Characterization of Alzheimer's β-Secretase Protein BACE

The cerebral deposition of amyloid β-peptide is an early and critical feature of Alzheimer's disease. Amyloid β-peptide is released from the amyloid precursor protein by the sequential action of two proteases, β-secretase and γ-secretase, and these proteases are prime targets for therapeutic intervention. We have recently cloned a novel aspartic protease, BACE, with all the known properties of β-secretase. Here we demonstrate that BACE is anN-glycosylated integral membrane protein that undergoes constitutive N-terminal processing in the Golgi apparatus. We have used a secreted Fc fusion-form of BACE (BACE-IgG) that contains the entire ectodomain for a detailed analysis of posttranslational modifications. This molecule starts at Glu46 and contains fourN-glycosylation sites (Asn153, Asn172, Asn223, and Asn354). The six Cys residues in the ectodomain form three intramolecular disulfide linkages (Cys216–Cys420, Cys278–Cys443, and Cys330–Cys380). Despite the conservation of the active site residues and the 30–37% amino acid homology with known aspartic proteases, the disulfide motif is fundamentally different from that of other aspartic proteases. This difference may affect the substrate specificity of the enzyme. Taken together, both the presence of a transmembrane domain and the unusual disulfide bond structure lead us to conclude that BACE is an atypical pepsin family member. The cerebral deposition of amyloid β-peptide is an early and critical feature of Alzheimer's disease. Amyloid β-peptide is released from the amyloid precursor protein by the sequential action of two proteases, β-secretase and γ-secretase, and these proteases are prime targets for therapeutic intervention. We have recently cloned a novel aspartic protease, BACE, with all the known properties of β-secretase. Here we demonstrate that BACE is anN-glycosylated integral membrane protein that undergoes constitutive N-terminal processing in the Golgi apparatus. We have used a secreted Fc fusion-form of BACE (BACE-IgG) that contains the entire ectodomain for a detailed analysis of posttranslational modifications. This molecule starts at Glu46 and contains fourN-glycosylation sites (Asn153, Asn172, Asn223, and Asn354). The six Cys residues in the ectodomain form three intramolecular disulfide linkages (Cys216–Cys420, Cys278–Cys443, and Cys330–Cys380). Despite the conservation of the active site residues and the 30–37% amino acid homology with known aspartic proteases, the disulfide motif is fundamentally different from that of other aspartic proteases. This difference may affect the substrate specificity of the enzyme. Taken together, both the presence of a transmembrane domain and the unusual disulfide bond structure lead us to conclude that BACE is an atypical pepsin family member. Alzheimer's disease amyloid precursor protein β-site APP-cleaving enzyme fluorescein 5-maleimide α-cyano-4-hydroxycinnamic acid high performance liquid chromatography matrix-assisted laser desorption ionization trypsin-endoproteinase Asp-N (3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate amyloid β-peptide polyacrylamide gel electrophoresis phenylthiohydantoin The hallmarks of Alzheimer's disease (AD)1 pathology are brain plaques and vascular deposits (1.Alzheimer A. Centralbl. Nervenheilk. Psychiatr. 1907; 30: 177-179Google Scholar) consisting of the 4-kDa amyloid β-peptide (Aβ) (2.Glenner G.G. Wong C.W. Biochem. Biophys. Res. Commun. 1984; 120: 885-890Crossref PubMed Scopus (4225) Google Scholar). Overproduction of the 42-amino acid form of Aβ, Aβ42, has been suggested to be the cause of all known cases of familial early onset AD (3.Younkin S.G. J. Physiol. (Paris). 1998; 92: 289-292Crossref PubMed Scopus (252) Google Scholar), and it is assumed that Aβ42 deposition plays an early and critical role in sporadic AD as well. Therefore, Aβ metabolism has attracted considerable interest. In 1987 it was shown (4.Kang J. Lemaire H.-G. Unterbeck A. Salbaum J.M. Masters C.L. Grzeschik K.-H. Multhaup G. Beyreuther K. Muller-Hill B. Nature. 1987; 325: 733-736Crossref PubMed Scopus (3951) Google Scholar) that formation of Aβ requires proteolytic cleavage of a large type I transmembrane protein, the β-amyloid precursor protein (APP), which is constitutively expressed in most cell types. Over the next decade the proteolytic processing of APP has been studied in great detail in a variety of systems by many groups. Taken together, these studies have shown that Aβ is generated at a low rate by most cells analyzed and that two different proteolytic activities are required for Aβ generation. First, β-secretase cleaves APP to generate the N terminus of Aβ, and second, γ-secretase cleaves the C terminus, leading to the release of Aβ (for review see Ref. 5.Haass C. Selkoe D.J. Cell. 1993; 75: 1039-1042Abstract Full Text PDF PubMed Scopus (740) Google Scholar). Studies with intact cells expressing APP and the endogenous secretases have led to conclusions about the properties of the β- and γ-secretases,e.g. their tissue distribution, subcellular localization, substrate requirements (see e.g. Ref. 6.Citron M. Teplow D.B. Selkoe D.J. Neuron. 