The structure of an immunoglobulin light chain fragment in mouse myeloma cells

J. Mol. Biol. (1975) 97, 11-20

T h e S t r u c t u r e o f an I m m u n o g l o b u l i n

Light Chain Fragment

in M o u s e M y e l o m a C e l l s SANDRA H. BRIDGES AND JULIAN B. ~LEISCH3~AN

Department of Microbiology Division of Biology and Biomedical Sciences Wazhington University School of Medicine St. Louis, Mo. 63110, U.S.A. (Received 18 March 1976) An immunoglobulin light chain fragment, which forms a relatively large proportion of the radioactivity associated with intracetlular immunologicaUy precipitable immunogtobulin after short incubations with radioactively labeled amino acid, has been described in mouse myeloma cells (Schubert & Cohn, 1970). A detailed characterization of this fragment from S176 and J558 mouse myeloma cells has been undertaken, including purification and subsequent pcptide mapping, in order to determine the possible significance of this fragment in the biosynthesis of the complete light chain. The purified material appears rather heterogeneous in length on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a modal molecular weight of 15,500. Peptide mapping of [3H]leucine-labeled material, with homologous light chain carrier, demonstrated that both variable and constant region peptides were present. These data, and the pattern of specific activity derived from the maps, were more compatible with the fragment being a collection of nascent chains and/or proteolytic fragments than a discrete variable region piece.

1. Introduction Immunoglobulin polypeptide chains are probably formed from at least two structural genes, coding for the variable and constant regions of the chain (Milstein & Mum'o, 1970). How these genes, or their products, are joined to form" the complete chain is an intriguing problem. Studies of growing immunoglobulin polypeptide chains (Fleischman, 1967; Knopf et al., 1973) suggested that joining at the protein level (post-translationally) is unlikely, and favored pre-translational joining at the DNA or messenger RNA level. The structure of certain "deletion" variants of heavy chains support this view (Frangione & Franklin, 1973). Recent studies on the primary structure of mRNA for light chains imply that nueleotide sequences coding for variable, "switch", and constant region peptides are present in the same RNA molecule (Milstein et al., 1974). This makes the strongest case thus far for pretranslational joining. Schubert & Cohn (1970) described a light chain-related polypeptide fragment (IF) in certain mouse plasmacytomas. The fragment was approximately half the size of a light chain and appeared to be a biosynthetic precursor of the complete chain. The authors did not study the structure of the fragment in detail, but they suggested 11

12

S . H . B R I D G E S AND J. B. F L E I S C H M A N

t h a t it m a y be a separately synthesized V t polypeptide. They proposed a model in which a small pool of newly synthesized V polypeptides could be joined to nascent C polypeptides, to yield a labeling pattern consistent with other studies on the growth of immunoglobulin polypeptide chains. They felt that their observations thus kept open the possibility of a post-translational joining mechanism. We felt it important to resolve the question raised b y the findings of Schubert & Cohn. We wished to characterize the fragment in terms of its size, heterogeneity and primary structure, and to determine whether or not it corresponded to a separately synthesized V polypeptide. We found t h a t the fragment is heterogeneous, contains both V and C peptides, and resembles a collection of nascent chains and/or proteolytic fragments rather than a discrete V polypeptide. Thus the structure of the fragment does not support the post-translational joining model proposed b y Schubert & Cohn (1970). 2. M a t e r i a l s a n d M e t h o d s

(a) Myeloma tumor8 Immtmoglobulin A-producing mouse myeloma tumors S176 and J558 were gifts from Martin Weigert, the Salk Institute. They were maintained as subcutaneous tumors in BALB/e AN mice for the production of myeloma protein-containing sera and as a source of S176 myeloma cells for labeling experiments. The J558 cells used for labeling experiments are a tissue culture cell line, adapted for growth in suspension culture. The secreted proteins of both myelomas have ~ heavy chains and ~ light chains. Protein S176 binds 5-acetouracfl and ~-l,3-dextran; protein J558 binds ~-l,3-dextran (Carson & Weigert, 1973).

