Prodomain Processing of Asp1 (BACE2) Is Autocatalytic

Generation of the amyloid peptide through proteolytic processing of the amyloid precursor protein by β- and γ-secretases is central to the etiology of Alzheimer's disease. The highly elusive β-secretase was recently identified as a transmembrane aspartic proteinase, Asp2 (BACE). The Asp2 homolog Asp1 (BACE2/DRAP) has also been reported to exhibit β-secretase cleavage of amyloid precursor protein. Most aspartic proteinases are generated as inactive proenzymes, requiring removal of the prodomain to generate active proteinase. Here we show that prodomain processing of Asp1 occurs between Leu62 and Ala63 and is autocatalytic. Asp1 cleaved a maltose-binding protein-Asp1 prodomain fusion protein and a synthetic peptide at this site. Mutation of one of the conserved catalytic aspartic acid residues in the active site of Asp1 to asparagine (D110N) abolished this cleavage. Mutation of P1′ and P2′ residues in the substrate to phenylalanine reduced cleavage at this site. Asp1 expressed in cells was the mature form, and prodomain processing occurred intramolecularly within the endoplasmic reticulum/early Golgi. Interestingly, a proportion of mature Asp1 was expressed on the cell surface. When full-length Asp1(D110N) was expressed in COS-7 cells, it was not processed, suggesting that no other proteinase can activate Asp1 in these cells. Generation of the amyloid peptide through proteolytic processing of the amyloid precursor protein by β- and γ-secretases is central to the etiology of Alzheimer's disease. The highly elusive β-secretase was recently identified as a transmembrane aspartic proteinase, Asp2 (BACE). The Asp2 homolog Asp1 (BACE2/DRAP) has also been reported to exhibit β-secretase cleavage of amyloid precursor protein. Most aspartic proteinases are generated as inactive proenzymes, requiring removal of the prodomain to generate active proteinase. Here we show that prodomain processing of Asp1 occurs between Leu62 and Ala63 and is autocatalytic. Asp1 cleaved a maltose-binding protein-Asp1 prodomain fusion protein and a synthetic peptide at this site. Mutation of one of the conserved catalytic aspartic acid residues in the active site of Asp1 to asparagine (D110N) abolished this cleavage. Mutation of P1′ and P2′ residues in the substrate to phenylalanine reduced cleavage at this site. Asp1 expressed in cells was the mature form, and prodomain processing occurred intramolecularly within the endoplasmic reticulum/early Golgi. Interestingly, a proportion of mature Asp1 was expressed on the cell surface. When full-length Asp1(D110N) was expressed in COS-7 cells, it was not processed, suggesting that no other proteinase can activate Asp1 in these cells. amyloid precursor protein maltose-binding protein matrix-assisted laser desorption/ionization mass spectrometric/spectrometry aminohexanoic acid 2,4-dinitrophenyl high performance liquid chromatography endoplasmic reticulum Alzheimer's disease is a neurodegenerative disorder characterized by the deposition of the amyloid β-peptide in the brain as amyloid plaques (1Glenner G.G. Wong C.W. Biochem. Biophys. Res. Commun. 1984; 120: 885-890Crossref PubMed Scopus (4169) Google Scholar, 2Masters C.L. Simms G. Weidemann N.A. Multhaup G. McDonald B.L. Beyreuther K. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4245-4249Crossref PubMed Scopus (3620) Google Scholar). The amyloid β-peptide is generated through sequential proteolytic processing of the amyloid precursor protein (APP)1 by β- and γ-secretases (3Selkoe D.J. Annu. Rev. 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Asp2 mRNA is abundant in the brain, and overexpression of Asp2 in cultured cells results in an increase in the level of β-secretase-derived soluble APP and intracellular C-terminal fragments. An increase in the level of secreted amyloid β-peptide has also been observed in cells overexpressing Asp2. In addition, Asp2 colocalizes with APP within intracellular Golgi compartments, where β-secretase cleavage is known to occur (10Haass C. Lemere C.A. Capell A. Citron M. Seubert P. Schenk D. Lannfelt L. Selkoe D.J. Nat. Med. 1995; 1: 1291-1296Crossref PubMed Scopus (439) Google Scholar). The homolog of Asp2, Asp1 (BACE2), has also been identified (11Saunders A.J. Kim T.-W. Tanzi R.E. Science. 