1995; 14: 661-670Abstract Full Text PDF PubMed Scopus (233) Google Scholar) etc., but until recently the identity of both β- and γ-secretase was unknown. This changed when we very recently identified the novel transmembrane aspartic protease BACE as the major β-secretase (7.Vassar 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 (3307) Google Scholar). Three subsequently published independent studies (8.Hussain 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 (1001) Google Scholar, 9.Sinha 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. Zhao J. Mc Conlogue L. Varghese J. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1482) Google Scholar, 10.Yan 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 (1339) Google Scholar) have confirmed this conclusion. Here we characterize the BACE protein. We show that BACE is an N-glycosylated integral membrane protein that undergoes constitutive N-terminal processing in the Golgi apparatus. We determine the processing and N-glycosylation sites and the disulfide bonds. Our results demonstrate that BACE is an unusual member of the pepsin family. Trypsin, pepsin, and endoproteinase Asp-N were obtained from Roche Molecular Biochemicals. Fluorescein 5-maleimide (FM) was purchased from Molecular Probes (Eugene, OR). 4-HCCA was from Sigma. Sialidase was obtained from Glyko (Novato, CA). N-and O-glycanases were from Genzyme (Cambridge, MA).N-Glycosidase F was purchased from Roche Molecular Biochemicals. Other chemicals are of high quality grade. Untransfected 293 cells or 293 cells stably expressing BACE were scraped into phosphate-buffered saline, and the cells were precipitated. The pellet was resuspended in 25 mm HEPES, pH 7.2, with protease inhibitors, and the cells were swollen on ice for 60 min. Cells were lysed by 3 freeze-thaw cycles at −80 °C and then centrifuged for 15 min at 1,000 × g to precipitate nuclei. The supernatant was centrifuged for 60 min at 100,000 × gto give a crude membrane pellet and a supernatant containing cytosolic proteins. Membranes were solubilized in 25 mm HEPES, pH 7.2, 2% CHAPS and centrifuged at 20,000 × g for 10 min. The resulting supernatant contained the membrane-bound proteins. To determine if BACE is an integral or peripheral membrane protein, crude membranes were washed with either 0.5 m NaCl or 100 mm Na2CO3, pH 11, to release peripherally bound proteins. A polyclonal antibody specific to the propeptide region of BACE was raised following standard procedures using as immunogen the peptide CGIRLPLRSGLGGAPLGLRLPR (comprising amino acids 25–45 of BACE and an N-terminal Cys residue for coupling). After metabolic labeling with 35Smethionine aliquots of the same cell lysates were immunoprecipitated using the previously described BACE C-terminal antiserum (7.Vassar 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 (3307) Google Scholar) and the propeptide antiserum following protocols described before (11.Haass C. Schlossmacher M.G. Hung A.Y. Vigo-Pelfrey C. Mellon A. Ostaszewski B.L. Lieberburg I. Koo E.H. Schenk D. Teplow D.B. Selkoe D.J. Nature. 1992; 359: 322-325Crossref PubMed Scopus (1763) Google Scholar). N-Glycosidase F treatment was performed after immunoprecipitation. For pulse-chase cells were for min and then for the as a mm in was used at in a were analyzed by by on a The containing the ectodomain of BACE and the Fc of amino was described previously (7.Vassar 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 (3307) Google Scholar). protein was from of stably cells with protein A The protein A of and a low of Fc In to the Fc this was by gel using a × in phosphate-buffered In to the of residues in the was with 10 mm in m pH at for were by using a × The protein was to proteolytic for peptide The and intact were with at °C for in m pH The was to to a with endoproteinase Asp-N the same The were to using a × of the protein was performed in pH for at °C with an of and the was by or pepsin were by using a × systems A and were A and The were with a from 2% to min and from to 10 min. rate was at The peptide was by at and or were with For of the protein was in mm pH and with for at were with in mm pH and were to were with the the same The was by for laser desorption ionization of the was performed using either a or The was in and then on the with acid or 4-HCCA as were analyzed using an using a The was with acid at The was with a of 0.5 The and standard were using analysis of and was performed on a from CA). For analysis of amino an was analysis was performed with the analysis for protein sites of the enzyme were identified by on analysis at the to the The were analyzed by the of after peptide of analysis was performed by using or The were with and 1998; PubMed Scopus Google Scholar). The were by for analysis by of the BACE protein that BACE is a transmembrane domain protein (7.Vassar 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 (3307) Google Scholar), and it has been shown that active enzyme be released from membrane after treatment with 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. Zhao J. Mc Conlogue L. Varghese J. