Cotranslational cleavage and glycosylation of the mineral oil-induced plasmacytoma-46B κ chain precursor by plasmacytoma microsomes

ARCHIVES OF BIOCHEMISTRY Vol. 199, No. 1. January,

AND BIOPHYSICS pp. 37-42, 1980

Cotranslational Cleavage Plasmacytoma-46B MICHAEL Depnrtment

of Microbiology,

and Glycosylation of the Mineral Oil-Induced K Chain Precursor by Plasmacytoma Microsomesl GREEN

St. Louis Received

AND

liniuersity St. Louis, June

WAYNE School

MiSSOlLri

11, 1979; revised

E. GLEIBER

qf‘kfedicine. 63104

1402 South

Gmtrd

Boulevard,

.July 30, 1979

The mineral oil-induced plasmacytoma-46B light chain precursor, PL”““~, is a cotranslational acceptor for the transfer of the endogenous lipid-linked core oligosaccharides present in microsomal membrane preparations from plasmacytoma tissue as well as those from canine pancreas and Krebs II ascites tumor cells. The membranes tested appear to first cleave then glycosylate PL”~“~. All the membrane preparations tested were capable of cleaving and glycosylating only a fraction of the ~IL\‘~“” synthesized. The predominant neM form observed in the presence of ascites membranes was the cleaved, glycosylated species, Ltjz”. while the plasmacytoma and pancreas membranes produced both L>!,‘!!” and the cleaved species L”4B’3. These studies describe a system suitable for the study of the core glgcosylation activity of lymphoid cell membranes.

We are interested in developing assays with which to measure membrane-associated immunoglobulin cleavage and glycosylation activities in lymphoid cells. We have recently described a cotranslational assay for the immunoglobulin precursor cleavage activity of murine plasmacytoma intracellular membrane preparations (1). This report describes the use of the precursor of the MOPC-46B’ glycosylated light chain (2) as a cotranslational acceptor for the core oligosaccharide in a cell-free system capable of the specific proteolytic cleavage and glycosylation of the newly synthesized polypeptide chains. ’ This research was supported by USPHS 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. y Abbreviations used: MOPC. mineral oil-induced plasmacgtoma; PL”~““, precursor of MOPC-46B protein; L”4fi’3, cleaved PL\‘~““; L>!zfifi. cleaved, glycosylated pL”“““; SDS, sodium dodecrl sulfate; Con A, concanavalin A.

Previous investigations of this type of transfer reaction have utilized either posttranslational assays with suitably prepared protein acceptors or cotranslational assays. Thus, Pless and Lennarz (3) have described the preparation of artificial substrates from normal proteins with unglycosylated AsnX-Ser sequences by sulfitolysis in the case of ovalbumin and RNase A. Similarly, Struck et al. (4) have used S-carboxymethylation or S-aminoethylation to prepare suitable substrates from cu-lactalbumin. The recognition sequences exposed as a result of denaturation were able to accept an oligosaccharide moiety transferred by added cellular membranes. Struck et al. (4) have also used a peptide isolated from a-lactalbumin as an acceptor. Other studies have led to the development of a cell-free system capable of the post-translational glycosylation of a presynthesized MOPC-46B light chain precursor by Ehrlich ascites membranes in a two-stage reaction (5). Another approach is exemplified by the specific, sequential glycosylation of the precursor of vesicular stomatitis virus G protein in a cell-free system containing canine pancreas membranes (6) or in a coupled transcription-translation system in

38

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extracts of HeLa cells (7). The canine pancreas membranes were also shown to be capable of the glycosylation of the cr-lactalbumin cell-free product (8) and both pancreas membranes and oviduct membranes have been reported to be able to glycosylate the MOPC-46B light chain precursor (9). Membranes from Krebs II ascites cells were used to accomplish the glycosylation of the a-subunit of human chorionic gonadotropin (10). Our own experience with the MOPC-46B precursor (1) had indicated that there were several advantages in its use as a substrate with which to develop a cotranslational assay of the core glycosylation activity of lymphoid cell membranes. MOPC-46B light chain mRNA is an extremely efficient template in the Krebs II ascites cell-free system being used for these experiments. In addition, the MOPC-46B related cell-free products can be clearly resolved by SDS-gel electrophoretic analysis (1,s). Furthermore, the MOPC46B precursor represents a homologous substrate for the lymphoid cell membranes under investigation. Our present results with the MOPC-46B precursor indicate that plasmacytoma membranes cleave the MOPC-46B precursor before glycosylating it and that there is significant variation among membrane preparations in their abilities to glycosylate newly synthesized protein chains.

