In Vivo Cleavage of α2,6-Sialyltransferase by Alzheimer β-Secretase (2004)

β-Site amyloid precursor protein-cleaving enzyme 1 (BACE1) is a membrane-bound aspartic protease that cleaves amyloid precursor protein to produce a neurotoxic peptide, Aβ, and is implicated in triggering the pathogenesis of Alzheimer disease. We previously reported that BACE1 cleaved rat β-galactoside α2,6-sialyltransferase (ST6Gal I) that was overexpressed in COS cells and that the NH2 terminus of ST6Gal I secreted from the cells (E41 form) was Glu41. Here we report that BACE1 gene knock-out mice have one third as much plasma ST6Gal I as control mice, indicating that BACE1 is a major protease which is responsible for cleaving ST6Gal I in vivo. We also found that BACE1-transgenic mice have increased level of ST6Gal I in plasma. Secretion of ST6Gal I from the liver into the plasma is known to be up-regulated during the acute-phase response. To investigate the role of BACE1 in ST6Gal I secretion in vivo, we analyzed the levels of BACE1 mRNA in the liver, as well as the plasma levels of ST6Gal I, in a hepatopathological model, i.e. Long-Evans Cinnamon (LEC) rats. This rat is a mutant that spontaneously accumulates copper in the liver and incurs hepatic damage. LEC rats exhibited simultaneous increases in BACE1 mRNA in the liver and in the E41 form of the ST6Gal I protein, the BACE1 product, in plasma as early as 6 weeks of age, again suggesting that BACE1 cleaves ST6Gal I in vivo and controls the secretion of the E41 form. β-Site amyloid precursor protein-cleaving enzyme 1 (BACE1) is a membrane-bound aspartic protease that cleaves amyloid precursor protein to produce a neurotoxic peptide, Aβ, and is implicated in triggering the pathogenesis of Alzheimer disease. We previously reported that BACE1 cleaved rat β-galactoside α2,6-sialyltransferase (ST6Gal I) that was overexpressed in COS cells and that the NH2 terminus of ST6Gal I secreted from the cells (E41 form) was Glu41. Here we report that BACE1 gene knock-out mice have one third as much plasma ST6Gal I as control mice, indicating that BACE1 is a major protease which is responsible for cleaving ST6Gal I in vivo. We also found that BACE1-transgenic mice have increased level of ST6Gal I in plasma. Secretion of ST6Gal I from the liver into the plasma is known to be up-regulated during the acute-phase response. To investigate the role of BACE1 in ST6Gal I secretion in vivo, we analyzed the levels of BACE1 mRNA in the liver, as well as the plasma levels of ST6Gal I, in a hepatopathological model, i.e. Long-Evans Cinnamon (LEC) rats. This rat is a mutant that spontaneously accumulates copper in the liver and incurs hepatic damage. LEC rats exhibited simultaneous increases in BACE1 mRNA in the liver and in the E41 form of the ST6Gal I protein, the BACE1 product, in plasma as early as 6 weeks of age, again suggesting that BACE1 cleaves ST6Gal I in vivo and controls the secretion of the E41 form. A characteristic feature of Alzheimer disease (AD) 1The abbreviations used are: AD, Alzheimer disease; Aβ, amyloid β-peptide; APP, amyloid precursor protein; BACE, β-site APP-cleaving enzyme; FPLC, first performance liquid chromatography; LEA, Long-Evans Agouti; LEC, Long-Evans Cinnamon; PSGL-1, P-selectin glyco-protein ligand-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MES, 4-morpholineethanesulfonic acid; LMW, lower molecular weight.1The abbreviations used are: AD, Alzheimer disease; Aβ, amyloid β-peptide; APP, amyloid precursor protein; BACE, β-site APP-cleaving enzyme; FPLC, first performance liquid chromatography; LEA, Long-Evans Agouti; LEC, Long-Evans Cinnamon; PSGL-1, P-selectin glyco-protein ligand-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MES, 4-morpholineethanesulfonic acid; LMW, lower molecular weight. is deposition of amyloid β-peptide (Aβ) in the brain, which is implicated in the pathogenesis of AD (1Selkoe D.J. Physiol. 