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1482) Google Scholar). We cell lysates of 293 cells stably BACE and and membrane by with the BACE C-terminal antiserum (7.Vassar 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 (3307) Google Scholar) confirmed that BACE is in the membrane but in the the membrane with 0.5 m or m pH 11, release the protein into the that BACE is an integral membrane protein BACE on at a from the amino acid that it may be (7.Vassar 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 (3307) Google Scholar). BACE is immunoprecipitated after min labeling from the 293 with the C-terminal an at is cells the same show this the is with F the at that the The same is obtained with a antibody raised to the propeptide region of BACE This antibody show a with cells but the same as the C-terminal and the same is F processing and of BACE from 293 cells after min labeling using a C-terminal antiserum or a propeptide 293 cells stably expressing from 293 cells stably expressing BACE with F. of BACE from stably 293 cells after min labeling by the in using the C-terminal of BACE from stably 293 cells after labeling by the in using the propeptide of the BACE by propeptide C-terminal of BACE from cells for 3 in the presence or of To the of BACE in the cell we performed a pulse-chase in which the cells were for min and then in the of lysates were at the and immunoprecipitated with the C-terminal antibody after labeling a the N-glycosylated is 3 this has and of the is as form that is BACE is and the N-glycosylated form is The N-glycosylated protein that is into the form that is in 293 We performed pulse-chase with the propeptide antibody the same is as with the C-terminal by most of the has see in a of the is at a 60 results that the BACE protein undergoes constitutive N-terminal processing and that the N-terminal processing in with the of residues of the in the Golgi apparatus. This was confirmed by a A treatment Cells for 3 in the of A show the protein that is with the C-terminal antibody but with the propeptide In cells with A for 3 show the form that is with both the C-terminal and the propeptide treatment of the cells with A both N-terminal processing and of the that propeptide cleavage in the Golgi apparatus. In to characterize the posttranslational of BACE in it is to a large of the protein to We have previously described a form of BACE that but be the transmembrane this protein it is assumed that the structure of the BACE ectodomain is in a major (7.Vassar 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 (3307) Google Scholar) and with the protein were we to for This protein has been and contains the domain of β-secretase and the Fc amino of as shown in A. the of the protein the Fc we to a molecule of the structure in which the two are by disulfide bonds. The protein was expressed in 293 cells and from the by protein A by gel on a at of protein was subsequently obtained by a with a of with the This the structure but 3 with the structure after treatment of with the at as described previously (7.Vassar 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 (3307) Google Scholar), and the at after treatment with and that BACE contains sites but of The was a gel with and and after with in the were from or of the enzyme. The protein was a with the was analyzed using a as described The at the and the at the BACE from cells (7.Vassar 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 (3307) Google Scholar) or from brain 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. Zhao J. Mc Conlogue L. Varghese J. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1482) Google Scholar) starts at cycles of analysis for the N-terminal were from the N-terminal domain of BACE from residues and to and We the as the of the enzyme after cleavage of the peptide and the as the active The from the to for 10 The of the was for BACE to as for (see 3 was for the presence of residues using The protein was with and endoproteinase The peptide analysis a of N-terminal the all be from the This that all residues in the BACE ectodomain form disulfide bonds. To the disulfide and the was with and endoproteinase The was performed to the or The peptide the protease or endoproteinase peptide was for the The results are in I. two and is to be a residue to the amino acid the that the were these a but of both confirmed the disulfide linkages as with of and these results we the disulfide as peptide consisting of the two and the presence of in the Fc we to determine all disulfide from the the protein was with pepsin The peptide is shown in analysis and the for disulfide linkages and N-glycosylation and the analyzed disulfide bond containing are shown in contained two and confirmed this the was the to of a residue This disulfide bond was by peptide (see and two and the This peptide us to determine at residue was by analysis of The difference the and may be to of the peptide was to The disulfide was by analysis of peptide containing two and The of from peptide was with the the disulfide of the Fc was to be peptide (see the peptide the linkages and in the Fc of from trypsin-endoproteinase Asp-N of residues were by but are from the protein (see 3 residues were by but are from the protein (see 3 in a of Cys of and were We determine which of the form the disulfide to the show the (see and were We determine which of the form the disulfide to the show the (see in a of of of was by using The was a with 4-HCCA as We have analyzed the for the of N-glycosylation sites (Asn153, Asn172, Asn223, and from BACE and site from are to the After analysis of all we that the N-glycosylation sites of BACE are by The that with the same amino acid were on that these N-glycosylation sites may have For the peptide containing is into and of a e.g. and to the analysis the has the of this peptide have and and of the are in to the of the of the structure is in but the that the may have high leading to the from sites were by of at the show The peptide contained two a disulfide sites were by of at the show The peptide contained two a disulfide in a This the of the recently identified β-secretase protein BACE at the We analysis by properties of the intact form of BACE that contains the transmembrane and we confirmed that the BACE protein is an integral membrane protein (7.Vassar 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 (3307) Google Scholar, 9.Sinha 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. Zhao J. Mc Conlogue L. Varghese J. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1482) Google Scholar). of the of BACE in 293 cells that BACE is constitutively to a form the propeptide this processing is as has been before 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. Zhao J. Mc Conlogue L. Varghese J. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1482) Google Scholar). in the 293 cells processing of BACE to β-secretase The BACE protein is and of the is as protein. We at this the of protein is an or a major of BACE is low as well. BACE is Our results show that BACE is The that is BACE which contains the propeptide and that A treatment processing that the cleavage of the propeptide in the Golgi apparatus. The of the propeptide processing enzyme is but an as for pepsin D.R. Biophys. Biophys. PubMed Scopus Google Scholar), if the specificity of BACE (7.Vassar 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 (3307) Google Scholar). To the of BACE in we of the previously described (7.Vassar 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 (3307) Google Scholar) containing the entire ectodomain of BACE, which be the transmembrane this form of the enzyme is active and the specificity of β-secretase (7.Vassar 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 (3307) Google Scholar), it to of BACE using this of the Glu46 previously described for the transmembrane form (7.Vassar 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 (3307) Google Scholar, 9.Sinha 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. Zhao J. Mc Conlogue L. Varghese J. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1482) Google Scholar) and a at that has the peptide but contains the We of this form when we analyzed membrane-bound BACE, that the propeptide cleavage of is as as that of this is to different or other the two is The ectodomain of BACE contains six to the it but all form disulfide bonds. BACE we by but we that all three disulfide are intramolecular BACE is a member of the pepsin family (7.Vassar 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 (3307) Google Scholar), that it have a structure to other aspartic proteases pepsin, or and proteases (for review see Ref. D.R. Biophys. Biophys. PubMed Scopus Google Scholar). we show that it has homology with other pepsin family in the disulfide shown in a aspartic protease J. J. A. A. J. 1999; PubMed Scopus Google Scholar), with β-secretase in the of the C-terminal may affect substrate specificity of the a detailed of the structure for β-secretase this prime for the treatment of Alzheimer's disease at the may to be for