FIG. 1. Cotranslational processing of pL”““” by added membranes. [YH]Leucine-labeled PL\‘~“~ synthesis was carried out in the absence of membranes (l), in the presence of 0.3 and 0.5 mgiml pancreas membranes (2, 3), 0.3 and 0.6 mgiml ascites membranes, 0.1, 0.2, 0.4, and 0.8 mgiml MOPC-315 membranes (4-g), 0.13. 0.26, and 0.5 mgiml TEPC-15 membranes (lo-12), 0.2,0.4, and 0.8 mgiml MOPC-315 membranes, (13-15)0.09,0.18, and0.36mgiml MOPC104E membranes (16- 18). Cell-free products were analyzed by SDS-polyacrylamide gel electrophoresis and visualized by autofluorography. The positions of the MOPC-46B L-chain precursor (PL”~~“), the presumptive cleaved precursor (Lh’4F,R), and the presumptive glycosylated and cleaved precursor (L’14”“) are indicated.

GLEIBER EXPERIMENTAL

PROCEDURES

Materials. L-[4,5-“H]Leucine (60 Wmmol) and L-[““Slmethionine (ea. 1000 Wmmol) were obtained from AmershamiSearle. Triton X-100 and concanavalin A-Sepharose were purchased from Sigma Chemical Company. S. griseus endoglycosidase H was purchased from Miles Laboratories. Rabbit liver tRNA (stripped) was purchased from Gibco. General procedures. Maintenance of plasmacytoma lines, preparation of immunoglobulin mRNA, fractionation of plasmacytoma tissue, preparation of EDTAstripped membranes, preparation of Krebs II ascites extract, cell-free protein synthesis, gel electrophoresis, analysis of gels by fluorography, and other analytical procedures were all performed exactly as described previously (1). To prepare the translation products for SDS-gel analysis, the cell-free products were precipitated by the addition of 2 vol of 10% (w/v) trichloroacetic acid. The precipitates were then collected by centrifugation, dried, and dissolved in gel sample buffer. Chromatography on Con A -Sepharose. Cell-free products were analyzed on 2 x 0.5cm columns of Con A-Sepharose prepared in glass-wool stoppered Pasteur pipets. Before being used for experiments the columns were washed with .5 ml of a solution containing 0.1 mgiml bovine serum albumin, 0.2 M a-methylmannoside, and in wash buffer (1.0% Triton X-100 in 50 mM Tris-HCI, pH 7.4, 100 mM KCl, 2..5 mM MgCl,). The columns were then equilibrated with wash buffer. Cellfree reaction mixtures were prepared for chromatography by the addition of Triton X-100 to a final concentration of 1% (w/v) and centrifugation at 2000g for 10 min. Aliquots (50 or 100 ~1) of the supernatants were loaded on the columns and the columns were washed with five 1.0.ml portions of the wash buffer. The first 3 ml of the wash was pooled and the protein in the fractions was precipitated by the addition of trichloroacetic acid to a final concentration of 10% (w/v). Cellthat bound to the column were free products specifically eluted with three 1.0.ml portions of 0.8 M cymethylmannoside in wash buffer. The eluate was pooled and the proteins were precipitated as described above. The precipitates were collected by centrifugation, dried, and dissolved in gel sample buffer for analysis by SDS-gel electrophoresis. The Con A-Sepharose columns were then equilibrated with storage buffer (0.1 M sodium acetate (pH 6.1), 1.0 M NaCl, 1 mM MnCl,, 1 mM M&l,, 1 mM Call,, 0.02% (w/r) sodium azide) and stored in this buffer at 4°C. Endoglycosidase H treatnzent. Cell-free reaction mixtures were brought to a final concentration of 0.5% SDS (w/v) and 1% 2.mercaptoethanol and heated in a boiling xvater bath for 1 min. An equal volume of 0.3 M sodium citrate was then added and the sample Leas divided into two parts. Endoglycosidase H was added at a concentration of O.OO.j-0.01 unit/50 ~1 to one portion and both samples were incubated at 37°C for 18 h. The cell-free products were then precipitated by the