(b) Preparation and incubation of cell suspensions S 176 cell suspensions were prepared in Dulbeeco's modified Eagle's medium, containing 1 × 10 -5 ~-T.-leueine, and 1 to 2 nag trypsin, by stirring for 30 rain at room temperature. Heat-inactivated fetal calf serum was added to a final concentration of 16%, followed by DNAase (10 pg/ml), for 10 to 15 min at 37°C. Tissue fragments were removed, and the cell suspension was further treated as described below for J558 cells, except that horse serum w a s n o t used. J558 cultured cells were washed once with leucine-free Dulbeeco's modified Eagle's medium containing 2.5% horse serum, and resuspended at 5 × 105 eells/mi for short incubations (1 to 3 rain) or 1 × 106 cells]ml for long incubations (5 to 6 h). The viability of all cell preparations by trypan blue exclusion was 90% or better. After equilibration at 37°C for 20 rain, [3HJleucine (New England Nuclear or Schwarz-Mazm, 30 to 60 Ci/mmol) was added to 100 to 500 t~Ci/ml for short incubations and to 50/~Ci/ml for long incubations. Final leucine concentrations were adjusted to 1 × 10 -3 ~, and 5× 10 -5 ~, for short and long incubations, respectively, with unlabeled L-leucine. The cells linearly incorporated [SH]leucine into hot trichloroacetic acid-insoluble material during the entire incubation. Labeling was terminated by pouring the cell suspensions over frozen medium lacking leucine. The cells were centrifuged at 4°C, resuspended in phosphate buffered saline, and frozen at -- 20°0. (c) Preparation, immunoprseipitation and gel eleetrophoreslz of labeled

chains and fragments Cell extracts were prepared by 4 cycles of freezing and thawing, homogenization in a motor-driven Potter-Elvehjem homogenizer and centrifugation at 10,000 g for 1 h at 4°C; superuatemts were analyzed as detailed below. Labeled intraeellular immunoglobulin chains and fragments were precipitated from short-incubation cell extracts by direct precipitation with rabbit antiserum to S176 light t Abbreviations used: V, variable region; C, constant region; L chain, light chain; H chain, heavy chain; IF, immunoglobulin fragment.

