Maturation and Endosomal Targeting of β-Site Amyloid Precursor Protein-cleaving Enzyme

The amyloidogenic Aβ peptide is liberated from the amyloid precursor protein (APP) by two proteolytic activities, β-secretase and γ-secretase. Recently, a type I membrane protein termed BACE (β-site APP cleaving enzyme) with characteristics of an aspartyl protease has been identified as the β-secretase. We undertook a series of biochemical and morphological investigations designed to characterize the basic properties of this protein. Initial studies indicated that BACE undergoes N-linked glycosylation at three of four potential sites. Metabolic pulse-chase experiments revealed that after core glycosylation, BACE is rapidly and efficiently transported to the Golgi apparatus and distal secretory pathway. BACE was also found to be quite stable, being turned over with a t 1 2 of ∼16 h. Retention of BACE in the endoplasmic reticulum by introduction of a C-terminal dilysine motif prevented complex carbohydrate processing and demonstrated that propeptide cleavage occurs after exit from this organelle. BACE exhibited intramolecular disulfide bonding but did not form oligomeric structures by standard SDS-polyacrylamide gel electrophoresis analysis and sedimented as a monomer in sucrose velocity gradients. Immunofluorescence studies showed a largely vesicular staining pattern for BACE that colocalized well with endosomal, but not lysosomal, markers. Measurable levels of BACE were also detected on the plasma membrane by both immunostaining and cell surface biotinylation, and cycling of the protein between the cell membrane and the endosomes was documented. A cytoplasmic dileucine motif was found to be necessary for normal targeting of BACE to the endosomal system and accumulation of the protein in this intracellular site.

[1]  G. Multhaup,et al.  Maturation and Pro-peptide Cleavage of β-Secretase* , 2000, The Journal of Biological Chemistry.

[2]  M. Citron,et al.  Expression Analysis of BACE2 in Brain and Peripheral Tissues* , 2000, The Journal of Biological Chemistry.

[3]  J. Tang,et al.  Human aspartic protease memapsin 2 cleaves the beta-secretase site of beta-amyloid precursor protein. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[4]  D. Selkoe,et al.  The Transmembrane Aspartates in Presenilin 1 and 2 Are Obligatory for γ-Secretase Activity and Amyloid β-Protein Generation* , 2000, The Journal of Biological Chemistry.

[5]  A Helenius,et al.  Setting the standards: quality control in the secretory pathway. , 1999, Science.

[6]  Alfredo G. Tomasselli,et al.  Membrane-anchored aspartyl protease with Alzheimer's disease β-secretase activity , 1999, Nature.

[7]  R. Barbour,et al.  Purification and cloning of amyloid precursor protein β-secretase from human brain , 1999, Nature.

[8]  David G. Tew,et al.  Identification of a Novel Aspartic Protease (Asp 2) as β-Secretase , 1999, Molecular and Cellular Neuroscience.

[9]  S. Sisodia Alzheimer's disease: perspectives for the new millennium. , 1999, The Journal of clinical investigation.

[10]  J. Treanor,et al.  Beta-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE. , 1999, Science.

[11]  M. Frosch,et al.  Presenilin 1 Facilitates the Constitutive Turnover of β-Catenin: Differential Activity of Alzheimer’s Disease–Linked PS1 Mutants in the β-Catenin–Signaling Pathway , 1999, The Journal of Neuroscience.

[12]  Sarah Tomlin,et al.  Microtechnology: Laying it on thick , 1999, Nature.

[13]  D. Selkoe,et al.  Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and γ-secretase activity , 1999, Nature.

[14]  C. Pitcher,et al.  Cluster of differentiation antigen 4 (CD4) endocytosis and adaptor complex binding require activation of the CD4 endocytosis signal by serine phosphorylation. , 1999, Molecular biology of the cell.

[15]  R. Rozmahel,et al.  Presenilin mutations associated with Alzheimer disease cause defective intracellular trafficking of β-catenin,a component of the presenilin protein complex , 1999, Nature Medicine.

[16]  Hans Clevers,et al.  Destabilization of β-catenin by mutations in presenilin-1 potentiates neuronal apoptosis , 1998, Nature.

[17]  P. Fraser,et al.  The Presenilin 1 Protein Is a Component of a High Molecular Weight Intracellular Complex That Contains β-Catenin* , 1998, The Journal of Biological Chemistry.

