Proc. Natl. Acad. Sci. USAVol. 75, No. 4, pp. 1768-1772, April 1978Biochemistry

mRNA-dependent synthesis of a glycosylated subunit of humanchorionic gonadotropin in cell-free extracts derivedfrom ascites tumor cells

(glycoprotein hormone/secretory protein/in vitro protein synthesis/membrane-dependent sugar transfer)

MALGORZATA BIELINSKA AND IRVING BOIMEDepartments of Obstetrics and Gynecology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110

Communicated by Oliver H. Lowry, February 8,1978

ABSTRACT Protein synthesized in ascites cell-free extractsin response to first trimester placental mRNA was immu-noprecipitated with antisera directed against the a subunit ofhuman chorionic gonadotropin. The immunoprecipitable pro-teins were resolved on sodium dodecyl sulfate/polyacrylamideslab gels. In membrane-depleted extracts placental mRNA di-rected the synthesis of the "preprotein" form of the a subunit.However, when membranes were added to the cell-free extractsa protein migrating more slowly than pre-a subunit was ob-served. This protein was specifically adsorbed to a concanavalinA column, and its migration on sodium dodecyl sulfate gels wasenhanced after treatment with a-mannosidase (EC 3.2.1.24) orendo-p-N-acetylglucosaminidase CII or H. Similar results wereobtained when mRNA was translated in lysates derived fromfirst trimester placenta instead of in ascites cell extracts. Kineticstudies of the glycosylation reaction revealed that sugar at-tachment can occur just prior to release of the protein. Thesedata show that the apoprotein of the a subunit can be glycosyl-ated in vitro. The data also suggest that the glycosylated proteincontains a sugar core consisting of di-N-acetylchitobiose andat least four mannose residues, some of which are a-linked. Inaddition, it a pears that this carbohydrate unit is present inhomologous p acental membranes as well as in the ascites tumormembranes.

Chorionic gonadotropin (hCG), a major peptide hormoneelaborated by the human placenta in early pregnancy, iscomposed of two glycosylated subunits, a and (3. However, thesesubunits are not produced in equal quantities in placenta, cul-tured choriocarcinoma cells, or various ectopic tumors (1-8).The a subunit is the predominant peptide synthesized in cell-free extracts derived from first trimester tissue and far exceedsthe subunit (9). This is consistent with the observation that themRNA encoding the a subunit represents a large fraction of themRNA in first trimester tissue (10).The a subunit of the circulating hormone contains two oli-

gosaccharide chains which represent 30% of its molecularweight (11, 12). The sugar units, which are linked through an

N-glycosidic bond to asparagine residues, contain N-acetyl-glucosamine and mannose residues in the core. Linked to thiscore structure are outer chains of sialic acid and galactoseresidues.

Evidence from several laboratories indicates that a di-N-acetylchitobiose-mannose unit is transferred to a variety ofapoproteins en bloc from a dolichol intermediate contained inmicrosomal membranes (13-18). The terminal sugars are thensequentially attached to the growing glycose chain as the pro-tein migrates through the secretory pathway (19, 20).The initial attachment of the oligosaccharide unit occurs on

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked"advertisement" in accordance with 18 U. S. C. §1734 solely to indicatethis fact.

unfinished polypeptide chains (21-24). Since membranes ofthe endoplasmic reticulum participate in another nascent-protein modification, i.e., the cleavage of the presegment of avariety of secretory proteins (25-27), these membranes pre-sumably play a role in the events of glycosylation. Because sugarattachment to a variety of exported proteins might be requiredfor their secretion, glycosylation may play a -critical role incontrolling the extracellular levels of the hormone. Studies re-lated to this point and to the role of glycosylation in the syn-thesis-secretion of hCG would be greatly facilitated if transferof sugars to the polypeptide chain could be monitored in amRNA-dependent, cell-free protein-synthesizing system. Re-cent studies have revealed that microsomal membranes canglycosylate vesicular stomatitis virus proteins in an in vitrosystem (24, 28). In the present work we show the membrane-dependent glycosylation of the a subunit of hCG in cell-freeextracts derived from ascites tumor cells and first trimesterplacenta. Data are also presented concerning the structure ofthe attached oligosaccharide.

