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Ceramide Synthase Inhibition by Fumonisin B1 Causes Accumulation of 1-Deoxysphinganine

Zitomer, Nicholas C., Mitchell, Trevor, Voss, Kenneth A., Bondy, Genevieve S., Pruett, Sarah T., Garnier-Amblard, Ethel C., Liebeskind, Lanny S., Park, Hyejung, Wang, Elaine, Sullards, M. Cameron, Merrill, Alfred H., Riley, Ronald T.·2009·Journal of Biological Chemistry
DOI 10.1074/jbc.m808798200
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Abstract

Fumonisin B(1) (FB(1)) is a mycotoxin that inhibits ceramide synthases (CerS) and causes kidney and liver toxicity and other disease. Inhibition of CerS by FB(1) increases sphinganine (Sa), Sa 1-phosphate, and a previously unidentified metabolite. Analysis of the latter by quadrupole-time-of-flight mass spectrometry assigned an m/z = 286.3123 in positive ionization mode, consistent with the molecular formula for deoxysphinganine (C(18)H(40)NO). Comparison with a synthetic standard using liquid chromatography, electrospray tandem mass spectrometry identified the metabolite as 1-deoxysphinganine (1-deoxySa) based on LC mobility and production of a distinctive fragment ion (m/z 44, CH(3)CH=NH (+)(2)) upon collision-induced dissociation. This novel sphingoid base arises from condensation of alanine with palmitoyl-CoA via serine palmitoyltransferase (SPT), as indicated by incorporation of l-[U-(13)C]alanine into 1-deoxySa by Vero cells; inhibition of its production in LLC-PK(1) cells by myriocin, an SPT inhibitor; and the absence of incorporation of [U-(13)C]palmitate into 1-[(13)C]deoxySa in LY-B cells, which lack SPT activity. LY-B-LCB1 cells, in which SPT has been restored by stable transfection, however, produce large amounts of 1-[(13)C]deoxySa. 1-DeoxySa was elevated in FB(1)-treated cells and mouse liver and kidney, and its cytotoxicity was greater than or equal to that of Sa for LLC-PK(1) and DU-145 cells. Therefore, this compound is likely to contribute to pathologies associated with fumonisins. In the absence of FB(1), substantial amounts of 1-deoxySa are made and acylated to N-acyl-1-deoxySa (i.e. 1-deoxydihydroceramides). Thus, these compounds are an underappreciated category of bioactive sphingoid bases and "ceramides" that might play important roles in cell regulation.

