Cell Surface Tumor Endothelium Marker 8 Cytoplasmic Tail-independent Anthrax Toxin Binding, Proteolytic Processing, Oligomer Formation, and Internalization
2003; Elsevier BV; Volume: 278; Issue: 7 Linguagem: Inglês
10.1074/jbc.m210321200
ISSN1083-351X
AutoresShihui Liu, Stephen H. Leppla,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoThe interaction of anthrax toxin protective antigen (PA) and target cells was assessed, and the importance of the cytosolic domain of tumor endothelium marker 8 (TEM8) in its function as a cellular receptor for PA was evaluated. PA binding and proteolytic processing on the Chinese hamster ovary cell surface occurred rapidly, with both processes nearly reaching steady state in 5 min. Remarkably, the resulting PA63 fragment was present on the cell surface only as an oligomer, and furthermore, the oligomer was the only PA species internalized, suggesting that oligomerization of PA63 triggers receptor-mediated endocytosis. Following internalization, the PA63 oligomer was rapidly and irreversibly transformed to an SDS/heat-resistant form, in a process requiring an acidic compartment. This conformational change was functionally correlated with membrane insertion, channel formation, and translocation of lethal factor into the cytosol. To explore the role of the TEM8 cytosolic tail, a series of truncated TEM8 mutants was transfected into a PA receptor-deficient Chinese hamster ovary cell line. Interestingly, all of the cytosolic tail truncated TEM8 mutants functioned as PA receptors, as determined by PA binding, processing, oligomer formation, and translocation of an lethal factor fusion toxin into the cytosol. Moreover, cells transfected with a TEM8 construct truncated before the predicted transmembrane domain failed to bind PA, demonstrating that residues 321–343 are needed for cell surface anchoring. Further evidence that the cytosolic domain plays no essential role in anthrax toxin action was obtained by showing that TEM8 anchored by a glycosylphosphatidylinositol tail also functioned as a PA receptor. The interaction of anthrax toxin protective antigen (PA) and target cells was assessed, and the importance of the cytosolic domain of tumor endothelium marker 8 (TEM8) in its function as a cellular receptor for PA was evaluated. PA binding and proteolytic processing on the Chinese hamster ovary cell surface occurred rapidly, with both processes nearly reaching steady state in 5 min. Remarkably, the resulting PA63 fragment was present on the cell surface only as an oligomer, and furthermore, the oligomer was the only PA species internalized, suggesting that oligomerization of PA63 triggers receptor-mediated endocytosis. Following internalization, the PA63 oligomer was rapidly and irreversibly transformed to an SDS/heat-resistant form, in a process requiring an acidic compartment. This conformational change was functionally correlated with membrane insertion, channel formation, and translocation of lethal factor into the cytosol. To explore the role of the TEM8 cytosolic tail, a series of truncated TEM8 mutants was transfected into a PA receptor-deficient Chinese hamster ovary cell line. Interestingly, all of the cytosolic tail truncated TEM8 mutants functioned as PA receptors, as determined by PA binding, processing, oligomer formation, and translocation of an lethal factor fusion toxin into the cytosol. Moreover, cells transfected with a TEM8 construct truncated before the predicted transmembrane domain failed to bind PA, demonstrating that residues 321–343 are needed for cell surface anchoring. Further evidence that the cytosolic domain plays no essential role in anthrax toxin action was obtained by showing that TEM8 anchored by a glycosylphosphatidylinositol tail also functioned as