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FEBS Lett. 2000; 468: 59-64Crossref PubMed Scopus (121) Google Scholar) and its expression is elevated in trisomic brains (16Motonaga K. Itoh M. Becker L.E. Goto Y. Takashima S. Neurosci. Lett. 2002; 326: 64-66Crossref PubMed Scopus (35) Google Scholar). A series of extensive studies showed that γ-secretase activity is performed by a protein complex that includes presenilins (PS1 or PS2) (17Li Y.M. Lai M.T. Xu M. Huang Q. DiMuzio-Mower J. Sardana M.K. Shi X.P. Yin K.C. Shafer J.A. Gardell S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6138-6143Crossref PubMed Scopus (494) Google Scholar). γ-Secretase is essential, not only for Aβ production, but also for neuronal development, due to its cleavage of a Notch receptor (3De 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 (1775) Google Scholar, 18Struhl G. Greenwald I. Nature. 1999; 398: 522-525Crossref PubMed Scopus (700) Google Scholar). Nevertheless, development of inhibitors to γ-secretase is a promising approach for treating Alzheimer disease, as is also true for β-secretase inhibitors (19Hong L. Koelsch G. Lin X. Wu S. Terzyan S. Ghosh A.K. Zhang X.C. Tang J. Science. 2000; 290: 150-153Crossref PubMed Scopus (693) Google Scholar). ST6Gal I (β-galactoside α2,6-sialyltransferase) is a type II membrane protein that is localized in the trans-Golgi network. It catalyzes α2,6-sialylation of Galβ1,4-GlcNAc structures on N-glycans. ST6Gal I is highly expressed in the liver and is expressed in most other tissues to some extent (20Kitagawa H. Paulson J.C. J. Biol. Chem. 1994; 269: 17872-17878Abstract Full Text PDF PubMed Google Scholar). The majority of serum ST6Gal I is secreted from the liver (21Weinstein J. Lee E.U. McEntee K. Lai P.H. Paulson J.C. J. Biol. Chem. 1987; 262: 17735-17743Abstract Full Text PDF PubMed Google Scholar, 22Colley K.J. Lee E.U. Adler B. Browne J.K. Paulson J.C. J. Biol. Chem. 1989; 264: 17619-17622Abstract Full Text PDF PubMed Google Scholar), and secretion is enhanced during acute-phase hepatic reactions (23Kaplan H.A. Woloski B.M. Hellman M. Jamieson J.C. J. Biol. Chem. 1983; 258: 11505-11509Abstract Full Text PDF PubMed Google Scholar, 24Dalziel M. Lemaire S. Ewing J. Kobayashi L. Lau J.T. Glycobiology. 1999; 9: 1003-1008Crossref PubMed Scopus (43) Google Scholar). We previously found that BACE1 is involved in the cleavage and secretion of ST6Gal I, at least in cultured cells (25Kitazume 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 (228) Google Scholar, 26Kitazume S. Tachida Y. Oka R. Kotani N. Ogawa K. Suzuki M. Dohmae N. Takio K. Saido T.C. Hashimoto Y. J. Biol. Chem. 2003; 278: 14865-14871Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), leading to an assumption that BACE1 cleaves ST6Gal I in the liver and triggers its secretion into plasma. To confirm in vivo cleavage of ST6Gal I by BACE1, in the present study we analyzed plasma ST6Gal I levels in BACE1-deficient and BACE1-transgenic mice. We also used a mutant animal strain, the Long-Evans Cinnamon (LEC) rat, to analyze the mechanisms of ST6Gal I secretion in hepatopathological conditions. The LEC rat, a model of Wilson disease, has a deletion in the gene for the copper-transporting ATPase gene (ATP7B) (27Yoshida M.C. Masuda R. Sasaki M. Takeichi N. Kobayashi H. Dempo K. Mori M. J. Hered. 1987; 78: 361-365Crossref PubMed Scopus (193) Google Scholar, 28Li Y. Togashi Y. Sato S. Emoto T. Kang J.H. Takeichi N. Kobayashi H. Kojima Y. Une Y. Uchino J. J. Clin. Invest. 1991; 87: 1858-1861Crossref PubMed Scopus (287) Google Scholar, 29Wu J. Forbes J.R. Chen H.S. Cox D.W. Nat. Genet. 