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• W2047052174 abstract "Polycystin-1 (PC1), the PKD1 gene product, plays a critical role in renal tubule diameter control and disruption of its function causes cyst formation in human autosomal dominant polycystic kidney disease. Recent evidence shows that PC1 undergoes cleavage at the juxtamembrane G protein-coupled receptor proteolytic site (GPS), a process likely to be essential for its biological activity. Here we further characterized the proteolytic cleavage of PC1 at the GPS domain. We determined the actual cleavage site to be between leucine and threonine of the tripeptide HLT3049 of human PC1. Cleavage occurs in the early intracellular secretory pathway and requires initial N-glycan attachment but not its subsequent trimming. We provide evidence that the cleavage occurs via a cis-autoproteolytic mechanism involving an ester intermediate as shown for Ntn hydrolases and EMR2. Polycystin-1 (PC1), the PKD1 gene product, plays a critical role in renal tubule diameter control and disruption of its function causes cyst formation in human autosomal dominant polycystic kidney disease. Recent evidence shows that PC1 undergoes cleavage at the juxtamembrane G protein-coupled receptor proteolytic site (GPS), a process likely to be essential for its biological activity. Here we further characterized the proteolytic cleavage of PC1 at the GPS domain. We determined the actual cleavage site to be between leucine and threonine of the tripeptide HLT3049 of human PC1. Cleavage occurs in the early intracellular secretory pathway and requires initial N-glycan attachment but not its subsequent trimming. We provide evidence that the cleavage occurs via a cis-autoproteolytic mechanism involving an ester intermediate as shown for Ntn hydrolases and EMR2. Polycystin-1 (PC1) 2The abbreviations used are: PC1, polycystin-1; PKD, polycystic kidney disease; ADPKD, autosomal dominant PKD; HEK cells, human embryonic kidney cells; MDCK cells, Madin-Darby canine kidney cells; WT, wild type; aa, amino acid(s); REJ, receptor for egg jelly; BFA, brefeldin A; GPS, G-protein-coupled receptor proteolytic site; uFL, uncleaved full-length; NTF, N-terminal fragment; CTF, C-terminal fragment; DOC, degree of cleavage; ETL, EGF-TM7-latrophilin-related protein.2The abbreviations used are: PC1, polycystin-1; PKD, polycystic kidney disease; ADPKD, autosomal dominant PKD; HEK cells, human embryonic kidney cells; MDCK cells, Madin-Darby canine kidney cells; WT, wild type; aa, amino acid(s); REJ, receptor for egg jelly; BFA, brefeldin A; GPS, G-protein-coupled receptor proteolytic site; uFL, uncleaved full-length; NTF, N-terminal fragment; CTF, C-terminal fragment; DOC, degree of cleavage; ETL, EGF-TM7-latrophilin-related protein. is encoded by the PKD1 gene, which is mutated in 85–90% of human autosomal dominant polycystic kidney disease (ADPKD), which affects 1 in 1000 people world-wide (1Gabow P.A. N. Engl. J. Med. 1993; 329: 332-342Crossref PubMed Scopus (838) Google Scholar). ADPKD is characterized by the progressive development of numerous fluid-filled cysts derived from tubular epithelial cells in both kidneys, which leads to end-stage kidney disease in ∼50% of affected individuals by the age of 60. ADPKD is a systemic disease with many extrarenal manifestations, including cyst formation in the liver and spleen and cardiovascular abnormalities (1Gabow P.A. N. Engl. J. Med. 1993; 329: 332-342Crossref PubMed Scopus (838) Google Scholar). PC1 is thought to function as a cell surface signaling receptor at cell-cell/cell-matrix junctions and as a mechano-sensor in renal primary cilia that activates signaling pathways involved in renal tubular differentiation (2Boletta A. Germino G.G. Trends Cell Biol. 2003; 13: 484-492Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 3Harris P.C. Torres V.E. Curr. Opin. Nephrol. Hypertens. 