S T R U C T U R E OF A L I G H T C H A I N F R A G M E N T

13

chains (Gateway Immunosora Co., Caholda, Ill.), and from long-incubati0n extracM with rabbit antiserum toS176 myeloma protein. Direct, rather than indirect prec~itation was used in order to minimize the amount of total protein to be loaded on the polyacryl~.m!de gels. Independent indirect immunopreeipitations with goat anti-rabbit immunogl0bul!ri G confirmed that the amount of rabbit antiserum used in the direct precipitations was su~cient to precipitate all the radioactive !mmunoglobn]in-related material in the extract, The rabbit antisera were incubated with the cell extracts for 45 rain at 37°0 and overnight at 4°C. The precipitates were washed 3 times with cold phosphate buffered saline (0.02 MKPO4, 0-15 M-lqaCl, p H 7-45) and solubilized in freshly prepared 9.8 M-urea, 1% sodium dodecyl sulfate, and 0.5 M-Tris.HC1, pH 8.0. Prior to analysis by sodium dodecyl sulfate--polyacryJamide gel electrophoresis (Laemmli~ 1970), the samples were completely reduced with 0.05 m-dithiothreitol (Calbiothem) for 1 h at 37°0, followed by heating at 100°C for 1 rnln. At the conclusion of the electrophoretic run, the gels were fractionated by an automatic gel divider (Savant), using distilled water as a diluent. After an overnight incubation at 37°C, samples of the fractious were counted in Kinard's solution (Kinard, 1957). We wished to rule out the possibility that I F was generated by proteolysis during the preparation and/or immunoprecipitation of the extracts. After homogenization of labeled J558 cells at 4°0, a sample of the homogenate was diluted in phosphate buffered saline containing Trasylol (Bayer), a protease inhibitor, at a final concentration of 50 Kallikrein inactivation units]ml, and a control sample was diluted into phosphate buffered saline. Both samples were centrifuged and immunoprecipitated as described above, maintaining a concentration of 50 Ka|lilrrein inactivation units of Trasylol/ml in the experimental sample. Samples were incubated at 4°0 overnight, and at 37°C for 1 h and 3 h, followed by overnight at 4°C. The proportion of I F on polyacrylamide gels of both experimental and control samples was identical; thus, there was no evidence that I F is generated prot~olytically under our preparative conditions. (d) Purifice~ion of l~h~ chain carrier Fractious of myeloma serum were prepared in 40% ammonium sulfate and mildly reduced and alkylated with iodoacetic acid (S176) or amlncethylated (J558) prior to purification (Bridges & Little, 1971). Ethylcneimine (Pierce) was added to a final 20-fold molar excess over dithiothreitel thiol groups in 3 equal amounts at 10-mln intervals. S176 was purified by preparative electrophoresis on agarose; J558 was purified on a dextran immunoabsorbent column (Carson & Weigert, 1973). H and L chains were separated by gel filtration after a second mild reduction and ~m~noethylation (Bridges & Little, 1971). (e) Pept~s amdyd, o$ chair** and/raam*~ Fractions containing the radioactively-labeled L chain or I F obtained from sodium dodecyl sulfate-polyacrylamido gels by olution with distilled water at 37°0 (section (e), above) were pooled appropriately, filtered through glass-fibers tO remove gel particles, and the filtrates lyophilized. The lyophl]i~.ed L chains or I F were redissolved in distilled water, purified light chain carrier (200 t~g) was added, and excess sodium dodecyl sulfate was removed by acetone]ether/1 ~r-HC1 (20:5:1) precipitation of the protein at 0°C. After overnight drying, the precipitate was dissolved in 8 M-urea, 0.5 ~r-Tris- HC1, p H 8-0. Additional purified light chain carrier (5 rag) was added, and reduction and am~noethylation were carried out as previously described, except that the reduction was for 1 h at room temperature in 0.05 M-dithiothreitol. Completely reduced and arnlnoethylated protein was suspended in 0.05 ~t-ammonium bicarbonate (pH 8"5), and digested with TPCK-trypsin (Worthington; 1:50) for 2 h at 37°C; the digestion was terminated by lyophilization. The sample was clarified by centrifugation and applied in p H 4.7 pyridlne acetate buffer (Weigert & Garen, 1965) to Whatman no. 3 paper (46 cm × 57 cm) and electrophoresed for 125 mln at 1-5 kV. The paper was dried overnight at room temperature and equilibrated for 6 h and chromatographed for 18 to 19 h in a perpendicular direction in the ascending system of Weigort & Garen (1965). After drying, the paper was sprayed with 0.025% ninhydrin, and the spots were cut out and rinsed once with acetone. The peptides were eluted from the paper with

S. H. B R I D G E S AND J . B. F L E I S C H M A N

14

I Y-acetic acid (Edstrom, 1968). Usually one.fifth of the sample was hydrolyzed for 24 h at ll0°C in 6 N-H(]I and analyzed on a Spinco model 120B A.mlrm acid analyzer, adapted for high sensitivity, l~our-fifths were counted after Iyophilization in the counting vial, subsequent resolubilization with 0.2 ml distilled water, and addition of 10 ml Bray's solution. All radioactivity was in leucine, since a count of fractions eluted from the 8.m~no acid analyzer demonstrated only one peak of radioactivity, coinciding with the ninhydrin absorbance peak of leucine. The specific activity of each peptide was expressed as the ratio of cts]min to nmol of leueine.

3. R e s u l t s

(a)

I~i~io~

and antigenic and biosy~hetic properties of immunogZobulin fragment

We confirmed the observation of Schubert & Cohn (1970) t h a t I F can be immunospecifically precipitated from cytoplasmic supernatant fractions of mouse m y e l o m a cell homogenates. Figure 1 shows the radioactivity profile on a 10% polyacrylamide gel of intraeellular H chain, L chain and immunoglobulin fragment immunospecifically precipitated from an extract of S176 t u m o r cells after incubation for three minutes with [3H]leucine. After longer incubations the proportion of I F decreases; after 30 minutes it is hardly detectable. F o r this reason, the I F required for peptide analysis (below) was isolated after short incubations. 1mmunospecific precipitation of I F was almost completely inhibited b y 200 Fg of non-radioactive L chain, confirming the antigenic relation to L chains (Schubert & Cohn, 1970).