1994; 7: 541-545Crossref PubMed Scopus (347) Google Scholar). Golgi-localized ATP7B is involved in copper secretion into the plasma, which is coupled with ceruloplasmin synthesis and biliary copper excretion (30Petris M.J. Mercer J.F. Culvenor J.G. Lockhart P. Gleeson P.A. Camakaris J. EMBO J. 1996; 15: 6084-6095Crossref PubMed Scopus (527) Google Scholar). Like patients with Wilson disease, LEC rats suffer from toxic accumulation of copper in the liver and eventually develop hepatitis and then hepatocellullar carcinoma (31Mori M. Yoshida M.C. Takechi N. Taniguchi N. The LEC Rat, A New Model for Hepatitis and Liver Cancer. Springer-Verlag, Tokyo1991Google Scholar). This rat strain is often utilized for studying the pathogenesis of hepatitis and hepatoma. On the assumption that ST6Gal I secretion is stimulated in the hepatopathological model of LEC rats and that such stimulation is related to the level of BACE1 activity, we analyzed the expression profiles of mRNAs of ST6Gal I and BACE1 in the liver of LEC rats and also the secretion of ST6Gal I into plasma. Materials—Male LEC and Wistar rats, maintained in specific-patho-gen-free conditions, were purchased from Charles River Japan Inc. (Yokohama, Japan) and the Shizuoka Agricultural Cooperative Association for Laboratory Animals (Shizuoka, Japan), respectively. Samples of LEA (Long-Evans Agouti) rats were kindly provided by Dr. Noriyuki Kasai, Tohoku University. BACE1 knock-out mice and their control mice from Amgen were maintained in the Charles River Laboratories, Inc. (Wilmington, MA). BACE1-transgenic mice were prepared by using an overexpression cassette, in which the chicken β-actin promoter drived the expression of human BACE1 (32Shitara H. Kaneda H. Sato A. Iwasaki K. Hayashi J. Taya C. Yonekawa H. FEBS Lett. 2001; 500: 7-11Crossref PubMed Scopus (44) Google Scholar). The sources of materials used in this work were as follows: tissue culture media and reagents, including Dulbecco's modified Eagle's medium and William's E medium, were from Invitrogen; Expre35S35S protein labeling mix was from PerkinElmer Life Sciences; recombinant N-glycosidase F from Roche Diagnostics, CMP-[14C]NeuAc and protein A-Sepharose Fast Flow were from Amersham Biosciences; affinity Gel-CDP from Merck/EMD Bioscience (San Diego, CA); protein molecular weight standards were from Bio-Rad; and all other chemicals from Sigma or Wako Chemicals (Osaka, Japan). Protein concentration was determined with Pierce BCA protein assay reagents. Anti-BACE1 antibody was purchased from MoBiTec Co. (Göttingen, Germany). We used anti-ST6Gal I polyclonal antibodies to detect ST6Gal I from plasma and to immunoprecipitate ST6Gal I proteins from cell lysates and media of hepatocytes (33Weinstein J. de Souza-e-Silva U. Paulson J.C. J. Biol. Chem. 1982; 257: 13845-13853Abstract Full Text PDF PubMed Google Scholar). E41 antibody that specifically recognizes ST6Gal I starting at Glu41 was obtained from IBL Co. (Fujioka, Japan). Pulse-Chase Analysis and Immunoprecipitation of ST6Gal I Using Rat Hepatocytes—Hepatocytes (∼5 × 106 cells), isolated from 10-week-old male Wistar rats by two-step collagenase perfusion (34Seglen P.O. Methods Cell Biol. 1976; 13: 29-83Crossref PubMed Scopus (5190) Google Scholar), were seeded on collagen type I-coated plates (100-mm, Iwaki, Funabashi, Japan) and were grown in William's E medium containing 5% fetal bovine serum. Metabolic labeling of cells and immunoprecipitation of expressed proteins were performed essentially as described previously, using Expre35S35S protein label (100 μCi/ml) (25Kitazume 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 (228) Google Scholar, 35Kitazume-Kawaguchi S. Dohmae N. Takio K. Tsuji S. Colley K.J. Glycobiology. 