MATERIALS AND METHODS[S5S]Methionine was obtained from Amersham and New En-gland Nuclear. Concanavalin A coupled to agarose, a-methyl-D-mannopyranoside, and D-galactose were purchasedfrom Sigma. Purified endo-fl-N-acetylglucosaminidase CII (29)was a gift from E. Li. Endo-f3-acetylglucosaminidase H wasobtained from Miles Laboratories. Antisera generated againsthighly purified reduced and carboxymethylated subunits ofhCG were prepared by S. Birken and R. Canfield and weregenerously supplied to us. a-Mannosidase (EC: 3.2.1.24) andfl-galactosidase (EC 3.2.1.23) were purchased from Boehrin-ger Mannheim.The preparation of cell sap (S-100), ribosomes, and mem-

branes derived from ascites tumor cells and the preparation ofplacental homogenates as well as placental RNA have beendescribed (25). Protein synthesis in the presence of 0.1 AM[35S]methionine was assayed in cell-free extracts according toSzczesna and Boime (25).

Immunoprecipitation of Cell-Free Products. The synthe-sized proteins were immunoprecipitated as follows: after in-cubation, the reaction mixtures were made 1% in Triton X-100and sodium deoxycholate and then centrifuged at 100,000 Xg for 60 min to remove ribosomes. Aliquots (100-150 Ml), theequivalent of about 200,000 cpm, were added to reactionmixtures containing 3-7 Ml of a subunit antisera or normalrabbit serum, and sufficient phosphate-buffered saline to bring

Abbreviations: hCG, human chorionic gonadotropin; NaDodSO4, so-dium dodecyl sulfate; buffer A, 30 mM Tris, pH 7.5/5 mMMg(OAc)2/120 mM NaCl; Con A, concanavalin A.

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the volume of the reaction to 300 ,A. After the mixtures. wereincubated for 16 hr at 40, 25 ,l of sheep anti-rabbit antisera wasadded; incubation was continued for an additional 2 hr at 24°.The precipitates were centrifuged at 3000 X g for 10 mfn andwashed three times with phosphate-buffered saline. The im-munoprecipitates were dissolved in sodium dodecyl sulfate(NaDodSO4) buffer (25) and analyzed on 20% NaDodSO4/polyacrylamide gels (25). The gels were dried, impregnatedwith 2,5-diphenyloxazole and exposed to Kodak RP-10 x-rayfilm (fluorography) (30).Concanavalin A Affinity Chromatography. Concanavalin

A (Con A) coupled to agarose was packed into 3-ml syringes andwashed extensively first with 30 mM Tris, pH 7.5/5 mMmagnesium acetate/120 mM NaCl (buffer A), with buffer Acontaining 0.2 M a-methyl-D-mannopyranoside according tomodifications of Stohlman et al. (31).The column was further washed with buffer A containing

0.1% Triton X-100, then 500 Ml of reaction mixture was appliedto the column and left for 30 min at room temperature. Thecolumn was then washed with buffer A containing 0.1% TritonX-100. The material bound to the column was eluted with 0.2M galactose or 0.2 M a-methyl-D-mannopyranoside. Thefractions obtained after elution were pooled and dialyzedovernight against 100 volumes of 1:6 dilution of buffer A. Thedialyzed material was lyophilized and dissolved in an amountof distilled water corresponding to 0.1 of the original dialyzedvolume. The products emerging from the column before andafter sugar elution were immunoprecipitated as describedabove.

