THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 32, Issue of November 15, pp. 19996-19999,199O Printed in U.S. A.

Eukaryotic and Prokaryotic Signal Peptides Direct Secretion of a Bacterial Endoglucanase by Mammalian Cells*

(Received for publication, July 10, 1990)

Judith Hall$& Geoffrey P. Hazlewood7l, M. Azim Suranin, Barry H. HirstQ, and Harry J. Gilbert+-)1 From the Departments of $Agricultural Biochemistry and Nutrition and §Physiological Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, United Kingdom and the llAgricultura1 and Food Research Council Institute of Animal Physiology and Genetics Research, Babrahum, Cambridge CB2 4AT, United Kingdom

It is well established that hydrophobic signal se- quences direct proteins into or across the endoplasmic reticulum membrane in eukaryotes and cell mem- branes in prokaryotes. Although it is recognized that eukaryote proteins are efficiently secreted by bacterial systems, the export of bacterial proteins by eukaryotes has received little attention.

To investigate membrane translocation of bacterial proteins by mammalian cells, the secretion of a bacte- rial endoglucanase (endoglucanase E) from stably transfected Chinese hamster ovary cells has been ex- amined. We report that a functional endoglucanase is secreted when fused to prokaryote or eukaryote signal peptides. Furthermore, the endoglucanase was post- translationally modified before secretion. Data pre- sented in this paper suggest that secretion of bacterial proteins by eukaryote cells may be a general phenom- enon and infer that there are no specific requirements with respect to the origin of the signal sequences.

Both eukaryote and prokaryote proteins contain similar N- terminal sequences, termed the signal peptide, which play a pivotal role in the export of the polypeptide. In general, three distinct physicochemical regions may be determined within signal peptides: a positively charged N terminus, a central hydrophobic region, and a more polar flexible C terminus ending in a signal cleavage site. Individual signal peptides vary from about 15 to 40 residues and show a low degree of sequence conservation (Von Heijne, 1988). Although exhibit- ing overall similarities, significant differences between eukar- yote and prokaryote sequences are evident, provoking the suggestion that there may be fundamental differences between eukaryotes and prokaryotes with respect to protein transport (Von Heijne, 1988; Gascuel and Danchin, 1986). In addition, protein secretion is more complex in eukaryotes than prokar- yotes because of post-translational modifications often asso- ciated with secretion and an increased number of subcellular compartments. Finally, there are differences in membrane lipid composition (Stryer, 1981), and signal recognition par- ticles are only found in eukaryotes (Savier et al., 1989).

Previous studies on the secretion of heterologous proteins have focused on the capacity of bacteria to secrete mammalian polypeptides. Chicken ovalbumin (Fraser and Bruce, 1978) is secreted efficiently into the Escherichia coli periplasm when

*This work was supported by Agricultural and Food Research Council Grant TAP 39. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

11 To whom correspondence should be sent.

fused to eukaryotic signal peptides. The transport of bacterial proteins across eukaryotic membranes has not been studied widely. Wiedmann et al. (1984) showed E. coli ,&lactamase to be secreted in Xenopus oocytes whereas the same enzyme synthesized in vitro had been shown earlier to be transported into canine pancreatic microsomes (Miiller et al., 1982). How- ever, secreted p-lactamase was not glycosylated or subjected to other post-translational modifications, and the percentage of the bacterial enzyme, which appeared in the medium, was not reported. In addition, there have been no reports in the literature of a comparison of the efficiency of prokaryotic and eukaryotic signal peptides in directing mammalian secretion of proteins. There have also been no studies on the secretion and post-translational modification, in mammalian cells, of bacterial proteins.

In view of the paucity of information on the secretion of bacterial proteins in mammalian cells, we have investigated the expression, post-translational modification, and secretion of a bacterial endoglucanase (endoglucanase E) from Clostri- dium thermocellum in Chinese hamster ovary (CHO)’ cells. The mature enzyme, consisting of 780 residues with an M, of 84,016 (Hall et al., 1988), is glycosylated in its endogenous host (Hazlewood et al., 1990) and contains two distinct func- tional regions: a catalytic domain consisting of the 340 N- terminal amino acids, and a distinct cellulose binding domain that is located between residues 430 and 671 of the mature protein (Hall et al., 1988; Durrant et al., 1990). The data presented in this report showed that a truncated form of the bacterial enzyme was secreted efficiently in CHO cells when fused to either a eukaryotic or prokaryotic signal peptide. The transported protein was post-translationally modified and functionally active.

