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Expression Analysis of BACE2 in Brain and Peripheral Tissues

2000; Elsevier BV; Volume: 275; Issue: 27 Linguagem: Inglês

10.1074/jbc.m002688200

1083-351X

Brian D. Bennett, Safura Babu‐Khan, Richard Loeloff, Jean-Claude Louis, Eileen Curran, Martin Citron, Robert Vassar,

Advanced Glycation End Products research

Beta-site amyloid precursor protein cleaving enzyme (BACE) is a novel transmembrane aspartic protease that possesses all the known characteristics of the β-secretase involved in Alzheimer's disease (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., and Citron, M. (1999) Science 286, 735–741). We have analyzed the sequence and expression pattern of a BACE homolog termed BACE2. BACE and BACE2 are unique among aspartic proteases in that they possess a carboxyl-terminal extension with a predicted transmembrane region and together they define a new family. Northern analysis reveals that BACE2 mRNA is expressed at low levels in most human peripheral tissues and at higher levels in colon, kidney, pancreas, placenta, prostate, stomach, and trachea. Human adult and fetal whole brain and most adult brain subregions express very low or undetectable levels of BACE2 mRNA. In addition, in situ hybridization of adult rat brain shows that BACE2 mRNA is expressed at very low levels in most brain regions. The very low or undetectable levels of BACE2 mRNA in the brain are not consistent with the expression pattern predicted for β-secretase. Beta-site amyloid precursor protein cleaving enzyme (BACE) is a novel transmembrane aspartic protease that possesses all the known characteristics of the β-secretase involved in Alzheimer's disease (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., and Citron, M. (1999) Science 286, 735–741). We have analyzed the sequence and expression pattern of a BACE homolog termed BACE2. BACE and BACE2 are unique among aspartic proteases in that they possess a carboxyl-terminal extension with a predicted transmembrane region and together they define a new family. Northern analysis reveals that BACE2 mRNA is expressed at low levels in most human peripheral tissues and at higher levels in colon, kidney, pancreas, placenta, prostate, stomach, and trachea. Human adult and fetal whole brain and most adult brain subregions express very low or undetectable levels of BACE2 mRNA. In addition, in situ hybridization of adult rat brain shows that BACE2 mRNA is expressed at very low levels in most brain regions. The very low or undetectable levels of BACE2 mRNA in the brain are not consistent with the expression pattern predicted for β-secretase. β-amyloid peptide amyloid precursor protein beta-site APP cleaving enzyme ventromedial hypothalamus mammilary body glycosyl-phospatidylinositol Amyloid plaques, composed of the 4-kDa β-amyloid peptide (Aβ),1 are hallmark lesions found in Alzheimer's disease brain (1.Glenner G.G. Wong C.W. Biochem. Biophys. Res. Commun. 1984; 120: 885-890Crossref PubMed Scopus (4146) Google Scholar). Evidence suggests that Aβ plays a central role in the pathogenesis of Alzheimer's disease (2.Selkoe D.J. Nature. 1999; 399: 23-31Crossref PubMed Scopus (1519) Google Scholar). Two major forms of Aβ differing at the carboxyl terminus are generated, Aβ40 and Aβ42, and overproduction of the longer peptide is highly correlated with early-onset familial Alzheimer's disease (3.Younkin S.G. J. Physiol. (Paris). 1998; 92: 289-292Crossref PubMed Scopus (247) Google Scholar).Aβ is formed by proteolysis of amyloid precursor protein (APP), a large type-I transmembrane protein (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 (3916) Google Scholar). The protease β-secretase initiates Aβ formation by cleaving at the amino terminus of the Aβ domain to generate the secreted APPsβ ectodomain and the membrane-bound carboxyl-terminal fragment C99 (see Fig. 1 of Ref. 5.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 (3256) Google Scholar). A second protease called γ-secretase then cleaves C99 to form the carboxyl terminus of the mature Aβ peptide. In a nonamyloidogenic pathway, APP is processed by the protease α-secretase, which cleaves within the Aβ domain to produce the soluble APPsα ectodomain and the membrane-bound C83 fragment. C83 is cleaved by γ-secretase to produce the nontoxic p3 fragment. The β- and γ-secretase cleavages of APP are both required for Aβ formation, and the inhibition of either or both of these secretases is a prime therapeutic goal for the treatment of Alzheimer's disease.Until recently, the identities of the secretases had been elusive. The metalloproteases TACE and ADAM 10 both appear to be involved in α-secretase processing of APP (6.Buxbaum 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) (7.Lammich 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). Presenilin is required for γ-secretase activity (8.De Strooper B. Saftig P. Craessaerts K. Vanderstichele H. Guhde G. Annaert W. Figura K.V. Leuven F.V. Nature. 