1999; 9: 1397-1406Crossref PubMed Scopus (31) Google Scholar). Cells were labeled with 18 h, or labeled with 2 h and chased for 6 or 18 h, in 4 ml of William's E medium containing 5% fetal bovine serum. The collected media were diluted with 4 ml of Pierce binding buffer, and the cells were lysed in immunoprecipitation buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 5 mm EDTA, 0.5% Nonidet P-40, 0.1% SDS). From the cell lysates and media, ST6Gal I proteins were immunoprecipitated with anti-ST6Gal I or E41 polyclonal antibodies. Immunoprecipitated proteins were denatured in Laemili sample buffer containing 5% β-mercaptoethanol by boiling for 5 min. Immunoprecipitated proteins were analyzed on SDS-polyacrylamide gels (5-20% gradient of polyacrylamide), and radiolabeled proteins were visualized with a BAS 2000 radioimage analyzer (Fuji Film, Tokyo). Real-time Quantitative PCR—Total RNA was isolated from rat livers using Trizol reagent (Invitrogen), and 5-10 μg of RNA was reverse-transcribed with random hexamers by using a Super Script II RT kit (Invitrogen) according to the manufacturer's protocol. The cDNA was then amplified with 900 nm forward primer, 900 nm reverse primer, 250 nm fluorogenic probe, and 25 μl of Universal PCR Master Mix (Applied Biosystems) in a total volume of 20 μl, using an ABI PRISM 7900HT sequence detection system (Applied Biosystems). The PCR conditions were 1 cycle at 50 °C for 2 min, 1 cycle at 95 °C for 10 min, and 40 cycles at 95 °C for 15 s and 50 °C for 1 min. All primers and probes were purchased from Applied The of primers and probes were as follows: ST6Gal I, and BACE2, BACE1 and primers and we used and were to the Universal PCR Master Mix (Applied which all for The probes for ST6Gal I, BACE1, and BACE2 were labeled with the The probes for were labeled with at the and at the were labeled with the The expression levels of were in and were to the expression of BACE1-deficient and BACE1-transgenic of BACE1-deficient and control mice were by PCR using prepared from livers and primers and A fragment was in mice, and a fragment was in the BACE1-deficient mice. We also the level of BACE1 protein in the livers of BACE1-deficient and BACE1-transgenic mice by livers were with buffer (50 mm MES, pH 1 mm EDTA, (50 or that was with buffer (50 mm Tris-HCl, pH NaCl, was with sample buffer Nature. PubMed Scopus Google Scholar), to SDS-polyacrylamide (5-20% and then to a The membrane was with was used as the and was used for detection (25Kitazume 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 (228) Google Scholar). of ST6Gal I from mice (50 were diluted with 10 of buffer A mm Tris-HCl, pH and then a ml of Amersham using an The was with 5 ml of buffer and protein was then with a gradient of for at a of were collected and the ST6Gal I was by anti-ST6Gal I ST6Gal were and with from rats μg of were by the kit from and then with To the level of soluble ST6Gal I, protein were in buffer mm pH NaCl, 0.1% to SDS-polyacrylamide (5-20% and then to a The membrane was with anti-ST6Gal I was used as the and was used for detection (25Kitazume 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 (228) Google Scholar). ST6Gal I by anti-ST6Gal I antibody was with a N-glycosidase F the sample with was in μl of N-glycosidase F buffer (50 mm pH 50 mm EDTA, Nonidet P-40, and and then into The were at °C for h in the or of 1 of N-glycosidase F. The were analyzed by with anti-ST6Gal I of ST6Gal I from Rat from Wistar rats was diluted with 10 of buffer A (50 mm Tris-HCl, pH and buffer A. The sample was then a mm × Amersham using an The was with 250 ml of buffer and protein was then with a gradient of for at a of 5 were collected and the ST6Gal I was by of the with anti-ST6Gal I The in ST6Gal I protein was buffer mm pH and then a was with buffer and was with a gradient of buffer mm pH NaCl, 10 mm were in the of 50 mm buffer, pH 0.5% 1 mm 1 mm CMP-[14C]NeuAc and 50 μl of enzyme in a total volume of μl, as described previously (33Weinstein J. de Souza-e-Silva U. Paulson J.C. J. Biol. Chem. 1982; 257: 13845-13853Abstract Full Text PDF PubMed Google Scholar). 