EXPERIMENTAL PROCEDURES

Plasmid Construction-Derivatives of the endoglucanase gene des- ignated celE’p and celE’e were constructed as follows. Truncated celE (Hall et al., 1988) encoding residues 4-367 of mature endoglu- canase E (essentially the catalytic domain) was excised from pHGB2 (Hall et al., 1988) on a Ll-kilobase HindIII/SstI restriction fragment. Using site-directed in vitro mutagenesis, a translational stop codon was created such that celE’ encoded a 364-amino acid protein. The internal EcoRI site at position 832 was deleted by site-directed in uitro mutagenesis without altering the primary structure of the en- coded enzime. Modified celE’ was cloned intd HindIII/SstI-cleaved ~h4TL22t1 (Chambers et al.. 1988) to generate pMST1. To create . celE’e and celE’p, DNA sequences enco&ng hum& growth hormone and endoglucanase E signal peptides were synthesized incorporating the following features: an NcoI site at the ATG translational start codon, a B&HI site immediately 5’ of the NcoI site, and an Hind111 site at the 3’ end of the simal peptide coding sequence. The plasmid pMST1 was digested witi Hindi and B&HI- (the latter knzyme

’ The abbreviations used are: CHO, Chinese hamster ovary; SDS, sodium dodecyl sulfate.

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Mammalian Secretion of a Bacterial Protein

cleaves the vector’s multiple cloning sequence upstream of the celE’ gene), and the signal peptide coding sequences, digested with the same enzymes, were cloned into the recombinant plasmid. A 1.9- kilobase BamHI restriction fragment containing the fl-globin poly- adenylation signal was inserted into the BglII site contained in the vector’s multiple cloning sequence downstream of the endoglucanase structural gene. Both celE’e and celE’p linked to the polyadenylation signal were excised on a 3.1-kilobase BamHI/NruI restriction frag- ment and cloned into pSV2DHFR that had been digested with EcoRI (filled in with T4 DNA polymerase) and BarnHI. Finally, a 330-base pair Hind111 DNA fragment, encoding the early SV40 enhancer/ promoter, was filled in with Sequenase and cloned into the BamHI site (also filled in with Sequenase) that had been created immediately upstream of the ATG translational start codon of the respective signal peptides. The celE coding regions of the two constructs are depicted in Fig. 1.

Cell Culture, DNA Transfection, and Cell Line Selection-Chinese hamster ovary cells (CHO) deficient in dihydrofolate reductase were maintained in a medium supplemented with 4 fig/ml hypoxanthine, 4 pg/ml thymidine, and 10% (v/v) fetal calf serum with gentamicin (25 rglml).

Plasmids celE’p, celE’e, and SVZDHFR weTe linearized with PuuII and precipitated with ethanol. CHODHFR- cells were transfected with either pCelE’p, pCelE’e, or pSV2DHFR (5 pg/106 cells) by electroporation (Bio-Rad electroporator; 1 pulse at 25 microfarads; 550 V). After transfection the cells were plated into medium contain- ing 10% fetal calf serum and hypoxanthine-thymidine supplements. Two days later the cells were washed and fed with medium containing 10% dialyzed fetal calf serum and lacking nucleosides. Cells were washed and fed with selective medium every 2-3 days. Transfected colonies appeared after 7-14 days. Initial transfectants were pooled and grown in increasing concentrations of methotrexate (0.1,0.2, and 0.5 /*M).

Synthesis of Radiolabeled Endoglucanase E-The synthesis of endoglucanase E’ was monitored by labeling cells in methionine-free medium containing 200 &i/ml [3”S]methionine (specific activity,

FIG. 1. ceZE’ genes designed to express in CHO cells. The fusion of the celE’ gene (@) to the SV40 promoter (0) and @globin polyadenylation sequence (W) is described under “Experimental Pro- cedures.” The primary structures of the two signal peptides and the nucleotide sequence between the ATG start codon and the 3’ nucleo- tide of the SV40 promoter are shown. The restriction sites BumHI (B), BglIII (Bg), Sal1 (Sa), SphI (S), EcoRI (R), and NcoI (N) are shown. The multiple cloning regions of pUC18, between the Sal1 and Hind111 sites, and pMTL22p, between the SstI and BglII restriction sites, are depicted as regions a and b, respectively.