Glycosidase Modifications of Cell-Free Products. Immu-noprecipitates obtained from 150 Ml of reaction mixture weredissolved in 50 mM sodium acetate, pH 6.0/0.1 M 2-mercap-toethanol/0.5% Triton X-100/0.8% NaDodSO4; this mixturewas heated for 2 min at 100°. After cooling, 25 Mg of a-man-nosidase (EC 3.2.1.24) was added and the mixture was incu-bated for 24 hr at room temperature. The proteins were thenprecipitated with 10% trichloroacetic acid and prepared forNaDodSO4/polyacrylamide gel electrophoresis as described.Experiments with endoglycosidases CII and H were performedunder the same conditions.

RESULTSAll reaction mixtures were immunoprecipitated with antiserumto a subunit. The immunoprecipitates were dissolved in Na-DodSO4 buffer and the labeled precipitable proteins were re-solved on NaDodSO4/polyacrylamide gels.

Although the molecular weight of the circulating form of theglycosylated a subunit is 15,500, it migrates on NaDodSO4 gelsas if it had a molecular weight of 22,000 (Fig. 1). This is con-sistent with previous reports that the presence of sugar retardsthe migration of proteins in NaDodS04 gels (32). First trimesterRNA, in an ascites membrane-free system, directed the syn-thesis of a protein with a molecular weight of about 14,000which was precipitated with a-subunit antisera (Fig. 1, lane 2).This protein was not observed when normal rabbit serum wasused in place of a-subunit antiserum (lane 1) or in the presenceof antiserum directed against the ,B subunit (data not shown).In addition, as expected, the appearance of labeled a-subunitprotein was eliminated when unlabeled purified a subunit wasadded to the immunoprecipitation reaction mixture (lane 4).Little, if any, competition was observed when # subunit wasadded (lane 3).

This labeled protein (band A) contains 24 additional aminoacids at the NH2 terminus of the mature form of the a-subunitapoprotein (molecular weight 10,500) and corresponds to the

2 3 4 5 6

a -*

Lyso--

-D-C

-A

FIG. 1. Fluorograph of a NaDodSO4polyacrylamide gel elec-tropherogram of [35S]methionine-labeled proteins isolated by im-munoprecipitation. Reconstituted S-100-ribosome extracts derivedfrom ascites tumor cells were incubated in the presence (lanes 5 and6) and absence (lanes 2-4) of membranes. The ribosomes were re-moved by centrifugation and aliquots from the supernatant fluid weretreated with antiserum to a subunit. The immunoprecipitates weredissolved in NaDodSO4 buffer and applied to NaDodSO4polyac-rylamide gels; 3000-6000 cpm were applied to the gel. a and Lyso referto the migrations, respectively, of mature a subunit and lysozyme(molecular weight 14,000). (Lane 1) Normal rabbit serum was sub-stituted for the a subunit antiserum. (Lane 2) Antiserum, no mem-brane; (lane 3) 40 pg of unlabeled (8 subunit added before a antiserum,no membrane; (lane 4) unlabeled subunit a added before a antiserum,no membrane; (lane 5) antiserum, plus membrane; (lane 6) unlabeleda subunit added before a antiserum, plus membrane.

preform of the subunit (S. Berkin, J. Fetherston, R. Canfield,and I. Boime, unpublished data). We attempted to cleave it withascites membranes as was demonstrated for human pre-pla-cental lactogen (25). However, instead of the appearance of asmaller form, proteins with apparently larger molecular weightswere observed (Fig. 1, lane 5). The migration of one majorprotein, band D, was similar (but not identical) to that of maturea subunit. These labeled proteins synthesized in the presenceof membranes were not observed on the NaDodSO4 gels whenpurified a subunit was added to the immunoprecipitationmixture (lane 6). (The appearance of the less prominent bandC was quite variable and its levels depend on the batch ofmembranes used.) Because of their migration, we suspected thatthese heavier proteins were glycosylated.