a PA receptor. Anthrax toxin, the major virulence factor of Bacillus anthracis, consists of three polypeptides: protective antigen (PA, 1The abbreviations used are: PA, protective antigen; TEM8, tumor endothelium marker 8; CHO, Chinese hamster ovary; LF, lethal factor; HA, hemagglutinin; aa, amino acid; MAPKK, several mitogen-activated protein kinase kinases; PI-PLC, phosphatidylinositol-specific phospholipase C; HBSS, Hanks' balanced salt solution; GPI, glycosylphosphatidylinositol; uPAR, urokinase plasminogen activator receptor; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide 83 kDa), lethal factor (LF, 90 kDa), and edema factor (EF, 89 kDa) (1Leppla S.H. Alouf J.E. Freer J.H. Comprehensive Sourcebook of Bacterial Protein Toxins. Academic Press, London1999: 243-263Google Scholar, 2Smith H. Stanley J.L. J. Gen. Microbiol. 1962; 29: 517-521Crossref PubMed Scopus (32) Google Scholar). These three proteins are individually non-toxic. To intoxicate mammalian cells, PA binds to a ubiquitously expressed, recently identified cellular receptor, tumor endothelium marker 8 (TEM8) variant 2 (3Bradley K.A. Mogridge J. Mourez M. Collier R.J. Young J.A. Nature. 2001; 414: 225-229Crossref PubMed Scopus (763) Google Scholar), and is cleaved at the sequence RKKR167 on the cell surface by furin or furin-like proteases (4Klimpel K.R. Molloy S.S. Thomas G. Leppla S.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10277-10281Crossref PubMed Scopus (409) Google Scholar, 5Molloy S.S. Bresnahan P.A. Leppla S.H. Klimpel K.R. Thomas G. J. Biol. Chem. 1992; 267: 16396-16402Abstract Full Text PDF PubMed Google Scholar). Proteolysis yields the amino-terminal 20-kDa fragment (PA20), which is released into the medium, and the carboxyl-terminal 63-kDa fragment (PA63), which remains bound to the receptor and self-associates to form a ring-shaped heptamer (6Milne J.C. Furlong D. Hanna P.C. Wall J.S. Collier R.J. J. Biol. Chem. 1994; 269: 20607-20612Abstract Full Text PDF PubMed Google Scholar, 7Petosa C. Collier R.J. Klimpel K.R. Leppla S.H. Liddington R.C. Nature. 1997; 385: 833-838Crossref PubMed Scopus (687) Google Scholar). The heptamer binds up to 3 molecules of LF or EF (8Mogridge J. Cunningham K. Collier R.J. Biochemistry. 2002; 41: 1079-1082Crossref PubMed Scopus (184) Google Scholar,9Mogridge J. Cunningham K. Lacy D.B. Mourez M. Collier R.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7045-7048Crossref PubMed Scopus (165) Google Scholar). The resulting oligomeric complex is then internalized into endosomes, where the decreased pH causes the PA63 heptamer to insert into the endosomal membrane and produce a channel through which LF and EF translocate to the cytosol (10Wesche J. Elliott J.L. Falnes P.O. Olsnes S. Collier R.J. Biochemistry. 1998; 37: 15737-15746Crossref PubMed Scopus (175) Google Scholar). Therefore, PA is the central part of anthrax toxin, serving as the delivery vehicle for binding and translocation of LF and EF into the cytosol of the cells. The combination of PA plus LF kills animals (11Ezzell J.W. Ivins B.E. Leppla S.H. Infect. Immun. 1984; 45: 761-767Crossref PubMed Google Scholar, 12Beall F.A. Taylor M.J. Thorne C.B. J. Bacteriol. 1962; 83: 1274-1280Crossref PubMed Google Scholar) and certain cells, including mouse macrophages (13Friedlander A.M. J. Biol. Chem. 1986; 261: 7123-7126Abstract Full Text PDF PubMed Google Scholar, 14Hanna P.C. Kochi S. Collier R.J. Mol. Biol. Cell. 1992; 3: 1269-1277Crossref PubMed Scopus (79) Google Scholar). LF is a zinc-dependent metalloprotease that cleaves several mitogen-activated protein kinase kinases (MAPKK) in their amino-terminal regions (15Duesbery N.S. Webb C.P. Leppla S.H. Gordon V.M. Klimpel K.R. Copeland T.D. Ahn N.G. Oskarsson M.K. Fukasawa K. Paull K.D. Vande Woude G.F. Science. 