Oxylipins in Marine Diatoms - MarBEF · Biosynthetic pahway of oxylipins in marine diatoms. ......
Transcript of Oxylipins in Marine Diatoms - MarBEF · Biosynthetic pahway of oxylipins in marine diatoms. ......
![Page 1: Oxylipins in Marine Diatoms - MarBEF · Biosynthetic pahway of oxylipins in marine diatoms. ... Adrianna Ianora Antonio Miralto Giovanna Romano Ylenia Carotenuto University of Salerno](https://reader031.fdocuments.in/reader031/viewer/2022020319/5c6891fd09d3f263648baf8b/html5/thumbnails/1.jpg)
Oxylipins in Marine Diatoms
Istituto di Chimica Biomolecolare
Stazione Zoologica “A. Dohrn”
![Page 2: Oxylipins in Marine Diatoms - MarBEF · Biosynthetic pahway of oxylipins in marine diatoms. ... Adrianna Ianora Antonio Miralto Giovanna Romano Ylenia Carotenuto University of Salerno](https://reader031.fdocuments.in/reader031/viewer/2022020319/5c6891fd09d3f263648baf8b/html5/thumbnails/2.jpg)
2-trans-4-trans-7-cis decatrienal 2-trans-4-cis-7-cis decatrienal
2-trans-4-trans decadienal
Miralto et al. Nature 1999,402:173-176
CHO
CHO
CHO
First report of oxylipins in marine diatoms
Aldehydes were correlated to the antiproliferative effect of diatomson copepod reproduction
![Page 3: Oxylipins in Marine Diatoms - MarBEF · Biosynthetic pahway of oxylipins in marine diatoms. ... Adrianna Ianora Antonio Miralto Giovanna Romano Ylenia Carotenuto University of Salerno](https://reader031.fdocuments.in/reader031/viewer/2022020319/5c6891fd09d3f263648baf8b/html5/thumbnails/3.jpg)
Detection of Short Detection of Short ChainChain AldehydesAldehydes in in DiatomDiatom ExtractsExtracts
The reaction was directly applied on diatom extracts
CHO
CHO
CHO
C8:2
C8:3
C7:2
CHO
CHO
Thalassiosira rotula
CHO CHO
C10:3
Skeletonema costatum
C8:2
C7:2C8:3
d’Ippolito et al. Tetrahedron Lett. 2002, 43: 6137
1
2
3
45
6CHO
R
1’
2’
3’
4’5’
6’
RCO2Et
CO2EtPh3P
![Page 4: Oxylipins in Marine Diatoms - MarBEF · Biosynthetic pahway of oxylipins in marine diatoms. ... Adrianna Ianora Antonio Miralto Giovanna Romano Ylenia Carotenuto University of Salerno](https://reader031.fdocuments.in/reader031/viewer/2022020319/5c6891fd09d3f263648baf8b/html5/thumbnails/4.jpg)
Skeletonema costatum: major diatom blooming species
C8:3
C8:2C7:2
StandardStandard
C8:3 C8:2C7:2
StandardStandard
Aldehydic pool of Skeletonema costatum
Aldehydic pool of Adriatic phytoplankton
Workshop Diatom-Copepod Interactions, Ischia 2002
Chemical data support:-phytoplankton composition-production and mortality
of copepod eggs
2001-2005
![Page 5: Oxylipins in Marine Diatoms - MarBEF · Biosynthetic pahway of oxylipins in marine diatoms. ... Adrianna Ianora Antonio Miralto Giovanna Romano Ylenia Carotenuto University of Salerno](https://reader031.fdocuments.in/reader031/viewer/2022020319/5c6891fd09d3f263648baf8b/html5/thumbnails/5.jpg)
1) Clarification of the biosynthetic pathwayleading to aldehydes
- To understand what the regulating factors of the process are
- To develop a molecular approach to study field and lab samples.
2) Distribution and diversity of oxylipin pathway in different diatoms
-To understand if aldehydes are the only molecules involved in diatom-copepod interactions
![Page 6: Oxylipins in Marine Diatoms - MarBEF · Biosynthetic pahway of oxylipins in marine diatoms. ... Adrianna Ianora Antonio Miralto Giovanna Romano Ylenia Carotenuto University of Salerno](https://reader031.fdocuments.in/reader031/viewer/2022020319/5c6891fd09d3f263648baf8b/html5/thumbnails/6.jpg)
Oxylipin pathway in plants
Aldehydes formed through lipoxygenase/hydroperoxidelyase pathway
“Oxylipins” : oxygenated compoundsbiosynthesized from fatty acids byoxygenase-catalyzed oxygenation
![Page 7: Oxylipins in Marine Diatoms - MarBEF · Biosynthetic pahway of oxylipins in marine diatoms. ... Adrianna Ianora Antonio Miralto Giovanna Romano Ylenia Carotenuto University of Salerno](https://reader031.fdocuments.in/reader031/viewer/2022020319/5c6891fd09d3f263648baf8b/html5/thumbnails/7.jpg)
S. costatum CHO2,4 octadienal
CHO2,4 heptadienal
CHO2,4 octatrienal
CDCl3
2H –22H-4
22HH--55
2H-NMR
1H-NMR
2H-1
d’Ippolito et al.,Org.Lett. 2003, 5: 885
d6-HDTA (16:3ω4)
COOH
D
D
D D D D
Biosynthesis of octadienal
Cell-freeS. costatumCell-free
S. costatumDerivatized
aldehydic poolDerivatized
aldehydic pool
O
O D
D D
D
1 2
4
5
EI-MS spectrum
![Page 8: Oxylipins in Marine Diatoms - MarBEF · Biosynthetic pahway of oxylipins in marine diatoms. ... Adrianna Ianora Antonio Miralto Giovanna Romano Ylenia Carotenuto University of Salerno](https://reader031.fdocuments.in/reader031/viewer/2022020319/5c6891fd09d3f263648baf8b/html5/thumbnails/8.jpg)
COOH
D
D
D D D D
COOH
D
D
D D DDHO-O
ODC
D D
D
d6-HTrA (16:3ω4)
d4-octadienal
d6-9-HpHTrE
9-LOX
HPL
9
?