2006; 15: 456-463Crossref PubMed Google Scholar, 4Nauli S.M. Alenghat F.J. Luo Y. Williams E. Vassilev P. Li X. Elia A.E. Lu W. Brown E.M. Quinn S.J. Ingber D.E. Zhou J. Nat. Genet. 2003; 33: 129-137Crossref PubMed Scopus (1585) Google Scholar). It is a 4302-amino acid (aa) 11-transmembrane glycoprotein with a large N-terminal extracellular region (ectodomain) of 3072 aa and a short cytoplasmic C-terminal tail of ∼200 aa (5Hughes J. Ward C.J. Peral B. Aspinwall R. Clark K. San Millan J.L. Gamble V. Harris P.C. Nat. Genet. 1995; 10: 151-160Crossref PubMed Scopus (760) Google Scholar) (Fig. 1A). The ectodomain contains a novel combination of motifs. One of them is the ∼1000-aa long receptor for egg jelly (REJ) domain, a structure of unknown function, which was originally described in sea urchin receptor for egg jelly protein (6Moy G.W. Mendoza L.M. Schulz J.R. Swanson W.J. Glabe C.G. Vacquier V.D. J. Cell Biol. 1996; 133: 809-817Crossref PubMed Scopus (217) Google Scholar). Situated between the REJ domain and the first transmembrane domain is the GPS (G protein-coupled receptor proteolytic site) domain of ∼50 aa (7Ponting C.P. Hofmann K. Bork P. Curr. Biol. 1999; 9: R585-R588Abstract Full Text Full Text PDF PubMed Google Scholar, 8Qian F. Boletta A. Bhunia A.K. Xu H. Liu L. Ahrabi A.K. Watnick T.J. Zhou F. Germino G.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16981-16986Crossref PubMed Scopus (234) Google Scholar). It was first demonstrated to be the internal cleavage site for the neuronal G protein-coupled receptor, latrophilin/CIRL, with the actual cleavage site at HL↓T (where ↓ identifies the position of cleavage) (9Krasnoperov V.G. Bittner M.A. Beavis R. Kuang Y. Salnikow K.V. Chepurny O.G. Little A.R. Plotnikov A.N. Wu D. Holz R.W. Petrenko A.G. Neuron. 1997; 18: 925-937Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). It was later identified in many proteins of the LNB-TM7 family, a group of seven-transmembrane receptors related to family-B G protein-coupled receptors (10Stacey M. Lin H.H. Gordon S. McKnight A.J. Trends Biochem. Sci. 2000; 25: 284-289Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). These proteins, like PC1, are extraordinary for having unusually large and complex N-terminal extracellular regions; they include EMR2, ETL, and Drosophila Flamingo. We have previously shown indirectly that PC1 undergoes cleavage at the GPS domain (8Qian F. Boletta A. Bhunia A.K. Xu H. Liu L. Ahrabi A.K. Watnick T.J. Zhou F. Germino G.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16981-16986Crossref PubMed Scopus (234) Google Scholar). This reaction results in the N-terminal fragment (NTF) and C-terminal fragment (CTF), which remain tethered noncovalently. One unique aspect of PC1 cleavage is that it is incomplete when expressed in various cell types. The degree of cleavage (DOC; (defined as the fraction of CTF over the sum of CTF and uncleaved full-length (uFL)) is typically ∼50% (i.e. an equal amount of CTF and uFL). Partial cleavage has also been found for endogenous PC1 (8Qian F. Boletta A. Bhunia A.K. Xu H. Liu L. Ahrabi A.K. Watnick T.J. Zhou F. Germino G.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16981-16986Crossref PubMed Scopus (234) Google Scholar). PKD1-associated missense mutations in the GPS domain and the neighboring REJ domain, as well as synthetic mutations at the predicted cleavage site, were found to disrupt the cleavage. They also resulted in loss of the functional properties of PC1 to activate the JAK2-STAT pathway and induce in vitro tubulogenesis of MDCK cells (8Qian F. Boletta A. Bhunia A.K. Xu H. Liu L. Ahrabi A.K. Watnick T.J. Zhou F. Germino G.