1998; 280: 734-737Crossref PubMed Scopus (898) Google Scholar, 16Vitale G. Pellizzari R. Recchi C. Napolitani G. Mock M. Montecucco C. Biochem. Biophys. Res. Commun. 1998; 248: 706-711Crossref PubMed Scopus (363) Google Scholar). How this cleavage triggers the lethal effects of the toxin and whether there are additional cellular substrates remains unclear. EF is a calmodulin-dependent adenylate cyclase that elevates intracellular cAMP concentrations (17Leppla S.H. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 3162-3166Crossref PubMed Scopus (773) Google Scholar), thereby causing diverse effects in cells including the impairment of phagocytosis (18O'Brien J. Friedlander A. Dreier T. Ezzell J. Leppla S. Infect. Immun. 1985; 47: 306-310Crossref PubMed Google Scholar). Previous studies on the interaction of PA with host cells have often used cytotoxicity assays to infer internalization mechanisms, or have used radiolabeled or chemically labeled PA that may behave differently due to modification. In the present studies, we directly assessed PA binding, proteolytic processing, and internalization by target cells using a highly sensitive and specific rabbit antiserum to PA. We found that following binding and processing by cell surface furin, the cleaved PA immediately forms the PA63 oligomer and that this oligomer is the only species of PA that is internalized. In addition, following internalization, the oligomer is quickly transformed into an SDS/heat-resistant form, a process coincident with insertion and channel formation in endosomal membranes. In related studies we extended the understanding of toxin internalization obtained in the recent breakthrough that identified TEM8 variant 2 as a PA receptor (3Bradley K.A. Mogridge J. Mourez M. Collier R.J. Young J.A. Nature. 2001; 414: 225-229Crossref PubMed Scopus (763) Google Scholar). Currently, there are three reported cDNAs that result from splicing variations in TEM8 (GenBankTM accession number NM_032208, NM_053034, and NM_18153). The physiological functions of these have not been studied. Beyond the fact that TEM8 variant 2 functions as a PA receptor, the only information available is that implicit in the initial identification that TEM8 expression is up-regulated in tumor endothelium (19St Croix B. Rago C. Velculescu V. Traverso G. Romans K.E. Montgomery E. Lal A. Riggins G.J. Lengauer C. Vogelstein B. Kinzler K.W. Science. 2000; 289: 1197-1202Crossref PubMed Scopus (1668) Google Scholar, 20Carson-Walter E.B. Watkins D.N. Nanda A. Vogelstein B. Kinzler K.W. St Croix B. Cancer Res. 2001; 61: 6649-6655PubMed Google Scholar). Thus, it remains unknown whether other TEM8 variants can also function as PA receptors, and whether TEM8 has functions beyond binding PA in anthrax toxin action. To answer these questions, in the present work, we constructed a series of TEM8 truncated mutants, transfected them into a PA receptor-deficient Chinese hamster ovary (CHO) cell mutant, and found that all constructs having a membrane anchor functioned as PA receptors. Protein toxins produced as described previously included PA (21Leppla S.H. Methods Enzymol. 1988; 165: 103-116Crossref PubMed Scopus (128) Google Scholar), PA-Δ FF (PA with 313FF314deleted) (22Singh Y. Klimpel K.R. Arora N. Sharma M. Leppla S.H. J. Biol. Chem. 1994; 269: 29039-29046Abstract Full Text PDF PubMed Google Scholar), diphtheria toxin (23Carroll S.F. Barbieri J.T. Collier R.J. Methods Enzymol. 1988; 165: 68-76Crossref PubMed Scopus (45) Google Scholar), PA-U7 (a non-cleavable variant of PA with the furin site RKKR replaced by PAA) (24Liu S. Bugge T.H. Leppla S.H. J. Biol. Chem. 2001; 276: 17976-17984Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), and FP59, a recombinant fusion toxin consisting of anthrax toxin LF amino acids 1–254 (LFn) fused to the ADP-ribosylation domain ofPseudomonas exotoxin A (25Arora N. Leppla S.H. Infect. Immun. 