Cotranslational cleavage of immunoglobulin light chain precursors by plasmacytoma microsomes

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 195, No. 2, July, pp. 368-37’7, 1979

Cotranslational

Cleavage of lmmunoglobulin Light Chain Precursors by Plasmacytoma Microsomesl MICHAEL GREEN with the technical assistance of BONNY

J.

ALPER

Department of Microbiology,

St. Louis University School of Medicine, 1402 South Grand Boulevard, St. Louis, Missouri 63101,

Received December 7, 1978; revised January 26, 1979 Murine plasmacytoma endoplasmic reticulum which has been freed of ribosomes by EDTA treatment is capable of the cotranslational proteolytic processing of representative A,, hp, and K immunoglobulin light chain precursors. Messenger RNA fractions from the MOPC-104E, MOPC-315, and MOPC-46B tumor lines were used to direct the synthesis of the light chain precursors in a cell-free system derived from Krebs II aseites cells. The precursor cleavage activity of the plasmacytoma membranes is comparable in activity and in characteristics to that of two well-defined membrane preparations: Krebs II ascites intracellular membranes (E. Szczesna and I. Boime, 1976, Proc. Nut. Acad. Sci. USA 73, 1179-1183) and EDTA-treated rough endoplasmic reticulum from canine pancreas (G. Blobel and B. Dobberstein, 1975, d. Cell Biol. 67, 852-862). The efficiency of the cleavage reaction appears to be dependent upon the precursor being utilized as a substrate. An assay suitable for a preliminary characterization of the plasmacytoma membrane preparations is described.

When a resting B lymphocyte, committed to the synthesis of cell surface immunoglobulin (1) is stimulated either specifically by a suitable antigen or nonspecifically by an appropriate mitogen (2,3), it differentiates into an actively secreting plasma cell with a well-defined endoplasmic reticulum (4, 5). Little is known about the mechanism of the synthesis and assembly of the intracellular membranes during this transition. An investigation of the proteolytic cleavage of immunoglobulin precursors and the glycosylation of newly synthesized immunoglobulin chains will serve as a tool for the elucidation of the mechanisms of assembly and possible regulation of the membrane-associated * This research was supported by U.S.P.H.S. Grant CA 20821 awarded by the National Cancer Institute, Department of Health, Education and Welfare. The cost of publication of this article was defrayed in part by the payment of page charges from funds made available to support the research which is the subject of this article. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. 1734 solely to indicate this fact. 0003.9861/79/080368-10$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

368

activities involved in the post-translational processing and eventual secretion of immunoglobulin chains. The plasmacytoma cell is a neoplastic analog of the monoclonal plasma cell, a terminally differentiated B lymphocyte (for review see (6)). It possesses a well-developed endoplasmic reticulum and is involved in the active secretion of a homogeneous immunoglobulin molecule. These features make this type an ideal source of cellular components with which to establish a cell-free system capable of the specific processing of immunoglobulin protein chains. Recent studies of cell-free systems which catalyze the coupled synthesis and processing of proteins have led to a better understanding of the mechanisms of membrane biogenesis and cellular secretion. It has been found, for example, that most proteins destined for secretion are synthesized as precursors with an amino terminal segment which is rapidly cleaved during protein synthesis. Studies describing the cell-free synthesis and, in several cases, the processing of the precursors of such diverse

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OF IMMUNOGLOBULIN

proteins as immunoglobulin L-chains’ (7- 13) and an immunoglobulin H-chain (14), parathyroid hormone (15), placental lactogen (16- 18), proinsulins (19, SO), prolactin (21-24), pancreatic zymogens (25), growth hormone (24, 26), and rat albumin (27) and egg proteins (28) have contributed to the determination of the mechanism of this reaction. In addition, specific cotranslational glycosylation of the precursors of the envelope glycoprotein (G protein) of vesicular stomatitis virus (29, 30) cw-lactalbumin (31), and human chorionic gonadotropin (32) have been accomplished in a cell-free system containing added membranes. Other studies have led to the development of a cell-free system capable of the glycosylation of the precursor for an immunoglobulin L-chain in the presence of Ehrlich ascites cellular membranes (33). These data have strengthened and expanded the signal hypothesis put forth to explain the segregation to the rough endoplasmic reticulum of polysomes synthesizing secretory proteins and the subsequent vectorial transfer of these proteins across the microsomal membrane (34, 35). These systems also provide the tools for the examination of the relationship between the primary sequenceand secondary structure of the amino terminal extension of precursor proteins to its function and to determine the relationship between membrane components that are responsible for the processing events. This report describes the use of the murine plasmacytoma as an experimental system for the assay and characterization of these membrane associated activities. Three tumor lines, MOPC-104E (E,L H-chain, A, L-chain), MOPC-315 ((Y H-chain, h2 L-chain), and MOPC-46B (glycosylated K L-chain) were used as sources of mRNA. 2 Abbreviations used: L-chain, immunoglobulin light chain; H-chain, immunoglobulin heavy chain; MOPC, mineral oil-induced plasmacytoma; TEPC, tetramethylpentadecane-induced plasmacytoma; L”l”“, light chain of the MOPC104E protein; LA’46B, light chain of the MOPC-46B protein; Lu315, light chain of the MOPC-315 protein; pL”‘O”, precursor of L”“)‘; of LMGER; PL~‘~‘~, precursor of pLM46R, precursor Ls1315; RER, rough endoplasmic reticulum; NP40, Nonidet-P40; SDS, sodium dodecyl sulfate, ConA, concanavalin A.

