6 of the furin family of proprotein processing enzymes

BIt ELSEVIER

Biochi~ic~a et BiophysicaAEta

Biochimica et BiophysicaActa 1246 (1995) 185-188

Amyloid precursor protein is not processed by furin, PACE 4, PC1/3, PC2, PC4 and PC5/6 of the furin family of proprotein processing enzymes Bart De Strooper a, John W . M . C r e e m e r s b Dieder Moechars a Danny Huylebroeck c Wim J.M. Van De Ven b, Fred Van Leuven a,*9 Herman Van den Berghe a,b 9

9

9

a Laboratory for Experimental Genetics, University ofLeuven, B-3000 Leuven, Belgium b Laboratory for Molecular Ontology, Center for Human Genetics, University of Leuven, Campus Gasthuisberg, 0 and N6, Herestraat 49, B-3000 Leuven, Belgium e Laboratory of Molecular Biology, University ofLeuven, B-3000 Leuven, Belgium

Received 7 July 1994; accepted19 September1994

Abstract

Proteolytic cleavage of the amyloid precursor protein (APP) has previously been shown to release its extracellular domain into the medium. The identification of the responsible proteinase(s), termed secretase(s), is a high priority in ongoing Alzheimer research. This is hampered by the unusual characteristics of these enzyme(s) and by the fact that they cleave only membrane associated APP.We report here, using a vaccinia virus based expression system, that pig kidney PK(15) ceils express full-length, membrane bound APP695, but that secretion of APP is low. This heterologous expression system allows to assay candidate secretases in a cellular context by simple co-transfection of the APP and candidate seeretase eDNA containing plasmids. Eight different members of the mouse and human furin family of proprotein processing enzymes were tested in this assay, but none of them enhanced the secretion of APP. Secretion of von Willebrand's factor was used as a positive control. Keywords: Furin; Amyloidprecursor protein; Proprotein convertase;Alzheimer's disease

1. Introduction

Cleavage of the amyloid precursor protein (APP) generates the amyloidogenic flA4-peptides that precipitate in the plaques of Alzheimer patients [1]. The normal cellular processing of APP includes several proteolytic steps [2] yielding either the flA4-peptide, or the p3-peptide and APPs, the soluble extracellalar domain of APP. Interestingly, all the known point mutations in the APP-gene linked to familial Alzheimer disease (AD), are clustered around the cleavage positions in APP [3]. This strongly suggests that abnormalities :in the proteolytic processing of APP may be directly related to the pathogenesis of AD. In the culture medium of cells, mainly APP~ is detected. The hypothetical a-secretase that releases this fragment, cleaves

* Corresponding author. E-mail:[email protected]: + 32 16 345871. 0167-4838/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0167-4838(94)00194-4

APP in the flA4-sequence and is therefore anti-amyloidogenic [4]. The identification of this enzyme is one of the main goals in current Alzheimer's research. Several observations indicate that a-secretase operates intracellularly in a compartment between the trans-Golgi network and the cell surface [5-7]. While APP is cleaved at the cell surface as well [8,9], this seems not to be the preponderant site because extraceUularly added proteinase inhibitors do not significantly inhibit APP processing [5]. In contrast, methylamine, a primary amine that accumulates in acid compartments such as the late compartments of the constitutive secretion pathway, inhibits in a dose-dependent way the secretion of APP~ [10]. Deletion of endocytosis and lysosomal sorting determinants in the cytoplasmic domain of APP does not interfere with cleavage and secretion, indicating that the a-cleavage does not occur in the endosomal/lysosomal pathways [10-12]. Recently, microsequencing of APP-fragments in different cell lines demonstrated heterogeneity at the a-secretase