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16981-16986Crossref PubMed Scopus (234) Google Scholar). These findings indicate that GPS cleavage likely plays a critical role for the biological function of PC1. The GPS or GPS-like sequence is present at a similar juxtamembrane position of all PC1 family members (11Li A. Tian X. Sung S.W. Somlo S. Genomics. 2003; 81: 596-608Crossref PubMed Scopus (61) Google Scholar, 12Yuasa T. Takakura A. Denker B.M. Venugopal B. Zhou J. Genomics. 2004; 84: 126-138Crossref PubMed Scopus (37) Google Scholar) (Fig. 1A). The cleavage property, however, varies among the few PC1 family members in which cleavage has been studied. Cleavage has also been demonstrated for suREJ3 (13Mengerink K.J. Moy G.W. Vacquier V.D. J. Biol. Chem. 2002; 277: 943-948Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), and PKDREJ and suREJ2 have recently been shown to be uncleaved (14Butscheid Y. Chubanov V. Steger K. Meyer D. Dietrich A. Gudermann T. Mol. Reprod. Dev. 2006; 73: 350-360Crossref PubMed Scopus (33) Google Scholar, 15Galindo B.E. Moy G.W. Vacquier V.D. Dev. Growth Differ. 2004; 46: 53-60Crossref PubMed Scopus (21) Google Scholar). The functional significance of cleavage or noncleavage for the PC1 family is currently unclear. Proteolytic cleavage at the extracellular juxtamembrane position occurs in many membrane proteins including receptors and channel proteins (16Hooper N.M. Karran E.H. Turner A.J. Biochem. J. 1997; 321: 265-279Crossref PubMed Scopus (556) Google Scholar). It serves a variety of functions ranging from precursor activation (e.g. ENac) (17Hughey R.P. Bruns J.B. Kinlough C.L. Harkleroad K.L. Tong Q. Carattino M.D. Johnson J.P. Stockand J.D. Kleyman T.R. J. Biol. Chem. 2004; 279: 18111-18114Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar) to receptor inactivation (e.g. V2 vasopressin receptor) (18Kojro E. Fahrenholz F. J. Biol. Chem. 1995; 270: 6476-6481Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) to creation of a high-affinity binding pocket for ligands (19Williams J.F. McClain D.A. Dull T.J. Ullrich A. Olefsky J.M. J. Biol. Chem. 1990; 265: 8463-8469Abstract Full Text PDF PubMed Google Scholar). It occurs generally by proteases along the secretory pathway (e.g. furin) (20Taylor N.A. Van De Ven W.J. Creemers J.W. FASEB J. 2003; 17: 1215-1227Crossref PubMed Scopus (190) Google Scholar) or at the plasma membrane (e.g. TACE sheddase) (21Malemud C.J. Front. Biosci. 2006; 11: 1696-1701Crossref PubMed Scopus (562) Google Scholar). However, several types of self-catalyzed protein modifications that do not require the intervention of other enzymes have been identified (22Perler F.B. Nat. Struct. Biol. 1998; 5: 249-252Crossref PubMed Scopus (38) Google Scholar). The best characterized mechanism among them is the cis-autoproteolysis, a self-catalyzed protein rearrangement that results in cleavage at the HX↓(T/S/C) (22Perler F.B. Nat. Struct. Biol. 1998; 5: 249-252Crossref PubMed Scopus (38) Google Scholar, 23Xu Q. Buckley D. Guan C. Guo H.C. Cell. 1999; 98: 651-661Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). This process is essential for the biological function of a diverse group of proteins that include Hedgehog, glycosylasparaginase, nucleoporin, and intein-containing proteins (24Paulus H. Annu. Rev. Biochem. 