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The ability of plasmacytoma membranes to process the cell-free translation products of the immunoglobulin mRNAs is examined. Additional experiments which further characterize the processing activities are also described. EXPERIMENTAL

PROCEDURES

Materials. L-[4,5-3H]Leucine (53 Ci/mmol) was obtained from AmershamSearle. S. aureus protease V8 was obtained from Miles Laboratories. Kyro EOB was the giR of Dr. D. H. Hughes of the Proctor and Gamble Co., Cincinnati, Ohio. Triton X-100 was purchased from Sigma, and Nonidet-P40 was obtained from Shell Oil Co. RNase-free sucrose was obtained from Schwam-Mann. Oligo(dT)-cellulose was purchased from Collaborative Research. Rabbit antiserum specific for murine A,, A,, or K L-chains was purchased from Gateway Immunosera Inc., St. Louis, Missouri. Glass-distilled, autoclaved water and sterile glassware were used for all procedures. Tumor lines and proteins. The plasmacytoma lines TEPC-15 and MOPC-104E were donated by Dr. J. Davie at Washington University. MOPC-315 was donated by Mr. E. Simms at Washington University. The MOPC-46B line was donated by Dr. S. Pestka at the Roche Institute of Molecular Biology. All plasmacytoma lines were maintained as solid tumors in Balb/c mice by serial, subcutaneous passage of 0.1-0.2 ml of minced tumor fragments at intervals of 14-20 days. Excised tumors were freed of necrotic material and either fractionated immediately or frozen in liquid nitrogen and then stored at -70°C. Radioactive authentic immunoglobulin markers were obtained from the protein secreted into the media of tumor cell suspensions according to previously published procedures (13). Preparation of mRNA. Unless otherwise stated, all manipulations were performed at 4°C. Two methods were used to extract immunoglobulin mRNA. In the first method, tumor tissue was disrupted in 0.25 M sucrose in 50 mM Tri-HCl (pH 7.4), 5 mM MgCl,, 50 mM KC1 (TMK buffer) for 30 s at half speed in a Waring Blendor. A ratio of 3 ml of fluid/g of tumor tissue was used. The tumor fragments were then homogenized by eight strokes in a loose-fitting Teflon-glass homogenizer (0.25.mm clearance). NP40 (lo%, v/v) was then added to a final concentration of 0.5%, and the homogenate was allowed to stand for 5 min at 4°C. The detergent-treated homogenate was centrifuged at 10,000 rpm for 10 min at 4°C in a SS-34 rotor of a RC-2B Sorvall centrifuge. The supernatant was removed and diluted fourfold with distilled water. SDS (20% w/v) was added to a final concentration of l%, and 0.1 vol of 1.0 M Tris-HCl (pH 9.0), 1.0 M NaCl, 0.01 M EDTA was added. The resulting mixture was extracted with 1 vol

