Electrochemiluminescence: A New Diagnostic and Research Tool

Yang, Hongjun; Leland, Jonathan K.; Yost, David A.; Massey, Richard J.

doi:10.1038/nbt0294-193

Cited by 123 publications

(71 citation statements)

References 3 publications

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“…Interestingly, the fractions with ␥-secretase activity (nos. [13][14][15][16] contain PS1 heterodimer as shown by immunodetection with antibodies versus PS1-NTF and PS1-CTF. SDS/PAGE͞immunoblot analysis of the other fractions indicated that the PS1 heterodimer was absent from fractions devoid of ␥-secretase activity (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Presenilin 1 is linked with γ-secretase activity in the detergent solubilized state

Lai²,

Xu³

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

527

412

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␥-Secretase is a membrane-associated protease that cleaves within the transmembrane region of amyloid precursor protein to generate the C termini of the two A␤ peptide isoforms, A␤40 and A␤42. Here we report the detergent solubilization and partial characterization of ␥-secretase. The activity of solubilized ␥-secretase was measured with a recombinant substrate, C100Flag, consisting largely of the C-terminal fragment of amyloid precursor protein downstream of the ␤-secretase cleavage site. Cleavage of C100Flag by ␥-secretase was detected by electrochemiluminescence using antibodies that specifically recognize the A␤40 or A␤42 termini. Incubation of C100Flag with HeLa cell membranes or detergent-solubilized HeLa cell membranes generates both the A␤40 and A␤42 termini. Recovery of catalytically competent, soluble ␥-secretase critically depends on the choice of detergent; CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate) but not Triton X-100 is suitable. Solubilized ␥-secretase activity is inhibited by pepstatin and more potently by a novel aspartyl protease transition-state analog inhibitor that blocks formation of A␤40 and A␤42 in mammalian cells. Upon gel exclusion chromatography, solubilized ␥-secretase activity coelutes with presenilin 1 (PS1) at an apparent relative molecular weight of approximately 2.0 ؋ 10 6 . Anti-PS1 antibody immunoprecipitates ␥-secretase activity from the solubilized ␥-secretase preparation. These data suggest that ␥-secretase activity is catalyzed by a PS1-containing macromolecular complex.

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Section: Resultsmentioning

confidence: 99%

Presenilin 1 is linked with γ-secretase activity in the detergent solubilized state

Lai²,

Xu³

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

527

412

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“…Cells were selected in maintenance medium without nucleosides (selection medium). Conditioned medium from individual colonies was assayed using an electrochemiluminescence detection method on an Origen analyzer (IGEN) the technology reviewed by Yang et al (17). A colony expressing high levels of Asp-2 was used to inoculate 100 ml of selection medium to generate conditioned medium from which Asp-2 Fc was purified by affinity chromatography on immobilized Protein A.…”

Section: Methodsmentioning

confidence: 99%

Characterization of the Glycosylation Profiles of Alzheimer's β-Secretase Protein Asp-2 Expressed in a Variety of Cell Lines