1999; 286: 1255aCrossref Google Scholar, 12Acquati F. Accarino M. Nucci C. Fumagalli P. Jovine L. Ottolenghi S. Taramelli R. FEBS Lett. 2000; 468: 59-64Crossref PubMed Scopus (121) Google Scholar). Like Asp2, Asp1 also cleaves APP at the β-site, resulting in an increase in the level of β-secretase-derived soluble APP and intracellular C-terminal fragments (13Hussain I. Powell D.J. Howlett D.R. Chapman G.A. Gilmour L. Murdock P.M. Tew D.G. Meek T.D. Chapman C. Schneider K. Ratcliffe S.J. Tattersall D. Testa T.T. Southan C. Ryan D.M. Simmons D.L. Walsh F.S. Dingwall C. Christie G. Mol. Cell. Neurosci. 2000; 16: 609-619Crossref PubMed Scopus (143) Google Scholar, 14Farzan M. Schnitzler C.E. Vasilieva N. Leung D. Choe H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9712-9717Crossref PubMed Scopus (345) Google Scholar). Thus, Asp1 represents a second β-secretase candidate. Recent reports suggest that the presenilins are novel aspartic proteinases that either exhibit γ-secretase activity (15Wolfe 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, 16Li 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, 17Esler 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, 18Steiner H. Kostka M. Romig H. Basset G. Pesold B. Hardy J. Capell A. Meyn L. Grim M.L. Baumeister R. Fechteler K. Haass C. Nat. Cell Biol. 2000; 2: 848-851Crossref PubMed Scopus (250) Google Scholar) or form part of a large multimeric complex that includes γ-secretase activity (19Sisodia S.S. Science. 2000; 289: 2296-2297Crossref PubMed Scopus (16) Google Scholar). Like all aspartic proteinases, Asp1 and Asp2 are generated as proenzymes; and in most cases, removal of the prodomain is required to produce mature active enzyme. Therefore, an analysis of the mechanism of prodomain cleavage and the identification of the protease(s) that catalyze this event are essential for further understanding of the amyloidogenic cascade. Clearly, any upstream activating proteinases could represent novel therapeutic targets through which APP processing might be modulated. Recently, it has been reported that activation of Asp2 is mediated by furin or a furin-like enzyme that recognizes the RXXR motif immediately upstream of the prodomain cleavage site (20Bennett B.D. Denis P. Haniu M. Teplow D.B. Kahn S. Louis J.-C. Citron M. Vassar R. J. Biol. Chem. 2000; 275: 37712-37717Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 21Creemers J.W.M. Dominguez D.I. Plets E. Serneels L. Taylor N.A. Multhaup G. Craessaerts K. Annaert W. De Strooper B. J. Biol. Chem. 2001; 276: 4211-4217Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 22Benjannet S. Elagoz A. Wickham L. Mamarbachi M. Munzer J.S. Basak A. Lazure C. Cromlish J.A. Sisodia S. Checler F. Chrétien M. Seidah N.G. J. Biol. Chem. 2001; 276: 10879-10887Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). Having previously shown that prodomain processing in Asp1 occurs between residues Leu62 and Ala63(13Hussain I. Powell D.J. Howlett D.R. Chapman G.A. Gilmour L. Murdock P.M. Tew D.G. Meek T.D. Chapman C. Schneider K. Ratcliffe S.J. Tattersall D. Testa T.T. Southan C. Ryan D.M. Simmons D.L. Walsh F.S. Dingwall C. Christie G. Mol. Cell. Neurosci. 2000; 16: 609-619Crossref PubMed Scopus (143) Google Scholar), we aimed in this study to determine whether this is an intra- or intermolecular event and to identify any enzyme(s) that could mediate this cleavage. Asp1 (base pairs 61–276) and Asp2 (base pairs 64–225) fragments spanning the prodomain cleavage site (Ala21–Tyr91) were amplified by polymerase chain reaction, in which the reverse oligonucleotide encoded a His6 tag followed by a termination codon at the 3′-end. The fragments were cloned in frame to the maltose-binding protein (MBP) sequence at the BamHI/HindIII sites in pMal2c (New England Biolabs, Inc.) to generate MBP-Asp1pro-His6and MBP-Asp2pro-His6. Sequences were confirmed by dideoxy nucleotide sequencing. Generation of the Asp1-Fc/Signal pIgPlus construct has been described previously (13Hussain I. Powell D.J. Howlett D.R. Chapman G.A. Gilmour L. Murdock P.M. Tew D.G. Meek T.D. Chapman C. Schneider K. Ratcliffe S.J. Tattersall D. Testa T.T. Southan C. Ryan D.M. Simmons D.L. Walsh F.S. Dingwall C. Christie G. Mol. Cell. Neurosci. 