1994; 62: 4955-4961Crossref PubMed Google Scholar). Rabbit anti-PA polyclonal antiserum (number 5308) and LF polyclonal antiserum (number 5309) were made in our laboratory by immunization with recombinant PA and LF. Polyclonal antibody against the amino-terminal sequence of MAPKK1 (MEK1-NT) was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Goat anti-rabbit IgG-HRP (sc2054) and goat anti-mouse IgG-HRP (sc2005) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Bafilomycin A1, saponin, and phosphatidylinositol-specific phospholipase C (PI-PLC) were purchased from Sigma. CHO cell clone 6 (CHO CL6) is a line recloned in this laboratory from CHO 10001, a subclone of CHO-S (26Gottesman M.M. Methods Enzymol. 1987; 151: 3-8Crossref PubMed Google Scholar), which was obtained from Dr. Michael Gottesman (National Institutes of Health, Bethesda). CHO FD11, a furin-deficient derivative of CHO CL6, was developed in our laboratory by chemical mutagenesis (27Gordon V.M. Klimpel K.R. Arora N. Henderson M.A. Leppla S.H. Infect. Immun. 1995; 63: 82-87Crossref PubMed Google Scholar). CHO PR230 is a spontaneous PA receptor-deficient mutant derived from CHO WTP4, which is derived from the thioguanine- and ouabain-resistant cell WTB111 (28Robbins A.R. Oliver C. Bateman J.L. Krag S.S. Galloway C.J. Mellman I. J. Cell Biol. 1984; 99: 1296-1308Crossref PubMed Scopus (65) Google Scholar), which was derived from CHO-K1. All CHO cells were grown in α-minimal essential medium supplemented with 5% fetal calf serum, 2 mm glutamine, 50 μg/ml gentamycin, and 25 mm HEPES. PA binding was assessed at both 37 and 4 °C. Cells were grown in 24-well plates to confluence. Cells were incubated with 1 μg/ml PA for different lengths of time and then washed five times with Hanks' balanced salt solution (HBSS) (Biofluids, Rockville, MD). The cells were lysed in 100 μl of modified RIPA lysis buffer (50 mm Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mm NaCl, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml each of aprotinin, leupeptin, and pepstatin). In measurements of PA internalization, the cells were first treated with 0.5 ml of 0.5 mg/ml trypsin in HBSS per well at 37 °C for 5 min to remove proteolytically the cell surface-bound PA, then washed, and lysed. The cell lysates were subjected to SDS-PAGE or native-PAGE using 4–20% Tris-glycine gradient gels (NOVEX, San Diego). Prior to loading, the cell lysates were boiled for 10 min in 1× SDS sample buffer (50 mm Tris-HCl, pH 6.8, 2% SDS, 100 mmdithiothreitol, 0.01% bromphenol blue, 6% glycerol) for SDS-PAGE or were incubated for 10 min at room temperature in 1× native buffer (NOVEX) for native-PAGE. The proteins were then transferred to nitrocellulose membranes, followed by Western blotting as described (29Liu S. Netzel-Arnett S. Birkedal-Hansen H. Leppla S.H. Cancer Res. 2000; 60: 6061-6067PubMed Google Scholar). PA was visualized by chemiluminescence using the West Pico Kit (Pierce). For the two-dimensional analysis, cell lysate was first separated on native-PAGE, and the gel strip was then sequentially equilibrated for 15 min each in Buffer I (125 mm Tris-HCl, pH 6.8, 1% SDS, 8.7% glycerol, 5 mm dithiothreitol) and Buffer II (125 mm Tris-HCl, pH 6.8, 1% SDS, 8.7% glycerol, 2% iodoacetamide) and subjected to SDS-PAGE, followed by Western blotting as described above. In measurements of LF translocation, CHO CL6 cells grown in 24-well plates were incubated with 1 μg/ml LF along with 1 μg/ml PA or PA-Δ FF for 1 h at 37 °C, washed once with HBSS, and treated with 0.5 ml 0.5 mg/ml trypsin in HBSS per well at 37 °C for 5 min to remove