2000; 69: 447-496Crossref PubMed Scopus (375) Google Scholar). It is initiated by a nucleophilic attack of the side chain hydroxyl group (T, S) or thiol group (C) of HX↓(T/S/C) on the penultimate α-carbonyl group. The process is followed by the reversible N–O or N–S acyl rearrangement that converts the peptide (amide) bond to a more reactive (thio)ester intermediate. The subsequent attack of the intermediate by a second nucleophile results in the irreversible cleavage of the scissile bond. Lin et al. (25Lin H.H. Chang G.W. Davies J.Q. Stacey M. Harris J. Gordon S. J. Biol. Chem. 2004; 279: 31823-31832Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) have recently described that the cleavage of EMR2, a LNB-TM7 member, occurs at its GPS domain through a similar cis-autoproteolytic mechanism. This result suggested that PC1 might be cleaved through the same mechanism. In this study, we confirmed the cleavage site within the GPS domain of PC1 as proposed previously and identified the intracellular location at which PC1 cleavage takes place. We have provided evidence that PC1 is cleaved at the GPS domain through a similar cis-autoproteolytic mechanism involving an ester intermediate. Cell Culture—The HEK293 cell line was obtained from the American Type Culture Collection (Manassas, VA). MDCK(hPKD1FLAG) with stable expression of hPKD1FLAG, human PKD1 cDNA with C-terminal FLAG epitope (8Qian F. Boletta A. Bhunia A.K. Xu H. Liu L. Ahrabi A.K. Watnick T.J. Zhou F. Germino G.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16981-16986Crossref PubMed Scopus (234) Google Scholar), was established as described by Boletta et al. (26Boletta A. Qian F. Onuchic L.F. Bhunia A.K. Phakdeekitcharoen B. Hanaoka K. Guggino W. Monaco L. Germino G.G. Mol. Cell. 2000; 6: 1267-1273Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). All cells were maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin in a humidified atmosphere at 37 °C under 5% CO2. Transfection of the cells was performed using Lipofectamine 2000 (Invitrogen). The HEK293 stable cell lines, HEK(hPKD1FLAG) and HEK(ED-Fc), were established using the Flp-In™ T-REx™ system (Invitrogen). Reagents and Antibodies—All chemicals and reagents were obtained from Sigma unless otherwise specified. Complete protease inhibitor mixture was from Roche Applied Science. Agarose-conjugated anti-FLAG M2 antibody was from Sigma. Anti-Fc was from Jackson ImmunoResearch (West Grove, PA). Anti-CT, the polyclonal antibody against human PC1 cytoplasmic C-terminal tail is described elsewhere (27Hanaoka K. Qian F. Boletta A. Bhunia A.K. Piontek K. Tsiokas L. Sukhatme V.P. Guggino W.B. Germino G.G. Nature. 2000; 408: 990-994Crossref PubMed Scopus (655) Google Scholar). Protein G-agarose beads were from GE Healthcare. Generation of PC1 and ETL Cleavage Mutant Constructs—The construct wild-type (WT) hPKD1FLAG was the base plasmid for the generation of the cleavage mutants (8Qian F. Boletta A. Bhunia A.K. Xu H. Liu L. Ahrabi A.K. Watnick T.J. Zhou F. Germino G.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16981-16986Crossref PubMed Scopus (234) Google Scholar). The cleavage mutants were generated in a two-step procedure by PCR using pfu DNA polymerase (Stratagene, La Jolla, CA), as described in (8Qian F. Boletta A. Bhunia A.K. Xu H. Liu L. Ahrabi A.K. Watnick T.J. Zhou F. Germino G.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16981-16986Crossref PubMed Scopus (234) Google Scholar). To generate expression vector for the soluble human PC1 or ETL ectodomain-human immunoglobulin Fc fusion proteins, the Fc region of human IgG1 was PCR-amplified from a human immunoglobulin G1 Fc fragment cDNA (GenBank™ accession number AF150959) and inserted in-frame at the 3′-end of the ectodomain of human PC1 (residues 1–3072) or of ETL, respectively, at AvrII site using primers Fc-F(AvrII) and Fc-R(AvrII). The sequences of the primers used are as follows (AvrII site is underlined): Fc-F(AvrII), 5′-AAAACCTAGGCAAATCTTGTGACAAAACTCA-3′; Fc-R(AvrII), 5′-TTTTCCTAGGTCATTTACCCGGAGACAGGG-3′. The AvrII site used for cloning