[18]  R. Doms,et al.  Detection of a Novel Intraneuronal Pool of Insoluble Amyloid β Protein that Accumulates with Time in Culture , 1998, The Journal of cell biology.

[19]  L. Kasturi,et al.  The amino acid following an asn-X-Ser/Thr sequon is an important determinant of N-linked core glycosylation efficiency. , 1998, Biochemistry.

[20]  Hugo Vanderstichele,et al.  Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein , 1998, Nature.

[21]  R. Doms,et al.  Alzheimer's Aβ(1–42) is generated in the endoplasmic reticulum/intermediate compartment of NT2N cells , 1997, Nature Medicine.

[22]  C. Masters,et al.  Distinct sites of intracellular production for Alzheimer's disease Aβ40/42 amyloid peptides , 1997, Nature Medicine.

[23]  R. Doms,et al.  Novel β-Secretase Cleavage of β-Amyloid Precursor Protein in the Endoplasmic Reticulum/Intermediate Compartment of NT2N Cells , 1997, The Journal of cell biology.

[24]  Mark M. Davis,et al.  Ligand-specific oligomerization of T-cell receptor molecules , 1997, Nature.

[25]  P. Greengard,et al.  Generation of Alzheimer beta-amyloid protein in the trans-Golgi network in the apparent absence of vesicle formation. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[26]  D. Teplow,et al.  Metabolism of the Swedish Amyloid Precursor Protein Variant in Neuro2a (N2a) Cells , 1996, The Journal of Biological Chemistry.

[27]  D. Selkoe,et al.  The Swedish mutation causes early-onset Alzheimer's disease by β-secretase cleavage within the secretory pathway , 1995, Nature Medicine.

[28]  B. Marsh,et al.  Molecular regulation of GLUT-4 targeting in 3T3-L1 adipocytes , 1995, The Journal of cell biology.

[29]  Joanne I. Yeh,et al.  Distinct signals in the GLUT4 glucose transporter for internalization and for targeting to an insulin-responsive compartment , 1995, The Journal of cell biology.

[30]  M. Jackson,et al.  A Role for Acidic Residues in Di-leucine Motif-based Targeting to the Endocytic Pathway (*) , 1995, The Journal of Biological Chemistry.

[31]  O. Bakke,et al.  Targeting of membrane proteins to endosomes and lysosomes. , 1994, Trends in cell biology.

[32]  S. Squazzo,et al.  Evidence that production and release of amyloid beta-protein involves the endocytic pathway. , 1994, The Journal of biological chemistry.

[33]  M. Birnbaum,et al.  A Leu-Leu sequence is essential for COOH-terminal targeting signal of GLUT4 glucose transporter in fibroblasts. , 1994, The Journal of biological chemistry.

[34]  J Alexander,et al.  Antigen analogs/MHC complexes as specific T cell receptor antagonists. , 1994, Annual review of immunology.

[35]  P. Lansbury,et al.  Seeding “one-dimensional crystallization” of amyloid: A pathogenic mechanism in Alzheimer's disease and scrapie? , 1993, Cell.

[36]  S. Kornfeld,et al.  The cytoplasmic tail of the mannose 6-phosphate/insulin-like growth factor-II receptor has two signals for lysosomal enzyme sorting in the Golgi , 1992, The Journal of cell biology.

[37]  G. Griffiths,et al.  Differential endocytosis of CD4 in lymphocytic and nonlymphocytic cells , 1991, The Journal of experimental medicine.

[38]  T. McGraw,et al.  Mutagenesis of the human transferrin receptor: two cytoplasmic phenylalanines are required for efficient internalization and a second- site mutation is capable of reverting an internalization-defective phenotype , 1991, The Journal of cell biology.

[39]  F. Maxfield,et al.  Human transferrin receptor internalization is partially dependent upon an aromatic amino acid on the cytoplasmic domain. , 1990, Cell regulation.

[40]  K. Grzeschik,et al.  The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor , 1987, Nature.

[41]  G. Glenner,et al.  Alzheimer's disease: Initial report of the purification and characterization of a novel cerebrovascular amyloid protein , 1984 .

[42]  R. Lake FURTHER CHARACTERIZATION OF THE F1-HISTONE PHOSPHOKINASE OF METAPHASE-ARRESTED ANIMAL CELLS , 1973, The Journal of cell biology.