Interrelations between Assembly and Secretion of Recombinant ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY Q 1993 by The American Society for Biochemistry andMolecular Biology, Inc.

Vol. 268, No. 1, Issue of January 5,pp. 180-184,1993 Printed in U.S.A.

Interrelations between Assembly and Secretion of Recombinant Human Acetylcholinesterase* (Received for publication, July 6, 1992)

Anat Keremj?, Chanoch Kronman, Shoshana Bar-Nun$, Avigdor Shafferman, and Baruch VelanQ From the Departmentof Biochemistry, Israel Instituteof Biological Research, Ness-Zona 70450 and the $Departmentof Biochemistry, Faculty of Life Sciences, Tel-Auiu University, Tel-Auiu 69978, Israel

Transport and secretionof recombinant human ace- subunit and the hydrophobic H-subunit (nomenclature actylcholinesterase(rHuAChE)werestudiedintranscording toMassoulie et al. (1992a)), both derived from a single fected human293 cells expressing either the oligomer- gene by alternative splicing (Schumacher et al., 1988; Li et ized soluble enzymeor a monomeric mutant derivative ab, 1991). The structural subunits include the collagen-like in which Cys-580 was substituted by alanine (C580A). Q-subunit (Krejci et al., 1991) and the 20-kDa lipid-linked I n cells expressing the wild-typeenzyme, the gradual hydrophobic P-subunit (Inestrosa et al., 1987). assembly of newly synthesized intracellular rHuAChE TheT-subunitis involved intheformation of several monomers into oligomersoccurs withinthe endo- multimeric AChE ectoenzyme configurations; secreted AChE plasmic reticulum. Secretion of mature wild-type en- is usually composed of soluble homo-oligomers (dimers and zyme into the medium is efficient and appears to be exclusive to multimeric forms. Consistently, intracel- tetramers), whereas cell-bound AChEs consist of T-type telular oligomers, but not monomers, are endoglycosi- tramers attached tocell-associated structural subunits (Massoulie et al., 1992b). Studies on the assembly of AChE have dase H-resistant, indicating that only oligomers shown that intracellular AChE monomers serve as precursors undergo terminal glycosylation in the wild-type enzyme. In contrast, in cells expressing the dimerization- for the secreted complex multimeric forms (Rotundo, 1984; defective C580A mutant, newly synthesized rHuAChELazar et al., 1984; Brockman et al., 1986). Intersubunit disulmonomers undergo terminal glycosylation and arese- fide bonds involving the most COOH-terminal cysteine of the creted into themedium as efficiently as wild-type mul- catalytic subunitwere shown to playa key role inthe assembly timers. No significant difference betweenthe intracel- of cholinesterase multimeric forms (Lockridge et al., 1979; lular transport rates of wild-type rHuAChE oligomers Roberts et al., 1991; Velan et al., 1991b; Heider and Brodbeck, and mutantC580A monomerswas revealed by probing 1992). Much less is known, however, about the relationship with specific lectins. In both systems, transport and between multimer assembly and efficiency of AChE export. processingpriortothetrans-Golgigalactosylation This information should be of relevance since transport and compartment appear to be rate-limiting, whereas the