was introduced by site-specific mutagenesis. All constructs were confirmed by sequencing. EMR2 constructs were kindly provided by Dr. Hsi-Hsien Lin (Oxford, UK). Purification of PC1 ED-Fc and N-terminal Sequencing—HEK cells with stable expression of ED-Fc, HEK(ED-Fc) were harvested and lysed in lysis buffer (20 mm sodium phosphate (pH 7.2), 150 mm NaCl, 1 mm EDTA, 10% glycerol, 0.5% Triton X-100) containing Complete protease inhibitor cocktails for 1 h on ice. The lysate was centrifuged at 9000 × g for 15 min at 4 °C. The cleared supernatant was subjected to immunoprecipitation using protein G-agarose beads overnight at 4 °C under constant and gentle rotation. The beads were washed three times with 10 ml of phosphate-buffered saline and eluted with 4 ml of 100 mm glycine (pH 2.5). The eluate was neutralized by adding 800 μl of 0.5 m sodium phosphate (pH 7.2) and concentrated using Centriprep-10 concentrators. The concentrated sample was loaded onto 10% SDS gel for electrophoresis and electrotransferred onto a polyvinylidene difluoride membrane. The membrane was stained with Coomassie Brilliant Blue R-250, and the protein band was excised for N-terminal sequencing. N-terminal sequence was determined by automated Edman degradation using a protein sequencer from Applied Biosystems (Midwest Analytical, Inc). Immunoprecipitation and Western Blot Analysis—Transfected HEK293 or MDCK cells were washed twice with phosphate-buffered saline and lysed in lysis buffer containing Complete protease inhibitor mixture for 1 h on ice. The lysate was centrifuged at 9000 × g for 15 min at 4 °C. The cleared supernatant was subjected to immunoprecipitation using agarose-conjugated anti-FLAG M2 antibody at 4 °C for 1 h under constant rotation. In the case of the Fc fusion proteins, protein G-agarose beads were used to bind the Fc fragment. The beads were washed three times with lysis buffer. The immunoprecipitated proteins were eluted in SDS loading buffer by incubation at 95 °C for 3 min. The eluted immunoprecipitation product was resolved on a 4% or 3–8% SDS gel and electroblotted to a polyvinylidene difluoride membrane. The membrane was probed with primary antibodies and then with the secondary horseradish peroxidase-conjugated antibody (GE Healthcare). The signal was detected with SuperSignal West Pico Chemiluminescent detection system (Pierce). In Vitro Cleavage Assay—HEK cells transfected with expression constructs of full-length WT or mutant PC1 were harvested for lysis as described as above. The PC1 proteins were bound to agarose-counjugated anti-FLAG M2 antibody. For in vitro cleavage reaction, the beads were evenly distributed to the tubes for different time points. For example, the beads were evenly distributed to nine tubes for four time points. Tube 1 was a negative control at time zero. 250 mm hydroxylamine was added to four of the remaining eight tubes, and the other four tubes were used as parallel negative controls. The time zero negative control was eluted from the beads by SDS loading buffer right after the distribution of the beads. Other samples remained at 4 °C until they were taken out at desired time points. After washing twice with cleavage buffer, the samples were eluted with SDS loading buffer and kept at –80 °C before loading onto 3–8% SDS-PAGE for Western blotting. For the soluble Fc fusion proteins isolated from the conditioned media, the in vitro cleavage reaction was carried out as described by Lin et al. (25Lin H.H. Chang G.W. Davies J.Q. Stacey M. Harris J. Gordon S. J. Biol. Chem. 2004; 279: 31823-31832Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Briefly, the fusion proteins bound to the protein G-agarose beads were evenly distributed to three tubes (tubes 1, 2, and 