Characterization of recombinant, soluble β-secretase from an insect cell expression system

William D. Mallender, Debra Yager, Luisa Onstead, Michael R. Nichols, Christopher Eckman, Kumar Sambamurti, Lisa M. Kopcho, Jovita Marcinkeviciene, Robert A. Copeland, Terrone L. Rosenberry

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

The β-site amyloid precursor protein-cleaving enzyme (BACE) cleaves the amyloid precursor protein to produce the N terminus of the amyloid β peptide, a major component of the plaques found in the brains of Alzheimer's disease patients. Sequence analysis of BACE indicates that the protein contains the consensus sequences found in most known aspartyl proteases, but otherwise has only modest homology with aspartyl proteases of known three-dimensional structure (i.e., pepsin, renin, or cathepsin D). Because BACE has been shown to be one of the two proteolytic activities responsible for the production of the Aβ peptide, this enzyme is a prime target for the design of therapeutic agents aimed at reducing Aβ for the treatment of Alzheimer's disease. Toward this ultimate goal, we have expressed a recombinant, truncated human BACE in a Drosophila melanogaster S2 cell expression system to generate high levels of secreted BACE protein. The protein was convenient to purify and was enzymatically active and specific for cleaving the β-secretase site of human APP, as demonstrated with soluble APP as the substrate in novel sandwich enzyme-linked immunosorbent assay and Western blot assays. Further kinetic analysis revealed no catalytic differences between this recombinant, secreted BACE, and brain BACE. Both showed a strong preference for substrates that contained the Swedish mutation, where NL is substituted for KM immediately upstream of the cleavage site, relative to the wild-type sequence, and both showed the same extent of inhibition by a peptide-based inhibitor. The capability to produce large quantities of BACE enzyme will facilitate protein structure determination and inhibitor development efforts that may lead to the evolution of useful Alzheimer's disease treatments.

Original languageEnglish (US)
Pages (from-to)619-626
Number of pages8
JournalMolecular Pharmacology
Volume59
Issue number3
StatePublished - 2001

Fingerprint

Amyloid Precursor Protein Secretases
Insects
Aspartic Acid Proteases
Alzheimer Disease
Amyloid beta-Protein Precursor
Peptides
Proteins
Enzymes
Cathepsin D
Pepsin A
Consensus Sequence
Brain Diseases
Drosophila melanogaster
Renin
Amyloid
Sequence Analysis
Therapeutics
Western Blotting
Enzyme-Linked Immunosorbent Assay
Mutation

ASJC Scopus subject areas

  • Pharmacology

Cite this

Mallender, W. D., Yager, D., Onstead, L., Nichols, M. R., Eckman, C., Sambamurti, K., ... Rosenberry, T. L. (2001). Characterization of recombinant, soluble β-secretase from an insect cell expression system. Molecular Pharmacology, 59(3), 619-626.

Characterization of recombinant, soluble β-secretase from an insect cell expression system. / Mallender, William D.; Yager, Debra; Onstead, Luisa; Nichols, Michael R.; Eckman, Christopher; Sambamurti, Kumar; Kopcho, Lisa M.; Marcinkeviciene, Jovita; Copeland, Robert A.; Rosenberry, Terrone L.

In: Molecular Pharmacology, Vol. 59, No. 3, 2001, p. 619-626.

Research output: Contribution to journalArticle

Mallender, WD, Yager, D, Onstead, L, Nichols, MR, Eckman, C, Sambamurti, K, Kopcho, LM, Marcinkeviciene, J, Copeland, RA & Rosenberry, TL 2001, 'Characterization of recombinant, soluble β-secretase from an insect cell expression system', Molecular Pharmacology, vol. 59, no. 3, pp. 619-626.
Mallender WD, Yager D, Onstead L, Nichols MR, Eckman C, Sambamurti K et al. Characterization of recombinant, soluble β-secretase from an insect cell expression system. Molecular Pharmacology. 2001;59(3):619-626.
Mallender, William D. ; Yager, Debra ; Onstead, Luisa ; Nichols, Michael R. ; Eckman, Christopher ; Sambamurti, Kumar ; Kopcho, Lisa M. ; Marcinkeviciene, Jovita ; Copeland, Robert A. ; Rosenberry, Terrone L. / Characterization of recombinant, soluble β-secretase from an insect cell expression system. In: Molecular Pharmacology. 2001 ; Vol. 59, No. 3. pp. 619-626.
@article{f936dfc2fa60420da51ddef9bdf6b53b,
title = "Characterization of recombinant, soluble β-secretase from an insect cell expression system",
abstract = "The β-site amyloid precursor protein-cleaving enzyme (BACE) cleaves the amyloid precursor protein to produce the N terminus of the amyloid β peptide, a major component of the plaques found in the brains of Alzheimer's disease patients. Sequence analysis of BACE indicates that the protein contains the consensus sequences found in most known aspartyl proteases, but otherwise has only modest homology with aspartyl proteases of known three-dimensional structure (i.e., pepsin, renin, or cathepsin D). Because BACE has been shown to be one of the two proteolytic activities responsible for the production of the Aβ peptide, this enzyme is a prime target for the design of therapeutic agents aimed at reducing Aβ for the treatment of Alzheimer's disease. Toward this ultimate goal, we have expressed a recombinant, truncated human BACE in a Drosophila melanogaster S2 cell expression system to generate high levels of secreted BACE protein. The protein was convenient to purify and was enzymatically active and specific for cleaving the β-secretase site of human APP, as demonstrated with soluble APP as the substrate in novel sandwich enzyme-linked immunosorbent assay and Western blot assays. Further kinetic analysis revealed no catalytic differences between this recombinant, secreted BACE, and brain BACE. Both showed a strong preference for substrates that contained the Swedish mutation, where NL is substituted for KM immediately upstream of the cleavage site, relative to the wild-type sequence, and both showed the same extent of inhibition by a peptide-based inhibitor. The capability to produce large quantities of BACE enzyme will facilitate protein structure determination and inhibitor development efforts that may lead to the evolution of useful Alzheimer's disease treatments.",
author = "Mallender, {William D.} and Debra Yager and Luisa Onstead and Nichols, {Michael R.} and Christopher Eckman and Kumar Sambamurti and Kopcho, {Lisa M.} and Jovita Marcinkeviciene and Copeland, {Robert A.} and Rosenberry, {Terrone L.}",
year = "2001",
language = "English (US)",
volume = "59",
pages = "619--626",
journal = "Molecular Pharmacology",
issn = "0026-895X",
publisher = "American Society for Pharmacology and Experimental Therapeutics",
number = "3",

