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• W2037083628 abstract "BACE1 is a membrane-bound aspartic protease that cleaves the amyloid precursor protein (APP) at the β-secretase site, a critical step in the Alzheimer disease pathogenesis. We previously found that BACE1 also cleaved a membrane-bound sialyltransferase, ST6Gal I. By BACE1 overexpression in COS cells, the secretion of ST6Gal I markedly increased, and the amino terminus of the secreted ST6Gal I started at Glu41. Here we report that BACE1-Fc chimera protein cleaved the A-ST6Gal I fusion protein, or ST6Gal I-derived peptide, between Leu37 and Gln38, suggesting that an initial cleavage product by BACE1 was three amino acids longer than the secreted ST6Gal I. The three amino acids, Gln38-Ala39-Lys40, were found to be truncated by exopeptidase activity, which was detected in detergent extracts of Golgi-derived membrane fraction. These results suggest that ST6Gal I is cleaved initially between Leu37 and Gln38 by BACE1, and then the three-amino acid sequence at the NH2 terminus is removed by exopeptidase(s) before secretion from the cells. BACE1 is a membrane-bound aspartic protease that cleaves the amyloid precursor protein (APP) at the β-secretase site, a critical step in the Alzheimer disease pathogenesis. We previously found that BACE1 also cleaved a membrane-bound sialyltransferase, ST6Gal I. By BACE1 overexpression in COS cells, the secretion of ST6Gal I markedly increased, and the amino terminus of the secreted ST6Gal I started at Glu41. Here we report that BACE1-Fc chimera protein cleaved the A-ST6Gal I fusion protein, or ST6Gal I-derived peptide, between Leu37 and Gln38, suggesting that an initial cleavage product by BACE1 was three amino acids longer than the secreted ST6Gal I. The three amino acids, Gln38-Ala39-Lys40, were found to be truncated by exopeptidase activity, which was detected in detergent extracts of Golgi-derived membrane fraction. These results suggest that ST6Gal I is cleaved initially between Leu37 and Gln38 by BACE1, and then the three-amino acid sequence at the NH2 terminus is removed by exopeptidase(s) before secretion from the cells. amyloid β-peptide amyloid precursor protein β-site APP-cleaving enzyme the hinge and constant region of IgG high performance liquid chromatography matrix-assisted laser desorption/ionization time-of-flight mass spectrometry The deposition of amyloid β-peptide (Aβ)1 in the brain is a hallmark of the pathogenesis of Alzheimer's disease (1Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5135) Google Scholar). Aβ, a 39–43-amino acid peptide, is a proteolytic product derived from the amyloid precursor protein (APP). The β-secretase initially generates the NH2 terminus of Aβ, cleaving APP to produce a soluble NH2-terminal fragment (APPsβ) and a 12-kDa COOH-terminal fragment (C99) that remains membrane bound. C99 is further cleaved by γ-secretase, resulting in the production of pathogenic Aβ peptide (2De Strooper B. Saftig P. Craessaerts K. Vanderstichele H. Guhde G. Annaert W. Von Figura K. Van Leuven F. Nature. 1998; 391: 387-390Crossref PubMed Scopus (1543) Google Scholar, 3Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1679) Google Scholar). As an alternative processing pathway, α-secretase cleaves within the Aβ sequence to produce a soluble NH2-terminal fragment (APPsα) and a 10-kDa membrane-bound COOH-terminal fragment (C83) (4Lammich S. Kojro E. Postina R. Gilbert S. Pfeiffer R. Jasionowski M. Haass C. Fahrenholz F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3922-3927Crossref PubMed Scopus (977) Google Scholar, 5Buxbaum J.D. Liu K.N. Luo Y. Slack J.L. Stocking K.L. Peschon J.J. Johnson R.S. Castner B.J. Cerretti D.P. Black R.A. J. Biol. Chem. 1998; 273: 27765-27767Abstract Full Text Full Text PDF PubMed Scopus (834) Google Scholar). C83 is also cleaved by γ-secretase to produce the nonpathogenic p3 peptide. BACE1 (β-amyloid-converting enzyme 1), a pepsin-like membrane-bound aspartic protease, has recently been identified as β-secretase (6Vassar R. Bennett B.D. Babu-Khan S. Kahn S. Mendiaz E.A. Denis P. Teplow D.B. Ross S. Amarante P. Loeloff R. Luo Y. Fisher S. Fuller J. Edenson S. Lile J. Jarosinski M.A. Biere A.L. Curran E. Burgess T. Louis J.C. Collins F. Treanor J. Rogers G. Citron M. Science. 