CLEAVAGE

AND

GLYCOSYLATION

OF IMMUNOGLOBULIN

addition of 2 vol of 10% (w/v) trichloroacetic acid. The precipitates were collected by centrifugation, dried, and dissolved in gel sample buffer for analysis. Preporation of f[35S]Met-tRNA,Mr’. AminoacytRNA synthetases and Met-tRNA>“’ transformylase from E. co/i K-12 were prepared exactly as described by Stanley (11). Specific aminoacylation and formylation of tRNA>‘” contained in a sample of crude rabbit liver tRNA was accomplished by the use of the E. coli enzymes cacodylic acid (pH 7.4 with NaOH). 10 mM ATP, 1 mM CTP. 15 ItIM MgCl,, 50 pM L-[:“S]methionine (30-50 Ci/mmol), 0.1 mM each of the other amino acids, 0.1 mM calcium leucororin. 25 mgiml crude rabbit liver tRNA. and 7.5 mg protein/ml E. coli enzyme fraction. After incubation at 37°C for 30 min. the fl”“S]Met-tRNAt”” was recovered by phenol extraction and gel filtration through Sephadex G-50 as described (11). RESIJLTS

Modification of the Cell-Free Translation Product of M46B mRNA by Added Membranes In a cell-free system derived from Krebs II ascites cells MOPC-46B mRNA has been shown to direct the synthesis of a single major product which migrates slightly faster than native MOPC-46B protein in SDSpolyacrylamide gels (1). Evidence derived from immunological analysis and limited proteolysis of this product have demonstrated that this species is the precursor form of the MOPC-46B light chain, pL”46H (1). When the cell-free protein synthesizing system is supplemented with various membrane preparations, two additional products appear, one migrating faster and one migrating more slowly than pLb’4fiB (Fig. 1). In the presence of EDTA-stripped canine pancreas membranes (slots 2,3) the faster migrating species (Lh’4fiH) is produced in greater amounts than the slower one (Lz<,GR), while in the presence of Krebs II ascites membranes (slots 4,5) L$B” is the predominant ne\v form. When various EDTAstripped plasmacytoma membrane preparations are tested as sources of processing activities both new protein species are tletectetl (slots (i-18). The relative amount of each, however, varies with the source of the membrane preparation. All the plasmacytoma membranes yield similar amounts. of L”‘“” but MOPC-315 membranes routinely produce the best yield of I~$;::“‘~.

LIGHT TIME

39

CHAIN

(MIN)

MEF;;:;ES 0

6 12 1825355075

A.

-,L;,4,68

ASCITES

0

6 12 1825355075

B. PANCREAS

0

6

Jr

LM46B my

-

pLM46B

\

~mLM46B

I2 18 25355075

LM46t7

C. /

G’y

\LM46B

FIG. 2. Time course of PL\“‘“~

processing by ascites membranes (0.3 mgiml) (A), pancreas membranes (0.3 mgiml) (B), and MOPC-315 membranes (0.4 mgiml) (C). [“H]Leucine-labeled pL ‘W synthesis was initiated in the presence of membranes. At the indicated times, 50.~1 aliyuots were removed, brought to 0.05% (w/w) Triton X-100, and incubated until a total of 75 min had elapsed. The cell-free products were analyzed by SDSpolyacrylamide gel electrophoresis and visualized by autofluorography.

In order to investigate the relationship between the different species observed in the presence of membranes, kinetic studies of the maturation of pLh’46Bwere performed. In these studies, pL”14’jB synthesis was begun in the presence of various membrane preparations, but at the indicated times after initiation of protein synthesis aliquots of the original mixture were brought to a final concentration of 0.05% (w/w) Triton X-100 and protein synthesis was allowed to continue until a total of 75 min had elapsed. Since the membrane-associated modification activities are inactivated by this concentration of Triton X-100 the final products reflect what has occurred in the presence of intact membranes up until the time of addition of detergent. Figure 2 shows an SDS-polyacrylamide gel analysis of typical experiments. In the presence of ascites membranes (A) no species moving more rapidly than PL M46Ris observed and LE,$!Hiq. detectable at 12 to 18 min. In the presence of pancreas membranes (B), detectable L4’4”B (12 min) precedes detectable L!i!’ (25 min) by about 13 min, while in the caseof M315 membranes (Cl L1’46B(12 min) precedes Lg&!B (18 min) by only 6 min.

