Allophycocyanin 1 as a Near-Infrared Fluorescent Tracer: Isolation, Characterization, Chemical Modification, and Use in a Homogeneous Fluorescence Resonance Energy Transfer System

Trinquet, Eric; Maurin, Fabrice; Préaudat, M.; Mathis, Gérard

doi:10.1006/abio.2001.5298

Cited by 38 publications

(35 citation statements)

References 27 publications

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“…7D). It is known that APC organization may change depending on the ionic environment and/or the APC concentration itself (45). When concentrated enough (A max Ͼ 0.1), APC was mostly in the trimeric form (␣␤) 3 and its absorption and fluorescence emission maxima were 651 and 661 nm, respectively (Fig.…”

Section: Resultsmentioning

confidence: 99%

Two Novel Phycoerythrin-Associated Linker Proteins in the Marine CyanobacteriumSynechococcussp. Strain WH8102

et al. 2005

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The recent availability of the whole genome of Synechococcus sp. strain WH8102 allows us to have a global view of the complex structure of the phycobilisomes of this marine picocyanobacterium. Genomic analyses revealed several new characteristics of these phycobilisomes, consisting of an allophycocyanin core and rods made of one type of phycocyanin and two types of phycoerythrins (I and II). Although the allophycocyanin appears to be similar to that found commonly in freshwater cyanobacteria, the phycocyanin is simpler since it possesses only one complete set of ␣ and ␤ subunits and two rod-core linkers (CpcG1 and CpcG2). It is therefore probably made of a single hexameric disk per rod. In contrast, we have found two novel putative phycoerythrin-associated linker polypeptides that appear to be specific for marine Synechococcus spp. The first one (SYNW2000) is unusually long (548 residues) and apparently results from the fusion of a paralog of MpeC, a phycoerythrin II linker, and of CpeD, a phycoerythrin-I linker. The second one (SYNW1989) has a more classical size (300 residues) and is also an MpeC paralog. A biochemical analysis revealed that, like MpeC, these two novel linkers were both chromophorylated with phycourobilin. Our data suggest that they are both associated (partly or totally) with phycoerythrin II, and we propose to name SYNW2000 and SYNW1989 MpeD and MpeE, respectively. We further show that acclimation of phycobilisomes to high light leads to a dramatic reduction of MpeC, whereas the two novel linkers are not significantly affected. Models for the organization of the rods are proposed.

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Section: Resultsmentioning

confidence: 99%

Two Novel Phycoerythrin-Associated Linker Proteins in the Marine CyanobacteriumSynechococcussp. Strain WH8102

et al. 2005

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“…Homogeneous Time-resolved Fluorescence (HTRF)-Monoclonal anti-CX3CR1 (clone 51637) was labeled with the different fluorescent donors or acceptors as described previously (24,25). The donor was a pyridine-bipyridine (PBP) europium cryptate (europium cryptate-PBP).…”

Section: Methodsmentioning

confidence: 99%

Functional Adhesiveness of the CX3CL1 Chemokine Requires Its Aggregation

Hermand

Pincet

Carvalho

et al. 2008

Journal of Biological Chemistry

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In its native form, the chemokine CX3CL1 is a firmly adhesive molecule promoting leukocyte adhesion and migration and hence involved, along with its unique receptor CX3CR1, in various inflammatory processes. Here we investigated the role of molecular aggregation in the CX3CL1 adhesiveness. Assays of bioluminescence resonance energy transfer (BRET) and homogeneous time-resolved fluorescence (HTRF) in transfected cell lines and in primary cells showed specific signals indicative of CX3CL1 clustering. Truncation experiments showed that the transmembrane domain played a central role in this aggregation. A chimera with mutations of the 12 central transmembrane domain residues had significantly reduced BRET signals and characteristics of a non-clustering molecule. This mutant was weakly adhesive according to flow and dual pipette adhesion assays and was less glycosylated than CX3CL1, although, as we demonstrated, loss of glycosylation did not affect the CX3CL1 adhesive potency. We postulate that cell surfaces express CX3CL1 as a constitutive oligomer and that this oligomerization is essential for its adhesive potency. Inhibition of CX3CL1 self-assembly could limit the recruitment of CX3CR1-positive cells and may be a new pathway for anti-inflammatory therapies.Migration of circulating leukocytes to injury sites is the first step of the inflammation process, which involves a sequence of coordinated interactions between leukocytes and endothelial cells (1). Central to this physiological and pathological event are chemokines, a family of low molecular weight soluble proteins, that function to attract leukocytes bearing the appropriate receptors (2). Chemokines trigger activation of leukocytes and their firm adhesion to inflamed endothelium, mainly through the mediation of integrins and their cognate ligands (3).Among chemokines, there are two exceptions: CXCL16 and CX3CL1 are type-I membranous proteins. In addition to their chemokine domain (CD), 4 they are composed of a long mucinlike stalk, a transmembrane domain, and a cytoplasmic tail (4, 5) (Fig. 1). The CX3CL1 molecule, with its unique CX3CR1 receptor (6), has been shown to be central in defenses against neurodegenerative disorders (7,8) and against several cancers in murine models (9, 10). The CX3CL1-CX3CR1 axis is also involved in various inflammatory diseases (2), including renal inflammation (11) and atherosclerosis (12, 13). Understanding the structure of this pair of molecules is necessary for exploring pharmacological methods to regulate their activity. The structure of CX3CR1, a receptor of the G protein-coupled receptor (GPCR) family, is relatively well known, but that of CX3CL1 much less so.CX3CL1-CD (76 residues), a globular protein domain 3 nm in diameter (14) and maintained by two disulfide bridges, is structurally similar to other chemokines. It is composed of a disordered N terminus up to the first cysteine (Cys-8), a long loop followed by a three-stranded antiparallel ␤-sheet (residues 24 -51) and a C-terminal ␣-helix (residues 56 -67) packed agains...

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“…It consists of two different subunits, a and ß, with apparent molecular masses of 19.5 and 17.4 kDa (Sun & Wang 2003). APC has uses not only as the particularly bright fluorescent protein commonly used in flow cytometry (Kronick 1986), but also as an acceptor in a fluorescence resonance energy transfer system (Trinquet et al 2001). In addition, our previous reports have revealed that rAPC could remarkably inhibit the S-180 carcinoma in mice (Tang et al 1996Zhao et al 2003;Ge et al 2005), which indicated that rAPC might be used as a potential pharmaceutical product.…”

Section: Introductionmentioning

confidence: 98%

Heterologous Expression and Purification of Recombinant Allophycocyanin in Marine Streptomyces sp. Isolate M097

Hou

Qin

et al. 2006

World J Microbiol Biotechnol

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Allophycocyanin 1 as a Near-Infrared Fluorescent Tracer: Isolation, Characterization, Chemical Modification, and Use in a Homogeneous Fluorescence Resonance Energy Transfer System

Cited by 38 publications

References 27 publications

Two Novel Phycoerythrin-Associated Linker Proteins in the Marine CyanobacteriumSynechococcussp. Strain WH8102

Two Novel Phycoerythrin-Associated Linker Proteins in the Marine CyanobacteriumSynechococcussp. Strain WH8102

Functional Adhesiveness of the CX3CL1 Chemokine Requires Its Aggregation

Heterologous Expression and Purification of Recombinant Allophycocyanin in Marine Streptomyces sp. Isolate M097

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