1999; 286: 735-741Crossref PubMed Scopus (3268) Google Scholar, 7Sinha S. Anderson J.P. Barbour R. Basi G.S. Caccavello R. Davis D. Doan M. Dovey H.F. Frigon N. Hong J. Jacobson-Croak K. Jewett N. Keim P. Knops J. Lieberburg I. Power M. Tan H. Tatsuno G. Tung J. Schenk D. Seubert P. Suomensaari S.M. Wang S. Walker D. John V. et al.Nature. 1999; 402: 537-540Crossref PubMed Scopus (1473) Google Scholar, 8Yan R. Bienkowski M.J. Shuck M.E. Miao H. Tory M.C. Pauley A.M. Brashier J.R. Stratman N.C. Mathews W.R. Buhl A.E. Carter D.B. Tomasselli A.G. Parodi L.A. Heinrikson R.L. Gurney M.E. Nature. 1999; 402: 533-537Crossref PubMed Scopus (1328) Google Scholar, 9Luo Y. Bolon B. Kahn S. Bennett B.D. Babu-Khan S. Denis P. Fan W. Kha H. Zhang J. Gong Y. Martin L. Louis J.C. Yan Q. Richards W.G. Citron M. Vassar R. Nat. Neurosci. 2001; 4: 231-232Crossref PubMed Scopus (943) Google Scholar, 10Cai H. Wang Y. McCarthy D. Wen H. Borchelt D.R. Price D.L. Wong P.C. Nat. Neurosci. 2001; 4: 233-234Crossref PubMed Scopus (946) Google Scholar). In the case of γ-secretase, functional presenilin is required (2De Strooper B. Saftig P. Craessaerts K. Vanderstichele H. Guhde G. Annaert W. Von Figura K. Van Leuven F. Nature. 1998; 391: 387-390Crossref PubMed Scopus (1543) Google Scholar, 3Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1679) Google Scholar), and recent reports have shown that Notch and ErbB4 are also its substrates (11De Strooper B. Annaert W. Cupers P. Saftig P. Craessaerts K. Mumm J.S. Schroeter E.H. Schrijvers V. Wolfe M.S. Ray W.J. Goate A. Kopan R. Nature. 1999; 398: 518-522Crossref PubMed Scopus (1792) Google Scholar, 12Struhl G. Greenwald I. Nature. 1999; 398: 522-525Crossref PubMed Scopus (700) Google Scholar, 13Ni C.Y. Murphy M.P. Golde T.E. Carpenter G. Science. 2001; 294: 2179-2181Crossref PubMed Scopus (756) Google Scholar, 14Lee H.J. Jung K.M. Huang Y.Z. Bennett L.B. Lee J.S. Mei L. Kim T.W. J. Biol. Chem. 2002; 277: 6318-6323Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). Nevertheless, inhibitors toward both β- and γ-secretases are still promising therapeutics for Alzheimer's disease (15Li Y.M. Xu M. Lai M.T. Huang Q. Castro J.L. DiMuzio-Mower J. Harrison T. Lellis C. Nadin A. Neduvelil J.G. Register R.B. Sardana M.K. Shearman M.S. Smith A.L. Shi X.P. Yin K.C. Shafer J.A. Gardell S.J. Nature. 2000; 405: 689-694Crossref PubMed Scopus (861) Google Scholar, 16Esler W.P. Kimberly W.T. Ostaszewski B.L. Diehl T.S. Moore C.L. Tsai J.Y. Rahmati T. Xia W. Selkoe D.J. Wolfe M.S. Nat. Cell Biol. 2000; 2: 428-434Crossref PubMed Scopus (503) Google Scholar, 17Doerfler P. Shearman M.S. Perlmutter R.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9312-9317Crossref PubMed Scopus (155) Google Scholar, 18Marcinkeviciene J. Luo Y. Graciani N.R. Combs A.P. Copeland R.A. J. Biol. Chem. 2001; 276: 23790-23794Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). We previously found that BACE1 is involved in the cleavage and secretion of a membrane-bound sialyltransferase, ST6Gal I (19Kitazume S. Tachida Y. Oka R. Shirotani K. Saido T.C. Hashimoto Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13554-13559Crossref PubMed Scopus (231) Google Scholar), and the secreted ST6Gal I starts at Glu41. Several reports show that BACE1 prefers Leu at position P1 (20Citron M. Teplow D.B. Selkoe D.J. Neuron. 1995; 14: 661-670Abstract Full Text PDF PubMed Scopus (232) Google Scholar, 21Turner III, R.T. Koelsch G. Hong L. Castanheira P. Ermolieff J. Ghosh A.K. Tang J. Castenheira P. Ghosh A. Biochemistry. 2001; 40: 10001-10006Crossref PubMed Scopus (196) Google Scholar, 22Gruninger-Leitch F. Schlatter D. Kung E. Nelbock P. Dobeli H. J. Biol. Chem. 2002; 277: 4687-4693Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Indeed, APP substitution of Leu at the P1 position for Met together with substitution of Asn at the P2 for Lys, a Swedish mutation of familial Alzheimer's disease, clearly facilitates BACE1-dependent cleavage and induces rapid progression of pathological symptoms. In this article we describe in vitro cleavage of ST6Gal I-derived peptide or protein by BACE1. Eight-week-old male Wistar rats, maintained in specific-pathogen free conditions, were purchased from the Shizuoka Agricultural Cooperative Association for Laboratory Animals (Shizuoka, Japan). Tissue culture media and reagents, including Dulbecco's modified Eagle's medium and Lipofectin, were purchased from Invitrogen. Protein A-Sepharose Fast Flow was purchased from Amersham