186

B. De Strooper et al. /Biochimica et Biophysica Acta 1246 (1995) 185-188

cleavage site [13]. This was interpreted to suggest that several proteinases are involved in the secretory cleavage of APP. While the alternative explanation of one single a-secretase cleavage, followed by proteolytic trimming by peptidyl peptidases was not excluded directly, the hypothesis of multiple a-secretases is attractive, since it explains the relative lack of sequence specificity of c~-secretase as observed in extensive mutagenesis studies [9,10,14]. Replacing lysine by aspartic acid at position P1 amino terminal of the ~-secretase cleavage site of mouse APP695, together with replacing arginine by glutamic acid at position P4, resulted in a 50% decrease of the rate of secretion of APPs in transfected COS-1 cells, indicating that at least one of the putative a-secretases requires a basic residue at either P1, P4 or at both positions [10]. Proteinases of the furin family of proprotein processing enzymes are potential candidates for a-secretase activity [15-17]. Furin, the mammalian prototype of this family, is an ubiquitously expressed subtilisin-like proteinase that is membrane anchored in the Trans Golgi Network, but which can also appear at the cell surface [18]. Substrates for this enzyme are membrane bound glycoproteins like the insulin receptor precursor and soluble precursors of secreted proteins as von Willebrand factor (vWF), the activin A and B precursor polypeptide, fl-NGF and complement pro-C3 [19-23]. Interestingly, cleavage and secretion of the later protein is inhibited by methylamine, similarly to what was observed for APP [24,25,10]. While furin cleaves substrates on the carboxyl side of the consensus sequence Arg-X-Lys/Arg-Arg which is not present in APP, the basic residue at the P2 position has been shown to be less important [26,27]. The neuroendocrine specific proprotein processing enzymes P C 1 / P C 3 and PC2 cleave usually carboxy-terminal of paired basic sequences Arg-Arg or Lys-Arg [15-17]. The sequence specificity of the other members of the furin family, namely PC4, PC5/PC6 (with two isoforms P C 5 / P C 6 A and PC5/PC6B) and PACE 4 has been studied less exhaustively [15-17]. Except for PACE4, their expression is more restricted (Table 1) and in addition to furin only PC5/PC6B contains a transmembrane domain [17]. The possibility that one or more members of the furin family display a-secretase activity, was tested towards APP expressed in vaccinia virus infected pig kidney PK(15) cells, which have been shown to contain low endogenous furin activity [23].

Table 1 The furin family of proprotein processingenzymes Enzyme Membrane Tissue associated expression Furin + ubiquitous PC1/PC3 (+ ) neuroendocrine cells PC2 (+ ) neuroendocrine cells PC4 germ line cells PACE 4 ubiquitous PC5/PC6A selected endocrine and non-endocrine cells PC5/PC6B + small intestine Furin and PC5/PC6B contain a hydrophobicregion that probablyfunctions as a transmembrane anchor. PC1/PC3 and PC2 have both an amphiphatic a-helical segment near their carboxylterminus that may be involved in membrane association. The other proteins are most likely soluble. See text for discussion and for references

pGEM3Zf(+ ) or 13Zf(+ ) (Promega), allowing expression under the T7-promoter, as described before [26,29]. The cDNA coding for pro-von Willebrand factor was cloned in pGEM 7zf( + ) [26,29]. The cDNAs containing all coding sequences of mouse PC1/PC3 and human PC2 were kindly provided by Dr. D. Steiners, Chicago. The cDNAs of mouse PC4, mouse PC5/PC6A, mouse P C 5 / 6 B and human PACE 4, were a generous gift of Dr. K. Nakayama, Tsukuba. 2.2. Cell culture and infection

Pig kidney PK(15) cells were cultured in DMEM supplemented with FCS and antibiotics as described previously [26]. Cells were infected with recombinant vaccinia virus, V.V.:TT, [30] at a m.o.i, of 5, in medium containing bovine serum albumin ( 2 0 / x g / m l ) . Recombinant V.V.:T7 produces T7 RNA polymerase [30]. After 1 h, fresh medium was added and the cells were transfected with 4 ~ g DNA, using the DOTAP transfection reagent and procedure (Boehringer Mannheim). Metabolic labeling of cells was performed 16-18 h after lipofection. Cells were starved for 1 h in methioninefree RPMI medium and subsequently labeled for 4.5 h in the presence of 1 0 0 / x C i / m l [35S]methionine. Media were collected and centrifuged for 5 min at 3000 rpm. Cells were washed with PBS and solubilized in RIPA-buffer [31]. Media and cell extracts were either processed directly or stored at - 2 0 ° C . 2.3. Immunoprecipitation o f A P P

2. Materials and methods 2.1. Plasmids and cDNAs

cDNA coding for mouse APP 695 and 770 [28] were cloned in p G E M 9 Z f ( - ) (Promega). The cDNA coding for human furin, PC1/PC3, PC2, PACE 4 and for mouse PC4, PC1/PC3, P C 5 / P C 6 A and P C 5 / 6 B were cloned in

Cell lysates and conditioned media were precleared with 3 × 5 0 / z l protein A Sepharose 4B (Pharmacia) saturated with Swine anti-Rabbit antibodies (DAKO). APP was immunoprecipitated with a polyclonal APP-antiserum B 2 / 3 as described [10]. Precipitated material was extensively washed and applied on a 6% polyacrylamide gel after solubilization in SDS-containing buffer [31]. After

187

B. De Strooper et al. /Biochimica et Biophysica Acta 1246 (1995) 185-188

o,w, h olh c,,3 hc2 h . CEIo C , C ,81

soaking in P P O / D M S O , the gels were exposed to Hyperfilm-MP (Amersham) for 2 to 7 days.

