radioactive peak corresponds to monomer light chains. Most of the labelled monomer L chain in Figs. 2a and b cannot have been 0.4 4oo present, non-covalently associated with heavy chain as immunoglobulin complexes contaminating the UJ light chain pool, since insufficient radioactivity to rep0.: resent the required amount of heavy chain was eluted associated with material of greater size than light L chain monomer. I Agar gel electrophoresis demonstrated that the 0 20 40 60 80 bulk of the radioactivity in the light chain pool of Fraction no. XzP1 is incorporated into light chain rather than Fig. 2. GI00 gel filtration in 1M propionic acid of (a) X2P1 amidomethylated light chain pool; and (b) P1 carboxy- other intracellular protein. This is shown by examinmethylated light chain pool. (O O) Absorbance ation of the autoradiograph of the iodoacetamide 280nm; (0 0) counts/min. M and D mark positions blocked light chain pool of X2PI (Fig. 3b, sample III). Most of the radioactivity is associated with a band of elution of monomer and dimer. electrophoretically coincident with carrier urinary light chains (Fig. 3a, sample IV). Moreover, since the electrophoretic conditions were non-dissociating this In the non-dissociating solvent used, the carrier confirms that the L chain is not extensively non-covamonomer light chains are known to behave as dimers lently associated with heavy chain. formed from non-covalently paired monomers, and In the autoradiograph of the iodoacetate blocked move as a single peak with covalently paired light chain pool of X2PI (Fig. 3b, sample II), the negamonomers. Thus selection of those fractions contain- tive charge introduced by the carboxymethylating ing carrier light chain ensured the inclusion of any agent has resulted in a shift of almost all the labelled intracellular light chain dimers in the pools. These material in the light chain band. This change in mobilight chain pools were refractionated, after the further lity corresponds to that found in earlier experiments 2000

0.6

1

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500 i

Fig. 3(a). Electrophoresis of alkylated samples and a mixture of carrier alkylated Bence Jones proteins. The samples in the slots were: i, carboxymethylated mouse Bence Jones carrier; ii, mixture of the two alkylated carrier light chains and the carboxymethylated 1"C labelled X2PI intracellular light chains; iii, mixture of the two alkylated carrier light chains and the amidomethylated 14C labelled X2P1 intracellular light chains; iv, amidomethylated mouse Bence Jones carrier. (b) Charge shift of 14C labelled X2PI light chains. Autoradiograph of (a).

|MM 14/8---~

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ANNE COOKE and ARNOLD FEINSTEIN

pH

(o)

(b)

(c)

(d)

Fig. 4. Charge shift of 14C labelled P1 products. Autoradiograph of alkylated '4C labelled PI products after gel-focusing, showing region of gel where light chains focus. (a) and (b), lysate--(a) amidomethylated, (b) carboxymethylated. (c) and (d), secreted into medium-(c) amidomethylated, (d) carboxymethylated. when a single sulphydryl group per urinary light chain is carboxymethylated (Feinstein, 1966, 1968). Thus virtually every free intracellular light chain carries a free sulphydryl group. A similar result was obtained with urinary light chains. The pattern of the iodoacetate treated urinary light chain (Fig. 3a, sample I) shows, when compared with Fig. 3a, sample IV, a new upper band which has undergone an identical shift. Thus after reduction the urinary light chains also carry a single sulphydryl. Further, when the intracellular and secreted light chains of P1 were examined in electrofOcusing gels (Fig. 4) an analogous charge shift was observed, the intracellular and secreted light chains of PI exhibiting an almost complete shift of isoelectric point following alkylation with iodoacetate (Figs. 4b and d). The charge shift analyses confirm Fig. 2 in showing all, or nearly all, free intracellular L chains to be monomers, since disulphide bridged dimers would have been unable to react with the iodoacetate. After electrophoresis in SDS acrylamide gels, t h e P1 urinary light chain was found in the monomer position. The reduced and alkylated sample behaved identically, confirming that the urinary protein is not

covalently dimerised. No charge shift is observed if the urinary protein is carboxymethylated without prior reduction so that the C-terminal sulphydryl is not free, presumably being bridged to a half cystine (Milstein, 1966). DISCUSSION

Covalently bonded light chain dimers have been isolated from myeloma cells obtained from various species (Namba & Hanaoka, 1969; Laskov & Scharff, 1970; Coil & Baglioni, 1967; Skortsov & Gurvich, 1968; Scharff et al., 1967). They have also been found in the culture fluids of several tumour lines (Baumal et al., 1971; Schubert et al., 1968; Laskov & Scharff, 1970; Scharff et al., 1967), and the urine of animals carrying such tumours frequently contains dimer light chains. Both the chromatographic and the alkylation results of our studies on X2P1 indicate however that only a small proportion, if any, of the light chains are in the dimer form in these cells. It would seem probable that those previous workers who carried out their isolations in the absence of an alkylating agent were observing an artifactual production of dimers.