370

MICHAEL

of a solution of distilled water-saturated phenol: chloroform:isoamyl alcohol (50:50:1, by volume) by vigorous shaking for 10 min at room temperature, then chilled in an ice-water bath for 5 min, and centrifuged at 2000 rpm for 30 min in a No. 259 rotor of a PR-6 centrifuge (International Equipment Co.). The upper aqueous layers were pooled and re-extracted with an equal volume of the phenol:chloroform:isoamyl alcohol mixture. One-tenth volume of 2.0 M potassium acetate (pH 5.5) and 2 vol of absolute ethanol were added to the final aqueous layers. The RNA was precipitated upon standing at -20°C overnight. The RNA pellet obtained after centrifugation for 30 min at 2000 rpm in the No. 259 rotor of the PR-6 was dissolved in water and stored at -70°C. In the second method, immunoglobulin mRNA was extracted from total plasmacytoma microsomes according to the procedures of Marcu et al. (36) with minor modifications. Briefly, tumor tissue was disrupted by homogenization in 0.88 M sucrose in TMK buffer (2 ml/g tumor tissue) containing cycloheximide (2 pg/ml) and polyvinylsulfate (20 fig/ml). The homogenate was centrifuged at 90009 to remove cell debris, nuclei, and mitochondria and the resulting supernatant was brought to 0.62 M sucrose by the addition of the appropriate volume of TMK buffer and to 200 pgiml heparin by the addition of solid heparin. Total microsomes were isolated by centrifugation at 100,OOOg for 45 min. The microsomes were resuspended in 0.1 M Tris-HCl (pH 9.0), 0.1 M NaCl, 1.0 mM EDTA containing 1% (w/v) SDS at a concentration of (approximately) 50-100 A,,, units/ml and extracted with phenol:chloroform:isoamyl alcohol as described above.3 The poly(A)-containing mRNA was obtained by chromatography of the RNA obtained from the above procedures on oligo(dT)-cellulose as previously described (13). Approximately 1500-2000 AzGa units were applied to a 1.5 x 5.0-cm column of oligo(dT)-cellulose. Further fractionation of the poly(A)-containing mRNA was accomplished in 5-20% (w/v) sucrose gradients in 0.02 M sodium acetate, pH 5.0. Approximately lo-20 A,, units of RNA in a 0.5-ml sample volume were heated at 80°C for 30 s, immediately cooled in an ice-water bath, and layered on 16.5-m] sucrose gradients in a Spinco SW 27.1 rotor. Centrifugation was performed at 26,500 rpm at 4°C for 16 h. Gradients were analyzed at 260 nm on a Gilford 250 spectrophotometer adapted to an ISCO flow cell. Fractionation of plaswmytoma tissue. Tumor tissue was fractionated according to described procedures (13, 34, 35). Briefly, tumor tissue was disrupted as described in the first RNA extraction method mentioned above and the homogenate was centrifuged at 10,000 rpm for 10 min at 4°C in a SS-34 rotor of a RC-2B s One A,,, unit is the amount of material that in 1.0 ml would yield a value of 1.0 for absorbance at 260 nm in a cuvette with a path length of 1.0 cm.

GREEN centrifuge (Sorvall). The resulting postmitochrondrial supernatant was layered over 5-ml layers of 2.3, 1.75, and 1.5 M sucrose in TMK buffer and centrifuged at 48,000 rpm for 19 h at 4°C in a 60 Ti Spinco rotor. The 1.75 M sucrose layer containing the membranebound polysomes was removed, diluted with 1 vol of TMK buffer, and layered over 5 ml of 1.3 M sucrose in TMK buffer and centrifuged for 50 min at 35,000 rpm in a 60 Ti Spinco rotor. The resulting pellets of membrane-bound polysomes were stored at -70°C. Preparation of EDTA-stripped membmnes. EDTAstripped membranes were prepared according to the procedures of Blobel and Dobberstein (35) with the following minor modifications. Plasmacytoma membrane-bound polysomes were suspended at 4°C in a buffer containing 50 mM Tris-HCl (pH 7.4) and 50 mM KC1 (TK buffer) to a concentration of 100 A&ml. A 0.2 M EDTA (pH 7.0) solution was added to a final concentration of 0.3 pmol EDTAIA,,, unit of membrane-bound polysomes. Aliquots (0.5 ml) of this suspension were layered onto lo-55% sucrose gradient in TK buffer. For larger preparations, a SW27 Spinco swinging bucket rotor was used, and centrifugation was 4 h at 25,000 rpm at 4°C. The turbid band of EDTA-stripped membranes were collected, diluted with 2 vol of TK buffer and centrifuged at 4°C for 50 min at 35,000 rpm in a Spinco 75 Ti rotor. The resulting pellets were stored frozen at -70°C. For use in protein synthesis assays the EDTA-stripped membranes were resuspended in a solution of 0.25 M sucrose in 50 mM Tris-HCl (pH 7.4), 2.5 mM MgCl,, 100 mM KCl, 7 mM 2-mercaptoethanol. Preparation of Krebs II ascites extract. The Krebs II ascites tumor was the generous gift of Dr. R. Thach. A preincubated 30,OOOgsupernatant fraction (S-30) was prepared from Krebs II ascites cells according to the procedure of Swan et al. (7). The supernatant fraction was further fractionated according to the procedures of Swan et al. (7) and Szczesna and Boime (17). Briefly, a ribosome-free supernatant was prepared from this extract by centrifugation of the preincubated S-30 at 50,000 rpm for 120 min in a 75 Ti Spinco rotor. A membrane-free ribosome pellet was obtained by overlaying an aliquot of the preincubated S-30 on a layer of 1.0 M sucrose in 30 mM Tris-HCl (pH 7.4), 5 mM MgCl,, 120 mM KCl, 7 mM 2-mercaptoethanol and centrifuging for 5 h at 50,000 rpm in a 60 Ti Spinco rotor. The ascites extract membrane fraction was prepared from the preincubated S-30 by collecting the material that accumulated at the 1.0 M sucrose interface, diluting it with 5 vol of 30 mM Tris-HCl (pH 7.4), 5 mM MgCl,, 120 mM KCl, 7 mM 2-mercaptoethanol and centrifuging at 40,000 rpm for 100 min in a type 75 Ti Spinco rotor. The membrane pellet was resuspended in 0.25 M sucrose in 50 mM Tris-HCl (pH 7.4), 2.5 mM MgCl,, 100 mM KCl, 7 mM 2-mercaptoethanol and stored at -70°C.