-pro vWF 200"

3. Results and discussion PK(15) cells infected with recombinant vaccinia virus (V.V.:T7), producing functional T7 R N A polymerase [30], were shown to express A P P when transfected with c D N A coding for A P P under the ogntrol of a T7 promoter (Fig. 1). A P P is synthesized as a 110 to 140 kDa glycoprotein in these cells, in accordance with previous findings in other cell lines [32,33], (Fig. 1). The lower molecular weight protein migrating slightly slower than the 103 kDa marker protein is the N-glycosylatedl precursor, while the smeared protein band with a mobi][ity around 140 kDa is the complex N- and O-glycosylated A P P [32,33]. After 4.5 h continuous labeling, however, most of the produced A P P was still associated with the infected PK(15) cells, while in

control APP 695

human furln

°I, ......

c

,

~

human

mock

PC1/3

trsnaf.

,I°

cl'

:~

~

~i~! ~ii~ ~ ~

103human PC2

cl,

mouse PC.4

c

,

mouse PCS/6A

mouse PC1/3

cl,

° ,

103-

I mou.. I huma:-] PO5/6. I PAOELJ

o1,1o Fig. 1. Proteinases of the furin family of proprotein processing enzymes do not enhance secretion of APP in PK(15) cells. Immunoprecipitated APP from cell extracts (C) or conditioned medium (S) of vaccinia virus V.V.:T7 [30] infected and pGEM9zf(-) mouse APP695 transfected PK(15) cells or from PSG5 mouse APP695 transfected COS-1 cells, metabolically labeled for 4.5 h. The first two lanes (control) show APP695 precursor (dense band) and glycosylated forms (smeared band) associated with the cells (C), but low APPs-secretionin the medium (S) of PK(15) cells. For comparison APP695 secretion by pSG5 mouse APP695 transfected COS-1 cells is illustrated at the lower right panel (labeled COS-I, APP695). Other lanes demonstrate that co-transfection with respectively human furin, human PC1/3, human PC2, mouse PC4, mouse PC5/PC6A, mouse PC1/3, mouse PC5/PC6B and human PACE 4 cDNA-containing TT-plasmid, does not increase secretion of APP695 into the medium. The lane labeled 'mock transl.' displays immunoprecipitated PK(15) cells which were virus-infected, followed by transfection with vectors (no insert) only. Autor~diographsare representative for three separate experiments. The position of a marker protein of 103 kDa is indicated on the left.

-vWF

Fig. 2. Secretion and processing of pro-vWF in PK(15) cells. Cultured PK(15) cells were infected with V.V:T7 and subsequently transfected with pGEM 7zf( + ) plasmid coding for pro-von Willebrand factor (provWF) and metabolically labeled with [35S]methionine for 30 min and chased for 4 h. Proteins were collected from the medium by TCA precipitation, dissolved in SDS-containing buffer, and electrophoresed under reducing conditions in 7.5% SDS-PAGE. Mock: infected PK(15) cells were transfected with T7 plasmids (no inserts); pro-vWF: cells transfected with pGEM 7zf( + ) pro-vWF-plasmid, secrete largely unprocessed pro-vWF into the medium. Co-transfection with human fufin (hfurin, lane 3) results in complete processing of pro-vWF to von Willebrand's factor (vWF). In the other lanes the effect of co-transfecting human PC1/3, human PC2, human PACE 4, mouse PC5/6A, and mouse PC5/6B is illustrated. The mobility of precursor (pro-vWF) and cleaved vWF (vWF) is indicated on the fight. The position of a 200 kDa marker protein is shown on the left hand side of the figure. Autoradiograph of a representative experiment is shown.