secretion of many multimeric ectoproteins depend upon prior following passage to the cell surface is rapid. In con- association into the assembled forms (reviewed by Rose and clusion, we suggest that in the presence of a free cys- Doms (1988), Hurtley and Helenius (1989), Pelham (1989), teine at theCOOH terminus of the rHuAChE polypep- and Gething and Sambrook(1992)). tide, secretionof monomers is not effectuated, whereas Free extracellular monomershave been identified in growth in itsabsence, monomers are exported from theendo- media of cell lines (Rieger et al., 1980; Lazar andVigny, 1980) plasmic reticulum and are capable of traversing the as well as in human plasma and cerebrospinal fluid (Atack et entire secretory pathway. al., 1987). These monomers are not necessarily indicative of effective secretion of unassembled AChEsince their molecular nature was not yet resolved. The secreted molecules could be Acetylcholinesterase (EC 3.1.1.7) (AChE)’ is a key enzyme genuineunassembled T-subunits,degradationproducts of in cholinergic systems where it serves as a potent terminator extracellular multimers, or, alternatively, products of the reof nerve impulse transmission. AChE occurs in multiple mo- cently identified mRNA species that encodes for AChE sublecular forms in different tissues of vertebrates and inverte- units devoid of the COOH-terminal cysteine (Liet al., 1991). We have previously reported the establishment of recombrates (reviewed by Massoulie and Bon (1982), Chatonnet and Lockridge (1989), and Taylor (1991)). This heterogeneity binant expression systems in which transfected human embryonic kidney 293 cell clones produce and secretehigh levels is generated throughassociations of various catalytic and structural subunits. Catalytic subunitsinclude the soluble T - of human AChE (Velan et al., 1991a; Kronman et al., 1992; Shafferman et al., 1992a, 1992b). Onesuchsystem, which * This work was supported in partby United StatesArmy Research expresses the cDNA coding for the wild-type human T-type and Development CommandContract DAMD17-89-C-9117 (to subunit (Soreq et al., 1990), allows production and secretion A. S.). The costs of publication of this article were defrayed in part of multimeric soluble AChE forms (Kronmanet al., 1992). An by the paymentof page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 alternative system, generatedby utilizing a mutant cDNA in which the COOH-terminal cysteinecodon was substituted by solely to indicate this fact. T o whom correspondenceshould be addressed:Israel Inst. of an alanine codon, was found to produce and secrete monoBiological Research, P. 0. Box 19, Ness-Ziona 70450, Israel. Tel.: meric human AChE (Velan et al., 1991b). In this study, we 972-8-381518 Fax: 972-8-401404. compare the pathways and kinetics of recombinant human The abbreviations used are: AChE, acetylcholinesterase (prefix AChE secretion in these two expression systems. We analyze H, endoglycosidase H; BFA, rHu indicates recombinant human); endo brefeldin A; ER, endoplasmic reticulum; PAGE, polyacrylamide gel the sequence of events involved in the assembly and processing of the wild-type homo-oligomeric AChE and the monoelectrophoresis.