2 or 6 h of at the were to to the from The profiles were by ST6Gal I in BACE1-deficient I is highly expressed in the liver (20Kitagawa H. Paulson J.C. J. Biol. Chem. 1994; 269: 17872-17878Abstract Full Text PDF PubMed Google Scholar), and a membrane-bound form of ST6Gal I is cleaved in the trans-Golgi network and then secreted into the plasma K.J. Lee E.U. Adler B. Browne J.K. Paulson J.C. J. Biol. Chem. 1989; 264: 17619-17622Abstract Full Text PDF PubMed Google Scholar, J. de Souza-e-Silva U. Paulson J.C. J. Biol. Chem. 1982; 257: 13845-13853Abstract Full Text PDF PubMed Google Scholar). We previously reported that secretion of ST6Gal I was increased by overexpression of BACE1 in COS cells (25Kitazume 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 (228) Google Scholar), indicating that BACE1 cleaves ST6Gal I at least in the cultured To cleavage of ST6Gal I by BACE1 also in vivo, we utilized BACE1-deficient mice for plasma ST6Gal I ST6Gal I cleavage and secretion to be or first we of BACE1-deficient and control mice by PCR and then the or of BACE1 protein in the prepared from the liver From plasma, ST6Gal I was or by of ST6Gal that ST6Gal I levels in BACE1-deficient mice were to that of control mice suggesting that BACE1 is a major protease that cleaves ST6Gal I to its secretion in vivo and that the plasma ST6Gal I in BACE1-deficient mice may be cleaved by some other We the elevated level of BACE1 the of the plasma ST6Gal I in vivo. To we have generated the of BACE1-transgenic mice that human BACE1 the control of promoter for We BACE1 expression in the liver of of of BACE1-transgenic mice elevated levels of plasma ST6Gal I as with the control mice lower mice elevated levels of plasma ST6Gal I. BACE1 was overexpressed the control of the promoter in BACE1-transgenic mice and BACE1 proteins in the (50 μg of prepared from liver of and control mice, were analyzed by with antibody ST6Gal I in plasma was and its level was by with anti-ST6Gal I ST6Gal I Secretion in Rat previously reported that rat ST6Gal I secreted from COS cells has a sequence of at its amino terminus (E41 form) (25Kitazume 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 (228) Google Scholar, 35Kitazume-Kawaguchi S. Dohmae N. Takio K. Tsuji S. Colley K.J. Glycobiology. 1999; 9: 1397-1406Crossref PubMed Scopus (31) Google Scholar). of BACE1 in COS cells increased the secretion of the E41 form (25Kitazume 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 (228) Google Scholar). We found that BACE1 first cleaved rat ST6Gal I and to the sequence at the amino and then the amino were by a to produce the E41 form that is secreted from the cells S. Tachida Y. Oka R. Kotani N. Ogawa K. Suzuki M. Dohmae N. Takio K. Saido T.C. Hashimoto Y. J. Biol. Chem. 2003; 278: 14865-14871Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). of the in vivo cleavage and secretion of ST6Gal I, we for the E41 form in rat plasma. using anti-ST6Gal I antibody of soluble ST6Gal I in the plasma of Wistar rats N-glycosidase F to the were as a of on an but with increased suggesting that the the is not due to the of structures but to the of the cleavage The were by were to in which we used an that specifically recognizes the E41 form (25Kitazume 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 (228) Google Scholar, 26Kitazume S. Tachida Y. Oka R. Kotani N. Ogawa K. Suzuki M. Dohmae N. Takio K. Saido T.C. Hashimoto Y. J. Biol. Chem. 2003; 278: 14865-14871Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). only with the molecular weight suggesting that is the E41 most by BACE1 The lower molecular weight form) has a cleavage by some other molecular weight at Glu41. a of for rat plasma sample is were from third or for with anti-ST6Gal I antibody the ST6Gal by the in were and with anti-ST6Gal I μg of protein was or antibody (50 μg of To we used which is often used for