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Figures

Image 1
CC-BY · From the PDF
FIGURE 2 ChangesinfreeSa(d18:0)andtheunidentifiedsphingoidbase(UnID)inconfluentcultures of LLC-PK1 cells treated with FB 1 and/or myriocin. Panel A showstheamountsofthesefree sphingoid bases in cells exposed to 35 𝛍 M FB 1 for various times ( white circles and squares ) or FB 1 plus myriocin ( black circles and squares ). Panel B shows the reversibility of these elevations when LLC-PK 1 cells were exposed for 48 h to FB 1 followed by a change to fresh medium without FB 1 (at time 0) but with ( black circles and squares ) or without myriocin ( white circles and squares ). The free sphingoid bases were analyzed by HPLC with fluorescence detection of the free sphingoid bases as OPA derivatives using C20-Sa (d20:0) as an internal standard (26). The data are the mean ± S.D. from three independent experiments.
FIGURE 2 ChangesinfreeSa(d18:0)andtheunidentifiedsphingoidbase(UnID)inconfluentcultures of LLC-PK1 cells treated with FB 1 and/or myriocin. Panel A showstheamountsofthesefree sphingoid bases in cells exposed to 35 𝛍 M FB 1 for various times ( white circles and squares ) or FB 1 plus myriocin ( black circles and squares ). Panel B shows the reversibility of these elevations when LLC-PK 1 cells were exposed for 48 h to FB 1 followed by a change to fresh medium without FB 1 (at time 0) but with ( black circles and squares ) or without myriocin ( white circles and squares ). The free sphingoid bases were analyzed by HPLC with fluorescence detection of the free sphingoid bases as OPA derivatives using C20-Sa (d20:0) as an internal standard (26). The data are the mean ± S.D. from three independent experiments.CC-BY · From the PDF
FIGURE 3 Liquid chromatography of LLC-PK1 cell extracts after 24 h ( A ) and 120 h ( B ) of treatment with FB1 with monitoring for Sa and a novel sphingoid base by electrospray ionization tandem mass spectrometry. Freesphingoidbaseswereextractedfrom100%confluentcultures(50-100 𝛍 gofprotein)usingthe method of Riley et al. (26) but without the addition of an internal standard. The chromatograms display the combined ion intensities for Sa ( m / z 302.2, the gray major peak ) and the unidentified sphingoid base ( m / z 286.3, the black major peak ), which eluted at retention times of ∼ 8 and 10 min, respectively. The insets are the product ion spectra for the eluted precursor m / z 302.2 ( inset in A ) and 286.3 ( inset in B ). Highlighted in A is a distinctive fragment for Sa ( m / z 60. 1) that is not produced from m / z 286.3 ( B ).
FIGURE 3 Liquid chromatography of LLC-PK1 cell extracts after 24 h ( A ) and 120 h ( B ) of treatment with FB1 with monitoring for Sa and a novel sphingoid base by electrospray ionization tandem mass spectrometry. Freesphingoidbaseswereextractedfrom100%confluentcultures(50-100 𝛍 gofprotein)usingthe method of Riley et al. (26) but without the addition of an internal standard. The chromatograms display the combined ion intensities for Sa ( m / z 302.2, the gray major peak ) and the unidentified sphingoid base ( m / z 286.3, the black major peak ), which eluted at retention times of ∼ 8 and 10 min, respectively. The insets are the product ion spectra for the eluted precursor m / z 302.2 ( inset in A ) and 286.3 ( inset in B ). Highlighted in A is a distinctive fragment for Sa ( m / z 60. 1) that is not produced from m / z 286.3 ( B ).CC-BY · From the PDF
FIGURE 4 Comparison of the MS 3 spectra for synthetic 1-deoxySa ( A ) and the deoxySa isolated from LLC-PK1 cell extracts ( B ) using the method of Riley et al. (26) with subsequent partial purification on a minicolumn of preparative C18 packing material (5 mm × 60 mm); fractions enriched in the deoxySa were pooled for analysis. The samples were introduced into a Thermo Electron Corporation LTQ XL via syringe pump and scanned across the mass range shown for the MS 3 fragments from the single dehydration product ( m / z 268) of deoxySa. Highlighted is the distinctive fragment for 1-deoxySa at m / z 44.
FIGURE 4 Comparison of the MS 3 spectra for synthetic 1-deoxySa ( A ) and the deoxySa isolated from LLC-PK1 cell extracts ( B ) using the method of Riley et al. (26) with subsequent partial purification on a minicolumn of preparative C18 packing material (5 mm × 60 mm); fractions enriched in the deoxySa were pooled for analysis. The samples were introduced into a Thermo Electron Corporation LTQ XL via syringe pump and scanned across the mass range shown for the MS 3 fragments from the single dehydration product ( m / z 268) of deoxySa. Highlighted is the distinctive fragment for 1-deoxySa at m / z 44.CC-BY · From the PDF
FIGURE 5 Structures of Sa and 1-deoxySa and a scheme for the biosynthesis of 1-deoxySa and its accumulation in cells exposed to FB 1 . Also shown are the known sites of inhibition by FB 1 and myriocin.
FIGURE 5 Structures of Sa and 1-deoxySa and a scheme for the biosynthesis of 1-deoxySa and its accumulation in cells exposed to FB 1 . Also shown are the known sites of inhibition by FB 1 and myriocin.CC-BY · From the PDF