Biosciences. CDP-hexanolamine-Sepharose was a gift from Dr. K. J. Colley (University of Illinois at Chicago). Columns for DNA purification were obtained from Qiagen Inc. (Chatsworth, CA). PCR was performed using LA Taq polymerase (Sigma). Protein molecular weight standards were purchased from Bio-Rad. BACE inhibitor, KTEEISEVN(Sta)VAEF (in which Leu was substituted with statine (Sta) for the P1 position of an APP analogue peptide), was purchased from Bachem (Bubendorf, Switzerland). A polyclonal antibody, Q38, which specifically recognizes the NH2 terminus of ST6Gal I-Q38 form, was prepared by immunizing a synthetic peptide, QAKEFQC, conjugated with keyhole limpet hemocyanin (23Saido T.C. Iwatsubo T. Mann D.M. Shimada H. Ihara Y. Kawashima S. Neuron. 1995; 14: 457-466Abstract Full Text PDF PubMed Scopus (504) Google Scholar). For the transient transfection experiment, ST6Gal I FLAG-pSVL, protein A ST6Gal I-pcDSA, and BACE Fc-pEF were constructed as described previously (19Kitazume S. Tachida Y. Oka R. Shirotani K. Saido T.C. Hashimoto Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13554-13559Crossref PubMed Scopus (231) Google Scholar, 24Kitazume-Kawaguchi S. Dohmae N. Takio K. Tsuji S. Colley K.J. Glycobiology. 1999; 9: 1397-1406Crossref PubMed Scopus (32) Google Scholar, 25Kitazume-Kawaguchi S. Kabata S. Arita M. J. Biol. Chem. 2001; 276: 15696-15703Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). To generate ST6Gal I FLAG-pcDSA, polymerase chain reaction was performed using ST6Gal I FLAG-pSVL templates with primer 1 (5′-CGCGAATTCAAGAAAGGGAGCGACTATGA-3′) and primer 2 (5′-GCGCTCGAGGCTCACTTGTCATCGTCGTCC-3′). PCR product purified with the QIAEX II Gel extraction system (Qiagen Inc.) was digested withEcoRI and XhoI and then ligated into the pcDSAEcoRI-XhoI site. STK40A FLAG-pcDSA was generated from ST6Gal I FLAG-pcDSA using a QuikChange site-directed mutagenesis kit (Invitrogen) with primer 3 (5′-CTTACACTGCAAGCAGCAGAGTTCCAGATGCCC-3′) and primer 4 (5′-GGGCATCTGGAACTCTGCTGCTTGCAGTGTAAG-3′) (24Kitazume-Kawaguchi S. Dohmae N. Takio K. Tsuji S. Colley K.J. Glycobiology. 1999; 9: 1397-1406Crossref PubMed Scopus (32) Google Scholar). COS-7 or rat hepatoma FTO2B cells maintained in Dulbecco's modified Eagle's medium, 10% fetal bovine serum were plated on 100- or 150-mm tissue culture dishes and grown in a 37 °C, 5% CO2 incubator until 50–70% confluent. Cells were transfected using Lipofectin and Opti-MEM I. Expression of proteins was typically allowed to continue for 24 to 48 h. To analyze the soluble secreted form of ST6Gal I-FLAG, COS cells were transfected with rat ST6Gal I FLAG-pSVL. At 48 h after transfection, soluble ST6Gal I-FLAG secreted in the media was pulled down with M2-agarose (Sigma) and analyzed by immunoblotting using either the E41 (1:500), Q38 (1:500), or anti-ST6Gal I polyclonal antibody (1:1000). Pre-absorption of the E41 antibody was performed using peptide EFQMPKC (10 μg/ml) or FQMPKC (10 μg/ml). Horseradish peroxidase-goat anti-rabbit IgG (Cappel, 1:1000) was used as a secondary antibody, and chemiluminescent substrate (Pierce) was used for detection (19Kitazume S. Tachida Y. Oka R. Shirotani K. Saido T.C. Hashimoto Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13554-13559Crossref PubMed Scopus (231) Google Scholar). Rat hepatoma FTO2B cells that endogenously express ST6Gal I at a high level were transfected with human BACE1-myc-pcDNA3.1 or the control vector. At 48 h after transfection, soluble ST6Gal I secreted in the media was immunoprecipitated with anti-ST6Gal I rabbit polyclonal antibody. One-third of the immunoprecipitant was used for detection with the anti-ST6Gal I antibody, and the rest of sample was used for detection with the E41 antibody. Each sample was treated with Laemmli sample buffer (26Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206602) Google Scholar), subjected to 4–20% gradient SDS-PAGE, and then transferred to a nitrocellulose membrane. The membrane was incubated with either anti-ST6Gal I or the E41 polyclonal antibody. Rat plasma (200 μl) was diluted with the buffer containing 10 mm sodium cacodylate, pH 6.5, 0.1% Triton CF-54, and 0.15m NaCl. Protein A-Sepharose (30 μl) was added to the mixture and rotated for 30 min to remove adhesive proteins. After the beads were removed by centrifugation, 20 μl of CDP-hexanolamine-agarose was then added to pull down sialyltransferase proteins. After