3). The protein in tube 1 was eluted immediately in the SDS loading buffer and served as control. The cleavage buffer was added to tubes 2 and 3. To tube 3, 250 mm hydroxylamine was also added. Tubes 2 and 3 were incubated at 37 °C for 6 h. The proteins were then eluted in the SDS loading buffer for detection. The signal intensity of the blots was quantified by scanning densitometry using NIH ImageJ software. DOC is the fraction of CTF over the sum of CTF and uncleaved full-length PC1. Determination of the Cleavage Site within the GPS Domain of PC1—The exact site of cleavage within the GPS domain of human PC1 was proposed to be at HL↓T3049 (7Ponting C.P. Hofmann K. Bork P. Curr. Biol. 1999; 9: R585-R588Abstract Full Text Full Text PDF PubMed Google Scholar, 8Qian F. Boletta A. Bhunia A.K. Xu H. Liu L. Ahrabi A.K. Watnick T.J. Zhou F. Germino G.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16981-16986Crossref PubMed Scopus (234) Google Scholar). We sought to confirm the cleavage site by N-terminal amino acid sequencing of the ∼150-kDa CTF fragment from HEK(hPKD1FLAG), which stably expresses human FLAG-tagged PC1 at the C terminus, but were unable to obtain sufficient quantity of the product. We therefore resorted to using ED-Fc, the human PC1 ectodomain (aa 1–3072) fused to human IgG Fc fragment (28Qian F. Wei W. Germino G. Oberhauser A. J. Biol. Chem. 2005; 280: 40723-40730Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) (Fig. 1B). ED-Fc was cleaved when expressed in cells, as evident by detection of a ∼34-kDa Stalk-Fc band by anti-Fc (Fig. 1C). The T/R(+1)-Fc, which contains the T to R mutation at the critical +1 position of HL↓T, known to disrupt cleavage of full-length PC1 (8Qian F. Boletta A. Bhunia A.K. Xu H. Liu L. Ahrabi A.K. Watnick T.J. Zhou F. Germino G.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16981-16986Crossref PubMed Scopus (234) Google Scholar), was not cleaved. This result confirmed that ED-Fc was cleaved at the same site as the full-length PC1. We isolated the ∼34-kDa Stalk-Fc for N-terminal sequencing following SDS-PAGE (Fig. 1D) and determined the sequence of five N-terminal residues as TAFGA (Fig. 1E). We therefore concluded that the cleavage site of PC1 is indeed at the predicted HL↓T3049, a site 23 aa N-terminal to the first transmembrane domain. Subcellular Localization of PC1 Cleavage—We sought to define the intracellular location where PC1 cleavage occurs by using inhibitors affecting protein trafficking (Fig. 2A). Brefeldin A (BFA) blocks membrane protein export out of ER by dissembling the Golgi complex and fusion of Golgi cisternae with ER, whereas monensin blocks intra-Golgi protein trafficking (29Dinter A. Berger E.G. Histochem. Cell Biol. 1998; 109: 571-590Crossref PubMed Scopus (316) Google Scholar). In HEK293 cells, PC1 cleavage was considerably reduced by BFA, as evident by reduced DOC (∼15%) (Fig. 2B, lane 2), compared with the untreated control (∼50%) (lane 1). However, it was not affected by monensin (lane 3). This result indicated that a modest amount of PC1 was cleaved in ER and a considerable amount was cleaved post-ER, probably in the ER-Golgi intermediate compartment (30Appenzeller-Herzog C. Hauri H.P. J. Cell Sci. 2006; 119: 2173-2183Crossref PubMed Scopus (306) Google Scholar). This result differs from that of EMR2, which is cleaved exclusively in ER (25Lin H.H. Chang G.W. Davies J.Q. Stacey M. Harris J. Gordon S. J. Biol. Chem. 2004; 279: 31823-31832Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). To assay whether the location of PC1 cleavage may differ between cell types, we performed the same analyses in MDCK cells with stable PC1 expression. We found that both BFA (up to 500 μg/ml) and monensin only minimally