THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 264, No. 26, Issue of September 15, pp. 15620-15623, 1989

Prrnted in U. S. A .

Fusion of a Cleavable Signal Peptide to the Ectodomain of Neutral Endopeptidase (EC 3.4.24.11) Results in the Secretion of an Active Enzyme in COS-1 Cells*

(Received for Publication, May 6, 1989)

Guy LemaySg, Gilles WaksmanSllIl , Bernard P. RoquesT, Philippe Crine$(I**, and Guy Boileau$ $$ From the SDepartement de Biochimie, Faculte de Medecine, Universite de Montreal, Montreal, Quebec H3C 357, Canada, the BDepartement de Chimie Organique, U 266 Znstitut de la Sante et de la Recherche Medicale, UA 498 and Centre National de la Recherche Scientifique, Faculte de Pharmacie, Paris, France, and the (IGroupe de Recherche en Transport Membranaire, Universite de Montreal, Montreal, Quebec H3C 357, Canada

Neutral endopeptidase (EC 3.4.24.11) is an integral membrane protein found at the plasma membrane of many cell types and is especially abundant at the apical “brush border” membrane of the kidney proximal tu- bules. The enzyme consists of a short amino-terminal cytosolic domain of 27 amino acids, a single hydropho- bic sequence which is believed to be responsible for anchoring the enzyme in the plasma membrane, and a large extracellular domain containing the active site. This model is consistent with the proposed function of neutral endopeptidase, which is believed to play an important role in the inactivation of small regulatory peptides at the cell surface. Site-directed mutagenesis has allowed the identification of 1 glutamic acid and 2 histidine residues essential for catalysis. All are located near the COOH terminus of the protein. We do not know, however, whether other segments of the protein are involved in the structure of the active site. The exact role of the cytosolic and transmembrane domains is also unknown. In this report, we have induced the secretion of a soluble form of recombinant neutral endopeptidase in COS-1 cells by fusing in-frame, the cDNA encoding the signal peptide of a secreted protein (pro-opiomelanocortin) to the cDNA sequences of the complete ectodomain of neutral endopeptidase. Char- acterization of the secreted recombinant protein indi- cated that it has the same catalytic properties as the membrane-bound recombinant enzyme or as the en- zyme extracted from kidney brush border membranes. Thus the extracellular domain alone is sufficient for conferring full catalytic activity to neutral endopepti- dase.

Neutral endopeptidase (EC 3.4.24.11) is a Zn-metalloen- dopeptidase found at the plasma membrane of several mam- malian cell types. The enzyme is particularly abundant in the kidney brush border membrane (Kenny, 1986). Neutral en-

* This work was supported in part by grants from the Medical Research Council of Canada and the Canadian Kidney Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Postdoctoral fellow of the Medical Research Council of Canada. **Supported by a scientist award from the Medical Research

Council of Canada. $$ “Chercheur-boursier” of the Fonds de la Recherche en SantC du

QuLbec. To whom correspondence should he addressed Dept. de Biochimie, Universiti! de Montreal, P. 0. Box 6128, Montreal, Quebec H3C 357, Canada.

dopeptidase is an ectoenzyme which cleaves small peptides rather than proteins. This suggests that neutral endopeptidase is involved in the inactivation of regulatory peptides at the cell surface (Bowes and Kenny, 1986). This role has been best documented in the mammalian brain where the enzyme has been shown to be involved in the inactivation of the opioid peptides Met- and Leu-enkephalins (Malfroy et al., 1978; Almenoff et aL, 1981). For this reason, neutral endopeptidase is often referred to as “enkephalinase.”

The complete primary structure of neutral endopeptidase has recently been deduced from the sequences of cDNA clones from human (Malfroy et al., 1988; Letarte et al., 1988), rabbit (Devault et al., 1987), and rat (Malfroy et al., 1987). Neutral endopeptidase is a 749-amino acid protein which has been proposed to consist of three distinct domains: a short NH2- terminal cytosolic sequence (27 residues), a single transmem- brane region (23 residues), and a large extracellular or ecto- domain responsible for the catalytic activity of the enzyme (Devault et al., 1987). Recent studies using site-directed mu- tagenesis have shown that 1 glutamic acid (residue 584) and 2 histidines (residues 583 and 587) located near the COOH terminus of the protein are essential for catalysis (Devault et al., 1988a, 1988b). The contribution of other regions to the catalytic activity is still completely unknown. Similarly we have very little information about the role of the cytosolic and membrane-spanning domains in either the transport of the protein to the cell surface or in the establishment and maintenance of a conformation suitable for catalytic activity.