It appears that glycosylation of a variety of proteins involvesthe attachment of a glycose core containing several mannoseresidues. Since Con A binds glycoprotein containing mannoseat the nonreducing ends of the oligosaccharide (33), we at-tempted to identify a possible glycosylated form of the a sub-unit using this lectin. Proteins synthesized in the presence ofmembranes were applied to columns containing Con A (Fig.2). After the columns were washed thoroughly, material ad-sorbed to the column was eluted with monosaccharides. Proteinseluted from the column were then precipitated with a-subunitantiserum and identified on NaDodSO4 gels. No detectableprotein corresponding to band D was present in the columnwash (lane 3). Elution was then attempted with 0.2 M galactose,which does not bind to Con A. This did not release detectableimmunoreactive protein (Fig. 2, lane 4). However, after sub-sequent elution with 0.2 M a-methylnannoside, labeled proteinemerged from the column, comigrating with the major productsynthesized in the membrane-supplemented system (lane 5).No detectable mannoside-releasable material was observed forprotein synthesized in the absence of membranes that was ap-plied to the column (lane 6). These data strongly suggest thatthe heavier a protein (protein D) is glycosylated and containsmannose residues.To investigate the sugar composition of protein D, we treated

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2 3 4 5 6

'01q

3 ;-

to

FIG. 2. Con A affinity chromatography of proteins synthesizedin ascites extracts. Proteins synthesized in the presence (lanes 2-5)or absence (lanes 1 and 6) of membranes were applied to columnscontaining Con A. The columns were washed with buffer A containing0.1% Triton X-100. The columns were then eluted with this buffercontaining 0.2 M galactose, which was followed by elution with 0.2Ma-methylmannoside. The eluates were dialyzed and Iyophilized andaliquots were immunoprecipitated with a antiserum. The precipi-tates were dissolved in NaDodSO4 and applied to NaDodSO4poly-acrylamide gels, which were subjected to fluorography; 2000-3000 cpmwere applied to the gel. Total protein synthesized in theabsence (lane1) and presence (lane 2) of membranes;immunoprecipitated with aantisera and not fractionated on the column. (Lane 3) Material elutedwith the first buffer wash containing no sugar. (Lane 4) Protein elutedwith galactose. (Lanes 5 and 6) Elution with a-methylmannoside.

it with a variety of glycosidases. a-Mannosidase apparentlyremoved most of the sugar from the glycosylated product, as

shown by its shift in migration (Fig. 3, lane 3). As a result of thistreatment, a new product (band B) migrating slightly more

slowly than the nonglycosylated product was observed. Themigration of band D was not sensitive to the action of p3-ga-lactosidase (data not shown), which suggests the absence ofnumerous galactose residues at the nonreducing end of the at-tached core. Similar enzymatic treatments of the nonglycosy-lated form (lane 6) did not alter its migration, which suggeststhat the enzymatic changes of the glycosylated product were

not related to possible proteolytic action. These results indicatethat protein D contains several mannose residues that are linkedtogether in a-anomeric configuration.

2 3 4 5

-.-BandB

FIG. 3. (Lanes 1-4) Effects of a-mannosidase and endoglycos-idase CII on the proteins synthesized in membrane-supplementedascites extracts. Products immunoprecipitated with a subunit anti-serum were dissolved in a sodium acetate buffer. After incubation witheither a-mannosidase (lane 3) or endoglycosidase CII (lane 4) theproducts were precipitated with trichloroacetic acid and prepared forNaDodSO4 gels, which were subjected to fluorography; 4000-6000cpm were applied to the gel. (Lane 2) Addition of a-mannosidase at0 time to a reaction mixture which was then immediately precipitatedwith acid. (Lane 1) Reaction mixture incubated in the absence ofenzymes. (Lanes 5 and 6) Immunoprecipitable protein synthesizedin the absence of membranes: (lane 5) protein incubated in the ab-sence of enzymes, (lane 6) incubation with a-mannosidase.