PROCESSING

OF IMMUNOGLOBULIN

Cell-free protein synthesis. The reaction mixtures contained the following components in a total volume (pH 7.4), 106 mM KCl, of 50 ~1: 35 mM Tris-HCl 2.5 mM MgCl,, 7 mM 2mercaptoethanol, 2.5-3.0 A,,,jml Krebs II ascites SlOO fraction, 4.0 A&ml Krebs II ascites ribosomes, 1.0-4.0 A,,,, units/ml plasmacytoma mRNA, 1 mM ATP, 0.1 mM GTP, 0.6 mM CTP, 5 mM creatine phosphate, 0.16 mgiml creatine phosphokinase, 5.0 &i [3H]leucine, and 40 pM concentrations of each of the unlabeled amino acids. Stripped-membrane preparations were added as indicated in the figure legends. Incubation was for 75 min at 30°C. Incorporation of radioactivity into hot trichloroacetic acid-precipitable material was determined on a 2 or 5.~1 aliquot from the reaction mixture by the procedure of Mans and Novelli (37). Gel elecfrophoresis and analysis of gels. Cell-free products were analyzed on discontinuous SDS-polyacrylamide gels according to previously described procedures (38). Samples were routinely analyzed in 12.5% polyacrylamide slab gels containing 0.1% SDS at 30 mA per gel (80-190 V during the run). The gel was prepared for autofluorogrdphy according to the method of Bonner and Lasky (39), dried, and then used to expose RP Royal X-omat X-ray film (Kodak) at -70°C. Quantition of radioactive bands on the autoradiograms or autofluorograms was accomplished by scanning the film at 550 nm in a Gilford 250 spectrophotometer equipped with a linear transport attachment. The area under the relevant peaks in a recording of the scan was then determined with the aid of a planimeter (Gelman Instrument Co). Partial proteolytic analysis oj’ cell-free products. Limited proteolysis of the light chain cell-free products by S. aureus V8 protease was performed according to the procedure of Cleveland et al. (40). In brief, the autoradiogram of a gel was overlayed on the dried gel, and the areas of the gel that contained the radioactive bands of interest were cut out with a scalpel. The dried piece of gel was rehydrated in 1.0 ml of sample buffer (0.125 M Tris-HCl (pH 6.8), 0.1% SDS, and 1 mM EDTA) for 30 min. The buffer was removed and the gel piece was frozen at -20°C until used. A gel slab was prepared with a 15% polyacrylamide resolving gel and a 4-cm stacking gel formed with 6-mm-wide slots. The sample slots were filled with sample buffer, and the thawed gel piece was pushed to the bottom of the well with a spatula. The spaces around the gel slice were filled by overlaying each slice with 10 ~1 of sample buffer containing201c glycerol. Finally, 10 ~1 of sample buffer containing 10% glycerol and the indicated amount of S. aureaus V8 protease was added to each sample well. Electrophoresis was begun at 30 mA constant current. When the bromphenol blue tracking dye reached the bottom of the stacking gel, the current was turned off for 30 min. Then electro-

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phoresis was resumed until the tracking dye reached the bottom of the resolving gel. Gels were prepared for autofluorography as described. Zmmunoprecipitation of cell-free products. Cell-free products were immunoprecipitated using antisera specific for the relevant light chain and S. aureus (Cowan I) cell walls as an immunoadsorbent according to the procedure of Kessler (41). Cell-free protein synthesis mixtures were brought to 0.5% NP40. After 5 min at 4”C, the samples were centrifuged at 5000g for 10 min, and the supernatant was saved. The appropriate antiserum was added to the sample (3 pl/lOO ~1 sample) and was incubated for 15 min at 25°C. An approximate amount (40 pl/lOO ~1 sample) of a 10% (v/v) suspension of S. aureus cell walls was then added, and the incubation was continued at 25°C for an additional 10 min. The mixtures were then centrifuged at 2OOOgfor 10 min. The supernatant was aspirated, and the pellet of cell walls was washed by three cycles of resuspension and centrifugation (2OOOgfor 10 min) in 0.05% NP40 in 50 mM Tris-HCl, 0.15 M NaCl, and 5 mM EDTA. For analysis of the immunoprecipitate by gel electrophoresis the pellet was resuspended in gel sample buffer and heated at 100°C for 2 min. The cell walls were removed by centrifugation at 20009 for 10 min, and the proteins in the supernatant were analyzed as described above. Analytical procedures. Protein concentrations were determined by the method of Lowry et al. (42). The A,,,, or A,,,, of membrane suspensions was determined from dilutions of an appropriate aliquot of the sample into a known volume of 0.1% (w/v) SDS in water. RESULTS