In the present study we have tried to determine whether a form of the enzyme, freed from its cytosolic and membrane- spanning domains, can still be transported to the cell surface and whether it is still active. For this purpose, we have substituted the intracytoplasmic and membrane-spanning do- mains of neutral endopeptidase for the signal peptide of a secretory protein (pro-opiomelanocortin) (POMC)’ and ex- pressed the chimeric protein in COS-1 cells using an SV40- derived vector, The protein is translocated into the endo- plasmic reticulum lumen where the heterologous signal pep- tide is cleaved. The neutral endopeptidase ectodomain also appears to acquire terminal sugars, suggesting that it is trans- ported to the Golgi apparatus. The enzyme is no longer

The abbreviations used are: POMC, pro-opiomelanocortin; BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle’s medium; endo F, endo-P-N-acetylglucosaminidase F; endo H, endo-6-N-ace- tylglucosaminidase H; EGTA, [ethylenebis(oxyethylenenitrilo)]te- traacetic acid; ER, endoplasmic reticulum; K,, Michaelis-Menten constant; RER, rough endoplasmic reticulum; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TBS, Tris-huff- ered saline.

15620

Soluble Form of Neutral Endopeptidase 15621

inserted in the plasma membrane but can be recovered as a soluble form of neutral endopeptidase in the culture medium. Finally this truncated iorm of neutral endopeptidase exhibits the same catalytic properties as the membrane-bound enzyme extracted from kidney ‘brush borders. These results therefore demonstrate that anchoring of the ectodomain into the plasma membrane is neither important for the catalytic activ- ity of neutral endopeptidase nor for its transport to the cell surface.

EXPERIMENTAL PROCEDURES A N D RESULTS~

DISCUSSION

As already reported for the kidney and intestinal hydrolases (Kenny and Maroux, 1982; Semenza, 1986), neutral endopep- tidase appears to be asymetrically inserted into the lipid bilayer with a large disulfide bond containing extracyto- plasmic domain and a very small anchoring tail (Booth and Kenny, 1980; Aubry et al., 1988). The disulfide bonds of the ectodomain appear to be important for stabilizing the tertiary structure of the proteins and thus for establishing and main- taining the structure of the active site (Tam et al., 1985). The rabbit kidney enzyme has been solubilized by treatment of the brush border membrane with toluene, followed by incu- bation with trypsin (Ken and Kenny, 1974). This method yielded a hydrophilic fo’rm of the enzyme in which the poly- peptide chain had been subjected to limited proteolysis, thus cleaving the hydrophilic protein from its hydrophobic anchor (Kenny and Maroux, 1982). This soluble form of the enzyme was still enzymatically active. The main purpose of this study was to determine wheth.er it would be possible to use genetic engineering techniques to promote the secretion of a soluble and active form of the enzyme from transfected eukaryotic cells. Obviously, this kind of enzyme, which can easily be purified from the incubation medium of cultured cells without the use of detergent would be very useful for further structural studies.

Neutral endopeptidasse is a class I1 integral membrane pro- tein. These membrane proteins have, near their amino ter- minus, a unique hydrophobic peptide acting both as a signal peptide to direct the translocation of the protein through the membrane of the RER and as a transmembrane domain for anchoring the protein in the plasma membrane of the cell (Garoff, 1985; Wickner and Lodish, 1985). Unlike class I membrane proteins which possess a cleavable signal peptide and are anchored in the membrane by an additional mem- brane-spanning hydrophobic sequence (also called “Stop Transfer Sequence”), class I1 proteins cannot be easily trans- formed into soluble forms by deleting the hydrophobic trans- membrane domain. In class I1 proteins, deletion of the an- choring segment also removes the signal peptide, thereby preventing the translocation of the protein in the RER and its transport to the cell surface. Theoretically, there could be two different approaches for transforming a membrane-bound class I1 protein into a sclluble form: (i) deletion of the intra- cytoplasmic domain could be introduced to transform the combined signal/anchor into a cleavable signal peptide; (ii) the extracellular domain of the protein could be fused to a heterologous cleavable signal peptide. Although the first ap- proach has been successfully used in vitro (Lipp and Dobber- stein, 1986; Szczesna-Skorupa et al., 1988; Schmidt and

Portions of this paper (including “Experimental Procedures,” “Results,” and Figs. 1-4) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

Spiess, 1988), there is no definitive evidence that these pro- teins will be actually secreted in uiuo. There are also many examples where deletion of the intracytoplasmic domain does not allow secretion of the protein (Davis et al., 1983; Jones et al., 1985).

In this study we have constructed a neutral endopeptidase secretion vector by fusing in-frame the sequences encoding the complete ectodomain of the rabbit kidney enzyme with the POMC signal peptide (Fig. l ) , these sequences being under the control of the SV40 early promoter. This vector was found to promote the secretion of a soluble form of neutral endo- peptidase from COS-1 cells that are normally devoid of neu- tral endopeptidase activity. To our knowledge, this is the first report of the design of a secreted soluble form of a class I1 membrane protein by fusing a heterologous signal peptide to the ectodomain of the protein.