COS-1 cells a significant amount of the produced A P P was already secreted into the medium (Fig. 1). Secretion of the precursor of the von Willebrand factor (pro-vWF), which does not contain a membrane anchor, was under similar experimental conditions easily demonstrated (Fig. 2, lane 2). The low level of secretion of A P P is thus not caused by a faulty secretion mechanism during the late phase of vaccinia virus infection. A decrease in endogenous furinlike proteolytic activity towards several proteins, including p r o - v W F (see Fig. 2, lane 2), has previously been demonstrated to occur under these experimental conditions [ 2 1 23]. W e suggest therefore that a decrease in proteolytic cleavage of A P P is the most likely explanation for the observed low rate of secretion of A P P s. Proteolytic processing activity towards p r o - v W F can be restored by cotransfecting plasmids coding for furin (Fig. 2, lane 3). The eight known members of the mouse and human furin family were tested for their ability to restore APPcleavage and secretion. However, no increase in secretion of APP was observed after co-transfection with plasmids containing the c D N A s for human furin, P C 1 / 3 , PC2, P A C E 4 and mouse PC4, P C 1 / 3 , P C 5 / P C 6 A and P C 5 / P C 6 B (Fig. 1). A s a control, proteolytic processing of p r o - v W F was also assayed (Fig. 2). Infection/transfection of PK(15) cells with a construct encoding p r o - v W F results in the secretion of mainly unprocessed p r o - v W F (Fig. 2, lane 2). Co-transfection with a plasmid coding for furin (Fig. 2, lane 3) results in complete cleavage [29]. The two other widely expressed convertases P A C E 4 and P C 5 / P C 6 A process p r o - v W F less completely. While expression of P C 1 / 3 and PC2 under the experimental conditions of the assay was certified (result not shown), these enzymes display no activity towards pro-vWF. In similar experiments low processing activity was detected for P C 1 / P C 3 (J.W.M. Creemers, unpublished data). No sub-

188

B. De Strooper et a l . / Biochimica et Biophysica Acta 1246 (1995) 185-188

strate processing activity for PC2, PC4 and P C 5 / 6 B could be detected in PK(15) cells. It should be mentioned that PC2 (and PC1/3) are involved in the processing of proproteins in the regulated secretory pathway [15-17] which is not operative in PK(15) cells. Differences in local pH, ion concentrations or the absence of cell specific chaperones might render these enzymes inactive in PK(15) cells. The activity of the germ line specific PC4 convertase and the intestine specific PC5/PC6B convertase might be cell specific as well. We conclude that co-transfection of APP695 with the respective members of the furin family of proprotein processing enzymes in PK(15) cells, does not lead to increased secretion of APP,. Since protein processing and secretion was active as illustrated with pro-vWF, and since the APP cDNA was expressed under the experimental conditions used, we further conclude that the tested proteinases are not involved in the proteolytic conversion of APP into its soluble form. Our experiments do not exclude that other members of this emerging family are responsible for the a-secretase-like conversion of APP in PK(15) cells. In this regard, the assay system developed here can directly test novel candidate APP secretases. Since the cellular configuration seems to be essential for the specificity of a-secretase [9], we will continue to rely on this current assay to screen further candidate APP-processing enzymes.

Acknowledgements This investigation was supported by grants 3.0069.89 and 3.0073.93 from the 'Fonds voor Geneeskundig Wetenschappelijk Onderzoek', by EC-contract BIOT-CT91-0302, by STW-contract NCH22.2726, by a grant 'Geconcerteerde Acties' of the 'Ministerie voor Onderwijs' of the Belgian Government, by a grant of the interuniversity-network for Fundamental Research (IUAP, 1991-1996). Part of this work is done under contract with the Action Program for Biotechnology of the Flemish government (VLAB, ETC-008). B.D.S. is a postdoctoral fellow of the 'Nationaal Fonds voor Wetenschappelijk Onderzoek'. We thank the 'IWONL' for a scholarship to D.M. and the 'Katholieke Universiteit van Leuven' for continuous support. The authors would also like to thank P. Groot Kormelink and E.W. Beek for their contributions to the vaccinia virus experiments.

References [1] Kang, J., Lemaire, H.G., Unterbeck, A., Salbaum, J.M., Masters, C.L., Grzeschik, K.H., Multhaup, G., Beyreuther, K. and Miiller-Hill, B. (1987) Nature 325, 733-736. [2] Haass, C. and Selkoe, D. (1993) Cell 75, 1039-1042. [3] Hardy, J. and Mullan, M. (1992) Nature, 268-269.