FIGURE 6 Incorporation of L-[U13 C ] alanine ( A ) and L-[U13 C]serine precursors ( B ) into the 1-deoxySa (m18:0), Sa (d18:0), and Sa1P (d18:0-P) in Verocells treated with 35 𝛍 MFB1 for 48 h and grown in medium containing increasing concentrations of either L-[U13 C]alanineor L-[U13 C]serine(100 𝛍 Mto500 𝛍 M). Thecontrol (0) contained 35 𝛍 MFB1 andboth [U12 C]alanine and [U12 C]serine were at 100 𝛍 M each. All other treatments contained both alanine and serine; however, while the concentration of L-[U13 C]alanine and L-[U13 C]serine ranged from 100 to 500 𝛍 M the other L-[ 12 C]amino acid (alanine or serine) was maintained at 100 𝛍 M. Free sphingoid bases and sphingoid base 1-phosphates were extracted from the proliferating (50-80% confluent after 48 h) cultures (50-150 𝛍 g of protein) using the method of Zitomer et al. (27). The 13 C-labeled deoxySa ( m / z 288), Sa ( m /z 304), Sa1P ( m / z 384), and [ 12 C, natural abundance 13 C] species of 1-deoxySa ( m / z 286), Sa ( m / z 302), and Sa1P ( m / z 382) were analyzed using LC ion trapESI-MSandthemeanoftheresultsfromfourreplicatedishesareexpressedas the ratio of the amount of 13 C-labeled compound/ 12 C-labeled compound. The standard deviations were all less than 5% of the mean values and are therefore not shown.
FIGURE 6 Incorporation of L-[U13 C ] alanine ( A ) and L-[U13 C]serine precursors ( B ) into the 1-deoxySa (m18:0), Sa (d18:0), and Sa1P (d18:0-P) in Verocells treated with 35 𝛍 MFB1 for 48 h and grown in medium containing increasing concentrations of either L-[U13 C]alanineor L-[U13 C]serine(100 𝛍 Mto500 𝛍 M). Thecontrol (0) contained 35 𝛍 MFB1 andboth [U12 C]alanine and [U12 C]serine were at 100 𝛍 M each. All other treatments contained both alanine and serine; however, while the concentration of L-[U13 C]alanine and L-[U13 C]serine ranged from 100 to 500 𝛍 M the other L-[ 12 C]amino acid (alanine or serine) was maintained at 100 𝛍 M. Free sphingoid bases and sphingoid base 1-phosphates were extracted from the proliferating (50-80% confluent after 48 h) cultures (50-150 𝛍 g of protein) using the method of Zitomer et al. (27). The 13 C-labeled deoxySa ( m / z 288), Sa ( m /z 304), Sa1P ( m / z 384), and [ 12 C, natural abundance 13 C] species of 1-deoxySa ( m / z 286), Sa ( m / z 302), and Sa1P ( m / z 382) were analyzed using LC ion trapESI-MSandthemeanoftheresultsfromfourreplicatedishesareexpressedas the ratio of the amount of 13 C-labeled compound/ 12 C-labeled compound. The standard deviations were all less than 5% of the mean values and are therefore not shown.CC-BY · From the PDF
FIGURE7 Absenceof1-deoxySabiosynthesisbyLY-BcellsandrestorationofitsformationinLY-B-LCB1 cells. Shown are the amounts of 1-[ 13 C]deoxySa ( panel A ) and 13 C-backbone-labeled ceramides and 1-deoxyDHCer ( panel B ) ( i.e. with [ 13 C] in the backbone only: labeled 13 C-base , or both the backbone and the fatty acid, labeled 13 C-base & fatty acid ) that are produced by LY-B and LY-B-LCB1 cells. The cells were incubated with 0.1 mM [U- 13 C]palmitate as the 1:1 complex with BSA for 24 h. For comparison with earlier findings with FB 1 treatment, the indicated groups were also treated with 25 𝛍 M FB 1 . Then the sphingolipids were extracted and analyzed by LC-ESI-MS/MS as described under 'Experimental Procedures.' Each treatment was conducted in triplicate, and the results shown are means ± S.D., n = 3.
FIGURE7 Absenceof1-deoxySabiosynthesisbyLY-BcellsandrestorationofitsformationinLY-B-LCB1 cells. Shown are the amounts of 1-[ 13 C]deoxySa ( panel A ) and 13 C-backbone-labeled ceramides and 1-deoxyDHCer ( panel B ) ( i.e. with [ 13 C] in the backbone only: labeled 13 C-base , or both the backbone and the fatty acid, labeled 13 C-base & fatty acid ) that are produced by LY-B and LY-B-LCB1 cells. The cells were incubated with 0.1 mM [U- 13 C]palmitate as the 1:1 complex with BSA for 24 h. For comparison with earlier findings with FB 1 treatment, the indicated groups were also treated with 25 𝛍 M FB 1 . Then the sphingolipids were extracted and analyzed by LC-ESI-MS/MS as described under 'Experimental Procedures.' Each treatment was conducted in triplicate, and the results shown are means ± S.D., n = 3.CC-BY · From the PDF
FIGURE 8 Endogenous N -acyl-sphingoid bases in LLC-PK 1 cells and after incubation of the cells with exogenously added 1-deoxySa with or without FB1. LLC-PK1 cells incubated for 48 h in growth medium supplemented with vehicle only, 10 𝛍 M1-deoxySa,50 𝛍 MFB1, or 10 𝛍 M1-deoxySaplus50 𝛍 M FB1 followed by analysis of the 1-deoxydihydroceramides ( panel A ) and ceramides ( panel B ) by LC ESI-MS/MS as described under 'Experimental Procedures.' Each treatment was conducted in triplicate, and the results shown are means ± S.D., n = 3.
FIGURE 8 Endogenous N -acyl-sphingoid bases in LLC-PK 1 cells and after incubation of the cells with exogenously added 1-deoxySa with or without FB1. LLC-PK1 cells incubated for 48 h in growth medium supplemented with vehicle only, 10 𝛍 M1-deoxySa,50 𝛍 MFB1, or 10 𝛍 M1-deoxySaplus50 𝛍 M FB1 followed by analysis of the 1-deoxydihydroceramides ( panel A ) and ceramides ( panel B ) by LC ESI-MS/MS as described under 'Experimental Procedures.' Each treatment was conducted in triplicate, and the results shown are means ± S.D., n = 3.CC-BY · From the PDF
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Research works printed but not linked: 8