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A.swrnhl>~ and Sccrction of AcPt~lcholincstcrasc

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m e r i c m u t a n t f o r m i n a n a t t e m p t to resolve possihle interdependence of AChE assemhlv and secretion. EXPERIMENTALPROCEDURES

Crll Linrs-The R11 cell line secretes wild-type rHuAChE, and the D9 line secretes the C58OA HuAChE mutant. The twolines were generated hy stahle transfection of human emhryonic kidney 29.1 cells (ATCC CR1,1547:3) usingexpression vectors pI,5CAN and pEmA58On, describedpreviously (Velan et al., 1991h; Kronman rt al.,

1992). Productionof AChE hv these lines reached levels of 10-20 p g / IO6 cells/24 h. Mrtnholic Lnhrling of Protrins-Cells were grown to confluence in :10-mm dishes in Dulhecco's modified Eagle's medium supplemented with 10% fetal calf serum. Cells were preincubated for 1 h in 250 pl (perdish) of methionine-freemedium, labeled for 1 h with ['''SI methionine (100pCi/ml, 1000Ci/mmol; Amersham Corp.) and chased in 1.25 ml of methionine-containing Dulhecco's modified Eagle's medium supplemented with 1 0 7 AChE-depleted fetal bovine serum (Shafferman rt ol., 1992a) and 100 units/ml aprotinin (Sigma). Hrefeldin A (HFA) ( 5 pg/ml; Epicentre) and/or nocodazole (20 pg/ml; Sigma) was included in the chase where indicated. At various chase periods, culture media were collected, and washed cells were lysed using a modification of a previously described method (Amitay rt a/., 1991). Lysis buffer consisted of phosphate-buffered saline containing 0.5% Nonidet 1'-40. freshly prepared 0.2 M iodoacetamide, 1 mM phenylmethylsulfonyl fluoride (Sigma), and 100 units/ml aprotinin. Whole cells and nuclei were removed from media andlysates by centrifugation at 4 "C for 15 min at 2000 X p; lysates were cleared hy a n additional centrifugation a t 4 "C for 30 min at 16,000 X g. Immunoprrcipitntion-rHuAChE was immunoprecipitated from cleared cell lysates and media with rabbit anti-rHuAChE antibodies (Shafferman et a/., 1992a) followed by protein A bacterial adsorbent (Hiomakor, Rehovot, Israel) as described previously (Amitay rt a/., 1991). In some experiments,immunoprecipitation was performed with Sepharose-bound anti-rHuAChE antihodies. Sepharose CL-4R was activatedwith p-nitrophenyl chloroformate (Wilchek and Miron. 1982). andcoupling of antibodies to the matrixwas carried out for 48 h at 4 "C in 0.2 M sodium carbonate (pH 8.5). Immunoadsorptionwas carriedout for 2 hat room temperature,andimmunoprecipitates were then washed three times with phosphate-buffered saline containing0.1'; SDS and 0.55 Nonidet P-40. Immunoprecipitated AChE was analyzed hy reducing or nonreducing SDS-PAGE followed by fluorography (Amitav rf a/., 1991). Fluorograms were quantified bv densitometry (UltraScan S L , I'harmacia LKH Hiotechnology Inc.). Trmtmrnt with Endoglycosidasr H-ImmunoprecipitatedrHuAChE was eluted with 50 mM Tris-HCI (pH 8.0) containing 1%SDS by boiling for 2 min, diluted with an equal volume of 50 mM sodium citrate (pH 5.5) containing 1 mM phenylmethvlsulfonvl fluoride and 100 units/ml aprotinin, and divided into two equal aliquots. Aliquots were incuhated for 18 hat 37 "C in the presence or absence of 0.3 ILJ/ ml endoglycosidase H (endo H) (Hoehringer Mannheim) as described previously (Amitav rt a/., 1991). Immunohbt and Ixctin Blot Analysrs-Immunoprecipitated rHuAChE was resolved hv nonreducing SDS-PAGF: and electrohlotted onto nitrocellulose filters (Towbin rt a/., 1979). Blots for lectin probing were immersed in boiling water for 5 min; blocked in 4 7 bovine serum albuminfor 2 h; and probed for 30 min with hiotinylated concanavalin A, hiotinylated wheat germ agglutinin, or biotinylated Ricinus communis agglutinin ( 1 pg/ml; Sigma). This was followed bv a 30-min incuhation with peroxidase-conjugated avidin (Sigma). Immunoblots were hlocked in 4 5 skimmed milk for 2 h and probed for 2 h with rahbit antibodies directed against hacteria-derived HuAChE polypeptides (Velan rt al., 1991h). followed by a 2 h-incubation with peroxidase-conjugated goat anti-rahhit IgG (Sigma). Peroxidase was visualized with 4-chloronaphthol (Hawkes rt a!.. 1982). R E S U L T SA N DD I S C U S S I O N

Recombinant Human Acptylcholinrsterase Is Spcretcd from Transfected Human Crlls in OligomericForm-Kineticsof assemhlyandsecretionofrHuAChEwereexaminedin a stahle cell line (R11) expressing high levels of the wild-t-ype enzyme. Cells were pulse-labeledfor 1 h with ["'Slmethionine, followed hv chase for various periods of time (Fig. 1). At the e n d of the pulse label, the intracellular pool of AChE molecules was composed of monomers(67 a n d 70 kDa) a n d disul-

dm.