40

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ASCITES

AND

GLEIBER

the results of treatment of the LM46Brelated products with endoglycosidase H from S. T KCH3 T KCH3 griseus (12), an enzyme which is specific for 0 FT 0 FT T MAN T the chitobiose linkage of the high mannose MAN LM46EI core oligosaccharide moieties (Fig. 4). It can /GLY be seen that while the enzyme has no effect pLM46B on pLM46B, it converts the LEp,GB species \ LM466 produced in the presence of ascites membranes to a form which migrates identically FIG. 3. Analysis of PL”‘~~” related products on Con with Lh’46R. Similar results were obtained A-Sepharose. [YH]Leucine-labeled PL”~~~ synthesis in the case of the L&‘$6B produced in the was carried out in the absence of membranes (slots presence of plasmacytoma membranes. l-3), in the presence of 0.3 mgiml ascites membranes These results further support the idea that (slots 4-6), and in the presence of 0.4 mg/ml MOPC-315 membranes (slots 7-9). Aliquots of the reaction mixLg‘““~ could proability of the cell-free products to bind to duce these results by interfering with the Con A-Sepharose was determined. As can cotranslational processing of the precursor. be seen in Fig. 3, a large fraction of all the MOPC-46B related products are retained on We have observed, however, that there is the columns. (Compare slots 1 to 2, 4 to 5, 7 to 8.) It has also been observed that the NONE ASC relative amount of retention of PL”~“~ and 7-x-T L”14’jB varies with each experiment. For (+E) (+ El these reasons, it was felt that it was necessary to observe that a protein can be bound M46B to the column and can be eluted from the LGLY f M46B column by a-methylmannoside in order to -PL classify it as a glycoprotein. The results in 7. LM46B Fig. 3 demonstrate that L$,‘zR is the only MOPC-46B related species that can both FIG. 4. Effect of S. griseus endoglycosidase H on bind to Con A-Sepharose and be specifically ]?H]Leucine-labeled PL”4fiU related cell-free products. eluted with a-methylmannoside. These was carried out in the absence (slots PL”““R synthesis findings are consistent with the presence of 1,2) or presence of ascites membranes (0.3 mgiml; mannose in an oligosaccharide moiety covaslots 3,4). The reaction mixtures were then divided lently attached to the MOPC-46B cell-free into two equal portions. Endoglycosidase H was added product. (0.005 unit/50 ~1) to one of the portions and the samples Additional evidence that Lg$B is the were incubated and prepared for SDS-gel electroglycosylated form of pLM4”B is provided by phoresis as described under Experimental Procedures. m5--i--Tm i KCH3 0 FT T MAN

M315

CLEAVAGE

AND

GLY’C‘OSYLATION

no significant difference between the distribution of MOPC-46B related forms produced in the presence of membranes when the precursor was synthesized in the presence of [“Hlleucine or in the presence of both [“Hlleucine and f[“~S]Met-tRNAr”’ (data not shown). We therefore consider this possibility unlikely. The results shown in Fig. 5 demonstrate that while all the MOPC-46B related forms can be detected Lvhen protein synthesis is carried out in the presence of leucine (slots l-5) only pL.L’4”B can be seen when the radioactive label is donated to the amino terminus of the primary translation product (slots 6- 10).

The present experiments describe the use of the MOPC-46B light chain precursor, acceptor for the PLS’-IHH>as a cotranslational transfer of the endogenous lipid-linked core oligosaccharides present in various microsomal membranes. The results presented here are entirely consistent with and extend the existing data obtained about similar processes in other systems (6-10). The data also demonstrate that pL114”B provides a readily available substrate with easily distinguishable modification products. The assay for core glycosylation developed as a result of the analysis of the effect of membranes on the cell-free translation of MOPC46B L-chain mRNA also provides useful insights into the properties of such membrane activities. The combination of proteolytic precursor cleavage and glycosylation could theoretically result in four different species related to a particular primary translation product: (a) the precursor itself and (b) the uncleaved glycosylatetl, (c) the cleaved but unglycosylated, and (d) the cleaved and glycosylated species. Only three of these possible forms were detected. The uncleaved, glycosylated form, which would be expected to migrate more slowly than the cleaved, glycosylated form, Ltj;V, was not detected. It IS possible, however, that this type of species could exhibit an unusual migration behavior on SDS-gels and move to a position where it would be hidden in the other bands. Aside from this, the simplest explanation for this