FIG. 4. Fluorograph of NaDodSO4Jpolyacrylamide gel electro-pherogram of proteins synthesized in ascites membrane-supple-mented extracts (lanes 2 and 3) and in placental S-30 extracts (lanes4-6) and immunoprecipitated with a subunit antiserum. (Lane 1)Placental extract, normal rabbit serum instead of a antiserum. a-

Mannosidase was added to immunoprecipitated proteins synthesizedin ascites (lane 3) and placental (lane 5) cell-free extracts. Additionof endoglycosidase H to immunoprecipitable proteins synthesizedin placental extracts is also shown (lane 6). No addition of enzymesto reaction mixtures containing protein synthesized in ascites (lane2) or placental (lane 4) extracts.

To further characterize the nature of the oligosaccharide wetreated the original labeled product with endoglycosidase CII(Fig. 3, lane 4). This enzyme splits the asparagine-bound di-N-acetylchitobiose, leaving behind a terminal asparaginyl-linked N-acetylglucosamine. Endoglycosidase C11 activity alsorequires the presence of at least four mannose residues linkedto the di-N-acetylchitobiose (34). Treatment with this enzymecaused a shift in the migration of the glycosylated protein (lane4). Significantly, the product of the endoglycosidase treatmentappears to migrate with the nonglycosylated protein. A minorprotein migrating slightly faster than A was observed (lane 4,arrow). Identical results were obtained with endoglycosidaseH, whose specificity is similar to that of endoglycosidase CII(34). The data suggest that the cell-free product contains di-N-acetylchitobiose and at least four mannose units.The question arises how the structure of the glycosylated

product synthesized in a heterologous cell-free system comparesto that synthesized in homologous extracts derived from firsttrimester placenta. Translation of first trimester RNA in a

placental 30,000 X g supernatant extract that contained ho-mologous membranes resulted in the appearance of a proteincomigrating with the ascites glycosylated protein synthesizedin the ascites cell-free system (Fig. 4, lane 4). It exhibited similarsensitivity to a-mannosidase (lane 5) and endoglycosidase H(lane 6). Thus, membranes derived from two dissimilar tissuesapparently contain similar, if not identical, glycose units whichcan be transferred to the appropriate protein acceptor sites.

There are several lines of in vio and in vitro evidence thatasparaginyl-linked glycosylation with N-acetylglucosamine andmannose occurs on nascent peptide chains (21-24). To inves-tigate this point we examined the time course for release ofglycosylated protein and related this to the time required forglycosylating the ribosomal bound nascent chains (Fig. 5). Fordetermining the time required to synthesize and release theglycosylated protein, placental mRNA was translated in thepresence of ascites membranes and at the indicated times ali-quots were removed. Cycloheximide was then added to preventfurther elongation, and the released proteins were examinedby immunoprecipitation (Fig. 5A). It takes about 12 min tosynthesize the complete protein. Also, it appears that the first

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A B

B 1

0 5 7 10 12 20Total incubation time. min

0 5 7 10 12Time of Tnton addition. min

FIG. 5. Time course of protein synthesis in the absence (A) andpresence (B) of Triton. (A) Aliquots from reaction mixtures con-taining membranes were taken at the indicated times, treated with10 mM cycloheximide, and processed for gel analysis. (B) Aliquotsfrom an ascites reaction mixture containing membranes were treatedwith 0.04% Triton X-100 at the indicated times. The Triton-treatedsamples were then incubated for a total of 40 min. The gel was thensubjected to fluorography.

