Human Lactase-Phlorizin Hydrolase Is Not Processed by Furin, PC1/PC3, PC2, PACE4 and PC5/PC6A of the Family of Subtilisin-Like Proprotein Processing Proteases

BB etBiochi~ic~a Biophysica AEta ELSEVIER Biochimica et Biophysica Acta 1311 (1996) 199-203

Human lactase-phlorizin hydrolase is not processed by furin, PC1/PC3, PC2, PACE4 and PC5/PC6A of the family of subtilisin-like proprotein processing proteases

Marcel Wiithrich a,b John W.M. Creemers c Wim J.M. van de Ven c Erwin E. Sterchi a,b,* a Institute of Biochemistry and Molecular Biology, Facul~, of Medicine, University of Berne, Berne, Switzerland b Department ofPaediatrics, Facul~' of Medicine, UniversiO' of Berne, Berne, Switzerland c Laboratory. of Molecular Oncology, Center of Human Genetics, Unicersi~ of Leuven, Leaven, Belgium

Received 11 July 1995; revised 24 November 1995; accepted 2 January 1996

Abstract

Human lactase-phlorizin hydrolase (LPH, EC 3.2.1.23/62) is synthesized as a single-chain precursor glycoprotein (pro-LPH) with a relative molecular mass of just over 200 kDa. Maturation to the mature enzyme (m-LPH, 160 kDa) occurs after passage of pro-LPH through the Golgi complex and involves the proteolytic removal of a 849 amino acid propeptide. The role of this propeptide as well as its removal is not fully understood and the proteolytic enzyme or enzymes involved are unknown. We studied the potential role of five different members of the family of subtilisin-like proprotein processing proteases in the maturation process of human LPH using a vaccinia virus based coexpression system in pig kidney PK(15) cells. Infected/transfected PK(15) cells expressed full-length pro-LPH but no maturation to m-LPH was observed. Coexpression of human pro-LPH with human furin, human PC1/PC3, human PC2, human PACE4 and mouse PC6A in PK(15) cells did not result in maturation of the enzyme. Cleavage and secretion of von Willebrand factor precursor (pro-vWF) was used as a positive control. None of the five proprotein processing proteases tested were capable of cleaving human pro-LPH, strongly suggesting that they are not involved in the maturation of this enzyme.

Keywords: Lactase-phlorizin hydrolase; Furin; Proprotein convertase; (Intestine)

1. Introduction brane protein. Biosynthetic studies in Caco-2 [6], transfected MDCK cells [7], and organ-cultured human small Small intestinal lactase-phlorizin hydrolase (LPH) (EC intestinal explants [8] have clearly shown that proteolytic 3.2.1.23/62) is responsible for the hydrolysis of lactose processing occurs after passage of pro-LPH through the during the suckling period. The structure of rabbit and Golgi complex. human LPH has been elucidated [1]. LPH is synthesized in A whole family of processing proteases has emerged in a pre-pro form in the lumen of the ER, where the N-termi- recent years, which are involved in maturation of bioactive nal signal peptide is removed. The pro-LPH (M r --215- peptides and proteins. The first enzyme of this novel 245 kDa) is proteolytically processed during transport to family to be described was the Kexin (EC 3.4.21.61) the microvillus membrane domain to yield the mature protease of yeast Saccharomyces cerevisiae. Kexin is a enzyme (Mr= 160 kDa) [2-5]. The mature enzyme is Ca 2÷- dependent serine protease with a subtilisin-like cat- inserted into the microvillus membrane as a type I mem- alytic domain, and is responsible for the processing of the pro-a-factor at dibasic sites [9]. Recently, seven mam- malian Kexin homologues, furin [10-12], PC2 [13-16] and Abbreviations: LPH, lactase-.phlorizin hydrolase; vWF, yon Wille- PCI/3 [14,16-20] PC4 [21], PACE4 [22], PC6A [23] and brand factor; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel PC6B [24] have been identified by cDNA cloning. Tissue electrophoresis; NP40, Nonidet ]?40; DOC, sodium deoxycholate; PBS, distribution of these processing endoproteases demon- phosphate-buffered saline; DEAE-dextran, o-(dimethylaminoethyl)-dextran; endo H, endoglycosidase It; PC, prohormone convertase. strated a unique pattern for each enzyme [13- * Corresponding author at address a. Fax: +41 31 6313737; e-mail: 17,19,21,23,25]. PC2 and PC1/3 preferentially cleave af- sterchi @ mci.unibe.ch. ter a pair of basic residues, Lys-Arg or Arg-Arg [26]. Furin