proteolytically the cell surface-bound toxin. The cells were then washed and permeabilized by saponin to allow efflux of cytosol as described (30Moskaug J.O. Sandvig K. Olsnes S. J. Biol. Chem. 1988; 263: 2518-2525Abstract Full Text PDF PubMed Google Scholar). Briefly, the cells were resuspended and incubated in 100 μl of phosphate-buffered saline containing 50 μg/ml saponin, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml each of aprotinin, leupeptin, and pepstatin for 30 min at 4 °C. The soluble fraction was separated from the particulate fraction by centrifugation at 15,000 × g for 5 min at 4 °C. The pellet was washed in HBSS and solubilized in RIPA lysis buffer. The samples from the soluble and pellet fractions were analyzed by native-PAGE followed by Western blotting using LF antiserum (number 5309). In measurements of MAPKK1 cleavage by LF, CHO CL6 cells grown in 24-well plate were incubated with 1 μg/ml LF along with 1 μg/ml PA or PA-Δ FF for 1 h at 37 °C, washed, lysed, and analyzed by SDS-PAGE followed by Western blotting using an antibody against the amino-terminal sequence of MAPKK1 (MEK1-NT). To assess the effect of vacuolar pH elevation on de novo formation of SDS/heat-resistant PA63 oligomer, CHO CL6 cells were incubated at 4 °C with 1 μg/ml PA for 3 h and then washed five times with ice-cold HBSS. The cells were then incubated in fresh medium in the presence or absence of 0.2 μm bafilomycin A1 at 37 °C. Incubations at 37 °C varied from 5 to 180 min. To assess the effect of vacuolar pH elevation on the preformed SDS/heat-resistant PA63 oligomer, cells were incubated with 1 μg/ml PA at 37 °C for 1 h and washed five times. One set of cells was then further incubated in fresh medium containing 0.2 μm bafilomycin A1 and the other in medium without the drug. The incubations at 37 °C varied from 10 to 180 min. The cells were lysed, and the lysates were processed by SDS-PAGE and Western blotting as described above. Human TEM8 variant cDNA fragments were isolated by reverse transcriptase-PCR from human fetal brain mRNA (catalog number 11438-017) purchased from Invitrogen. First-strand cDNA was synthesized by using the SuperScript First-strand Synthesis System (catalog number 11904-018) purchased from Invitrogen. We used 5′ primer PR5 (AAGTGTACA ATGGCCACGGCGGAGCGGAGAGCCCTCGGCATCGGCT, the start codon ATG is underlined and the BsrGI site for cloning is in boldface) in combination with various 3′ primers to amplify different carboxyl-terminal truncated TEM8 variants, as diagramed in Fig. 5 A). Thus, primer 115 aa (CCCACAAGGCATCGAGTTTTCCCTT, stop codon provided by the expression vector) was used to obtain the TEM8 variant TEM8-115 aa, having a 115-residue cytosolic tail. Similarly, primer 26 aa (CGGGATCC TA AGCGTAATCTGGAACATCGTATGGGTAACCATCATCATCTTCTTCCTCACTCTCCTCGGCA, the antisense of stop codon is underlined, the BamHI site is in boldface, and the sequence encoding for an influenza virus hemagglutinin (HA) tag is in italic) was used to obtain TEM8–26 aa. Primer 16 aa (CGGGATCC TA AGCGTAATCTGGAACATCGTATGGGTAGGCAGGGGGTGGAGGGACCTCCTTGATAAT, underlining, etc., as above) was used to obtain TEM8–16 aa. Primer 0 aa (CGGGATCC TACCAGAACCACCAGAGGAGAGCCAGGGCTA, underlining, etc., as above, and having no HA tag) was used to obtain TEM8–0 aa. Primer ED (CGGGATC CTAACCGTCAGAACAGTGTGTGGTGGTGATGATGACA, underlining, etc., as above) was used to obtain TEM8-ed, the variant having only the extracellular domain, residues 1–320. Finally, primer v3 (CTATTCCATGCAAGCAGCTGTTGTGGGGCCTGATGCAATTTTGTGGAGGCTACAGTGTGTGGTGGTGATGATGACAGAACTGGA, the antisense of stop codon is underlined) was used to obtain TEM8 variant 3. We found it is difficult to amplify full-length cDNA for TEM8 variant 1 due to its exceptionally high GC content, and