[4] Esch, F.S., Keim, P.S., Beattle, E.C., Blacher, R.W., Culwell, A.R., Oltersdorf, T., McClure, D. and Ward, P.J. (1990) Science 248, 1122-1124. [5] De Strooper, B., Van Leuven, F. and Van Den Berghe, H. (1992) FEBS Lett. 308, 50-53. [6] Sambamurti, K., Shioi, J., Anderson, J.P., Pappolla, M.A. and Robakis, N. (1992) J. Neurosci. Res. 33(2), 319-329. [7] Kuentzel, S., Ali, S., Altman, R., Grcenberg, B. and Raub, T. (1993) Biochem. J. 295, 367-378. [8] Haass, C., Koo, E., Mellon, A., Hung, A. and Selkoe, D. (1992) Nature 357, 500-502. [9] Sisodia, S. (1992) Proc. Natl. Acad. Sci. USA 89, 6075-6079. [10] De Strooper, B., Umans, L., Van Leuven, F. and Van den Berghe, H. (1993) J. Cell Biol. 121, 295-304. [11] Caparoso, G., Gandy, S., Buxbaum, J. and Grcengard, P. (1992) Proc. Natl. Acad. Sci. USA 89, 2252-2256. [12] Da Cruz E Silva, OAB., Iverfeldt, K., Oltersdorf, T., Sinha, S., Lieberburg, I., Ramabhadran, T., Suzuki, T., Sisodia, S., Gandy, S. and Greengard, P. (1993) Neuroscience 57, 873-877. [13] Zhong, Z., Higaki, J., Murakami, K., Wang, Y., Catalano, R., Quon D. and Cordell, B. (1994) J. Biol. Chem. 269, 627-632. [14] Maruyama, K., Kametani, F., Usami, M., Yamao-Harigaya, W. and Tanaka, K. (1991) Biochem. Biophys. Res. Commun. 179, 16701676. [15] Van De Ven, W.J.M., Van Duynhoven, J.L.P. and Roebroek, A.J.M. (1992) Crit. Rev. Oncogen 4, 115-136. [16] Steiner, D.F., Smeekens, S.P., Ohagi, S. and Chan, S.J. (1992) J. Biol. Chem. 267, 23435-23438. [17] Halban, P.A. and Irminger, J.C. (1994) Biochem. J. 299: 1-18. [18] Molloy, S.S., Thomas, L., Van Slyke, J.K., Stenberg, P.E. and Thomas, G. (1994) EMBO J. 13, 18-33. [19] Van De Ven, W.J.M., Voorberg, J., Fontijn, R., Pannekoek, H., Van den Ouweland, A.M.W. and Siezen, R.J. (1990) Mol. Biol. Rep. 14, 265-275. [20] Wise, R.J., Barr, p.j., Wong, P.A., Kiefer, M.C., Brake, A.J. and Kaufman, R.J. (1990) Proc. Natl. Acad. Sci. USA 87, 9378-9382. [21] Roebroek, A.J.M., Creemers, J.W.M., Pauli, I.G.L., Bogaert, T. and Van De Ven, W.J.M. (1993) EMBO J. 12, 1853-1870. [22] Misumi, Y., Oda, K., Fujiwara, T., Takami, N., Tashiro, K. and Ikerhara, Y. (1991) J. Biol. Chem. 266, 16954-16959. [23] Huylebroeck, D., Verschueren, K. and De Wade, P. (1994) in Inhibin and inhibin-related proteins (Burger, H.G., ed.), pp. 271-287, Ares Serona Symposia Publications, Rome. [24] Oda, K. and Ikehara, Y. (1985) Eur. J. Biochem. 152, 605-609. [25] Oda, K., Koriyama, Y., Yamada, Y. and Lkehara, Y. (1986) Biochem. J. 240, 739-745. [26] Creemers, J.W.M., Siezen, R.J., Roebroek, A.J.M., Ayoubi, T.A.Y., Huylebroeck, D. and Van de Ven, W.J.M. (1993) J. Biol. Chem. 268, 21826-21834. [27] Molloy, S.S., Brenahan, P.A., Leppla, L., Klimpel, K. and Thomas, G. (1992) J. Biol. Chem. 267, 16396-16402. [28] De Strooper, B., Van Leuven, F. and Van Den Berghe, H. (1991) Biochim. Biophys. Acta 1129, 141-143. [29] Creemers, J.W.M., Groot Kormelink, P.J., Roebroek, A.J.M., Nakayama, K. and Van de Ven, W.J.M. (1993) FEBS Lett. 336, 65-69. [30] Feurst, T.O., Niles E.G., Studier, F.W., Moss, B. and /_,oh, Y.P. (1986) Proc. Natl. Acad. Sci. USA 83, 8122-8126. [31] De Strooper, B., Van Leuven, F., Carmeliet, G., Van den Berghe, H. and Cassiman, J.J. (1991) Eur. J. Biochem. 199, 25-33. [32] Weidemann, A., K6nig, G., Bunke, D., Fischer, P., Salbaum, J.M., Masters, C.L. and Beyreuther, K. (1989) Cell 57, 115-126. [33] Pahlson, P., Shakin-Eshleman, S., and Spitalnik, S. (1992) Biochem. Biophys. Res. Commun. 189, 1667-1673.