H 6--

~4 ¢)

2

0

2.0

40

60

80

Fraction no.

FIO. 1. Radioaotivity profile, on 10% polyaerylamide gel, of intracellular H chain, L chain and IF hnmunospecifically precipitated from an extract of S176 tumor cells after a 3-rain incubation with [aH]leucine. The rabbit antiserum to S176 L chains, used in the immunoprecipitation, also contained antibodies to H chain.

STRUCTURE

OF A LIGHT

CHAIN

FRAGMENT

i5

(o 24

'16

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I

I

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(b o

6

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~

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(c) 6O

40

20

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40

60

Fraction no.

FIG. 2. Polyacrylamide gel electrophoresis of J558 I F a n d L chain. Intracellular material was immunospecifieally precipitated from a n e x t r a c t of J558 cells i n c u b a t e d for 1"~ m i n w i t h [SH]. leucine. I F a n d L chains were e]uted from 10% sodium dodeoyl sulfate-polya~rylamide gels, concentrated, a n d r e r u n as follows. (a) I F on 10% gel, (b) I F o n 15% gel, (e) L chain o n 15%

gel.

We also confirmed that IF behaves as a biosynthetic precursor for newly synthesized light chains (Schubert & Cohn, 1970). In a pulse-chase experiment YF rapidly disappeared from the immunospecifieally precipitable pool after the chase was initiated. Radioactivity in both H and L chains increased; however, inhibitio~ of precipitation of IF by excess L chain suggested that most of the IF is L chain-related. (b) Heterogeneity and molecular weight of immunoglobulin fragment In previous studies, the homogeneity and molecular weight of I F had not been rigorously assessed. We felt it important to study these parameters further. In gels

S. H. BRIDGES AND J. B. FLEISCHMAN

16

of polyacrylamide content of 10~/o or less, the immunoglobulin fragment runs very close to the dye front and appears quite homogeneous (Fig. 2(a)). However, when I F purified from 10~/o polyacrylamide gels is rerun on 15~/o gels, it is clearly heterogeneous (Fig. 2(b)). The molecular weight of IF shown in Figure 2 was determined on 15~/o sodium dodecyl sulfate-polyacrylamide gels, calibrated with 14C-labeled 1~IOPC173 ~ and K chains and with cytochrome c (Laemmli, 1970) as markers. The modal molecular weight of the fragment was 15,500 with a shoulder of molecular weight 13,400. (c) Peptide composition of immunoglobulin fragment In order to determine whether the fragment contained material from the variable region of the L chain, the constant region, or both, radioactively labeled I F was examined by peptide mapping. We assumed that the I F was sufficiently small to become uniformly labeled during the incubation times in these experiments (1 to 3 rain) (Schubert & Cohn, 1970). Radioactive I F purified on 1 0 ~ sodium dodecyI suffate-polyacrylamide gels was mixed with unlabeled homologous light chain carrier, completely reduced and aminoethylated, trypsin digested, and fingerprinted as described in Materials and Methods. In control experiments, radioactive light chains purified from acrylamide gels were analyzed in the same way. Our pcptide maps of the S176 and J558 light chains yielded seven well.separated lcucine-containing peptides, T1, T2, T4, TT, TS, T9 and T17, accounting for 11 of the 17 leucyl residues in the ~ chain. The other leucine-containing peptides, T5, T6 and T13, were not identified on the map, and were assumed to be in the insoluble residue remaining after try~tic digestion. S176 L chain differs from J558 L chain by only one residue (Asn for Ser at position 25; Cesari & Weigert, 1973). The J558 peptides, their residue positions, and specific activities derived from both IF and L TABLE 1

Specific activities of tryptic peptides from J558 immunoglobulin fragment and light chains Peptide

Residue positions

Ti 1-23 T2 24-56 T4 64-72 T7 106-113 T8 114-132 T9 133-137 T17 208-211 Total recovered Total applied % recovered ,,,,,,,