OF

1MMUNOGLOBI:LIN

NAAPMNAAPM O’SSA3OSSA3 N C CN E I2 3 4

LIGHT

I 5 5

NCCN E 678910

41

CHAIS

I 5 M46B

J- rLGLYp~M46B -L

LM46B

FIG. 3. Labeling of p1,“““~ by f[:‘“S]Met-tRNA~““. Synthesis of PL”~“” \vas carried out using [:‘H]leucine (slots l-3) or ~‘“S]methionine (100.000 cpm/X ~1; slots 6- 10) as the labeled precursor. Cell-free synthesis was carried out in the absence of membranes (slots l,(i), in the presence of 0.3 or 0.5 mg/ml ascites membranes (slots Z,‘i ant1 3,8, respectively), 0.3 mgiml pancreas membranes (slots 4,9), and 0.35 mg/ml MOPC-315 membranes (slots 5,lO). Cell-free products were prepared for analysis by gel electrophoresis as tiescribed under Experimental Procedures.

finding is that the nascent precursor is cleaved first upon its interaction with the membranes and then is subsequently glycosylated while, or after, it is transported through the membranes. The results of the experiments designed to investigate the kinetics of modification of pL”4”D (see Fig. 2) as well as the observation that the original amino terminus is only found in pL\‘““’ (see Fig. 5) are consistent with this explanation. It is also possible that the rate of cleavage of the putative glycosylated precursor is much faster than that of the unglycosylated precursor. Thus, if this species formed it would be immediately cleaved to Ltj;t$” and go undetected upon analysis of the cell-free products by SDS-gel electrophoresis. This less attractive explanation cannot be formally disproved by the present studies. The results of experiments investigating analogous reactions in other systems, ho\vever, are also consistent with the former, simpler explanation ((i-10). The results of screening membrane preparations for the rate of and extent of PL”“~~’ modification showed that all the membrane preparations tested were capable of cleaving and glycosylating only a fraction of the synthesized. In the case of the ascites PL h14fiR membranes the predominant modified form observed was Ltjz;E3. This is probably because

42

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the rates of cleavage and glycosylation are nearly equal in the ascites membrane preparations. On the other hand the plasmacytoma and pancreas membranes produced approximately equal amounts of L”146B and Lg$jR. It is likely that the rate of glycosylation in these membranes is limiting. The observed variation in the relative rates and amounts of cleavage and glycosylation could be due to: (a) differences in the concentration of endogenous lipid-linked oligosaccharide intermediate, (b) differences in the concentration or activity of the membrane-associated core oligosaccharide transferase, (c) a combination of both of these factors, or (d) the existence of two kinds of nascent protein modification sites, one bearing only precursor cleavage activity and the other bearing both cleavage and glycosylation activity. These possibilities could also explain the observed variability between membrane preparations from different plasmacytoma lines and among membrane preparations from the same tumor line. Further studies with this and similar systems are necessary to resolve these questions. These studies were designed to develop an assay for the core glycosylation activity of lymphoid cell membranes. This assay provides a basis for the further analysis of the organization of membrane components

GLEIBER

responsible modification

for this type of cotranslational of immunoglobulin chains. REFERENCES

1. GREEN, M. (1979) Arch. Biochem. Biophys. 195, 368-377. 2. MELCHERS, F. (1971) Biochem%stry 10, 653-659. 3. PLESS, D. P., AND LENNARZ, W. J. (1977) PTOC. Nat. Acad. Sci. USA 174, 134-138. 4. STRUCK, D. K., LENNARZ, W. J., AND BREW, K. (1978) J. Biol. Chew 253, 5786-5794. 5. TUCKER, P., AND PESTKA, S. (1977) J. Biol. Chem. 252, 4474-4486. 6. ROTHMAN, J. E., AND LODISH, H. F. (1977) Nature (London) 269, 775-780. 7. IRVING, R. A., TONEGUZZO, F., RHEE, S. H., HOFMANN, T., AND GHOSH, H. P. (1979) Proc. Nat. Acad. Sci. USA 76, 570-574. 8. LINGAPPA, V. R., LINGAPPA, J. R., PRASAD, R., EBNER, K. E., AND BLOBEL, G. (1978) Proc. Nat. Acad. Sci. USA 75, 2338-2342. 9. BRINKLEY, S. A., DAS, R. C., AND HEATH, E. C. (1979) Fed. Proc. 38, 620 (Abstr. 2070). 10. BRELINSKA, M., AND BOIME, I. (1978) PrOC. Nat. Acad. Sci. USA 75, 1768-1772. 11. STANLEY, W. M., JR. (1972) Anal. Biochem. 48, 202-216. 12. TARENTINO, A. L., AND MALEY, F. (1974)J. Biol. Chem. 249, 811-817. 13. HOUSMAN, D., JACOBS-LORENA, M., RAJBHANDARY, U. L., AND LODISH, H. F. (1970) Nature (London) 277, 680-685.