FIG. 1. Assembly of rHuAChE mommers into dimers and acquisition of endo H resistance. H I 1 cells were grown to conlluence in 30-mm dishes. metabolically laheled for 1 h w i t h [ '"Slrnethionine.and chased in methionine-containing medium as tlewrihed under"Experimental l'rocedurc~s." A t the indicated chase pericwls. cells and medium were separated. cells were lysed. and rlluA('hE was immunoprecipitated.rHuAChE was analyzed hy nonreducing I O ' ; SDS-PAGE followed by fluorography. Eluted samples were Ioarltd dirertlv on gels (11) o r following encio H analysis t H I. For enrlr1 11 treatment, sampleswere divided Into t w o equal aliquots anti inruhted with ( + ) or without I-) entln H as desrrlhed under "Experimental Procedures." Each lane contains AChE recovered from -2 X IO' w l l ~ (Ivsate or medium). Hands m, monomers; hands d, S-S-bonded d l mers: hands d g - m . deglvcosylated monomers: hands dg-d, deglycosylated dimers; hands tg-m, terminally glyrosylatctf mature monomers: hands tp-d. terminally glycosylated mature dimcrs. r also represent fide-honded dimers (1.70 k D a ) . T h e l a t t ecould r H u A C h E t e t r a m e r s ( V e l a n c t 01.. 1991h) generated hy noncovalent interaction of dimeric forms. A gradual decrease in the relativeamountofintracellularmonomer as well as a concomitant increase in the multimeric forms were nllserved during the chase (Fig. 1). The multimeric forms were secreted 4..i h.onlysmallamounts nf intothemedium:andafter laheled rHuAChE were found the in cell. Comparison hetween intracellularenzymelevelsimmediatelyafterlahelingand 4-6 h of chase (Fig. 1 ) testifies t o a extracellular levels at recovery of 79 5 7 (average of four pulse-chase experiments: densitometry not shown). The high recnvery in the recomhin a n t 29.1 system is significantly different from the 2 0 7 recnveryohservedinavianmuscle cells (Rotundo, 19R8). T h i s probably reflects a specific intracellular tlegradation mechanism that has evolved in excitatory cells. of rHuAChErecoveredfrommediumatvariousperiods chase was composed mainly of S-S-honded multimers (Fig. I ) , although a minor fraction of monomeric forms was detected occasionally (see helow). The preferential secretion of oligomeric rHuAChE forms was further supported hy analysis with endo H (Fig. 1R). As expected, all secreted rHuX('hF: molecules are endo H-resistant due to their terminal glycosylation. In contrast, the monomeric and most of the multimeric intracellular rHuAChEs were sensitiveto e n d o H. T h e intracellular pool of endo H-resistant AChE molecules ronsistedexclusively of theoligomericspecies(Fig. 1H). Together, the presenceof terminally glycosylated oligomers and the absence of processed monomers within the cells indicate that oligomerization occurs prior to terminal glycosylation o f the carhohydrate moieties of r H u A C h E . T h i s c o u l d also suggest that the endo H-resistant monomers occasionally found

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Assemblv and Spcretion of Acctvlcholinesterase