2000; 16: 609-619Crossref PubMed Scopus (143) Google Scholar). The Asp2-Fc construct was generated by subcloning the Asp2 cDNA (base pairs 61–1359) into the HindIII/XhoI sites in the Signal pIgPlus and the prodomain construct at the prodomain cleavage site were generated by and and Asp2-Fc were expressed in COS-7 cells and the as described previously (13Hussain I. Powell D.J. Howlett D.R. Chapman G.A. Gilmour L. Murdock P.M. Tew D.G. Meek T.D. Chapman C. Schneider K. Ratcliffe S.J. Tattersall D. Testa T.T. Southan C. Ryan D.M. Simmons D.L. Walsh F.S. Dingwall C. Christie G. Mol. Cell. Neurosci. 2000; 16: 609-619Crossref PubMed Scopus (143) Google Scholar). The fusion were expressed in and protein was in of with for at The cells were by at for and by in of of and at The cell was at for and the was with at for The was into a and with of and The protein was with and protein were and and fusion were with or Asp2-Fc in in a of at and an was at the An of was to the reaction, and were on were by with were as described of the were with a the by the were the on to the with acid in were the The was immediately into a was on a and were on an mass with a for was with or Asp2-Fc in and for in a of The was by the of of The were a in acid and with a of acid in of over The on the full-length peptide and cleavage was followed by at are not at to of the determine the enzyme and substrate were at for and by the of and the of peptide cleaved was by as described were to the with were as described of with Asp1 prodomain peptide or APP β-secretase at for COS-7 cells overexpressing the were with or cells were at the or in the of or for which cell were generated as described previously (4Hussain I. Powell D. Howlett D.R. Tew D.G. Meek T.D. Chapman C. Gloger I.S. Murphy K.E. Southan C.D. Ryan D.M. Smith T.S. Simmons D.L. Walsh F.S. Dingwall C. Christie G. Mol. Cell. Neurosci. 1999; 14: 419-427Crossref PubMed Scopus (997) Google Scholar). in the cell were by as described by the Cell of were on for analysis an as described previously (4Hussain I. Powell D. Howlett D.R. Tew D.G. Meek T.D. Chapman C. Gloger I.S. Murphy K.E. Southan C.D. Ryan D.M. Smith T.S. Simmons D.L. Walsh F.S. Dingwall C. Christie G. Mol. Cell. Neurosci. 1999; 14: 419-427Crossref PubMed Scopus (997) Google Scholar) or an COS-7 cells overexpressing the were with cells were with to with at in for at were with and for as described previously (4Hussain I. Powell D. Howlett D.R. Tew D.G. Meek T.D. Chapman C. Gloger I.S. Murphy K.E. Southan C.D. Ryan D.M. Smith T.S. Simmons D.L. Walsh F.S. Dingwall C. Christie G. Mol. Cell. Neurosci. 1999; 14: 419-427Crossref PubMed Scopus (997) Google Scholar). were and in were on followed by analysis with an of the amyloid β-peptide of APP (4Hussain I. Powell D. Howlett D.R. Tew D.G. Meek T.D. Chapman C. Gloger I.S. Murphy K.E. Southan C.D. Ryan D.M. Smith T.S. Simmons D.L. Walsh F.S. Dingwall C. Christie G. Mol. Cell. Neurosci. 1999; 14: 419-427Crossref PubMed Scopus (997) Google Scholar), or with prodomain processing of we generated a prodomain fusion protein that to Asp1 the prodomain cleavage with a His6 tag at the the sequence of Asp1 and sequence were to the of the The protein was expressed in E. and as a of on and was with both an and an We shown by that both the the in the is most to be in the of residues at the of these followed by peptide mass was The results that both to the fusion protein. the as as the C-terminal were not observed in the mass The to these been by known to occur in the analysis of Chem. PubMed Scopus Google Scholar). of mass was also observed that was with the not with the suggesting that it represents a form of the substrate or of at the prodomain cleavage site is to generate cleavage a large and a determine Asp1 or Asp2 can mediate cleavage at this was with active Asp1 or Asp2, both expressed as soluble fusion of with in a in the mass of the prodomain substrate to with cleavage at the site and of the which was not analysis that was with both and the cleavage was with the to of the C-terminal His6 tag cleavage analysis of with was to the the of one and in the that were the cleavage analysis of the cleavage was sequence that the of the processing of occurred at the the peptide with In addition, of the cleavage of to with and The of Asp1 to mediate prodomain cleavage at the site in the of a fusion protein that in this proteinase prodomain to generate the mature enzyme. In cleavage of by Asp2 is suggesting that it is to activate determine whether the Asp2-Fc or fusion protein can the Asp2 we generated an fusion protein in which of Asp2 were to with a C-terminal His6 protein was also expressed and of this protein with or Asp2-Fc not in a in mass in to prodomain cleavage of Asp2 is to be and it not to be mediated by is with reports suggesting that prodomain processing of Asp2 is mediated by proteinase (20Bennett B.D. Denis P. Haniu M. Teplow D.B. Kahn S. Louis J.