TABLE I

Expression of endoglucanase E (EGE) in CHO cells Enzyme activity was determined as described by Hall et al. (1988).

The quantity of enzyme synthesized was based on a specific activity of 220 pmol of glucose-reducing equivalents of pure endoglucanase E’ uroduced ner min at 60 “C.

Cellulase gene Total EGE in EGE in medium BO-kDa form of

in CHO cells culture media” EGE in media

ngf IO5 cells 5% of total %

celEp 23 78 88

celEe 25 65 93

a The culture medium was aspirated and cell lysates prepared as described under “Experimental Procedures.” At least 95% of the cells were lysed by the detergent, as judged by microscopy.

50-

1 2 3 4 5 6 1 2345 6

c d

19997

b

I

123 4 5 6 1 2.3 4 5 6

FIG. 2. Synthesis of radiolabeled endoglucanase E’. Panels a and c contain endoglucanase E from cell lysates and media, respec- tively, of CHO cells (4 x lo5 cells/lane) containing celEp. Endoglu- canase E’ from cell lysates and media of CHO cells (10” cells/lane) containing celEe are shown in panels b and d, respectively. Lane 6, contains proteins immunoprecipitated with endoglucanase E’ anti- serum from medium and cell lysates of CHO cells containing pSV2DHFR only. The M, values x 10” of immunoprecipitated pro- teins are shown.

a b

,. *c - .-.

zw 50-

3?- -tih

.~ - --

-_ 1 2 3 4 I 2 3 4

FIG. 3. Methionine chase of radiolabeled endoglucanase E’. Panels a and b contain endoglucanase E’ from cell lysates and media, respectively. Lane 1 contains endoglucanase E from cells labeled for 2 h; lanes 2,3, and 4 contain endoglucanase E expressed by cells that had been chased with cold methionine for 1, 2, and 3 h, respectively. The M, values x 10” of immunoprecipitated proteins are shown.

1000 Ci/mmol). Cells were incubated for 20, 40,60, 120, and 180 min. Media were harvested and cell lysates prepared using a lysis buffer consisting of 50 mM Tris/HCl buffer, pH 8.0, containing 150 mM NaCl, 0.5 mM phenylmethylsulfonyl fluoride, and 1% NonidetP-40 detergent (Sambrook et al., 1989). Immunoprecipitation of endoglu- canase E’ in media and cell lysates was performed as described by

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b

FIG. 4. Effect of tunicamycin on endoglucanase E. Panels a and b contain cell lysates and media, respectively. Cells were radio- labeled for 2 h with [?S]methionine in the absence (lane I) or presence (lane 2) of tunicamycin. The iV, values x lo” of immunoprecipitated radiolabeled endoglucanase E’ are shown.

Gilbert et al. (1983) using antiserum specific for endoglucanase E’. When performing the immunoprecipitations, 20 pg of nonradioactive endoglucanase E was added to each reaction mix as carrier. The immunoprecipitates were dissolved in 2% (w/v) SDS containing 10% (v/v) mercaptoethanol and subjected to SDS-polyacrylamide gel elec- trophoresis (Laemmli, 1970) in an 8% (w/v) acrylamide gel. The gel was fluorographed and exposed to x-ray film for 48 h.

Methionine Chase of Radiolabeled Endoglucame E’-CHO cells (4 X 10’ cells) containing ce& were radiolabeled in methionine-free medium containing 200;Ci/ml [““Slmethionine for 2 h. The radio- active medium was removed and replaced with complete medium containing an excess of unlabeled methionine (2 mM). Radiolabeled endoglucanase E’ was immunoprecipitated from cells and media and subsequently analyzed by SDS-polyacrylamide gel electrophoresis as described above.

Tunicamycin Treatment of CHO Cells-Approximately 4 x 101 cells containing celEp were either untreated or pretreated with 5 rg/ml tunicamycin for 2 h. The cells were then radiolabeled for 2 h in 1 ml of methionine-free medium containing either 200 &i of [35S]methi- onine or [“‘Slmethionine and 5 pg/ml tunicamycin. Media were collected, cell lysates prepared, and immunoprecipitated endoglucan- ase E’ analyzed as described above.