Cells in the medium (Figs. 1R and 2) are degradation products of Tme [ h l ~ 3 i ' 4 5~ ~ secreted oligomers rather than an authentic secreted mono-~ .__ C B N N B ' C B N N B C B NNB C B N N B I . meric species. In this respect, the experimental fluctuations i n t h e levels of monomers in the various preparations may dreflect different degreesof proteolytic cleavage. Indeed, inclumsion of protease inhibitors, such as iodoacetamide in the cell lysate and aprotinin in the medium, strongly attenuated formation of monomers. Moreover, proteolytic cleavage of AChE disulfide-linked dimers to monomeric products has heen reMedlurn ported previously (Rotundo and Fambrough, 1979; Allmand Trmem)' o 1 I5 3 1 4S-. et al., 1981). &NBl C B N N B , C B N NB C B N N E Subunit Dimerization Occurs in Endoplasmic Reticulumd-" - o w T o determine the cellular compartment in which rHuAChE i s oligomerized, R11 cells were pulse-labeled and chased for m' 0 various periodsof time in the presence of RFA, which inhibits vesicular export of proteins from the endoplasmic reticulum FIG. 3. rHuAChEmonomersareassembledintodimers (ER) (Klausner et al., 1992). No extracellular rHuAChE was within endoplasmic reticulum. R11 cells were metaholically Iadetected in the presence of RFA, yet intracellular oligomeriheledwithI'.'S]methinnineandchased in methionine-containing zationwasnotimpaired(Fig.2).Thissuggeststhatthe medium with no additions (control, C), with RFA ( H I . with nocndaassembly process occurs within the ER. Rrefeldin is A known zole ( N ) , or with HFA andnocodaznle (.VH) as descrihedunder to cause a retrograde-dependent resorption of Golgi enzymes "Experimental Procedures." Analyses of AChE from cell lysates and into the ER, resulting ainmixed ER-Golgi compartment that medium were performed as indicated in the legend t n Fig. 2. I3nnd.s m, monomers; hands d, S-S-honded dimers. has a different milieu than the ER (Klausner et al., 1992). To demonst,rate that, oligomers were formed within an authentic bly is a prerequisite for export from the ER and for subsequent E R , nocodazole, a specific inhibitorof the microtuble-depend- secretion. Previous observations have indicated that transalso included in the chase. In the fected cells harhoring the dimerization-impaired ent retrograde pathway, was C58OA AChE presence of both RFA and nocodazole, intracellular dimerimutant produce and secrete rHuAChE monomers that display zationwasdetected(Fig. 3). Theseresultsprovethatin authentic characteristics of HuAChE (Velan et al., 199lb). transfected 293 cells, rHuAChE homo-oligomerization occurs The nonrestricted secretion of CFiROA monomers was demin the ER. This observationis in accordance with studies on onstrated in two independent cell lines, the embryonic 293 theassembly of asymmetricAChEinavianmusclecells line and the neuroblastoma SK-N-SH line. To analyze further (Rotundo, 1984) and agrees with our results that oligomerithe interrelations between assemhly and transport of AChE, zation precedes terminal glycosylation in the Golgi complex. the secretion rate and passage of the mutant and wild-type Mutant Monomers of Dimerization-defective AChE Displayenzymes through the secretory pathway were compared. D9 of Wild-t-vpe Enz-vmeSecretion Kinetics Similar to Those cells,expressinghighlevels of C580AAChE,werepulseThe early conversionof intracellular monomers into secreted labeled and chased for various periods of time (Fig.4A ). After multimeric forms raises the question whether subunit assem- 6 h of chase, only traces of AChE monomers were discerned 68 10% of thelabeledmoleculeswere inthecell,and - BFA recovered in the medium (averageof three pulse-chase exper4 24 2 0 6 iments;densitometrynotshown).Itappearsthat C.580A lime (h) monomersaresecretedfromcellsatefficienciesthatare C M C M C M C M C M essentially similar to those of t h e wild-t-ype oligomeric forms (Fig. 1). d- 0 -0 0 The secreted C580A monomers, like the wild-type enzvme under reducing conditions (data not shown), display a reduced m. I 0 " electrophoretic mobility in comparison with the intracellular enzyme (Fig. 4R,compare bands m and t g - m ) ,indicating that en route to secretion, their carbohydrate moieties are being + BFA processed.Accordingly,extracellularrHuAChEmonomers secreted by D9 cells were resistant to endo H, as expected for 4 24 2 6 0 lime (h) terminally glycosylated enzyme(bands tg-m). All intracellular C M C M C M C M C M monomers (hands m ) weresensitivetoendoH.exhibiting Hincompleteprocessing.Interestingly,intracellularendo resistant molecules could not be identified in D9 cells. In this d* 0 " R11 cells, in whichendo H aspect, D9 cellsdifferfrom ', resistant multimeric molecules were detected (Figs. 1H and m - 0 0 4R, compare 0-h lanes). This could indicate that the transit of newlysynthesized C.58OA monomers through the transGolgi apparatus and the trans-Golgi network is more rapid FIG. 2. AssemblyofrHuAChEmonomersintodimersin presence of hrefeldin A. 1311 cells were metaholically laheled with t h a n t h a t of the assembled wild-type AChE. I:'%]methionine and chased in methionine-containing medium with rHuAChE Oligomers as Well as C'580A Monomer.< Are Rapo r without HFA as described under "Experimental Procedures." At idly Shuttled through trans-Golgi Apparatus and trans-Golgi the indicated chaseperiods.cells ((') and medium ( M )were separated; Networh-The relative rates of transport of wild-type and cells were lysed; and rHuAChE was immunoprecipitatedfrom cleared of 293 lysates and medium and analyzed hy nonredncing 1 0 7 SDS-PAGE C58OA rHuAChEs through the central vacuolar system cellsweredeterminedbvlectinblotanalysis.Steadv-state followed by fluorography. Bonds m , monomers; hnnds d, S-S-honded pools of intracellular and extracellular rHuAChEs from R11 dimers. ~~

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