-C. Citron M. Vassar R. J. Biol. Chem. 2000; 275: 37712-37717Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 21Creemers J.W.M. Dominguez D.I. Plets E. Serneels L. Taylor N.A. Multhaup G. Craessaerts K. Annaert W. De Strooper B. J. Biol. Chem. 2001; 276: 4211-4217Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 22Benjannet S. Elagoz A. Wickham L. Mamarbachi M. Munzer J.S. Basak A. Lazure C. Cromlish J.A. Sisodia S. Checler F. Chrétien M. Seidah N.G. J. Biol. Chem. 2001; 276: 10879-10887Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). determine the for was with at or the cleaved at with of the substrate into within cleavage was of the substrate was to within cleavage was with of substrate within The catalytic activity of aspartic proteinases is on the of aspartic acid residues in the active site. processing of be abolished either of the catalytic aspartic acid residues in Asp1 is The catalytic aspartic acid in was to asparagine and the protein was expressed in COS-7 cells and the protein In the cleavage was to at the prodomain cleavage site no in mass was of that of the protein expressed in COS-7 cells was and had the sequence (13Hussain I. Powell D.J. Howlett D.R. Chapman G.A. Gilmour L. Murdock P.M. Tew D.G. Meek T.D. Chapman C. Schneider K. Ratcliffe S.J. Tattersall D. Testa T.T. Southan C. Ryan D.M. Simmons D.L. Walsh F.S. Dingwall C. Christie G. Mol. Cell. Neurosci. 2000; 16: 609-619Crossref PubMed Scopus (143) Google Scholar). Thus, it that the is not in COS-7 cells to to was through the Asp1 prodomain cleavage site to determine the on prodomain in were to and the were with of the to be as as that of the substrate expressed as the proportion of substrate cleaved analysis of the cleavage that of the P1′ and P2′ residues reduced the level of the peptide with the sequence and the level of peptide with the sequence In at the and had no on cleavage. that the active site of Asp1 is to large residues at either the P1′ or P2′ We on to determine whether Asp1 can a synthetic peptide spanning the prodomain cleavage site of of with peptide in cleavage to a a and analysis that this cleavage occurred at the prodomain cleavage between and Asp2-Fc also cleaved the Asp1 prodomain peptide at this in with not An of cleavage of the synthetic peptide with to substrate is shown in at of determine the Asp1 prodomain has any were in the of an Asp1 prodomain peptide of active with a of this peptide in of cleavage of synthetic Asp1 cleavage peptide with of cleavage by APP β-secretase that the prodomain is a of determine Asp1 in and to identify the intracellular in which prodomain processing COS-7 cells overexpressing the were with or Asp1 cells were at reduced which been shown to to the of in of the J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, K. Cell. Full Text PDF PubMed Scopus Google Scholar). Cell were generated and to with to analysis with the Asp1 as a protein of a in to a mass of to removal of the and of Asp1 in the cell cells at which the of the endoplasmic reticulum to the of and The protein to the form of the protein is the mature proteinase that the at prodomain processing of Asp1 is resulting in the of the In to was it as the of at and and Thus, processing is abolished in the enzyme. further of this is in these cells, is no other enzyme that is of processing The of mature as as at a and that processing can occur in the When cells were at which the of protein to the cell K. Cell. Full Text PDF PubMed Scopus Google Scholar), that prodomain processing occurs in further the identity of the intracellular site where prodomain processing of Asp1 cells were in the of known to with the of in the of the which a of that in the Golgi into the Cell. Full Text PDF PubMed Scopus Google Scholar), the of both and mature of In in the of the which Golgi and P. S. R. J. Biol. Chem. Full Text PDF PubMed Google Scholar), the of mature the of prodomain processing of Asp1 not occur in compartments, in the Golgi The results described for the processing of the enzyme that no other intracellular proteinase is of the prodomain of Asp1 and that prodomain cleavage is an this was with analysis of the cell with the that of both Asp1 was the Asp1 is to the prodomain of the mature