Two observations lead us to believe that sec-neutral endo- peptidase was actually routed through the secretory apparatus of the COS1 cells; 1) sequencing of the NH2 terminus of the protein shows that the POMC signal peptide has been re- moved (Figs. 1 and 3); 2) treatment of the recombinant protein found in the secretion medium with endoglycosidases H and F indicates that it has acquired terminal sugars most probably during its transit through the Golgi apparatus (Fig. 2).

Cleavage of the signal peptide was found to occur after the last amino acid of the POMC signal peptide (Figs. 1 and 3). This result is in perfect agreement with the rules predicting the cleavage site of a signal peptide (von Heijne, 1986) which gave the highest score for the peptide bond located at the junction of the signal peptide and the valine residue intro- duced by the polylinker. Since amino acids on the carboxyl side of the signal peptide have little influence on the selection of the cleavage site (von Heijne, 1986), we believe that the same cleavage site will be used regardless of the restriction site used for cloning or of the sequences introduced.

The sec-neutral endopeptidase enzyme is mostly found in an endoglycosidase H-resistant form present in the culture medium (Fig. 2, lane 12); this resistance to endoglycosidase H indicates that the protein has actually transited through the Golgi. However, intracellular forms of sec-neutral endo- peptidase sensitive to endoglycosidase H were still detected by immunoblotting (Fig. 2, lane I 1 ). This intracellular species represents about 20% of total see-neutral endopeptidase (in- tracellular + secreted) synthesized by COS-1 cells. This is in contrast with the results obtained with the wild-type enzyme where no species sensitive to endoglycosidase H could be detected (Fig. 2, lane 1 ). This observation can be explained either by a slight impairment of sec-neutral endopeptidase transport that retards all the molecules or by the misfolding of some of the molecules that are then retained in the RER. However, the ratio of enzyme activity recovered from the culture medium (95%) compared to the intracellular activity (5%) suggests that most of the cell-retained molecules are misfolded and inactive neutral endopeptidase molecules. The same ratio of secreted (95%) to intracellular (5%) forms was also found for the synthesis by COS-1 cells of POMC, a secretory protein (Noel et al., 1987).

The secreted recombinant neutral endopeptidase has also conserved its normal catalytic properties including K, (Fig. 4A) and sensitivity to the neutral endopeptidase-specific in- hibitor phosphoramidon (Fig. 4B). Therefore, anchoring of the enzyme in the plasma membrane does not appear to be important for the catalytic activity of neutral endopeptidase. A similar conclusion has already been reached by Kenny and Fulcher (1983). However, this hydrophilic form of neutral endopeptidase prepared by the combined action of toluene

15622 Soluble Form of Neutral Endopeptidase

and trypsin on kidney membranes had never been fully char- acterized. A well-characterized soluble form of neutral endo- peptidase will be of considerable interest for further structural studies of its active site, and might hopefully provide a useful material for its eventual crystaliization. Furthermore, this truncated form of the enzyme will be a valuable tool in the near future to study the role of intracytoplasmic and the membrane anchor region in the targeting of the protein to the apical membrane of epithelial cells.

Acknowledgment-We thank Dr. Nabil Seidah for performing the Edman degradation of the tritiated substrate. We thank Rahel Wholde-Giorgis and Claire V6zina for their help in preparing the secretion vector, Nathalie Labonti and Mireille Fyfe for their expert technical assistance, Dr. Lyse Zollinger for her help with polyclonal antiserum preparation, and Christine Ostiguy for her excellent pho- tographic work.

REFERENCES Almenoff, J., Wilk, S., and Orlowski, M. (1981) Biochem. Biophys.

Res. Commun. 102,206-214 Aubry, M., Berteloot, A., Beaumont, A,, Roques, B. P., and Crine, P.