1998; 391: 387-390Crossref PubMed Scopus (1541) Google Scholar, 9.De 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 (1789) Google Scholar), and it has been suggested that presenilin 1 itself is γ-secretase (10.Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1676) Google Scholar). However, direct demonstration that presenilin 1 possesses γ-secretase activity is so far lacking. We and others have recently identified a novel transmembrane aspartic protease, beta-site APP cleaving enzyme (BACE), that exhibits all the known properties of β-secretase (5.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 (3256) Google Scholar, 11.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 (997) Google Scholar, 12.Yan R. Bienkowski M.J. Shuck M.E. Miao H. Tory M.C. Pauley A.M. Brashler 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, 13.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. McConlogue L. John V. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1472) Google Scholar, 14.Lin X. Koelsch G. Wu S. Downs D. Dashti A. Tang J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1456-1460Crossref PubMed Scopus (737) Google Scholar). The BACE polypeptide sequence contains two active site motifs (D(T/S)G(T/S)) that are characteristic of aspartic proteases, and BACE appears most closely related to the pepsin aspartic protease family (for review, see Ref. 15.Rawlings N.D. Barrett A.J. Methods Enzymol. 1995; 248: 105-136Crossref PubMed Scopus (139) Google Scholar). However, BACE differs from members of the pepsin family in that it possesses a carboxyl-terminal extension of ∼80 amino acids that contains a predicted transmembrane region and a short cytosolic tail. Thus, BACE is predicted to be a type-1 transmembrane protein with the active site in the lumen and represents the first reported example of a transmembrane aspartic protease.Recently, we and others have identified a homolog of BACE termed BACE2 (or Asp1; GenBank™ accession number AF204944) by searching expressed sequence tag data bases (12.Yan R. Bienkowski M.J. Shuck M.E. Miao H. Tory M.C. Pauley A.M. Brashler 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, 16.Saunders A.J. Kim T.-W. Tanzi R.E. Fan W. Bennett B.D. Babu-Khan S. Luo Y. Louis J.-C. McCaleb M. Citron M. Vassar R. Richards W.G. Science. 1999; 286: 1255aCrossref Google Scholar). The BACE2 gene resides on chromosome 21 in the obligate Down's syndrome region at 21q22.3 (16.Saunders A.J. Kim T.-W. Tanzi R.E. Fan W. Bennett B.D. Babu-Khan S. Luo Y. Louis J.-C. McCaleb M. Citron M. Vassar R. Richards W.G. Science. 1999; 286: 1255aCrossref Google Scholar). This region contains the APP gene and is triplicated in the genomes of Down's syndrome patients, suggesting that the additional copy of the APP gene is responsible for the Alzheimer's-like dementia and the amyloid plaques that invariably develop in Down's syndrome patients (17.Tanzi R.E. Gusella J.F. Watkins P.C. Bruns G.A.B. St. George-Hyslop P.H. Van Keuren M.L. Patterson D. Pagan S. Kurnit D.M. Neve R.L. Science. 1987; 235: 880-884Crossref PubMed Scopus (1210) Google Scholar). The high amino acid similarity of BACE and BACE2, together with the localization of theBACE2 gene within the obligate Down's syndrome region, suggest that BACE2 may be a β-secretase candidate.Here, we present a detailed comparison of the polypeptide sequences of BACE and BACE2 with each other and with members of the pepsin family. Our analysis indicates that BACE and BACE2 are members of a novel family of transmembrane aspartic proteases. We have analyzed the expression of BACE2 mRNA in human peripheral tissues and brain by Northern blot and in rat brain by in situ hybridization. The levels of BACE2 mRNA are very low or undetectable in the brain and are not consistent with the predicted expression pattern for β-secretase. Amyloid plaques, composed of the 4-kDa β-amyloid peptide (Aβ),1 are hallmark lesions found in Alzheimer's disease brain (1.Glenner G.G. Wong C.W. Biochem. Biophys. Res. Commun. 1984; 120: 885-890Crossref PubMed Scopus (4146) Google Scholar). Evidence suggests that Aβ plays a central role in the pathogenesis of Alzheimer's disease (2.Selkoe D.J. Nature. 1999; 399: 23-31Crossref PubMed Scopus (1519) Google Scholar). Two major forms of Aβ differing at the carboxyl terminus are generated, Aβ40 and Aβ42, and overproduction of the longer peptide is highly correlated with early-onset familial Alzheimer's disease (3.Younkin S.G. J. Physiol. (Paris). 1998; 92: 289-292Crossref PubMed Scopus (247) Google Scholar). Aβ is formed by proteolysis of amyloid precursor protein (APP), a large type-I transmembrane protein (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 (3916) Google Scholar). The protease β-secretase initiates Aβ formation by cleaving at the amino terminus of the Aβ domain to generate the secreted APPsβ ectodomain and the membrane-bound carboxyl-terminal fragment C99 (see Fig. 1 of Ref. 5.