visible products contained both unglycosylated and glycosylatedforms. This suggests that sugar attachment occurs before or justat the time of release.To elucidate this point, we used Triton X-100 to disrupt the

membranes. We have previously shown that Triton inhibits thecleavage of the amino-terminal fragment from nascent pre-proteins without significantly affecting the rate of proteinsynthesis, and it was used as a tool for examining the cleavagereaction (35). Later, Triton was used in a similar manner toexamine the membrane-dependent sequestration and glycos-ylation of vesicular stomatitis viral glycoproteins (24). From a

translation reaction mixture containing membranes, aliquotswere removed at the indicated times and placed in tubes withTriton (Fig. 5B). The reaction mixtures were then incubatedfor an additional 30 min. It was clear that Triton added withinthe first 7 min prevented the formation of band D. At 10 min,however, band D was observed (arrow) despite Triton addition(Fig. 5B). Since it took about 12 min to synthesize and releasethe glycosylated protein, it appeared that glycosylation occurredabout 2 min before the release of the protein. It was not unex-

pected that glycosylation of the a protein occurred soon beforerelease since the two sugar acceptor asparagine residues werelocated 40 and 14 amino acids from the carboxyl end of themolecule (11). When Triton was added within the first fewminutes of incubation a protein comigrating with band B wasobserved. However, it disappeared when Triton was added atlater times. In addition, this detergent-induced protein was notobserved when Triton was added to a reaction mixture con-

taining no membranes (data not shown).

DISCUSSION

The data presented indicate that the apoprotein of the a subunitof hCG is glycosylated during translation of first trimesterplacental mRNA in cell-free extracts derived from ascites tumorcells. Glycosylation was dependent on the addition of mem-branes to the cell-free system. Evidence for glycosylation was:

(i) the major cell-free product was bound to Con A and was

released from the lectin with a-methylmannoside but not withgalactose; and (ii) the migration of this protein on NaDodSO4gels was sensitive to the action of a variety of glycosidases.

While these data do not define the entire structure of theattached oligosaccharide, the experiments with the glycosidasesare quite revealing. The glycosylated product (protein D) ismarkedly sensitive to the action of a-mannosidase and a new

protein (band B) appears which is apparently heavier than the

nonglycosylated product (band A) (Fig. 3). The results show thatprotein D contains v-mannose residues and in addition suggestthat no terminal sialic acid or galactose residues are present inthis attached oligosaccharide.

Experiments with endoglycosidase CII and H suggest thatthe cell-free product contains di-N-acetylchitobiose. In contrastto that seen with exoglycosidases, endoglycosidase activity leadsto protein that migrates with the unglycosylated protein (bandA). The apparent removal of most of the sugar by this enzymeis consistent with its known specificity, i.e., the substrate shouldcontain at least four mannose residues attached to di-N-acetylchitobiose (33). From these data then, we propose thefollowing structure for the attached oligosaccharide:

(Mn) > 3

GlcNAcIGlcNAc

I-Asn

Previous studies on the structure of the oligosaccharide chaindid not reveal the presence of di-N-acetylchitobiose (11, 12).In view of the observations that the dolichol unit may contain5-10 mannose residues (16) and the marked sensitivity of bandD to a-mannosidase, the glycosylated protein synthesized invitro may initially contain more mannose residues than thatfound in the mature form. Recent studies by Tabas et al. on thematuration of vesicular stomatitis viral glycoproteins in vivostrongly suggest conversion of a mannose-rich "nascent" unitto a core structure containing fewer mannose residues (36).

In the presence of Triton, the formation of band D was in-hibited, but a protein comigrating with the product (band B)of a-mannosidase treatment was observed. Recent preliminaryexperiments suggest that this protein contains N-acetylglu-cosamine. It is possible that this detergent-induced protein isan intermediate that results from the trimming of the man-nose-rich, nascent core to its corresponding stable form. Chenand Lennarz (37) have observed that, in the presence of dena-tured lactalbumin and deoxycholate-treated membranes, anoligosaccharide unit was added that contained one mannoseresidue attached through a a linkage to di-N-acetylchitob-iose.The structure of the nascent oligosaccharide core attached