instead we synthesized the 3′ cDNA region of variant 1 chemically and ligated it into TEM8-115 aa, resulting in full-length TEM8 variant 1. The TEM8 variant cDNA fragments were digested by BsrGI alone orBsrGI and BamHI and then cloned between theBsrGI and EcoRV or BsrGI andBamHI sites of pIRESHgy2B (catalog number 6939-1, Clontech Laboratories, Inc., Palo Alto, CA). This bicistronic mammalian expression vector contains an attenuated version of the internal ribosome entry site of the encephalomyocarditis virus, which allows both the gene of interest and the hygromycin B selection marker to be translated from a single mRNA. We also constructed a glycosylphosphatidylinositol (GPI)-anchored TEM8 by fusion of the TEM8 extracellular region to the GPI anchoring sequence of urokinase plasminogen activator receptor (uPAR) (31Ploug M. Ronne E. Behrendt N. Jensen A.L. Blasi F. Dano K. J. Biol. Chem. 1991; 266: 1926-1933Abstract Full Text PDF PubMed Google Scholar). To do so, the GPI sequence of human uPAR was amplified by using primers U5 (TATCGTACGTTGTAACCACCCAGACCTGGATGTCCAGT, theBsiWI cloning site is in boldface) and U3 (AATTCCAGCACACTGG TTAGGTCCAGAGGAGAGTGCCT, the antisense of the stop codon is underlined, and the BstXI site is in boldface), with a template of uPAR plasmid phuPAR (kind gift from Dr. Thomas H. Bugge, National Institutes of Health, Bethesda). The PCR product was digested by BsiWI and BstXI and cloned between the BsiWI and BstXI sites of the plasmid encoding TEM8-ed, resulting in an expression plasmid encoding the TEM8 extracellular part (residues 1–317) and the GPI anchoring sequence of uPAR (residues 293–335) linked by short tripeptide IVR. All the expression plasmids were confirmed by DNA sequencing and were transfected into CHO PR230 cells using LipofectAMINE Plus Reagent (Invitrogen), and stably transfected cells were selected by growth in hygromycin B (500 μg/ml) for 2 weeks. Hygromycin-resistant colonies were either isolated individually or pooled for further analysis. Cells were grown in 96-well plates to ∼50% confluence. Serial dilutions of PA (0–1000 ng/ml) combined with FP59 (100 ng/ml) were added to the cells to give a total volume of 200 μl/well and were incubated for 48 h. Cell viability was then assayed by adding 50 μl of 2.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) in α-minimal essential medium. The cells were incubated with MTT for 45 min at 37 °C; the medium was removed, and the blue pigment produced by viable cells was solubilized with 100 μl/well of 0.5% (w/v) SDS, 25 mm HCl, in 90% (v/v) isopropyl alcohol. The plates were vortexed, and the oxidized MTT was measured asA 570 using a microplate reader. Total RNA was isolated from exponentially growing CHO cells by using TRIzol Reagent (Invitrogen), separated on 1.0% agarose, 6.66% formaldehyde gels and then transferred onto nylon membranes (Immobilon-N, Millipore). Membranes were hybridized with a 32P-labeled 1.0-kb CHO TEM8 cDNA fragment isolated by reverse transcriptase-PCR by using 5′ primer TTCTGCCAGGAGGAGACACTTACATGC, and 3′ primer CCCACAAGGCATCGAGTTTTCCCTT. DNA sequencing analysis showed that this fragment was 90 and 89% identical to the corresponding mouse and human TEM8 sequences, respectively. Hybridization was performed in QuikHyb hybridization solution (catalog number 201220, Stratagene, La Jolla, CA) containing denatured salmon sperm DNA (0.5 mg/ml) at 60 °C overnight. Membranes were then washed in 2× SSC (catalog number 750020, Research Genetics, Huntsville, AL), 0.1% SDS at room temperature for 5 min, and then twice with 0.1× SSC, 0.1% SDS at 60 °C for 25 min. We used a high titered polyclonal anti-PA serum (number 5308) to assess PA binding and its subsequent processing and internalization by CHO cells. Analysis of unmodified PA eliminated concerns that radiolabeled or chemically labeled PA might behave differently from native PA due to the modification. The results showed that PA bound to CHO CL6 cells and was rapidly cleaved to PA63. More than half the PA bound was cleaved to PA63 within 5 min (Fig. 1 A, CL6 lanes). Because furin is the major cell surface protease that cleaves PA (4Klimpel K.R. Molloy S.S. Thomas G. Leppla S.