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 (3256) Google Scholar). A second protease called γ-secretase then cleaves C99 to form the carboxyl terminus of the mature Aβ peptide. In a nonamyloidogenic pathway, APP is processed by the protease α-secretase, which cleaves within the Aβ domain to produce the soluble APPsα ectodomain and the membrane-bound C83 fragment. C83 is cleaved by γ-secretase to produce the nontoxic p3 fragment. The β- and γ-secretase cleavages of APP are both required for Aβ formation, and the inhibition of either or both of these secretases is a prime therapeutic goal for the treatment of Alzheimer's disease. Until recently, the identities of the secretases had been elusive. The metalloproteases TACE and ADAM 10 both appear to be involved in α-secretase processing of APP (6.Buxbaum 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) (7.Lammich 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). Presenilin is required for γ-secretase activity (8.De Strooper B. Saftig P. Craessaerts K. Vanderstichele H. Guhde G. Annaert W. Figura K.V. Leuven F.V. Nature. 1998; 391: 387-390Crossref PubMed Scopus (1541) Google Scholar, 9.De 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 (1789) Google Scholar), and it has been suggested that presenilin 1 itself is γ-secretase (10.Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1676) Google Scholar). However, direct demonstration that presenilin 1 possesses γ-secretase activity is so far lacking. We and others have recently identified a novel transmembrane aspartic protease, beta-site APP cleaving enzyme (BACE), that exhibits all the known properties of β-secretase (5.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 (3256) Google Scholar, 11.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 (997) Google Scholar, 12.Yan R. Bienkowski M.J. Shuck M.E. Miao H. Tory M.C. Pauley A.M. Brashler 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, 13.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. McConlogue L. John V. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1472) Google Scholar, 14.Lin X. Koelsch G. Wu S. Downs D. Dashti A. Tang J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1456-1460Crossref PubMed Scopus (737) Google Scholar). The BACE polypeptide sequence contains two active site motifs (D(T/S)G(T/S)) that are characteristic of aspartic proteases, and BACE appears most closely related to the pepsin aspartic protease family (for review, see Ref. 15.Rawlings N.D. Barrett A.J. Methods Enzymol. 1995; 248: 105-136Crossref PubMed Scopus (139) Google Scholar). However, BACE differs from members of the pepsin family in that it possesses a carboxyl-terminal extension of ∼80 amino acids that contains a predicted transmembrane region and a short cytosolic tail. Thus, BACE is predicted to be a type-1 transmembrane protein with the active site in the lumen and represents the first reported example of a transmembrane aspartic protease. Recently, we and others have identified a homolog of BACE termed BACE2 (or Asp1; GenBank™ accession number AF204944) by searching expressed sequence tag data bases (12.Yan R. Bienkowski M.J. Shuck M.E. Miao H. Tory M.C. Pauley A.M. Brashler 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, 16.Saunders A.J. Kim T.-W. Tanzi R.E. Fan W. Bennett B.D. Babu-Khan S. Luo Y. Louis J.-C. McCaleb M. Citron M. Vassar R. Richards W.G. Science. 1999; 286: 1255aCrossref Google Scholar). The BACE2 gene resides on chromosome 21 in the obligate Down's syndrome region at 21q22.3 (16.Saunders A.J. Kim T.-W. Tanzi R.E. Fan W. Bennett B.D. Babu-Khan S. Luo Y. Louis J.-C. McCaleb M. Citron M. Vassar R. Richards W.G. Science. 1999; 286: 1255aCrossref Google Scholar). This region contains the APP gene and is triplicated in the genomes of Down's syndrome patients, suggesting that the additional copy of the APP gene is responsible for the Alzheimer's-like dementia and the amyloid plaques that invariably develop in Down's syndrome patients (17.Tanzi R.E. Gusella J.F. Watkins P.C. Bruns G.A.B. St. George-Hyslop P.H. Van Keuren M.L. Patterson D. Pagan S. Kurnit D.M. Neve R.L. Science. 1987; 235: 880-884Crossref PubMed Scopus (1210) Google Scholar). The high amino acid similarity of BACE and BACE2, together with the localization of theBACE2 gene within the obligate Down's syndrome region, suggest that BACE2 may be a β-secretase candidate. Here, we present a detailed comparison of the polypeptide sequences of BACE and BACE2 with each other and with members of the pepsin family. Our analysis indicates that BACE and BACE2 are members of a novel family of transmembrane aspartic proteases. We have analyzed the expression of BACE2 mRNA in human peripheral tissues and brain by Northern blot and in rat brain by in situ hybridization. The levels of BACE2 mRNA are very low or undetectable in the brain and are not consistent with the predicted expression pattern for β-secretase. We thank Desiree Olivares for help with figure preparation.