rotation for 16 h, beads were washed with phosphate-buffered saline. Sialyltransferase proteins immobilized to the beads were analyzed by immunoblotting with either anti-ST6Gal I or the E41 polyclonal antibody. When protein A-ST6Gal I-FLAG or protein A-STK40A FLAG was used as a substrate, both BACE1-Fc and protein A-ST6Gal I fusion proteins were purified from 20 ml of culture media of COS cells that transiently expressed these proteins by absorbing them in 20 μl of protein A-Sepharose and IgG-Sepharose (50% suspension in phosphate-buffered saline), respectively. These preparations of BACE1-Fc and protein-A-ST6Gal I were mixed, resulting in a final volume of 20 μl, which comprised 50 mm sodium acetate buffer (pH 4.5) and protease inhibitors for possible contaminating proteases associated with Sepharose beads (Complete (Roche), 10 μm pepstatin, 1 μm leupeptin, 1 mg/ml pepstatin, and 2 μm amastatin). The mixture was incubated at 37 °C for 2 h with rotation. The reaction was then terminated, and the product was analyzed by immunoblotting with anti-FLAG (M2) antibody. When peptides were used as substrates for assay, BACE1-Fc was purified from 50 ml of culture medium of COS cells. Each peptide (0.1 mm) was incubated for 16 h in 50 μl of the reaction mixture as described above. KTEEISEVN(Sta)VAEF (0.1 μm, Bachem) was used as a β-secretase inhibitor. After the incubation the reaction mixture was centrifuged to remove immobilized BACE1-Fc. The products were separated on a reversed-phase HPLC C30-UG-5 column (4.6-mm i.d. × 250 mm, Nomura Chemical Co., Japan) using a Waters model 600E HPLC system, equipped with a Senshu model SSC-5200 (Tokyo, Japan) UV detector. The sample, applied to the column equilibrated in 10% acetonitrile, was then eluted with a gradient of 10–50% acetonitrile for 40 min. The elution rate was 1 ml/min. For MALDI-TOF MS analysis, peptide DYEALTLQAKEFQMPKSQE was incubated with BACE1-Fc in a 5-fold scale, and each peak, separated by HPLC, was collected manually. Protein A-ST6Gal I-FLAG, prepared from 120 ml of culture medium, was cleaved by BACE1-Fc, and the soluble proteins yielded were precipitated with 75% ice-cold ethanol at −20 °C for 16 h. An aliquot of the precipitant was analyzed by SDS-PAGE and stained with silver nitrate (Daiichikagaku, Tokyo, Japan). The rest of the sample was subjected to SDS-PAGE followed by electrical blotting to an Immobilon membrane (Millipore). After staining with Coomassie Blue, the band of soluble ST6Gal I-FLAG (∼49 K) was excised, and its amino-terminal amino acid sequence was determined with a Procise 492 cLC protein sequencer (Applied Biosystems). ST6Gal I- or ST6Gal IK40A-Q38 form that starts at Gln38 was prepared by cleaving protein A-ST6Gal I-FLAG or protein A-ST6Gal IK40A-FLAG by BACE1 in vitro as described above. The Q38 form protein as a substrate was mixed with a microsomal fraction (10 μg of protein) or rat Golgi membrane (10 μg of protein) pretreated with 1% of Triton X-100. The mixture was incubated at 37 °C for 2 h in 20 μl of 50 mm sodium acetate buffer (pH4.5) containing 0.1 mg/ml bovine serum albumin. Incubation was terminated by the addition of a Laemmli sample buffer (25Kitazume-Kawaguchi S. Kabata S. Arita M. J. Biol. Chem. 2001; 276: 15696-15703Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). An aliquot of the incubation mixture was subjected to 4–20% gradient SDS-PAGE, and then the separated proteins were transferred onto a nitrocellulose membrane. The ST6Gal I-FLAG protein was detected by anti-FLAG antibody, and E41 form was detected by the E41 antibody. Several protease inhibitors such as amastatin, bestatin, 1,10-phenanthrolin, and EDTA were added to the reaction mixture to for the purpose of examining their inhibitory potency. Previously, we used ST6Gal I as a model molecule for studying how glycosyltransferases are cleaved and secreted from the cells (24Kitazume-Kawaguchi S. Dohmae N. Takio K. Tsuji S. Colley K.J. Glycobiology. 