}

TY - JOUR

T1 - Characterization of recombinant, soluble β-secretase from an insect cell expression system

AU - Mallender, William D.

AU - Yager, Debra

AU - Onstead, Luisa

AU - Nichols, Michael R.

AU - Eckman, Christopher

AU - Sambamurti, Kumar

AU - Kopcho, Lisa M.

AU - Marcinkeviciene, Jovita

AU - Copeland, Robert A.

AU - Rosenberry, Terrone L.

PY - 2001

Y1 - 2001

N2 - The β-site amyloid precursor protein-cleaving enzyme (BACE) cleaves the amyloid precursor protein to produce the N terminus of the amyloid β peptide, a major component of the plaques found in the brains of Alzheimer's disease patients. Sequence analysis of BACE indicates that the protein contains the consensus sequences found in most known aspartyl proteases, but otherwise has only modest homology with aspartyl proteases of known three-dimensional structure (i.e., pepsin, renin, or cathepsin D). Because BACE has been shown to be one of the two proteolytic activities responsible for the production of the Aβ peptide, this enzyme is a prime target for the design of therapeutic agents aimed at reducing Aβ for the treatment of Alzheimer's disease. Toward this ultimate goal, we have expressed a recombinant, truncated human BACE in a Drosophila melanogaster S2 cell expression system to generate high levels of secreted BACE protein. The protein was convenient to purify and was enzymatically active and specific for cleaving the β-secretase site of human APP, as demonstrated with soluble APP as the substrate in novel sandwich enzyme-linked immunosorbent assay and Western blot assays. Further kinetic analysis revealed no catalytic differences between this recombinant, secreted BACE, and brain BACE. Both showed a strong preference for substrates that contained the Swedish mutation, where NL is substituted for KM immediately upstream of the cleavage site, relative to the wild-type sequence, and both showed the same extent of inhibition by a peptide-based inhibitor. The capability to produce large quantities of BACE enzyme will facilitate protein structure determination and inhibitor development efforts that may lead to the evolution of useful Alzheimer's disease treatments.

AB - The β-site amyloid precursor protein-cleaving enzyme (BACE) cleaves the amyloid precursor protein to produce the N terminus of the amyloid β peptide, a major component of the plaques found in the brains of Alzheimer's disease patients. Sequence analysis of BACE indicates that the protein contains the consensus sequences found in most known aspartyl proteases, but otherwise has only modest homology with aspartyl proteases of known three-dimensional structure (i.e., pepsin, renin, or cathepsin D). Because BACE has been shown to be one of the two proteolytic activities responsible for the production of the Aβ peptide, this enzyme is a prime target for the design of therapeutic agents aimed at reducing Aβ for the treatment of Alzheimer's disease. Toward this ultimate goal, we have expressed a recombinant, truncated human BACE in a Drosophila melanogaster S2 cell expression system to generate high levels of secreted BACE protein. The protein was convenient to purify and was enzymatically active and specific for cleaving the β-secretase site of human APP, as demonstrated with soluble APP as the substrate in novel sandwich enzyme-linked immunosorbent assay and Western blot assays. Further kinetic analysis revealed no catalytic differences between this recombinant, secreted BACE, and brain BACE. Both showed a strong preference for substrates that contained the Swedish mutation, where NL is substituted for KM immediately upstream of the cleavage site, relative to the wild-type sequence, and both showed the same extent of inhibition by a peptide-based inhibitor. The capability to produce large quantities of BACE enzyme will facilitate protein structure determination and inhibitor development efforts that may lead to the evolution of useful Alzheimer's disease treatments.

UR - http://www.scopus.com/inward/record.url?scp=0035129455&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0035129455&partnerID=8YFLogxK

M3 - Article

VL - 59

SP - 619

EP - 626

JO - Molecular Pharmacology

JF - Molecular Pharmacology

SN - 0026-895X

IS - 3

ER -