Leueine (mnol) IF t L~ 63.3 141-1 64-8 102.1 109-2 55-5 39"6 576 ND§ ND

96.0 87.1 65.5 90.0 62-5 44.5 57'0 503 ND ND

Radioactivity (ets/min) IF L 1751 7890 4561 2644 3595 564 1232 22,237 90,000 25

Specific activity (ors/rain per nmol leueine) IF L

2829 4744 3039 4594 3254 1636 2999 23,095 68,000 34 ,,,,,

,,,,,,,,

t IF from J558 cells incubated 1-5 rain with [aH]leueine. Intra~llu~r I~ chain from J558 cells incubated Gh with [SH]leueine. § Not determined.

27.7 55.9 70.4 25.9 32.9 10.2 31.1

29.5 54.5 46.4 51.0 52-1 36.8 52-6

STRUCTURE

OF A LIGHT

CHAIN

FRAGMENT

17

chain controls, are listed in Table 1. T1, T2 and T4 are V 9eptides; T7 (residues 106 to 113) corresponds to the "switoh peptide" (V + C) residues in ~ chains; T8, T9 and T17 are C peptides (corresponding respectively to T9, T10 and T18 of Appella, 1971). The amlno acid compositions of all of our peptides agreed very closely with those reported by Cesari & Weiger~ (1973) and AppeUa (1971). The relative specific activities of the peptides derived from labeled ~ and L chain controls are compared graphically in Figures 3 and 4. It is immediately evident that (a) 10

D

T2

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I 180

215 C

FIQ. 3. Specific activities of poptides from S176 I F and L ohain. I F was isolated from an extract of S176 tumor evils incubated 3 mln with [aH]leueine ~nd L chain was isolated from secreted immunoglobulin after 6 h incubation. After rniTing with ,nlabeled S176 oarrier L ohain, the labeled material was completely reduced and aminoethylated, digested with trypsin, and fingerprinted as detailed in Materials and Methods. Portions of the eluted peptides were run On the Arni~o acid ~n~lyzer and counted, and the speeifio activity was expressed as ors/rain per nine! leueine. The peptides are arr~mged according to their positions in the sequenee of t h e L ohain; the width of each bar oorresponds to the length of the peptide, and the small arrow on the absoissa indioates the hypothetical "switch point" between V and C regions. (a) I F (3 rain intraoollular), (b) L ehain (6 h extracellular). 2

18

S. H . B R I D G E S 80

AND J, B. FLEISCHMAN

.................

(o)

60-

T2

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"6

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== 20 .S E

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I

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100

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(b)

:.,.= ci. (D

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I

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Residue position

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Fie. 4. Specific activities of peptides from J558 I F a n d L chain. I F was isolated from a n e x t r a c t of J558 cultured cells i n c u b a t e d 1-5 rain with [3H]leucine; L chain was isolated from intracollular immunoglobulin after 5 h incubation. J558 L chains were added as carrier. See t h e legend to Fig. 3 for descriptions of t h e subsequent isolation procedure a n d graph. (a) I F (1.5 rnin intracellular), (b) L chain (5 h intracellular).

the I F from both S176 and J558 cells contain both variable and constant region peptides, although the V peptides are more prominently represented. These results are more compatible with the I F being a collection of nascent, or proteolytically degraded light chains, rather than a discrete variable region (Schubert & Cohn, 1970). The radioactivity in the control L chains was distributed more uniformly, as would be .expected. The specific activityof T1 from intracellular I F and L chains, and of T9 from all labeled chains appeared to be unusually low. Possible explanations for this will be considered in the Discussion.