to the apoprotein of the a subunit appears to be independentof the source of membranes. Membranes derived from tumorcells or first trimester placenta resulted in the formation ofglycosylated proteins with identical mobilities on NaDodSO4gels. These results support the view that membranes fromvarious tissues contain a common mannose-rich sugar core thatis linked to asparagine residues during translation. It has beenproposed that the triplet amino acid segment Asn-X-Thr(Ser)represents a signal for the attachment of the core (38). Perhapsthe triplet is responsible for the initial commitment by theendoplasmic reticulum for adding the dolichol sugar core.Subsequently, specificity may be exerted by later trimming ofthe nascent core at other points in the secretory pathway, suchas the smooth endoplasmic reticulum and the Golgi network.The ultimate signal may involve some unique structural con-figuration in specific segments of the protein.The properties of in vitro glycosylation reaction reported

here and by others are reminiscent of the pre-protein cleavagereaction (25-27). Both are membrane dependent and utilizeribosome-bound nascent peptides as substrates (24-27). In ad-dition, as reported by others, cleavage and glycosylation pre-

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sumably require penetration and sequestration into membranes(24, 26, 39). This raises the question of whether cleavage andglycosylation are coupled reactions. For the a subunit, we haveevidence that the 24-amino-acid pre-segment had been re-moved from the glycosylated product (band D) (S. Birken, J.Fetherston, R. Canfield, and I. Boime, unpublished data)..Thisraises the possibility that the pre-a subunit is cleaved prior tosugar attachment. However, there is evidence that the peptidechain must contain 60-80 amino acid residues before cleavagecan occur (24, 35, 40), and since the acceptor-asparagines in thepre-a subunit are located 76 and 102 residues from the aminoterminus, cleavage and glycosylation may be difficult to dis-tinguish temporally. Until they can be separated it is not clearif the two reactions represent simultaneous or tandem events.However, we would favor the latter since unlike cleavage,which requires a nascent-peptide substrate of a relatively fixedlength, carbohydrate-coupling seems to depend upon thelocation of the acceptor asparagines and hence the length ofpolypeptide chain. Although the membrane-dependentcleavage and glycosylation are cotranslational processes, sugarattachment in vitro is not a priori restricted to nascent-chains.It has been shown that denatured forms of completed apopro-teins can be glycosylated in -the presence of detergents andmembranes (37, 41). Although the nature of the glycose unitis apparently not the same as the nascent core observed here andby others, it suggests that in vitro some sugar attachment cantake place independently of translation.

We thank Drs. Luis Glaser and Stuart Kornfeld for their helpfulsuggestions. We also thank Ms. Kathy Neely for her excellent typingassistance in preparing this manuscript. This work wassupported bygrants from the Public Health Service (PO-HD-08235) and from thePopulation Council of the Rockefeller University. M.B. is a Fellow ofthe Ford Foundation and I.B. is a recipient of a Research Career De-velopment Award (AM-00174) from the National Institutes ofHealth.

1. Pattillo, R. A. & Gey, G. 0. (1968) Cancer Res. 28, 1231-1238.

2. Kohler, P. O. & Bridson, W. F. (1971) J. Clin. Endocrinol. Metab.32,683-689.

3. Dawood, M., Saxena, B. B. & Landesman, R. (1977) Obstet.Gynecol. 50, 172-181.

4. Rosen, S. & Weintraub, B. (1974) N. Engl. J. Med. 290, 1441-1447.

5. Vaitukaitis, J. (1974) J. Clin. Endocrinol. Metab. 38,755-760.6. Pastorfide, G. B., Goldstein, D. P. & Kosasa, T. S. (1974) Am. J.

Obstet. Gynecol. 120,1025-1028.7. Ashitaka, Y., Mochizuki, M. & Tojo, S. (1972) Endocrinol 90,

609-617.8. Hagen, C. & McNeilly, A. S. (1975) J. Clin. Endocrinol. Metab.

41,466-470.9. Landefeld, T., Boguslawski, S., Corash, L. & Boime, I. (1976)

Endocrinology 98,1220-1227.