Cell-free Translation mRNA

of Phsmacytoma

The poly(A)-containing mRNA from the MOPC-104E, MOPC-315, and MOPC-46B tumor lines are complex mixtures of mRNAs ranging in size from approximately 9 to 18 S. The L-chain mRNAs were partially purified by fractionation of the total mRNA on sucrose density gradients. The mRNAs in the 11-14 S regions of the gradients were pooled, concentrated, and used to direct protein synthesis in a cell-free proteinsynthesizing system devoid of endogenous membranes derived from Krebs II ascites cells. Analysis of the cell-free products by electrophoresis in polyacrylamide slab gels is shown in Fig. 1. The L-chain mRNA preparations from the various tumor lines each direct the synthesis of a prominent protein species migrating in the region of

372

MICHAEL I mRNA

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FIG. 1. Cell-free synthesis and processing of L-chain precursors. Only the L-chain region of the gel is depicted. A. [3H]Leucine-labeled pLMLo4synthesis was carried out in the absence of membranes (Slots 1, 2) in the presence of 0.66 mg protein/ml Krebs II ascites cell membranes (Slots 3, 4) and in the presence of 0.74 mg protein/ml MOPC-315 EDTAtreated membranes (Slots 5, 6). Slot M shows the position of authentic [aH]L”ln4. Slots 2, 4, 6 (+Ab) depict the results of immunoprecipitation with antiserum to A, L-chains. B. [SH]Leucine-labeled pLM315 synthesis was carried out as described in A. Slot M shows the position of authentic [3H]LM315.Slots 2, 4, 6 (+ Ab) depict the results of immunoprecipitation with antiserum to X2 L-chains. C. [3H]Leucine-labeled PL”46B synthesis was carried out as described in A. Slot M shows the position of unlabeled urinary LM46B.

the homologous marker L-chain, The MOPC-104E (Fig. 1, Panel A, Slot 1) and the MOPC-315 (Fig. 1, Panel B, Slot 1) cellfree products, designated pL”lo4 and pL315, respectively, migrate more slowly than their homologous marker L-chains, while the MOPC-46B (Fig. 1, Panel C, Slot 1) cellfree product, pLM46B, migrates slightly faster than its homologous marker. Evidence for the identity of the relevant cell-free products to their homologous L-chains is provided by the analysis of the results of immunoprecipitation experiments employing antisera specific for the different classes of immunoglobulin L-chains (see Fig. 1). An antiserum specific for h1 L-chains, precipitates the pL”lo4 species (Fig. 1, Panel A,

GREEN

Slot 2) while an antiserum specific AZ L-chains precipitates the pLM315 species (Fig. 1, Panel B, Slot 2). Additionally an antiserum against LM46Bprecipitates the PL M46Bspecies (Fig. 1, Panel C, Slot 2). Further evidence for the identity of the cell-free products was provided by the results (data not shown) of partial proteolysis by S. aureus V8 protease of L”lo4 and PL M104of LM315and pLM315. The related proteins shared a similar pattern of leucinecontaining peptides. In other experiments, the peptide pattern obtained from the pLM46Bspecies was similar to that observed when unlabeled LM46Bisolated from the urine of tumor-bearing mice was digested with S. aureus V8 protease. All of the above data are consistent with the precursorproduct relationship of these pairs of proteins. Initial studies of the effects of the addition of membranes on the synthesis and processing of pL-chain species utilized two sources of processing activities: a membrane fraction from Krebs II ascites cells (43) and an EDTA-stripped RER preparation from the MOPC-315 tumor line. The EDTA treatment of the MOPC-315 RER removed >95% of the polysomes and resulted in a membrane preparation with no endogenous protein synthesis activity. Both these membrane preparations were capable of the specific cleavage of each of the precursors (see Fig. 1, Panels A, B, C; Slots 3, 5). It can also be seen that these processed forms can be immunoprecipiated by the appropriate antiserum (Fig. 1, Panels A, B, C; Slots 4, 6). In the case of the LM4’jBrelated cell-free products, it can be seen that in addition to a faster moving species, LM46B,the presence of ascites membranes results in the appearance of a more slowly migrating species (Fig. 1, Panel C, Slot 3). This more slowly migrating species can also be detected in the presence of EDTA-stripped RER preparations from various plasmacytoma lines (see Fig. 2, Panel C). Preliminary experiments have shown that this species binds to Con A-Sepharose absorbent and can be eluted with a-methylmannoside. These findings are consistent with terminal mannose covalently attached to this LM4’jB-related protein species. Whether the putative

PROCESSING

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glycosylation is of the pLM46Bor the LM46B form and the kinetic relationship between the different species observed in the presence of membranes are under investigation.

LIGHT

NAPMMT OSA31 NCNIOS E c54 I23456

mRNA

I

A.