STRUCTURE OF A LIGHT CHAIN FRAGMENT

19

4. Discussion The principal conclusion of our work is that the IF observed b y Schubert & Cohn (1970) is heterogeneous, is larger than they had suggested, and contains both variable and constant region peptides. It is evidently not the discrete variable region proposed in their model for post-translational joining. The IF resembles in size and struchlre a heterogeneous collection of nascent chains; its apparent uniformity on 10% polyacrylamide gels is evidently an artifact. Thus the structure of the fragment appears to be consistent with other evidence supporting a pre-translational joining mechanism for light chain biosynthesis. The specific activities of certain IF peptides (Figs 3 and 4) deserve comment. That of peptide T17, near the COOH-terminal of the light chain, is unexpectedly high for a population of nascent chains with a modal molecular weight of only 15,500. This might be explained by the presence of constant region fragments (Kuehl & Scharff, 1974), or by intracellular proteolysis of newly synthesized light chains, which were not resolved from the rest of the ~ on polyacrylamide gels. Peptide T9 had an anomalously low specific activity, even in control light chains labeled for long periods (Figs 3 and 4). We note that T9 is homologous to the COOHterminal sequence of peptide I-9 in the MOPC46 (K) chain, some of which also had unusually low specific activity in labeling studies (Knopf eta/., 1973). The authors ascribed this electrophoretic heterogeneity to possible deamidation of a portion of peptide I-9. However, our peptide T9 contains no amide residues; thus this explanation cannot apply. During preparation, labeled chains were exposed to urea longer than the carrier light chains were; possible carbamylation of the COOH-terminal cysteine residue in T9 at this stage may have interfered with subsequent aminoethylation. We also noted a low specific activity for amino-terminal peptide T1 in all preparations except extraceUular light chains (Fig. 3(b)). Some intracellular chains may conrain additional residues at the NH2-terminal which are removed during maturation of the light chain (Milstein et al., 1972). This would result in electrophoretic heterogeneity between labeled intracellular and unlabeled carrier chains, and may account for the low specific activity of T1. Our molecular weight determinations on 15% polyacrylamide gels assume that IF contains only protein. We did not test for attached transfer RNA which could have affected its mobility on the gel. However, most attached tRNA probably would have been released from IF during the incubations at 37°C and 100°C (Materials and Methods, section (c)) before loading on the gel. We are indebted to Mrs Donna Thurmond for carrying out the amino acid analyses, to Dr L. Keay for culturing the J558 tumor line, and to Dr M. Weigert for advice on myeloma protein and peptide isolation. We acknowledge the support of U.S. Public Health Service grants AI00257 and AI10810 and American Cancer Society fellowship no. PF659. REFERENCES Appella, E. (1971). Prec. Nat. Acad. Sci., U.S.A. 68, 590-594. Bridges, S. H. & Little, J. R. (1971). Bioehemiatxy, lO, 2525-2530. Carson, D. & Weige~; M. (1973). Prec. JVat. Acad. Sei., U.S.A. 70, 235-239. Cesari, I. M. & Weigert, M. (1973). Prec. Nat. Acad. Sci., U.S.A. 70, 2112-2116. Edstrom, 1%. D. (1968). Anal. Biochem. 26, 204-205.

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S.H.

B R I D G E S AND J. B. F L E I S C H M A N

Fleischman, J. B. (1967). Biochemistry, 6, 1311-1320. Frangione, B. & Fr~nl~lln, E. C. (1973). Semir~re in HematoZogy, 10, 53--64. ~ n o x d , F. E. (1957). R6v. Se~. Ir~t~u. 28, 293-294. Knopf, P, M , Munro, A. J. & Lennox, E. S. (1973). Arch. Biochem. Bio~hya. 157, 288302. Kuehl, W. M. & Soharff, M. D. (1974). J. Mol. BIOL 89, 409-421. Laemmll, U. K. (1970). Nature (London), 227, 680-685. Milatein, C. & Munro, A. J. (1970). Annu. Rev. Microbiol. 24, 335-358. Milstein, C., Brownlee, G. G., T4arrison, T. M. & Matthews, M. B. (1972). _Nat/ure (London), 289, 117-120. Milstein, C., Brownleo, G. O., Cartwright, E. M., Jarvis, J.M. & Proudfoot, N. J. (1974). Nature (Z,ond~n), 252, 384-359. Schubert, D. & Cohn, M. (1970). J. Mol. Biol. 53, 305-320, Weigert, M. G. & Garen, A. (1965). J. Mol. Biol. 12, 448-455,