Synthesis of fluorescence-Labeled sphingosine and sphingosine 1-phosphate; effective tools for sphingosine and sphingosine 1-phosphate behavior

Hakogi, Toshikazu; Shigenari, Toshihiko; Katsumura, Shigeo; Sano, Tomoaki; Kohno, Takayuki; Igarashi, Yasuyuki

doi:10.1016/s0960-894x(02)00999-x

Cited by 28 publications

(13 citation statements)

References 19 publications

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“…When NBD-Sph was added to erythrocytes, it was slowly incorporated into the erythrocytes and readily phosphorylated to NBD-S1P (Fig. 1B), as reported previously for Chinese hamster ovary (CHO) cells (23). Synthesized NBD-S1P was immediately exported to the extracellular buffer at a linear rate for at least 2 h (Fig.…”

Section: Nbd-s1p Is An S1p Transporter Substrate In Rat Erythrocytessupporting

confidence: 70%

“…1B, C). In CHO cells, NBDSph is incorporated into the cells and converted into several NBD-labeled sphingolipids, including NBD-S1P, NBD-ceramide, and NBD-sphingomyelin, similarly to sphingosine metabolism (23). The simplicity of sphingosine metabolism in erythrocytes facilitates interpreting changes in the fluorescence intensity derived from NBD-S1P; thus, changes in the amount of NBD-S1P were determined in terms of NBD-S1P transport and/or sphingosine kinase activity.…”

Section: Discussionmentioning

confidence: 99%

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Fluorescence-based rapid measurement of sphingosine-1-phosphate transport activity in erythrocytes

Kobayashi

Otsuka

Yamaguchi

et al. 2016

Journal of Lipid Research

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This article is available online at http://www.jlr.org S1P recruits lymphocytes from the thymus and secondary lymphoid organs to the circulatory system by activating S1P receptors on lymphocytes (5). Many components of blood, such as erythrocytes, platelets, and neutrophils, express sphingosine kinases and produce S1P (6). Platelets contain large amounts of S1P and release it only when activated by stimuli, such as thrombin, whereas erythrocytes constitutively release S1P in the absence of stimuli. Because erythrocytes constitute a large proportion of the blood cells in the body and because they lack S1P-degrading activity catalyzed by S1P lyase (SGPL1), S1P phosphatases (SGPP1 and SGPP2), and phospholipid phosphatases (PLPP1, PLPP2, and PLPP3) (7), they contribute substantially to plasma S1P. In addition to blood cells, endothelial cells constitutively release S1P into the blood without stimuli (8). Thus, plasma S1P is derived mainly from erythrocytes and endothelial cells (9). When the S1P supply from erythrocytes has been disrupted, the plasma S1P level and the number of the lymphocytes in the blood decrease substantially (10). Therefore, plasma S1P supplied by erythrocytes is essential for the correct distribution of lymphocytes in the blood.Erythrocytes transport sphingosine, which is supplied by other cells, to the intracellular side of the membrane and produce S1P by phosphorylation (6,7,11). In addition to the extracellular source of sphingosine, intracellular alkaline ceramidase also produces sphingosine used for S1P synthesis in human erythrocytes (12). Previously, we reported that erythrocytes export intracellularly synthesized S1P into the extracellular space via an ATP-dependent transporter in a time-dependent manner without any Abstract Sphingosine-1-phosphate (S1P) is present in the blood plasma and acts as a pivotal intercellular signal transmitter in the immune system by recruiting lymphocytes from the thymus and secondary lymphoid tissues. The plasma S1P concentration is maintained by the supply of S1P from erythrocytes. Previously, we showed that S1P release from erythrocytes is mediated by an ATP-dependent transporter. In this study, we attempted to establish a rapid and reliable method for measuring the S1P transport activity in erythrocytes by using a fluorescent S1P analog, 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled S1P. NBD-S1P was released from erythrocytes in a time-dependent manner. The NBD-S1P release was reduced after exposure to glyburide, which is an inhibitor of the S1P transporter in erythrocytes. Moreover, the release of NBD-S1P and S1P from erythrocytes was competitively inhibited by intracellular S1P and NBD-S1P, respectively. These results showed that the erythrocyte S1P transporter exports NBD-S1P. We optimized the sample-preparation conditions and lipid extraction to increase the sensitivity of the assay. Furthermore, we successfully measured NBD-S1P release without lipid extraction by decreasing the concentration of BSA in the assay buffer to 0.1%. This method will be ...