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10277-10281Crossref PubMed Scopus (409) Google Scholar,5Molloy S.S. Bresnahan P.A. Leppla S.H. Klimpel K.R. Thomas G. J. Biol. Chem. 1992; 267: 16396-16402Abstract Full Text PDF PubMed Google Scholar), we compared processing of PA by CHO FD11, a derivative of CHO CL6 cells that lacks furin (27Gordon V.M. Klimpel K.R. Arora N. Henderson M.A. Leppla S.H. Infect. Immun. 1995; 63: 82-87Crossref PubMed Google Scholar). The FD11 cells bound PA efficiently, but cleavage to PA63 was very slow (Fig. 1 A, FD11 lanes). In addition to intact PA and PA63, another PA species revealed on SDS-PAGE was an SDS/heat-resistant PA63 oligomer that migrated very slowly (Fig. 1 A). Because the formation of the PA63 oligomer requires proteolysis, the PA63 oligomer was hardly detected in cell lysates from FD11 (Fig. 1 A). The cell lysates were further analyzed by native-PAGE. The FD11 cell lysates contained mainly the intact PA (Fig. 1 B, FD11 lanes), as expected, whereas the lysates from CHO CL6 cells contained intact PA as well as two higher order PA63 oligomers, but no monomeric PA63 (Fig. 1 B, CL6 lanes). When the binding assay was performed by using trypsin-nicked PA in which the furin site was pre-cleaved by limited trypsin digestion (22Singh Y. Klimpel K.R. Arora N. Sharma M. Leppla S.H. J. Biol. Chem. 1994; 269: 29039-29046Abstract Full Text PDF PubMed Google Scholar), as expected the major PA species detected were the oligomers, and just a negligible amount of monomer PA was shown (Fig. 1 C). The nature of these two distinct PA63 oligomers is unclear. The faster migrating species, termed oligomer A in this study (Fig. 1,B and C), is probably free PA63 heptamer, whereas the more slowly migrating species, termed oligomer B (Fig. 1,B and C), may be a complex of the PA63 heptamer with cellular components such as the PA receptor or detergent-resistant membrane structures. When these oligomeric species (in Fig. 1 C, lane 1h) were subjected to second dimension SDS-PAGE, interestingly, oligomer A dissociated to PA63 monomer, whereas oligomer B turned out to be the mixture of both SDS-sensitive and -resistant oligomers (Fig. 1 D). Together these results showed not only that bound PA is rapidly cleaved by furin but also that the resulting PA63 monomer very rapidly oligomerizes. Thus, the cell surface-associated PA63 mimics the behavior of PA63 produced in vitro, which forms the heptamer in neutral aqueous solutions (21Leppla S.H. Methods Enzymol. 1988; 165: 103-116Crossref PubMed Scopus (128) Google Scholar). These heptamers have been visualized previously by electron microscopy (6Milne J.C. Furlong D. Hanna P.C. Wall J.S. Collier R.J. J. Biol. Chem. 1994; 269: 20607-20612Abstract Full Text PDF PubMed Google Scholar), x-ray diffraction (7Petosa C. Collier R.J. Klimpel K.R. Leppla S.H. Liddington R.C. Nature. 1997; 385: 833-838Crossref PubMed Scopus (687) Google Scholar), and electrophoresis (22Singh Y. Klimpel K.R. Arora N. Sharma M. Leppla S.H. J. Biol. Chem. 1994; 269: 29039-29046Abstract Full Text PDF PubMed Google Scholar, 32Singh Y. Klimpel K.R. Goel S. Swain P.K. Leppla S.H. Infect. Immun. 