1999; 9: 1397-1406Crossref PubMed Scopus (32) Google Scholar). We found that BACE1 was responsible for the cleavage and secretion of ST6Gal I, i.e. overexpression of BACE1 together with rat ST6Gal I in COS cells increased the secretion of a soluble ST6Gal I, which had a Glu41-Phe42-Gln43-Met44-Pro45-Lys46sequence at the NH2 terminus of soluble ST6Gal I (19Kitazume S. Tachida Y. Oka R. Shirotani K. Saido T.C. Hashimoto Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13554-13559Crossref PubMed Scopus (231) Google Scholar). We prepared a polyclonal antibody, E41, that recognized the NH2-terminal sequence of soluble ST6Gal I. In the present study, the specificity of the E41 antibody was further characterized by a pre-absorption experiment showing that the antibody was absorbed by the peptide EFQMPK but not by FQMPK (Fig.1A). Using the E41 antibody, we also examined the secretion of endogenous ST6Gal I in rat hepatoma FTO2B cells, which express high levels of the ST6Gal I protein. Soluble ST6Gal I, immunoprecipitated with anti-ST6Gal I antibody from the media of FTO2B cells, was detected with the E41 antibody (Fig.1B). With BACE1 overexpression in FTO2B cells, the ST6Gal I secretion markedly increased, and the increased soluble enzyme was also recognized by the E41 antibody. A similar form of soluble ST6Gal I was also present in rat plasma. The plasma enzyme was partially purified using CDP-hexanolamine-agarose resin and subjected to immunoblotting analysis. The plasma enzyme reacted with the E41 antibody as well as anti-ST6Gal I antibody (Fig. 1C). By pre-absorption of E41 antibody with peptide EFQMPK, E41 staining of the plasma enzyme was reduced (data not shown). These data suggest that in vivocleavage and secretion of endogenous ST6Gal I are also mediated by BACE1. We confirmed that the secreted ST6Gal I starts at Glu41in vivo as well as in cultured cells (19Kitazume S. Tachida Y. Oka R. Shirotani K. Saido T.C. Hashimoto Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13554-13559Crossref PubMed Scopus (231) Google Scholar), but in vitro studies by others (21Turner III, R.T. Koelsch G. Hong L. Castanheira P. Ermolieff J. Ghosh A.K. Tang J. Castenheira P. Ghosh A. Biochemistry. 2001; 40: 10001-10006Crossref PubMed Scopus (196) Google Scholar, 22Gruninger-Leitch F. Schlatter D. Kung E. Nelbock P. Dobeli H. J. Biol. Chem. 2002; 277: 4687-4693Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar) on BACE1 cleavage site preference showed that Lys40 residue at the P1 position is not a preferable amino acid for BACE1 cleavage. We therefore analyzed BACE1-dependent cleavage of a peptide substrate, DYEALTLQAKEFQMPKSQE, which corresponds to Asp31∼Glu49 of ST6Gal I sequence. The peptide substrate was incubated with purified BACE1-Fc chimera, and the products yielded were analyzed by reversed-phase HPLC. We detected two peptide peaks as products, the retention times of which corresponded to those of authentic peptides, DYEALTL and QAKEFQMPKSQE (Fig.2, A andB). These products were subjected to MALDI-TOF MS analysis, and their protonated molecular ions, [M+H]+, were observed at m/z 824 and 1451 (TableI and Fig. 2, C andD). Several other peaks (marked with an asterisk) in the HPLC chromatogram were also observed in the control reaction mixture without peptide substrate and shown by MALDI-TOF MS analysis to be non-peptide components. We did not detect peptide peaks corresponding to DYEALTLQAK and EFQMPKSQE on HPLC and MALTI-TOFMS analyses. This result indicates that BACE1-Fc cleaves the peptide substrate exclusively between Leu37 and Gln38.Table ISummary of MALDI-TOF MS analysisPeptide substrate DYEALTLQAKEFQMPKSQEFragmental pattern 1DYEALTLQAKEFQMPKSQE Calculated mass [M + H]+8241-aAtomic mass unit.14501-aAtomic mass unit. Observed mass [M + H]+DetectedDetectedFragmental pattern 2DYEALTLQAKEFQMPKSQE Calculated mass [M + H]+11511-aAtomic mass unit.11231-aAtomic mass unit. Observed mass [M + H]+Not detectedNot detected1-a Atomic mass unit. Open table in a new tab We further characterized the cleavage of the ST6Gal I peptide, by comparison with those of APPwt (KTEEISEVKMDAEFRHDSG) and APPsw (KTEEISEVNLDAEFRHDSG) peptides, which cover the β-cleavage site of APP. The APPsw peptide was the more preferable substrate (Fig. 3). BACE1-Fc cleaved ST6Gal I peptide with higher efficiency than APPwtpeptide. By the addition of a BACE inhibitor at 0.1 μm, cleavage of the STGal I peptide, as well as that of the APPsw peptide, was inhibited nearly 50%. When mutant substrate ST-LA was used, in which Leu at the P1 position was replaced by Ala, cleavage efficiency was reduced to 40% compared with the wild-type substrate (ST-WT), suggesting that the Leu residue in the ST6Gal I peptide is recognized as a preferable amino acid at the P1 position. This result is consistent with those reported previously by others (21Turner III, R.T. Koelsch G. Hong L. Castanheira P. Ermolieff J. Ghosh A.K. Tang J. Castenheira P. Ghosh A. Biochemistry. 