Characteristics and Assay of the Cleavage Activity of Plasmacytoma Membranes

Further studies designed to characterize the precursor cleavage activity of plasmacytoma cells included three sources of processing activities: the membrane fraction from the Krebs II ascites cells described above, an EDTA-stripped RER preparation from canine pancreas (35), and EDTAstripped RER preparations from the MOPC-104E, MOPC-315, and TEPC-15 tumor lines. Representative preparations of membranes were assayed for cleavage activity in cell-free systems synthesizing PL M104,pLM315, and pLM46B. The results shown in Fig. 2 indicate that EDTA-stripped membranes from any of the plasmacytoma lines are capable of the specific processing of any of the cell-free L-chain precursors. In general, all of the plasmacytoma membrane preparations inhibit total protein synthesis to a greater extent than do the Krebs II ascites or the canine pancreas membrane preparations. Additionally, differences can be seen in the efficiency of the assay depending upon which L-chain mRNA is used to direct protein synthesis and the L-chain precursor that is undergoing cleavage. It appears that pL”lo4 is synthesized relatively efficiently even in the presence of membranes and is the most efficiently cleaved to its respective mature L-chain by all the membrane preparations (Fig. 2, Panel A). Furthermore, while the synthesis of pLM315is relatively resistant to the addition of membranes, pLM315itself appears to be more resistant to cleavage than pL”lo4. Finally, although the synthesis of pL”.=B is also relatively resistant to the addition of membranes, only a small fraction of the pL”46B is converted to the LM4”Bform, and it appears that only some of the membrane preparations are capable of converting the pLM4’jB into the more slowly migrating possibly glycosylated form.

373

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NAPMMT OSA3 NCNIOS E c54 I23456

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L-chain FIG. 2. Processing of [3H]leucine-labeled precursors by exogenous membranes. Only the L-chain region of the autofluorogram is shown. A. pL”lo4 synthesis was carried out in the absence of membranes (Slot 1) and in the presence of 0.66 mg protein/ml Krebs II ascites membranes (Slot Z), 0.60 mg protein/ml pancreas membranes (Slot 3), and 0.80 mg protein/ml MOPC-315, 0.72 mg protein/ml MOPC-104E, and 0.56 mg protein/ml TEPC-15 EDTAtreated membranes (Slots 4, 5, 6). B. PL~‘“‘~ synthesis was carried out as described in A. C. PL”“‘~~ synthesis was carried out as described in A. The positions of the authentic L-chains and the pL-chains are indicated.

Additional experiments designed to characterize the membrane-associated plasmacytoma precursor cleavage activity demonstrated that like the well-characterized canine pancreas and Krebs II ascites membrane preparations it acts upon the growing nascent chain and is incapable of cleaving a finished protein. Efficient cleavage was obtained only when the membranes were added at the start of the incubation. Furthermore, it was observed that the proteolytic activity present in plasmacytoma membranes was sensitive to low concentrations (0.03%, w/w) of the nonionic detergents Triton X-100, Nonidet-P40, and Kyro EOB.

374

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FIG. 3. Membrane concentration dependence of pL”lo4 cleavage. Synthesis of pL”lo4 was carried out in the presence of increasing amounts of EDTA-stripped MOPC-104E membranes in a 25-~1 reaction mixture. The [3H]leucine-labeled cell-free products were analyzed by SDS-polyacrylamide gel electrophoresis and autofluorography. The results of scanning the PL”‘“~-L~‘~~ area of the film at 550 nm are as follows: trace A, no membranes added; trace B, 3.0 pg membrane protein added; added. trace C, 6.0 wg membrane protein added; trace D, 12.0 wg membrane protein was measured by planimetry. The percentage The amount of pL”lo4 plus L”‘04 recovered inhibition of protein synthesis (A), the absolute amount of LM104 recovered (x), and the relative amount of LMLo4 recovered (0) are depicted in the inset as a function of the amount of membrane protein added. The calculations for the inset were as follows: percentage inhibition of protein synthesis = (PL Ml04 + LM104)presence ,,f membranes/(~L2f104 + LM104)abScnce nf mrmhranus x 100 and, as an example: relative amount of L”‘“4 recovered at 3.0 -g _ protein of added membrane = (L~104),,,,,,=S.Ulrl,/ (PL”‘04 + LM104hlemb=3.0~~l.

These results are consistent with the results obtained in other laboratories studying cotranslational processing systems. In an attempt to quantitate the precursor cleavage activity present in various plasmacytoma membrane fractions an assay was devised to measure the relative fraction of mature light chain produced in a cell-free protein synthesis incubation when membranes were present during protein synthesis. Since pL”lo4 was the precursor species that appear to be the most efficiently cleaved, it was chosen as the standard substrate for these assays. A typical assay is shown in Fig. 3. Synthesis of pL”lo4 is carried out in the presence of increasing amounts of MOPC104E EDTA-stripped membranes. The autofluorogram produced from the gel of the cell-free products was scanned in a Gilford spectrophotometer, and the percentage

inhibition of protein synthesis, the absolute amount of L”lo4, and the relative fraction of L”lo4 were determined. These results are displayed in the inset in Fig. 3. There is a marked inhibition of protein synthesis dependent upon the concentration of membranes added. This is reflected in the slight increase and then decrease in the absolute amount of L”lo4 recovered. When this inhibition of protein synthesis is taken into account, a positive correlation is found between the relative fraction of L”lo4 recovered and the amount of MOPC-104E membranes added. When these measurements are used to compare membrane preparations from the same tumor lines as well as from different tumor lines, the results summarized in Table I are obtained. The activities of all the different preparations of membranes vary only twofold when measured in this manner.