Charlwood

Dingwall

Matico

et al. 2001

Journal of Biological Chemistry

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Amyloid 39 -42 ␤ -peptides are the main components of amyloid plaques found in the brain of Alzheimer's disease patients. Amyloid 39 -42 ␤-peptide is formed from amyloid precursor protein by the sequential action of ␤-and ␥-secretases. Asp-2 is a transmembrane aspartic protease expressed in the brain, shown to have ␤-secretase activity. Mature Asp-2 has four N-glycosylation sites. In this report we have characterized the carbohydrate structures in this glycoprotein expressed in three different cell lines, namely Chinese hamster ovary, CV-1 origin of SV40, and baculovirus-infected SF9 cells. Biantennary and triantennary oligosaccharides of the "complex" type were released from glycoprotein expressed in the mammalian cells, whereas mannose-rich glycans were identified from glycoprotein synthesized in the baculovirus-infected cells. Site-directed mutagenesis of the asparagine residues at amino acid positions 153, 172, 223, and 354 demonstrate that the protease activity of Asp-2 is dependent on its glycosylation.One of the key pathological features of Alzheimer's disease is the formation of brain plaques primarily due to the fibrilization of amyloid 39 -42 ␤-peptides (A␤) 1 (1, 2). The formation of these peptides by the action of proteases on amyloid precursor protein (APP) has been the subject of several studies (3, 4), as the identification of these proteases could lead to the discovery of inhibitors useful in the treatment of Alzheimer's disease. Over the last year there have been a number of reports detailing the discovery, purification, and characterization of a transmembrane aspartic proteinase (Asp-2). This glycoprotein has been shown to cleave the APP at the ␤-secretase site (5-9). High levels of this proteinase were identified in the human brain, and the enzyme activity was found in cells associated with the central nervous system (7) and in cell lines known to produce A␤ via cleavage of APP (5).Asp-2 contains four potential N-linked glycosylation sites at the following asparagine residues: Asn 153 , Asn 172 , Asn 223 , and Asn 354 . The close proximity of some of these glycosylated sites to the three intramolecular disulfide linkages and the catalytic site of Asp-2 has been the subject of more recent interest (10), especially because the type and extent glycosylation of a protein can have a profound effect on its physico-chemical properties (11). For instance, it is well known that N-linked oligosaccharides can influence glycoprotein folding in the endoplasmic reticulum (12) and can protect a protein from protease attack (13).The N-linked oligosaccharide structures on Asp-2 have not yet been characterized in detail, although it has been reported that all four asparagine sites have a heterogeneous mixture of carbohydrate attached (10). Another report has indicated that the glycans in mature Asp-2 are of the complex type, as removal of this carbohydrate is not possible by endoglycosidase H treatment (14).In this study we have released and analyzed the N-linked glycans from Asp-2 expressed as an Fc fusion in ...

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“…The amounts of IL-4 in culture supernatants and cell lysates were determined with a double-Ab method, using an ORIGEN Analyzer (IGEN, Gaithersburg, MD), which is based on an ECL detection technique (21). For the ORIGEN analysis, sheep anti-mouse IgG-coated magnetic beads (Dynabeads M-280, final dilution of 1/80; Dynal Biotech) were mixed with anti-IL-4 mAb (8D4-8, IgG1; BD PharMingen) at 50 ng/ml, biotinylated goat anti-IL-4 (polyclonal; R&D Systems) at 250 ng/ml, and 300 ng/ml of streptavidin, labeled with ruthenium (II) Tris-bipyridine chelate (TAG; IGEN) according to the manufacturer's recommendations.…”

Section: Ecl-based Detection Of Il-4mentioning

confidence: 99%

Germinal Center B Cells Constitute a Predominant Physiological Source of IL-4: Implication for Th2 Development In Vivo

Johansson‐Lindbom

Borrebaeck

2002

The Journal of Immunology

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Protective immunity depends upon the capability of the immune system to properly adapt the response to the nature of an infectious agent. CD4+ Th cells are implicated in this orchestration by secreting a polarized pattern of cytokines. Although Th2 development in animal models and in human cells in vitro to a large extent depends on IL-4, the nature of the cells that provide the initial IL-4 in vivo is still elusive. In this report, we describe the anatomical localization as well as the identity of IL-4-producing cells in human tonsil, a representative secondary lymphoid organ. We demonstrate that IL-4 production is a normal and intrinsic feature of germinal center (GC) B cells. We also show that expression of IL-4 is highly confined to the GCs, in which the B cells constitute the prevalent cellular source. Furthermore, immunofluorescence analysis of colon mucosa reveals a strikingly similar pattern of IL-4-expressing cells compared with tonsils, demonstrating that IL-4 production from GC B cells is not a unique feature of the upper respiratory tract. Our results show that GCs provide the most appropriate microenvironment for IL-4-dependent Th2 polarization in vivo and imply a critical role for GC B cells in this differentiation process.

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Electrochemiluminescence: A New Diagnostic and Research Tool

Cited by 123 publications

References 3 publications

Presenilin 1 is linked with γ-secretase activity in the detergent solubilized state

Presenilin 1 is linked with γ-secretase activity in the detergent solubilized state

Characterization of the Glycosylation Profiles of Alzheimer's β-Secretase Protein Asp-2 Expressed in a Variety of Cell Lines

Germinal Center B Cells Constitute a Predominant Physiological Source of IL-4: Implication for Th2 Development In Vivo

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