0167-4889/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PI1 S0167-4889(96)00007-9 200 M. Wiithrich et al. / Biochimica et Biophysica Acta 1311 (1996) 199-203 on the other hand has been shown to be involved in Recombinant vaccinia virus V.V.:T7 produces T7 RNA precursor cleavage at the consensus sequence Arg-X- polymerase [35]. After infection, medium was replaced Arg/Lys-Arg (RXK/RR) [27,28], but is also capable of with fresh medium containing 4 #g DNA and DOTAP cleaving the minimal cleavage site Arg-X-X-Arg (RXXR) transfection-reagent (Boehringer Mannheim Biochemical). [29,30]. Lipofectamine-mediated transfection was for 16-18 h ac- The N-terminal Ala (Alas69) [1] of mature LPH from cording to the suppliers instructions. human small intestine is preceded by an Arg residue (Arg868) and thus constitutes a typical trypsin-like cleav- 2.3. Metabolic labelling of PK(15) cells age site. An identical Arg-Ala site is present in rabbit LPH [31,32]. In addition, pro-LPH contains potential cleavage Biosynthesis and processing of pro-vWF- and pro-LPH sites for furin-like processing proteases upstream from the substrates were studied 16-18 h after cotransfection with N-terminal amino acid residue of mature LPH. There is a the different proprotein processing proteases. Prior to la- potential furin motif (RXXR) at positions belling of PK(15) cells, medium was removed and cells R829T830Ps31R832 . R832 is also part of a dibasic site were incubated for 1 h in RPMI medium that lacked (Args32Lys833) and an additional dibasic site is present at methionine. Subsequently, cells were labelled for 30 rain positions Lyss53Arg854 . There are also monobasic sites at in the presence of [35S]methionine (100 /xCi/ml, spec. positions Arg844, Args5 o, Lys866 and the Arg868 immedi- act. 800 Ci/mmol) (Du Pont NEN), followed by a chase ately in front of the N-terminal Alas69 of m-LPH. It is thus of 4 h with unlabelled methionine (final concentration i conceivable that pro-LPH is cleaved at one of these sites mM). In the case of the vWF samples, the medium was before the poly-peptide is further trimmed by an exopepti- spun down for 5 min at 3000 rpm and then concentrated dase to yield the mature LPH which may be isolated from by TCA-precipitation in the presence of bovine serum the enterocyte microvillus membrane. albumin (50 /xg/ml). Human LPH was immunoprecip- In this paper, we report the coexpression of a full-length itated from lysed PK15 cells as described below. cDNA for human pro-LPH together with cDNA for human furin, PC2, PC1/PC3, PACE4 and mouse PC6A in pig 2.4. Culture and metabolic labelling of Caco-2 cells kidney PK(15) cell lines in order to test the ability of these proprotein processing enzymes to cleave this precursor Caco-2 cells were cultured as previously described [6]. molecule. Metabolic labelling with 150 /xCi of L-[35S]methionine was continuous for 30 rain and 24 h, respectively. Labelled LPH was immunoprecipitated and analyzed by SDS-PAGE 2. Materials and methods as described.

2.1. Plasmids and cDNAs 2.5. Immunoprecipitation of human LPH

The full length cDNA coding for human LPH (kindly Labelled PK(15) cells were washed three times with 5 provided by Dr. Ned Mantei and Rahel B~inzinger, Swiss ml ice-cold PBS and lysed by adding 1 ml of lysis buffer Federal Institute of Technology, Ziirich) [1] was subcloned (25 mM Tris-HC1, pH 8.0, 50 mM NaCI) supplemented in pGEM3Zf + (Promega). Human von Willebrand factor with 1% NP 40, 1% DOC, 100 /zg/ml PMSF, 10 /xg/ml cDNAs (kindly provided by Dr. van Mourik, Central Lab- leupeptin, 2 /xg/ml pepstatin, 34.8 /xg/ml benzamidine, oratory of the Netherlands Red Cross Blood Transfusion and 2 /zg/ml aprotinin (all from Sigma). The lysates were Service) were cloned in pGEM7zf + [29,33]. Recombinant spun down at 100000 × g for 30 min prior to immuno- cDNA constructs coding for human furin, human precipitation. Solubilized membrane proteins were pre- PC1/PC3, human PC2, human PACE4 and mouse PC6A cleared twice with protein A-Sepharose CL-4B beads under the control of the T7 promoter, have been described (Pharmacia) and transferred to tubes with 100 /xl of anti- elsewhere [12,34,17,22,23,33] and were cloned in body-protein A-Sepharose (containing 3 /xl of the mono- pGEM3Zf + or 13Zf +. clonal antibody against lactase [36]). After an incubation period of at least 2 h at 4°C, the beads were washed three 2.2. Culture, infection and transfection of cells times with 1 ml PBS containing 0.5% NP-40, 0.05% DOC and 0.05% SDS and then twice with 125 mM Tris-HCl Pig kidney PK(15) cells were propagated in DMEM (pH 8.2), 500 mM NaC1, 1 mM EDTA, 0.5% NP-40. (Gibco BRL) supplemented with foetal calf serum (10% v/v) (Biological Industries), glutamine (2 mM) and antibi- 2.6. SDS-PAGE otics (penicillin (100 U/ml) and streptomycin (100 /xg/ml) (Gibco BRL). Cells were infected for 1 h with Immunoprecipitates were solubilized with 30 /xl of recombinant vaccinia virus V.V.:T7 at an m.o.i, of 5 in 2-fold concentrated electrophoresis sample buffer contain- medium containing bovine serum albumin (20 /xg/ml). ing 4% SDS, 20% glycerol, 5% 2-mercaptoethanol or 5 /xl M. Wi~thrich et al. / Biochimica et Biophysica Acta 1311 (1996) 199-203 201 of 2 M M-dithiothreitol (all from BioRad Laboratories), A boiled for 4 rain and submitted to electrophoresis on 6% a. uJ