2001; 40: 10001-10006Crossref PubMed Scopus (196) Google Scholar, 22Gruninger-Leitch F. Schlatter D. Kung E. Nelbock P. Dobeli H. J. Biol. Chem. 2002; 277: 4687-4693Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar); Leu at the P1 position in the APP sequence is recognized preferentially by BACE1. We also examined BACE1-dependent cleavage of protein A-ST6Gal I-FLAG chimera, which lacked a transmembrane domain, instead containing a signal peptide plus protein A and COOH terminally tagged with FLAG. The Protein A-ST6Gal I chimera was reported to have sialyltransferase activity (25Kitazume-Kawaguchi S. Kabata S. Arita M. J. Biol. Chem. 2001; 276: 15696-15703Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), suggesting that the catalytic domain was correctly folded as a native enzyme. The chimeric protein was incubated with BACE1-Fc. The cleaved protein was purified to near homogeneity (Fig. 4) and then transferred onto a polyvinylidene difluoride membrane for sequencing. Micro-sequence analysis revealed that the NH2-terminal sequence of the soluble protein was exclusively qAKEFQMpks, where lowercase letters indicate ambiguous identification (the first cycle of amino acid also contained Gly, most likely derived from the transfer buffer which contained a large amount of glycine). Thus, protein A-ST6Gal I-FLAG was cleaved exclusively between Leu37 and Gln38, as was the case with the peptide substrate. We also examined whether BACE1 recognized Lys40at P3′ in the ST6Gal I sequence. We constructed a mutant substrate in which Lys40 of protein A-ST6Gal I-FLAG was replaced with Ala. BACE1-Fc cleaved the protein A-ST6Gal IK40A-FLAG with efficiency similar to the wild type protein (Fig. 5). When we took a time course to compare the cleavage efficiency between these protein A-ST6Gal I proteins in detail, protein A-ST6Gal IK40A-FLAG was cleaved at almost the same efficiency as the wild type (data not shown). These results suggest that Lys40 of ST6Gal I is not critical for BACE1 recognition. Our results described above suggest that BACE1 cleaves ST6Gal I between Leu37 and Gln38; the cleaved product (Q38 form) was three amino acids longer than the secreted one (E41 form). We speculated that the three-amino acid QAK sequence of the Q38 form was removed by endogenous exopeptidase(s) before secretion, and hence we set up an assay for detecting possible exopeptidase activity. As a substrate for the assay we used the Q38 form of ST6Gal I, which had been prepared by cleavage of protein A-ST6Gal I-FLAG with BACE1-Fc. When the substrate was mixed with detergent extracts of a microsomal fraction of COS cells, its NH2-terminal QAK sequence was trimmed to generate the E41 form of ST6Gal I, which was detected by the E41 antibody (Fig. 6A). This suggests that the extracts contained protease activity, as we had expected. The protease activity was not detected by adding the microsomal fraction without detergent treatment. As shown in Fig.6B, we also detected such a protease activity in the detergent-solubilized Golgi fraction prepared from rat livers (27Leelavathi D.E. Estes L.W. Feingold D.S. Lombardi B. Biochim. Biophys. Acta. 1970; 211: 124-138Crossref Scopus (155) Google Scholar). These results suggest that the protease was localized mainly in the Golgi apparatus and its catalytic domain faces the luminal side. To rule out the possibility that the protease has endoprotease activity, we added protein A-ST6Gal I-FLAG as substrate to the detergent-solubilized Golgi fraction as the enzyme fraction to see whether the ST6Gal I E41 form was produced. Because we did not detect the production of ST6Gal I E41 form, we surmised that the protease in the Golgi fraction is a kind of exopeptidase that acts on ST6Gal I-Q38 form. We also confirmed that BACE1 itself has no trimming activity, because we did not detect production of the ST6Gal I-Q38 form without detergent-solubilized Golgi fraction (Fig. 6B). Thus we demonstrate the presence of luminal exopeptidase activity that trims the NH2-terminal QAK-sequence of the Q38 form. To further characterize the trimming activity, we tested the sensitivity of the putative peptidase to the various aminopeptidase inhibitors (Table II). As both EDTA and 1,10-pheananthroline significantly inhibited the peptidase