1999; 67: 1853-1859Crossref PubMed Google Scholar). Absence of PA63 monomer further indicated that oligomerization of PA63 is effectively irreversible. PA63 detected by SDS-PAGE (Fig. 1 A) evidently resulted from the resolution of PA63 oligomer by boiling in SDS loading buffer. The data above indicated that cells exposed to PA contain intact PA and PA63 oligomers on their surface (Fig. 1 B). To explore whether these PA species are equally internalized, we performed a PA trypsin protection assay. After incubation with PA at 4 or 37 °C, cells were treated with trypsin to remove the cell surface-bound PA, allowing identification of those materials internalized by endocytosis. Remarkably, PA63 oligomer constituted the major protected PA species at 37 °C (Fig. 1, A and B, CL6 lanes), indicating that the PA63 oligomer was the only form of PA to be internalized. Also present were small amounts of a PA fragment, probably the carboxyl-terminal 47-kDa receptor-binding portion remaining bound to receptor after incomplete cleavage by trypsin (33Novak J.M. Stein M.P. Little S.F. Leppla S.H. Friedlander A.M. J. Biol. Chem. 1992; 267: 17186-17193Abstract Full Text PDF PubMed Google Scholar). Endocytosis is temperature-dependent (10Wesche J. Elliott J.L. Falnes P.O. Olsnes S. Collier R.J. Biochemistry. 1998; 37: 15737-15746Crossref PubMed Scopus (175) Google Scholar), and therefore all surface-bound PA should be removed by trypsin from cells incubated at 4 °C. Thus, in a control for the previous experiment, we showed that trypsin removed all the cell-associated PA (Fig. 1 E), with the exception of the 47-kDa fragment mentioned above. Further evidence that intact, monomeric PA is not internalized into cells was obtained using PA-U7, an uncleavable variant of PA that can bind but cannot be proteolytically activated by cellular furin (24Liu S. Bugge T.H. Leppla S.H. J. Biol. Chem. 2001; 276: 17976-17984Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). This PA mutant was not internalized to a trypsin-resistant site even when incubated with cells at 37 °C for 4 h (Fig. 1 F). These results demonstrated that the proteolytic cleavage of receptor-bound PA is an absolute prerequisite not only for the biochemical property of self-assembly but also for its subsequent biological activity of undergoing endocytosis. Previous studies showed that in solution, purified PA63 can form two types of oligomers, an SDS-sensitive type, which forms at neutral pH and can be resolved into PA63 monomer by SDS, and the SDS-resistant type, which forms at acidic pH and persists in the presence of SDS (34Miller C.J. Elliott J.L. Collier R.J. Biochemistry. 1999; 38: 10432-10441Crossref PubMed Scopus (227) Google Scholar). This suggested that the SDS/heat-resistant oligomer shown above (Fig. 1 A) may be the counterpart of this SDS-resistant oligomer formed in acidic solution, and therefore may be produced following endocytosis and delivery to acidic compartments. In fact, when CHO CL6 cells were incubated with PA, the SDS/heat-resistant oligomer was formed at 37 °C (Fig. 1 A, CL6 lanes) but not at 4 °C, a temperature at which endocytosis does not occur (Fig. 1 E, 1h lane). Moreover, native-PAGE analysis revealed that the PA63 oligomer A formed at both 4 and 37 °C (Fig. 2 A). Based on these observations we hypothesized that the PA63 oligomer formed on the cell surface encounters a progressively more acidic environment along the endocytic pathway and undergoes conformational changes and membrane insertion at acidic pH that renders it resistant to SDS/heat. To verify this hypothesis, we incubated CHO CL6 cells with PA at 4 °C, washed, and then shifted to 37 °C for various lengths of time in the absence or presence of bafilomycin A1, a potent and specific inhibitor of the vacuolar (H+)-ATPase proton pumps that maintain the pH gradients of acidic compartments (35Beauregard K.E. Lee K.D. Collier R.J. Swanson J.A. J. Exp. Med. 1997; 186:
Referência(s)