PROCESSING OF IMMUNOGLOBULIN TABLE I PRECURSORCLEAVAGEACTIVITY OFPLASMACYTOMA MEMBRANE PREPARATIONS~

Source of membranes

Concentration of membranes necessary for 50% cleavage of pLM’O4 (pg protein/25 ~1 incubation mix)

Experiment 1 MOPC-104E Prep. 1 MOPC-104E Prep. 2 MOPC-315 Prep. 1 MOPC-315 Prep. 2

6.5 7.5 5.0 6.7

Experiment 2 MOPC-104E Prep. 2 MOPC-315 Prep. 1 MOPC-315 Prep. 2

6.0 4.2 4.5

(I Synthesis of pLmo4 was carried out with [“Hlleucine in the presence of increasing concentrations of the designated membrane preparations. SDS-polyacrylamide electrophoresis and autofluorography were performed to visualize the cell-free products. The relative amounts of Lh’lo4in each sample were determined as described in Fig. 3. The results for each membrane preparation were graphed as depicted in the inset in Fig. 3, and the membrane protein concentration necessary for 50% cleavage of pLmo4 was determined by extrapolation.

DISCUSSION

These studies were designed to establish methods with which to investigate development of membrane-associated post-translational modification activities in lymphoid cells. The use of the plasmacytoma as a model of a terminally differentiated B lymphocyte provides a basis for the further analysis of the organization of membrane components responsible for cotranslational modification of immunoglobulin. The present experiments describe the specific proteolytic maturation of representative immunoglobulin light chain precursors performed by exogenous plasmacytoma membranes. The data are entirely consistent with and extend the existing data obtained about analogous processes in other systems. For example, the plasmacytoma membranes must be present at the start of incubation in order for cleavage to occur. Thus, they

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are not active on completed light chain per-cursors, but cleave only nascent protein chains. Additionally, the cleavage activity is extremely sensitive to low concentrations of nonionic detergents. Membranes prepared from each of the plasmacytoma lines are capable of cleaving each of the L-chain precursors. It appears, however, that the efficiency of processing is dependent upon which precursor is being used as the substrate. Partial, as well as complete sequences of the amino terminal precursor segments of several L-chains (44-48) and an H-chain (14) have been determined. These data, together with the results of experiments similar to the ones described in this report should allow a determination of the number of different immunoglobulin precursor cleavage sequencesand their relative cleavage efficiency. The cotranslational protein processing system depends upon the intrinsic activity of the mRNA used to direct polypeptide synthesis and upon the efficiency of the resultant precursor nascent chain as a substrate for the cleavage activity. Mixing experiments designed to examine competition between different precursor nascent chains for the cleavage activity would be useful in attempts to correlate the structure of different precursor segments with their substrate activity. Such experiments would be best carried out with more purified mRNA preparations in order to avoid possible complications due to competing non-L-chain mRNA. One of the major goals of this work is the identification of the plasmacytoma membrane components responsible for proteolytic processing of nascent protein chains. A simple, reproducible assay for precursor cleavage would greatly facilitate the accomplishment of this task. In order for processing to occur, membranes must be present at the start of the protein synthesis reaction. Thus, production of mature light chain depends upon de novo synthesis of light chain precursor. The assay for the measurement of the precursor cleavage activity of various membrane preparations is therefore, of necessity, a cotranslational assay. Several difficulties arise in efforts to use this assay for a detailed study of this