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Section: Nbd-s1p Is An S1p Transporter Substrate In Rat Erythrocytessupporting

confidence: 70%

Section: Discussionmentioning

confidence: 99%

Fluorescence-based rapid measurement of sphingosine-1-phosphate transport activity in erythrocytes

Kobayashi

Otsuka

Yamaguchi

et al. 2016

Journal of Lipid Research

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“…BODIPY 540 sphingosine enabled imaging unacylated sphingolipids, which cannot be accomplished with the existing sphingolipid analogs that contain a fl uorescent fatty acid side chain. Furthermore, unlike previously reported fl uorescent sphingosine analogs (7)(8)(9), BODIPY 540 sphingosine can be visualized in parallel with GFP without the use of UV excitation. By exploiting these capabilities, we verifi ed that mammalian cells rapidly internalized BODIPY 540 sphingosine, transported it to the secretory pathway where it was metabolized to more complex fl uorescent sphingolipids, and eventually catabolized these fl uorescent sphingolipids.…”

Section: Discussionmentioning

confidence: 89%

A new, long-wavelength borondipyrromethene sphingosine for studying sphingolipid dynamics in live cells

Kim¹,

Lou²,

Kraft

2013

Journal of Lipid Research

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apoptosis, and other critical cellular processes during normal cell function and disease ( 1-4 ). Insight into sphingolipid biosynthesis, transport, and subcellular distribution has been acquired by observing fl uorescent sphingolipid analogs within living cells ( 5 ). Sphingolipid derivatives that contain a fl uorophore-labeled N -acyl fatty acid are often used to investigate dynamic processes that involve acylated sphingolipids ( 5 ). Fatty acids that contain a polyene fl uorophore are especially attractive for this purpose because the structure and behavior of polyene-containing lipids are very similar to the native lipid ( 6 ). However, to study the bioactive, unacylated sphingolipids, sphingosine and sphingosine-1-phosphate, the fl uorophore must be incorporated into the sphingosine backbone. Studies have confi rmed that such fl uorescent sphingosine analogs can be metabolized to more complex fl uorescently labeled sphingolipids in living cells ( 7-10 ). Despite their potential utility, only a limited number of fl uorophores, such as pyrene, borondipyrromethene (BODIPY), and nitrobenzo-2-oxa-1,3-diazole, have been incorporated into the sphingosine backbone ( 7-9 ).To increase the utility of fl uorescent sphingosine analogs for investigating sphingolipid dynamics in living cells, derivatives with a wider range of fl uorescence properties must be developed. Lacking in particular is a bioactive fl uorescent sphingosine that has neither an excitation maximum that is in the UV range nor emission that interferes with detecting green fl uorescent protein (GFP), the most common genetically encoded fl uorescent protein label ( 11 ). A probe with these properties is expected to Abbreviations: GFP, green fl uorescent protein; BODIPY, borondipyrromethene; rt, room temperature; ER, endoplasmic reticulum. 1 R. Kim and K. Lou contributed equally to this work.

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“…[5,9,10] Opting for a short and easily reproducible route, we used a method which we recently described for efficient synthesis of backbone-labeled sphingolipids. [7,11] Olefin cross metathesis of allyl alocohol 4 [12] and N-Boc-undec-10-enylamine (5) [13] under standard conditions using Grubb's catalyst (2nd generation) gave 6 in excellent yields (Scheme 1) under exclusive formation of the (E)-double bond, as previously observed with similar analogues by us [7,11] and others.…”

Section: Resultsmentioning

confidence: 99%

Synthesis and Immobilization of erythro‐C14‐ω‐Aminosphingosine‐1‐phosphate as a Potential Tool for Affinity Chromatography

et al. 2008

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A sphingosine-1-phosphate (S1P) analogue containing a terminal alkyl chain amino group is synthesized in a few steps via olefin cross-metathesis of an optically resolved intermediate and subsequent phosphorylation. Regioselective acylation of this intermediate at its N terminus in solution is demonstrated as a model reaction and provides a biologically active derivative. Finally, the omega-amino intermediate is immobilized on an affinity matrix. The choice of a UV-active phosphate protecting group allows for quantification of resin loading after cleavage which amounted to 66 %.

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Synthesis of fluorescence-Labeled sphingosine and sphingosine 1-phosphate; effective tools for sphingosine and sphingosine 1-phosphate behavior

Cited by 28 publications

References 19 publications

Fluorescence-based rapid measurement of sphingosine-1-phosphate transport activity in erythrocytes

Fluorescence-based rapid measurement of sphingosine-1-phosphate transport activity in erythrocytes

A new, long-wavelength borondipyrromethene sphingosine for studying sphingolipid dynamics in live cells

Synthesis and Immobilization of erythro‐C14‐ω‐Aminosphingosine‐1‐phosphate as a Potential Tool for Affinity Chromatography

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