affected PC1 cleavage (lanes 9, 11 and 12), indicating that cleavage occurred predominantly in the ER in MDCK cells. Our results indicate that PC1 cleavage takes place in the early secretory pathway but the location may differ between cell types. Role of N-Glycosylation of PC1 for Cleavage—PC1 is heavily N-glycosylated (31Boletta A. Qian F. Onuchic L.F. Bragonzi A. Cortese M. Deen P.M. Courtoy P.J. Soria M.R. Devuyst O. Monaco L. Germino G.G. Am. J. Kidney Dis. 2001; 38: 1421-1429Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 32Newby L.J. Streets A.J. Zhao Y. Harris P.C. Ward C.J. Ong A.C. J. Biol. Chem. 2002; 277: 20763-20773Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). We examined the role of N-glycosylation for PC1 cleavage. We found that inhibition of N-glycosylation of PC1 by tunicamycin (TM), as evident by increased mobility of the full-length PC1, abolished cleavage in both HEK293 and MDCK cells (Fig. 2B, lanes 4 and 10), indicating that the N-glycan addition to PC1 is required for cleavage, likely for correct folding of the protein. We examined whether the subsequent processing of the N-glycan plays a role for its cleavage using glycosidase inhibitors in HEK293 cells (Fig. 2A). Hepatocystin is the regulatory subunit of glucosidase II that carries out the initial glucose trimming and thus the ER quality control of newly synthesized glycoproteins in concert with lectin chaperones (33Drenth J.P. Martina J.A. Te Morsche R.H. Jansen J.B. Bonifacino J.S. Gastroenterology. 2004; 126: 1819-1827Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 34Trombetta E.S. Fleming K.G. Helenius A. Biochemistry. 2001; 40: 10717-10722Crossref PubMed Scopus (73) Google Scholar, 35Trombetta E.S. Parodi A.J. Annu. Rev. Cell Dev. Biol. 2003; 19: 649-676Crossref PubMed Scopus (356) Google Scholar). Mutations of hepatocystin cause autosomal dominant polycystic liver disease (36Drenth J.P. te Morsche R.H. Smink R. Bonifacino J.S. Jansen J.B. Nat. Genet. 2003; 33: 345-347Crossref PubMed Scopus (180) Google Scholar, 37Li A. Davila S. Furu L. Qian Q. Tian X. Kamath P.S. King B.F. Torres V.E. Somlo S. Am. J. Hum. Genet. 2003; 72: 691-703Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). We found that castanospermine (CAS) and 1-deoxynojirimycin (DNJ), the inhibitors of both glucosidase I and II (38Herscovics A. Biochim. Biophys. Acta. 1999; 1473: 96-107Crossref PubMed Scopus (237) Google Scholar), did not affect PC1 cleavage (Fig. 2B, lanes 5 and 6), indicating that glucose trimming and thus hepatocystin are not required for PC1 cleavage. In agreement with this result, inhibitors of mannosidases of the Golgi, 1-deoxymannojirimycin (ManDNJ) and swainsonine (SW), did not affect PC1 cleavage (Fig. 2B, lanes 7 and 8). Our results show that PC1 cleavage occurs after the transfer of the oligosaccharide precursor but does not require its subsequent processing. Evidence for cis-Autoproteolytic Mechanism for PC1 Cleavage—The sequence of the GPS domain in PC1 does not match the consensus cleavage site of any known intracellular processing proteases. We noticed that the cleavage tripeptide HL↓T of PC1 matches the cleavage site sequence HX↓(T/S/C) of the cis-autoproteolytic proteins, although no overall sequence similarity outside of it could be recognized between them (Fig. 3A). To determine whether PC1 cleavage might occur through a similar cis-autoproteolytic mechanism, we first examined the requirement of the +1 position residue for cleavage (Fig. 3, B and C). Such a mechanism requires that only Thr, Ser, and Cys, which contain a nucleophile side chain (-OH or -SH group), can support cleavage. We found that this was indeed the case. Substitution of Thr by Ser or Cys did not disrupt the cleavage, although the latter reduced the DOC (3–5%). Cleavage also occurred when the whole HL↓T was replaced by the cleavage tripeptide HF↓S of nucleoporin (39Rosenblum J.S. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11370-11375Crossref PubMed Scopus (94) Google Scholar). In contrast, substitution of Thr to Val, Gly, or Arg blocked cleavage (Fig. 3C). We next examined the formation of the postulated peptide ester, the hallmark of the cis-autoproteolysis, in the FLAG-purified WT PC1 expressed in HEK293 cells. The sample represents the steady-state condition of the cells and contains ∼50% of uFL available for the study. Formation of an ester intermediate in the known cis-autoproteolytic proteins is usually inferred from the increased cleavage rate in vitro by strong nitrogen nucleophiles such as hydroxylamine (23Xu Q. Buckley D. Guan C. Guo H.C. Cell. 1999; 98: 651-661Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 25Lin H.H. Chang G.W. Davies J.Q. Stacey M. Harris J. Gordon S. J. Biol. Chem. 2004; 279: 31823-31832Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 40Guan C. Liu Y. Shao Y. Cui T. Liao W. Ewel A. Whitaker R. Paulus H. J. Biol. Chem. 1998; 273: 9695-9702Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). This effect is a result of the facilitated hydrolysis of the (thio)ester intermediate (the rate-limiting step in the cis-autoproteolysis) by virtue of the high reactivity of hydroxylamine against peptide (thio)ester. We found no cleavage of the uFL PC1 occurring in vitro at various temperatures (4, 25, or 37 °C) regardless of the presence or absence of hydroxylamine as judged by the unchanged DOC (∼50%) (Fig. 3D, shown for reactions at 4 °C). While our kinetic study was under way, Lin et al. (25Lin H.H. Chang G.W. Davies J.Q. Stacey M. Harris J. Gordon S. J. Biol. Chem. 2004; 279: 31823-31832Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) reported that EMR2 is cleaved at its GPS domain via the cis-autoproteolytic mechanism. We reexamined PC1 cleavage using the condition as described in that study but still found no cleavage (not shown). Our result suggests that no detectable amount of stable ester intermediate was present in the sample during the incubation. Alternatively, the postulated oxygen ester intermediate might not be susceptible to hydroxylamine under our neutral conditions. We therefore performed the same analysis with FLAG-purified T/C(+1) mutant, in which the presumed thioester intermediate is known to be much more reactive to hydroxylamine than the oxygen ester in WT, as found for protein splicing reaction (41Shao Y. Xu M.Q. Paulus H. Biochemistry. 1996; 35: 3810-3815Crossref PubMed Scopus (70) Google Scholar). We found that T/C(+1) uFL underwent slow cleavage at 4 °C (to minimize activity of potential contaminating proteases) in the absence of hydroxylamine, with no cleavage apparent in the first 5 h of incubation (DOC ∼3%; Fig. 3E). However, cleavage consistently proceeded to a DOC of ∼11% after 16 h of incubation, a ∼4-fold of progression. In the presence of hydroxylamine, this DOC was reached after only 1 h of incubation and remained unchanged over time. Higher temperatures (25 or 37 °C) did not result in a higher DOC (not shown). Therefore, hydroxylamine shortened the t½ (defined as the time to reach half of the maximal DOC) from >5 h to <15 min, which is a >20-fold increase of the cleavage rate (Fig. 3F). In the presence of iodoacetamide, a thiol-alkylation reagent, this T/C(+1)" @default.
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• W2047052174 title "Characterization of cis-Autoproteolysis of Polycystin-1, the Product of Human Polycystic Kidney Disease 1 Gene" @default.
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