10. Landefeld, T., McWilliams, D. & Boime, I. (1976) Biochem.Biophys. Res. Commun. 72,381-390.

11. Bahl, 0. M. (1977) Fed. Proc. Fed. Am. Soc. Exp. Biol. 36,2119-2127.

12. Kennedy, J. F. & Chaplin, M. F. (1976) Biochem. J. 155,303-315.

13. Waechter, C. & Lennarz, W. (1976) Annu. Rev. Biochem. 45,95-112.

14. Pless, D. & Lennarz, W. (1977) Proc. Natl. Acad. Sci. USA 74,134-138.

15. Eagon, P. C., Hsu, A. F. & Heath, E. C. (1975) Fed. Proc. Fed.Am. Soc. Exp. Biol. 34, 678.

16. Spiro, M., Spiro, R. & Bhoyroo, V. (1976) J. Biol. Chem. 251,6400-6408.

17. Caccam, J., Jackson, J. & Eylar, E. (1969) Biochem. Biophys. Res.Commun. 35,505-511.

18. Parodi, A., Behrens, N., Leloir, L. & Carminatti, H. (1972) Proc.Nati. Acad. Sci. USA 71, 2391-2395.

19. Choi, Y. S., Knopf, P. M. & Lennox, E. S. (1971) Biochemistry10,659-667.

20. Hunt, L. A. & Summers, D. F. (1976) J. Virol. 20,637-645.21. Melchers, F. (1973) Biochemistry 12, 1471-1475.22. Kiely, M., McKnight, G. & Schimke, R. (1976) J. Biol. Chem. 251,

5490-5495.23. Bergman, L. W. & Kuehl, W. (1977) Biochemistry 16, 4490-

4497.24. Rothman, J. B. & Lodish, H. F. (1977) Nature 269,775-780.25. Szczesna, E. & Boime, I. (1976) Proc. Natl. Acad. Sci. USA 73,

1179-1183.26. Lingappa, V. R., DeVilliers-Thiery, A. & Blobel, G. (1977) Proc.

Nati. Acad. Sci. USA 74,2432-2436.27. Shields, D. & 1liobel, G. (1977) Proc. Nati. Acad. Sci. USA 74,

2059-2063.28. Tonneguzzo, F. & Ghosh, H. P. (1977) Proc. Nati. Acad. Sd. USA

74, 1516-1520.29. Ito, S., Muramatsu, T. & Kobata, A. (1975) Arch. Biochem. Bio-

phys. 171,78-86.30. Laskey, R. A. & Mills, A. D. (1975) Eur. J. Biochem. 56,335-

341.31. Stohlman, S., Eyler, S. & Wisseman, C. (1976) J. Virol. 18,

132-140.32. Morgan, F., Birken, S. & Canfield, R. (1975) J. Biol. Chem. 250,

5247-5248.33. Lis, H. & Sharon, N. (1973) Annu. Rev. Blochem. 42, 541-

574.34. Tai, T., Yamashita, K. & Kobata, A. (1977) Biochem. Blophys.

Res. Commun. 78,434-441.35. Boime, I., Szczesna, E. & Smith, D. (1977) Eur. J. Biochem. 73,

515-520.36. Tabas, I., Schlessinger, S. & Kornfeld, S. (1978) J. Biol. Chem.

253,716-722.37. Chen, W. & Lennarz, W. (1976) J. Biol. Chem. 251, 7802-

7809.38. Marshall, R. D. (1974) Biochem. Soc. Symp. 40,17-26.39. Smith, D. & Boime, I. (1977) FEBS Lett. 84, 115-118.40. Palmiter, R., Gagnon, J., Ericsson, L. H. & Wash, K. (1977) J. Biol.

Chem. 252,6386-6393.41. Tucker, P. & Pestka, S. (1977) J. Biol. Chem. 252, 4474-4486.

Proc. Natl. Acad. Sci. USA 75 (1978)

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