200 kDa I III I 1 ~ ~ -- pro-LPH 3. Results and discussion

Recently obtained evidence from the intestinal Caco-2 97 kDa m cell line and from polarized MDCK cells transfected with human pro-LPH eDNA [7,38] has shown that proteolytic 68 kDa cleavage of human pro-LPH is a post-Golgi event, possibly 1 2 3 4 5 6 7 occurring in the trans-Golgi network. However, the protease or proteases involved still remain unknown. To investigate the potential involvement of subtilisin- B like proprotein processing proteases in the maturation of MW 1 2 human lactase-phlorizin hydrolase, PK(15) cells were cotransfected with pro-LPH eDNA and cDNAs encoding pro-LPHc human furin (Hufurin), human PC1/PC3 (HuPC1/PC3), 205 kDa ..... ~! = pro-LPHh human PC2 (HuPC2), human PACFA (HuPACE4), and mouse PCA (MoPC6A). In parallel control experiments, i!!!i!iiiiiiiiiii ii ; PK(15) cells were transfected with eDNA encoding the m __ - m-,p, precursor of von Willebrand factor (pro-vWF) together with cDNAs encoding proprotein processing proteases. Metabolic labelling of pro-LPH-transfected PK(I 5) cells 0.5 24 h pulse with [35S]methionine for 30 min, followed by a 4 h chase Fig. 1. (A) Analysis of endoproteolytic processing of human pro-LPH by resulted in the synthesis of pro-LPH species comparable to different convertases. PK (15) cells were first infected with recombinant those in intestinal epithelial cells (Fig. 1A, lanes 2-7). vaccinia virus V.V.:T7, which encodes T7 RNA polymerase (lane 1) and subsequently cells were lipofected with 2 /xg of pGEMLPH DNA (lane For comparison, LPH synthesis in human small intesti- 2). Other lanes show cotransfection with human furin (lane 3), human nal epithelial cells (Caco-2) yielded a 215 kDa high man- PCI/PC3 (lane 4), human PC2 (lane 5), human PACE4 (lane 6) and nose glycoprotein which was subsequently converted to a mouse PC6A (lane 7). Biosynthesis and processing of LPH-related pro- 225 kDa complex glycosylated form (Fig. 1B, lanes 1 and teins were analysed in labelling experiments using [35S]methionine. The 2). Proteolytic processing of pro-LPH then resulted in the transfected cells were labelled for 30 rain and subsequently chased for 4 h. The relative position of pro-LPH and the molecular weight markers are development of the 160 kDa mature LPH found in the indicated. (B) Biosyntheticlabelling of LPH in Caco-2 cells. Caco-2 cells microvillus membrane (Fig. 1B, lane 2). No mature LPH were labelled with [35S]methioninefor 30 min (lane 1) and 24 h (lane 2). was observed in PK(15)cells transfected with pro-LPH After the indicated intervals LPH was immunoprecipitatedby the mono- cDNA only (Fig. 1A, lane 2). clonal antibody HBB 1/909/34/74. Immunoprecipitateswere subjected None of the cotransfected proprotein processing pro- to SDS-PAGE on 6% gels, followedby fluorography.Exposure time of lane 1 was 21 days for lane 2 it was 7 days. teases human furin, human PC1/PC3, human PC2, human PACE4 and mouse PC6A were capable of cleaving the expressed pro-LPH in PK(15) cells (Fig. 1A, lanes 3-7). pro-vWF. This may be due to the fact that these enzymes Control experiments of infected PK(15) cells transfected are thought to be involved in the processing of proteins in with the protease cDNAs together with von Willebrand the regulated secretion pathway [40,41], a pathway not factor (pro-vWF) eDNA are shown in Fig. 2. Expression present in PK(15) cells. Coexpression of pro-LPH with of vWF alone resulted in the synthesis and subsequent HuPACE4 and MoPC6A resulted in substantial cleavage secretion of mainly uncleaved pro-vWF (Fig. 2, lane 2). of pro-vWF. Cotransfection with Hufurin eDNA resulted in a quantita- In conclusion, cotransfection of human pro-LPH with tive conversion of pro-vWF to the mature vWF (Fig. 2, the different members of the furin family of proprotein lane 3). Insignificant cleavage was observed in cells ex- processing enzymes in PK(15) cells did not result in pressing HuPC 1/PC3 (Fig. 2, lane 4) and HuPC2 (Fig. 2, cleavage of pro-LPH. As proprotein synthesis and secre- lane 5). Although expression of HuPC1/PC3 and HuPC2 tion were functional in PK(15) as illustrated with vWF, under the experimental conditions used has been verified and the pro-LPH species expressed under the experimental [39] these enzymes exhibited little or no activity towards conditions used corresponded to those in intestinal epithe- 202 M. Wiithrich et al. /Biochimica et Biophysica Acta 1311 (1996) 199-203

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