form of this protein be with the or analysis with the that as the in these cells prodomain processing of Asp1 occurs through an of APP and Asp2 been shown to be on the cell A. G. D. P. C.L. Beyreuther K. Cell. Full Text PDF PubMed Scopus Google Scholar, T. Selkoe D.J. J. Cell Biol. 1995; PubMed Scopus Google Scholar, J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). determine whether Asp1 is also expressed at the cell we on COS-7 cells overexpressing the and with cells were with to the Cell were and the were with level of Asp1 was at the cell a the and intracellular cell were also for which is known to on the cell A. G. D. P. C.L. Beyreuther K. Cell. Full Text PDF PubMed Scopus Google Scholar, T. Selkoe D.J. J. Cell Biol. 1995; PubMed Scopus Google Scholar), and for the endoplasmic and D. J. Fuller Nature. PubMed Scopus Google Scholar). with we that APP was at the cell and and the endoplasmic were within the cell determine Asp1 at the cell is the mature proteinase the the cell were to with the Asp1 at a mass with the mature form of the protein Thus, prodomain processing of Asp1 occurs through the to the cell and the protein expressed on the cell is the processed, active form of the enzyme. prodomain processing is not a for to the cell as which was also expressed at the cell not known aspartic proteinases are generated as proenzymes, requiring removal of the prodomain to generate mature proteinase J. Wong J. Cell. Biochem. PubMed Scopus Google Scholar). most aspartic proteinases as and others as a proteinase activity for prodomain cleavage. has recently been reported that Asp2 not and that removal of prodomain is mediated by furin or a furin-like enzyme (20Bennett B.D. Denis P. Haniu M. Teplow D.B. Kahn S. Louis J.-C. Citron M. Vassar R. J. Biol. Chem. 2000; 275: 37712-37717Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 21Creemers J.W.M. Dominguez D.I. Plets E. Serneels L. Taylor N.A. Multhaup G. Craessaerts K. Annaert W. De Strooper B. J. Biol. Chem. 2001; 276: 4211-4217Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 22Benjannet S. Elagoz A. Wickham L. Mamarbachi M. Munzer J.S. Basak A. Lazure C. Cromlish J.A. Sisodia S. Checler F. Chrétien M. Seidah N.G. J. Biol. Chem. 2001; 276: 10879-10887Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). We that in to Asp2, Asp1 is of prodomain to generate mature Asp1 cleaved both an prodomain fusion protein and a synthetic peptide at the prodomain cleavage site in full-length cleavage was abolished one of the conserved aspartic acid residues in the active site of was to In addition, of either the P1′ or P2′ in the prodomain substrate to phenylalanine reduced cleavage at this site. The other observed was with are to be as secreted cells had one In the of Asp1 expressed in cells was the mature form the Asp1(D110N) was expressed in cells, the to to soluble secreted COS-7 cells the was processing of Asp1 occurred as of Asp1(D110N) with full-length Asp1 not in prodomain processing of not that prodomain cleavage is also that no other activity can for the of prodomain processing of the Asp1(D110N) in these cells. we in that Asp2 could both and a synthetic cleavage was with suggesting that Asp2 is to be a enzyme in In addition, we in cultured cells that of Asp1 occurred in the Golgi. Asp1 was observed in cells at or in the of A. is in to prodomain processing of Asp2, which is an enzyme that in the (20Bennett B.D. Denis P. Haniu M. Teplow D.B. Kahn S. Louis J.-C. Citron M. Vassar R. J. Biol. Chem. 2000; 275: 37712-37717Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). activity of aspartic proteinases is known to be by J. Wong J. Cell. Biochem. PubMed Scopus Google Scholar). the of the Golgi is at C.A. D.B. W. S. J. Cell Biol. PubMed Scopus Google Scholar, J. A. Proc. Natl. Acad. Sci. U. S. A. 1995; Scopus Google Scholar), we in that Asp1 activity at this which be to prodomain processing in The prodomain of Asp1 not exhibit any activity in suggesting that the prodomain be required for of the as has been reported for Asp2 X.-P. E. Yin K.-C. S. Lai M.-T. Y.-M. M. Register R.B. Sardana M.K. Tang J. T. Shafer J.A. Gardell S.J. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). prodomain processing of Asp1 and Asp2 is Asp1 Asp2 a activity to The of Asp1 a of activity with Asp2. it is that other intracellular that the activity of We for on fusion protein for and Smith for on We also and Howlett for of the