(1987) Biochem. Cell Biol. 65, 398-404 Aubry, M., Zollinger, M., Fortin, S., Vhien, C., LeGrimeIlec, C. , and

Crine, P. (1988) Biochim. Biophys. Acta 967, 56-64 Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., and Smith,

J. A. (1988) Current Protocols in Molecular Biology, Wiley Intersci- ence, New York

"

Booth. A. G.. and Kennv. A. J. (1980) Biochern. J. 187.31-44 Bowes; M. A:, and Kenny, A. J.'(1986) Biochem. J. 236,801-810 Crine, P., LeGrimellec, C., Lemieux, E., Labonti, L., Fortin, S.,

Blachier, A., and Aubry, M. (1985) Biochern. Biophys. Res. Com- mun. 131,255-261

Davis, A. R., Bos, T. J., and Nayak, D. P. (1983) Proc. Natl. Acad. Sci. U. S. A. 80,3976-3980

Devault, A,, Lazure, C., Nault, C., Le Moual, H., Seidah, N. G., Chritien, M., Kahn, P., Powell, J., Mallet, J., Beaumont, A., Roques, B. P., Crine, P., and Boileau, G. (1987) EMBO J. 6, 1317- 1322

Devault, A., Nault, C., Zollinger, M., Fournie-Zaluski, M.-C., Roques, B. P., Crine, P., and Boileau, G. (1988a) J . Biol. Chem. 263,4033- 4040

Devault, A., Sales, V., Nault, C., Beaumont, A., Roques, B. P., Crine, P., and Boileau, G. (1988b) FEBS Lett. 231, 54-58

Fulcher, I. S., Matsas, R., Turner, A. J., and Kenny, A. J. (1982) Biochem. J. 203, 519-522

Garoff, H. (1985) Annu. Reu. Cell Biol. 1,403-445 Gluzman, Y. (1981) Cell 28,51-59 Graham, F. L., and van der Eb, A. J. (1973) Virology 52,456-467 Jones, L. V., Compans, R. W., Davis, A. R., Bos, T. J., and Nayak,

Kenny, A. J., and Maroux, S. (1982) Physiol. Reu. 62,91-128 Kenny, A. J., and Fulcher, 1. S. (1983) Ciba Found. Symp. 95, 12-33 Kenny, A. J., Stephenson, S. L., and Turner, A. J. (1987) in Cell

Surface Peptidases (Kenny, A. J., and Turner, A. J., eds) pp. 169- 210, Elsevier Science Publishers B. V., Amsterdam

D. P. (1985) Mol. Cell Biol. 5, 2181-2189

Kerr, M. A,, and Kenny, A. J. (1974) Biochem. J. 137,477-488 Laemmli, U. K. (1970) Nature 227,680-685 Letarte, M., Vera, S., Tran, R., Addis, J. B., Onizaka, R. J., Quaken-

bush, E. J.. Jongeneel, C. V.. and McInnis, R. R. (1988) J. Enp. Med.' 168,.124711253

Lion J.. and Dobberstein. B. (1986) Cell 46, 1103-1112 Malfroy, B., Swerts, J . PI, Guyon, A., Roques, B. P., and Schwartz,

J. C. (1978) Nature 276 , 523-526 Malfroy, B., Schofield, P. R., Kuang, W. J., Seeberg, P. H., Mason,

A. J., and Henzel, W. S. (1987) Biochem. Biophys. Res. Commun.

Malfroy, B., Kuang, W.-J., Seeberg, P. H., Mason, A. J., and Scho- field, P. R. (1988) FEBS Lett. 229,206-210

Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

Mellon, P., Parker, V., Gluzman, Y., and Maniatis, T. (1981) Cell 27,

Murakami, H., Masui, H., Sato, G. H., Sueoka, N., Chow, T. P., and Kano-Sueoka, T. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 1158- 1162

Nakanishi, S., Teranishi, Y., Noda, M., Notak, M., Watanabr, Y., Kakidani, H., Jingami, H., and Numa, 5. (1980) Nature 287,752- 755

Noel, G., Zollinger, L., Lariviere, N., Nault, C., Crine, P., and Boileau, G. (1987) J . Biol. Chem. 262, 1876-1881

Schmid, S. R., and Spiess, M. (1988) J . Biol. Chem. 263, 16886- 16891

Semenza, G. (1986) Annu. Reu. Cell Biol. 2, 255-313 Szczesna-Skorupa, E., Browne, N., Mead, D., and Kemper, B. (1988)

Proc. Natl. Acad. Sci. U. S. A. 85, 738-742 Tam, L.-T., Engelbrecht, S., Talent, J. M., Gracy, R. W., and Erdos,

E. G. (1985) Biochem. Biophys. Res. Cornnun. 133 , 1187-1192 Taylor, J. W., Ott, J., and Eckstein, F. (1985) Nucleic Acids Res. 13,

Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. A d .

van Heijne, G. (1986) Nucleic Acids Res. 14,4683-4690 Wickner, W. T., and Lodish, H. F. (1985) Science 230,400-407

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FUSION OF A CLEAVABLE SIGNAL PEPTIDE TO THE ECTODOMAIN OF NEUTRkL ENDOPEPTIDASE (EC 3 4 24 1I)RESULTS IN THE

SECRETION OF AN kCTIYE ENZYME IN COS-I CELLS

EYPLRIMENTAL PROCEDURES

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