MICHAEL

type of membrane-associated activity. The dependence of this assay on de novo protein synthesis could make the results difficult to interpret in a situation where there is a high degree of nonspecific inhibition of protein synthesis. The likelihood of this happening is great when cellular membrane fractions and any contaminants contained therein are being added to protein synthesis incubations. The dependence upon de novo protein synthesis also necessitates the constant preparation of mRNA fractions enriched for L-chain mRNAs. It would clearly be easier to have on hand a supply of presynthesized L-chain precursors to use whenever necessary. The recent results of Jackson and Blobel (49) have described methods by which to overcome the above difficulties by the development of a post-translational assay using a detergent extract of canine pancreas EDTA-stripped microsomes as a source of precursor cleavage activity. The adaptation of these or similar methods to the plasmacytoma system should overcome the present dependence on the cotranslation assay employing intact membranes. However, the presently utilized cotranslational assay is adequate for a preliminary characterization of membrane preparations. REFERENCES 1. ANDERSSON, J., LAFLEUR, L., AND MELCHERS, F. (19’74) Eur. J. Immunol. 4, 170-180. 2. MELCHERS, F., AND ANDERSSON, J. (1974a) Transplant. Rev. 14, 76-130. 3. MELCHERS, F., AND ANDERSSON, J. (197413) Eur. J. Immunol. 4, 181-188. 4. SHANDS, J. W., PEAVY, D. L., AND SMITH, R. T. (1973) Amer. J. Pathal. 70, l-24. 5. SHOHAT, M., JANOSSY, G., AND DOURMASHKIN, R. R. (1973) Eur. J. Immunol. 3, 680-687. 6. POTTER, M. (1972) Physiol. Rev. 52, 631-719. 7. SWAN, D., AVIV, H., AND LEDER, P. (1972) PTOC. Nat. Acad. Sci. USA 69, 1967-1971. 8. MILSTEIN, C., BROWNLEE, G., HARRISON, T., AND MATHEWS, M. (1972) Nature New Biol. 239, 117- 120. 9. MACH, B., FAUST, C., AND VASSALLI, P. (1973) Proc. Nat. Acad. Sci. USA 70, 451-455. 10. SCHECHTER, I. (1973) Proc. Nat. Acad. Sci. USA 70, 2256-2260. 11. TONEGAWA, S., AND BALDI, I. (1973) Biochem. Biophys. Res. Commun. 51, 81-87. 12. SCHMECKPEPER, B. J., CORY, S., AND ADAMS, J. M. (1974) Mol. Biol. Rep. 1, 355-363.

GREEN 13. GREEN, M., ZEHAVI-WILLNER, T., GRAVES, P. N., MCINNES, J., AND PESTKA, S. (1976) Arch. Biochem. Biophys. 172, 74-89. 14. JILKA, R., AND PESTKA, S. (1977) Proc. Nat. Acad. Sci. USA 74, 5692-5696. 15. KEMPER, B., HABENER, J., ERNST, M. D., POTTS, J. T., AND RICH, A. (1976) Biochemistry 15, 15-19. 16. BOIME, I., MCWILLIAMS, D., SZCZESNA, E., AND CAMEL, M. (1976) J. Biol. Chem. 251, 820-825. 17. SZCZESNA, E., AND BOIME, I. (1976) Proc. Nat. Acad. Sci. USA 73, 1179-1183. 18. Cox, G., WEINTRAUB, B. D., ROSEN, S. W., AND MAXWELL, E. (1976) J. Biol. Chem. 251, 1723-1730. 19. CHAN, S. J., KEIM, P. L., AND STEINER, D. F. (1976) Proc. Nat. Acad. Sci. USA 73, 1964-1968. 20. SHIELDS, D., AND BLOBEL, G. (1977) Proc. Nat. Acad. Sci. USA 74, 2059-2063. 21. MAUER, R. A., STONE, R., AND GORSKI, J. (1976) J. Biol. Chem. 251, 2801-2807. 22. EVANS, G. A., AND ROSENFELD, M. G. (1976) J. Biol. Chem. 251, 2842-2847. 23. DANNIES, P. S., AND TASHJIAN, A. H., JR. (1976) Biochem. Biophys. Res. Commun. 70, 1180-1189. 24. LINGAPPA, V. R., DEVILLERS-THIERY, A., AND BLOBEL, G. (1977) Proc. Nat. Acad. Sci. USA 74, 2432-2436. 25. DEVILLERS-THIERY, A., KINDT, T., SCHEELE, G., AND BLOBEL, G. (1975) Proc. Nat. Acad. Sci. USA 72, 5016-5020. R. S., AND 26. SUSSMAN, P. L., TUSHINSKI, BANCROFT, F. C. (1976) Proc. Nat. Acad. Sci. USA 73, 29-33. 27. STRAUSS, A. W., DONOHUE, A. M., BENNETT, C. D., RODKEY, J. A., AND ALBERTS, A. W. (1977)Proc. Nat. Acad. Sci. USA 74,1358-1362. 28. PALMITER, R. D., GAGNON, J., ERICSSON, L. H., AND WALSH, K. A. (1977) J. Biol. Chem. 252, 6386-6393. 29. KATZ, F. N., ROTHMAN, J. E., LINGAPPA, V. R., BLOBEL, G., AND LODISH, H. F. (1977) PTOC. Nat. Acad. Sci. USA 74, 3278-3282. 30. ROTHMAN, J. E., AND LODISH, H. F. (1977) Nature (London) 269, 775-780. 31. LINGAPPA, V. R., LINGAPPA, J. R., PRASAD, R., EBNER, K. E., AND BLOBEL, G. (1978) Proc. Nat. Acad. Sci. USA 75, 2338-2342. 32. BRELINSKA, M., AND BOIME, I. (1978) Fed. Proc. 37, 1685 (Abstr.). 33. TUCKER, P., AND PESTKA, S. (1977) J. Biol. Chem. 252, 4474-4486. 34. BLOBEL, G., AND DOBBERSTEIN, B. (1975a) J. Cell Biol. 67, 835-851. 35. BLOBEL, G., AND DOBBERSTEIN, B. (1975b) J. Cell Biol. 67, 852-862.

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