activity at 1 mm, the enzyme seems to have metallopeptidase-like character. Moreover, we found that bestatin, but not amastatin, significantly inhibits the activity. These results suggest that the protease that trims NH2-terminal QAK sequence of ST6Gal I-Q38 form belongs to the bestatin-sensitive metalloaminopeptidase.Table IIEffect of various protease inhibitors on the exopeptidase activityInhibitorConcentrationResidual activitymm%Amastatin1.0265Bestatin1.0671,10-Phenanthroline1.062EDTA1.085ST6Gal I-FLAG Q38 form was incubated with detergent-solubilized Golgi membrane fraction (10 μg of protein) in the presence or absence of 1 mm protease inhibitors. The reaction products were then analyzed by immunoblotting with both the anti-FLAG and the E41 antibodies. The amounts of total ST6Gal-FLAG substrate and the product E41 form were quantitated using an LAS 1000 chemiluminescence analyzer (Fuji). Values are the average of relative residual activity obtained from the three independent experiments when E41 form/total substrate was taken as 100% in the control sample. Open table in a new tab ST6Gal I-FLAG Q38 form was incubated with detergent-solubilized Golgi membrane fraction (10 μg of protein) in the presence or absence of 1 mm protease inhibitors. The reaction products were then analyzed by immunoblotting with both the anti-FLAG and the E41 antibodies. The amounts of total ST6Gal-FLAG substrate and the product E41 form were quantitated using an LAS 1000 chemiluminescence analyzer (Fuji). Values are the average of relative residual activity obtained from the three independent experiments when E41 form/total substrate was taken as 100% in the control sample. We show here that BACE1 cleaves ST6Gal I between Leu37 and Gln38. Although amino-terminal sequencing analysis showed that most of soluble secreted ST6Gal I started at Glu41, there might be small amount of soluble ST6Gal I starting at Gln38. We prepared an antibody that specifically recognizes the NH2 terminus of ST6Gal I-Q38 form. This Q38 antibody failed to detect soluble secreted ST6Gal I from COS cells, even though E41 antibody did detect the soluble ST6Gal I (Fig. 7). The results indicate that secreted ST6GalI start mostly at Glu41. A simple explanation for the efficient conversion from ST6Gal I-Q38 form to E41 form is that the amount of exopeptidase activity exceeds that of the substrate. An alternative idea is that exopeptidase-dependent trimming may be a prerequisite for secretion. The latter speculation is supported by our previous cellular experiment (19Kitazume S. Tachida Y. Oka R. Shirotani K. Saido T.C. Hashimoto Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13554-13559Crossref PubMed Scopus (231) Google Scholar) in which a ST6Gal IK40A mutant expressed in COS cells was poorly secreted (40% of wild type level), although this mutation did not affect in vitro BACE1-dependent cleavage (Fig. 5). The mutation may affect exopeptidase-dependent trimming and then the secretory process. We therefore compared the exopeptidase activity toward ST6Gal I-Q38 form with that for its K40A mutant. Exopeptidase cleavage rate of the K40A mutant was half of wild type protein over a particular time course; i.e.when we set the cleavage rate of wild type after a 2-h incubation as 100%, the cleavage rate of K40A was 46%. After a 1-h incubation, the cleavage rate of the wild type was 40% and that of K40A was 17% (Fig. 8). These results suggest that K40A mutation reduces the efficiency of the exopeptidase activity. In the present experiment, we used ST6Gal I peptide and protein A-ST6Gal I-FLAG as substrates for BACE1, both of which were cleaved between Leu37 and Gln38. The observation fits other previous reports (20Citron M. Teplow D.B. Selkoe D.J. Neuron. 1995; 14: 661-670Abstract Full Text PDF PubMed Scopus (232) Google Scholar, 21Turner III, R.T. Koelsch G. Hong L. Castanheira P. Ermolieff J. Ghosh A.K. Tang J. Castenheira P. Ghosh A. Biochemistry. 2001; 40: 10001-10006Crossref PubMed Scopus (196) Google Scholar, 22Gruninger-Leitch F. Schlatter D. Kung E. Nelbock P. Dobeli H. J. Biol. Chem. 2002; 277: 4687-4693Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar) describing residue preferences for subsites of BACE1; i.e. P1 site is most stringently recognized by BACE1 and only large hydrophobic residues such as Leu, Phe, Met, and Tyr are accepted at this position. Previous reports (22Gruninger-Leitch F. Schlatter D. Kung E. Nelbock P. Dobeli H. J. Biol. Chem. 2002; 277: 4687-4693Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar,28Hong L. Koelsch G. Lin X. Wu S. Terzyan S. Ghosh A.K. Zhang X.C. Tang J. Science. 