RESULTS AND DISCUSSION

The 5’ region of the C. thermocellum celE gene encoding residues 4-364 of mature endoglucanase E was fused to DNA sequences specifying either the human growth hormone (DeNoto et al., 1981) or endoglucanase E (Hall et al., 1988) signal peptides. The resultant structural genes were linked 5’ to the early SV40 enhancer/promoter and 3’ to P-globin polyadenylation sequences (Fig. 1). Each construct was cloned into pSV2DHFR. The resultant plasmids were linearized and transfected into CHO dihydrofolate reductase- cells. Trans- fectants were selected by the withdrawal of hypoxanthine- thymidine from the medium. Confirmation that the selected dihydrofolate reductase+ mammalian cells contained celE’ was obtained by Southern hybridization of chromosomal DNA using celE’ as a probe (data not shown). Transfected cells were grown in the presence of methotrexate in order to amplify the dihydrofolate reductase gene and adjacent se- quences. Analysis of culture media and cell lysates demon- strated the presence of endoglucanase activity in cells con- taining celE’ fused to DNA encoding prokaryote as well as eukaryote signal sequences, consistent with the correct syn- thesis, translocation, and secretion of the bacterial protein (Table I). No cellulase activity was detectable in culture media

or cell lysates from cells transfected with just the dihydrofo- late reductase-encoding plasmid.

Secretion of endoglucanase E was confirmed by [3”S]methi- onine cell labeling experiments (Fig. 2). Synthesis and secre- tion of endoglucanase E’ by transfected cells were time de- pendent irrespective of the origin of the signal peptide. Three forms of immunoprecipitable endoglucanase E were identified in cell lysates from each cell line with M, values of 37,000, 46,000, and 50,000, respectively. The 50-kDa protein co-mi- grated with the extracellular form of endoglucanase E’ whereas the 37-kDa protein was comparable with endoglucan- ase synthesized by E. coli harboring truncated celE (Hazle- wood et al., 1990). When the radiolabeled cells were chased with 2 mM cold methionine there was a rapid (<l h) disap- pearance of the radiolabeled 50-kDa protein from the cells and its appearance in the media. The cellular level of the two smaller labeled proteins remained constant (Fig. 3). This result suggests that the two smaller proteins are not precur- sors of the secreted enzyme but are probably derivatives of endoglucanase E which have undergone incorrect post-trans- lational modification or are incorrectly folded and so remain trapped within the cell. The increased molecular size of en- doglucanase E secreted by CHO cells when compared with the E. coli product is indicative of glycosylation of the endog- lucanase. Inspection of the primary structure of endoglucan- ase E revealed three potential sites for N-glycosylation (Asn- X-Thr/Ser) and a threonine-serine-proline-rich region with potential for 0-glycosylation toward the C terminus of the protein. To investigate the nature of the post-translational modification of the bacterial proteins, CHO cells expressing endoglucanase E’ were incubated with tunicamycin, which inhibits N-linked glycosylation (Schwartz and Datema, 1980). The data (Fig. 4) showed that cells treated with the antibiotic synthesized an endoglucanase E derivative of M, 42,000 which was secreted. There was no evidence of the intracellular 46- and 50-kDa forms of the enzymes. These results are consistent with both N-linked and O-linked glycosylation of endoglucan- ase E by CHO cells.

Previous studies to investigate prokaryote secretion in higher eukaryote cells (Xenopus oocytes) have utilized p- lactamase as the model bacterial protein. It could be argued that it is difficult to make generalizations on protein secretion based on studies using a single prokaryote protein that may be atypical (Gascuel and Danchin, 1986) in a nonmammalian eukaryote cell. In addition, the secretion of /3-lactamase by an unusual pathway (Bunker and Moore, 1988) cannot be ignored especially as the enzyme is not glycosylated, a characteristic typical of proteins that are transported through the endo- plasmic reticulum and Golgi apparatus. This report describes the post-translational modification and competent secretion of a bacterial protein in a mammalian cell fused to either a bacterial or eukaryotic signal peptide. The fact that both endoglucanase E and p-lactamase originate from taxonom- ically unrelated bacteria and are secreted in diverse higher eukaryote cells argues in favor of the generality of eukaryotic secretion of bacterial proteins and is contrary to the views expressed by Gascuel and Danchin (1986).

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J Hall, G P Hazlewood, M A Surani, B H Hirst and H J Gilbertendoglucanase by mammalian cells.

Eukaryotic and prokaryotic signal peptides direct secretion of a bacterial

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