  1. Marasas, 2001, Environ. Health Perspect., 109, 239
  2. Voss, 2001, Environ. Health Perspect., 109, 259
  3. Riley, 2001, Environ. Health Perspect., 109, 301
  4. Merrill, 2001, Environ. Health Perspect., 109, 283
  5. Tolleson, 1999, Int. J. Oncol., 14, 833
  6. Riley, 1998, Rev. Méd. Vét., 149, 617
  7. Hornemann, 2008, Chem. Phys. Lipids., 154S, S63
  8. Urs, 2006, Mol. Cell. Endocrinol., 265–266, 174

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Metadata

Authors
Zitomer, N. C., Mitchell, T., Voss, K. A., Bondy, G. S., Pruett, S. T., Garnier-Amblard, E. C., Liebeskind, L. S., Park, H., Wang, E., Sullards, M. C., Merrill, A. H., & Riley, R. T.
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2009-02
Volume
284
Issue
8
Pages
4786-4795
ISSN
0021-9258
DOI
10.1074/jbc.m808798200
Cited by
235
References
40
Language
en
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CoChat. (2026). Citation check: Ceramide Synthase Inhibition by Fumonisin B1 Causes Accumulation of 1-Deoxysphinganine. https://app.cochat.ai/papers/10.1074/jbc.m808798200
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Zitomer, N. C., Mitchell, T., Voss, K. A., Bondy, G. S., Pruett, S. T., Garnier-Amblard, E. C., Liebeskind, L. S., Park, H., Wang, E., Sullards, M. C., Merrill, A. H., & Riley, R. T. (2009). Ceramide Synthase Inhibition by Fumonisin B1 Causes Accumulation of 1-Deoxysphinganine. Journal of Biological Chemistry. https://doi.org/10.1074/jbc.m808798200

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