2000; 290: 150-153Crossref PubMed Scopus (698) Google Scholar) showing that BACE1 prefers bulky hydrophobic residues at the P3 position also correspond well with the presence of Leu at the P3 of ST6Gal I, suggesting that BACE1-Fc preferably recognizes the Leu35-Thr36-Leu37 sequence of ST6Gal I and cleaves exclusively at this site. This cleavage is also supported by our own previous cellular experiment, in which replacement of Leu37 with Ala (ST6Gal IL37A), an unfavorable substitution for BACE1 cleavage in vitro, significantly reduced the secretion from the cells (61 ± 20% of the control level, p < 0.05). Taken together, we speculate that BACE1 cleaves ST6Gal I between Leu37 and Gln38 inside the cells and generates the Q38 form of soluble ST6Gal I as an initial product. Our data suggest that luminal exopeptidase activity is involved in the trimming of the NH2-terminal flanking sequence of Q38 form (Fig. 6). A proposed model for ST6Gal I processing and secretion is summarized in Fig. 9. In some cases, exopeptidase activities are critical for the processing of luminal proteins as well as physiologically important peptides (29Chesneau V. Pierotti A.R. Barre N. Creminon C. Tougard C. Cohen P. J. Biol. Chem. 1994; 269: 2056-2061Abstract Full Text PDF PubMed Google Scholar, 30Serwold T. Gaw S. Shastri N. Nat. Immunol. 2001; 2: 644-651Crossref PubMed Scopus (164) Google Scholar, 31Stoltze L. Schirle M. Schwarz G. Schroter C. Thompson M.W. Hersh L.B. Kalbacher H. Stevanovic S. Rammensee H.G. Schild H. Nat. Immunol. 2000; 1: 413-418Crossref PubMed Scopus (214) Google Scholar). A recent report by Komlosh et al. (32Komlosh A. Momburg F. Weinschenk T. Emmerich N. Schild H. Nadav E. Shaked I. Reiss Y. J. Biol. Chem. 2001; 276: 30050-30056Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) has identified a novel luminal endoplasmic reticulum exopeptidase that is involved in the trimming of major histocompatibility complex class I antigenic peptides. It is notable that the main portion of soluble ST6Gal I secreted from the cells is the E41 form (24Kitazume-Kawaguchi S. Dohmae N. Takio K. Tsuji S. Colley K.J. Glycobiology. 1999; 9: 1397-1406Crossref PubMed Scopus (32) Google Scholar); we did not detect the Q38 form in the culture medium (Fig. 7), suggesting that the Q38 form generated by BACE1 is efficiently converted to the E41 form before secretion. Moreover, our previous observation, in which a ST6Gal IK40A mutant expressed in COS cells was poorly secreted (24Kitazume-Kawaguchi S. Dohmae N. Takio K. Tsuji S. Colley K.J. Glycobiology. 1999; 9: 1397-1406Crossref PubMed Scopus (32) Google Scholar), supports the idea that the mutation may affect exopeptidase-dependent trimming and then the secretory process. Indeed, the present data indicate that the K40A mutation somewhat reduces exopeptidase trimming efficiency. At present, however, we cannot exclude the possibility that an additional unveiled mechanism other than the ST6Gal I cleavage process exists to regulate the ST6Gal I secretion. The data presented here suggest that the exopeptidase has a bestatin-sensitive metalloprotease-like character. Because we used a crude Golgi membrane fraction as an enzyme source in this study, purification of this exopeptidase will be required for further characterization in the future. Identification and characterization of the exopeptidase will be important for a better understanding of the molecular mechanisms underlying the cleavage and secretion of ST6Gal I. We thank Dr. Tae-Wan Kim (Harvard Medical School, Boston, MA) for providing human BACE1 myc-pcDNA and Dr. Carolyn Bruzdzinski (University of Illinois, Chicago) for FTO2B rat hepatoma cells. We also thank Dr. Marcos Milla (University of Pennsylvania School of Medicine, Philadelphia), Dr. Shoichi Ishiura (University of Tokyo, Japan), and Drs. Masafumi Tsujimoto and Akira Hattori (RIKEN) for helpful discussions on the assay for exopeptidase activity and Drs. Akemi Suzuki and Tamio Yamakawa (RIKEN Frontier Research System) for encouragement throughout the study. Kazuko Hashimoto is also acknowledged for invaluable secretarial assistance." @default.
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