Intracellular and Secreted Mouse Plasmacytoma Light Chains The results of Sutherland et al. (1970) which demonstrate spontaneous assembly of IgG2~ during P1 tumour cell lysis in the absence of iodoacetamide would support this possibility. The elution of radiolabelled monomer light chain as physical dimers in Figs. la and b indicates that the light chains of both XzP1 and PI show a strong tendency to associate as dimers in vitro. As this occurs with concentrations of light chains certainly no greater than those found in intracellular pools, it is likely that this dimerisation will also occur in vivo. From our studies on X2PI it would seem that alkylation of intracellular and reduced urinary light chains results in a charge shift equivalent to one unit charge. This indicates that they both have only one free - S H group. Since the intracellular and urinary light chains display nearly identical homogenous electrophoretic mobility, it would appear that the light chains of X2P1 do not undergo any charge change on secretion, as would occur for example with the addition of sialic acids to the carbohydrate portion of light chains (Melchers et al., 1966). As P1 like X2P1 has a free SH group on the L chain, it would appear that the P1 intracellular light chains are not temporarily blocked in a covalent disulphide bridge either to another light chain or to a cysteine residue. It would therefore appear that control of the assembly of P1 immunoglobulin does not involve temporary blocking of the L chain. This was also found (Stott & Feinstein, 1973) in the case of IgM assembly in the ceils of tumour MOPC 104E, where the IgM assembly pathway is known to be H + L---~HL---~ H2L 2 (Parkhouse, 1971). The assembly of P1 immtmoglobulin occurs via the intermediate H 2 and H2L, little HL being found in this tumour line (Baumal et al., 1971 ; Sutherland et al., 1970). The monomeric urinary light chains of mice bearing either tumour carried no free SH group, but were reversibly blocked, presumably by a bridge to a halfcystine residue (Milstein, 1966), as judged by charge

631

shift analysis. Since the chains secreted in vitro carried a free SH group, the half-cysfine blocking in vivo must occur in the extracellular fluid or during renal excretion. REFERENCES

Askonas B. A. & Williamson A. R. (1966) Nature 211,369. Awdeh Z. L., Williamson A. R. & Askonas B. A. (1968) Nature 219, 66. Baumal R., Potter M. & Scharff M. D. (1971) J. exp. Med. 134, 1316. Baumal R. & Scharff M. D. 0973) Transplantation Rev. 14, 163. Coli D. & Baglioni C. (1967) Nobel Symposium Vol. 3. Gamma Globulins (Edited by Killander J.) p. 401. Interscience, New York. Feinstein A. (1966) Nature 210, 135. Feinstein A. (1968) Chromatographic & Electrophoretic Techniques--II (Edited by Smith I.) p. 207. Pitman PresS, London. Feinstein A. & Stott D. I. (1972) J. Physiol., Lond. 226, 34. Laskov R., Lanzerotti R. & Scharff M. D. (1971) J. molec. Biol. 56, 327. Laskov R. & Scharff M. D. (1970) J. exp. Med. 131, 515. Melchers F., Lennox E. S. & Facon M. (1966) Biochem. biophys. Res. Commun. 24, 244. Milstein C. (1966) Biochem. J. 101, 339. Namba Y. & Hanaoka M. (1969) J. Immun. 102, 1486. Parkhousc R. M. E. (1971) Biochem. J. 123, 635. Salaman M. R. & Williamson A. R. (1971) Biochem. J. 122, 93. Scharff M. D., Shakiro A. L. & Ginsberg B. (1967) Cold Spring Harb. Syrup. quant. Biol. 32, 235. Schubert D. & Cohn M. (1968) J. molec. Biol. 38, 273. Schubert D., Munro A. & Ohno S. (1968) J. malec. Biol. 38, 253. Skortsov V. T. & Gurvich A. E. (1968) Nature 218, 377. Stott D. I. & Feinstein A. (1973) Eur. J. lmmun. 3, 235. Summers D. F., Maizel J. V., Jr. & Darnall J. E., Jr. 0965) Proc. natn. Acad. Sci., U.S.A. 54, 505. Sutherland E. W., Zimmerman D. H. & Kern M. (1970) Proc. natn. Acad. Sci., U.S.A. 66, 987. Wieme R. J. (1959) Clinica chim. Acta 4, 317.