Plausible mechanism of octadienal formation
d’Ippolito et al.,Org.Lett. 2003, 5: 885
![Page 9: Oxylipins in Marine Diatoms - MarBEF · Biosynthetic pahway of oxylipins in marine diatoms. ... Adrianna Ianora Antonio Miralto Giovanna Romano Ylenia Carotenuto University of Salerno](https://reader031.fdocuments.in/reader031/viewer/2022020319/5c6891fd09d3f263648baf8b/html5/thumbnails/9.jpg)
First proof of 9-Lipoxygenase (9-Lox)
COOH
D
D
D D D D
COOH
D
D
D D DDHO-O
ODC
D D
D
d6-HTrA (16:3ω4)
d4-octadienal
d6-9-HpHTrE
9-LOX
HPL
9
LC-MS of d6-9-HHTrE
COOH
D
D
D D DDHO
d6-9-HHTrEd’Ippolito et al., Phytochemistry, submitted
![Page 10: Oxylipins in Marine Diatoms - MarBEF · Biosynthetic pahway of oxylipins in marine diatoms. ... Adrianna Ianora Antonio Miralto Giovanna Romano Ylenia Carotenuto University of Salerno](https://reader031.fdocuments.in/reader031/viewer/2022020319/5c6891fd09d3f263648baf8b/html5/thumbnails/10.jpg)
HPLC3H10-EPA (20:5 ω3)
2 µCi
COOH
Cell-freeS. costatumCell-free
S. costatumDerivatized
aldehydic poolDerivatized
aldehydic pool
H2
Pd/C 5%1037 cpmheptadienal
24 cpmoctadienal
149 cpmC7:0
16 cpmC8:0
Biosynthesis of heptadienal
S. costatum CHO2,4 octadienal
CHO2,4 heptadienal
CHO2,4 octatrienal
![Page 11: Oxylipins in Marine Diatoms - MarBEF · Biosynthetic pahway of oxylipins in marine diatoms. ... Adrianna Ianora Antonio Miralto Giovanna Romano Ylenia Carotenuto University of Salerno](https://reader031.fdocuments.in/reader031/viewer/2022020319/5c6891fd09d3f263648baf8b/html5/thumbnails/11.jpg)
CHO2,4 octadienalCHO
2,4 heptadienalCHO2,4 octatrienal
CHO2,4,7 decatrienal
Thalassiosira rotula
H2
Pd/C 5%
HPLC
BiosynthesisBiosynthesis of of decatrienaldecatrienal
3H10-EPA (20:5 ω3)2 µCi
Cell-freeT. rotula
Cell-freeT. rotula
Derivatized aldehydic poolDerivatized
aldehydic pool
C10:01200 cpmC8:0
14 cpmC7:018 cpm
decatrienal4800 cpmoctadienal
15 cpmheptadienal20 cpm
COOH
![Page 12: Oxylipins in Marine Diatoms - MarBEF · Biosynthetic pahway of oxylipins in marine diatoms. ... Adrianna Ianora Antonio Miralto Giovanna Romano Ylenia Carotenuto University of Salerno](https://reader031.fdocuments.in/reader031/viewer/2022020319/5c6891fd09d3f263648baf8b/html5/thumbnails/12.jpg)
CO2HCO2H
CO2H
HOO
CO2H
HOO
OHC OHC
9-LOXS. costatumT. rotula
HPL
11 14
11-LOXT. rotula
14-LOXS. costatum
9
Lipoxygenase Pathway
HPL HPL
20:5 ω3
2,4,7 decatrienal 2,4 heptadienal 2,4 octadienal
16:3 ω4
CO2H
HOO
OHC
LOX= Lipoxygenase HPL=Hydroperoxidelyase
![Page 13: Oxylipins in Marine Diatoms - MarBEF · Biosynthetic pahway of oxylipins in marine diatoms. ... Adrianna Ianora Antonio Miralto Giovanna Romano Ylenia Carotenuto University of Salerno](https://reader031.fdocuments.in/reader031/viewer/2022020319/5c6891fd09d3f263648baf8b/html5/thumbnails/13.jpg)
CO2H
HOO
CO2HCO2H
CO2H
HOO
OHC OHC
9-LOXS. costatumT. rotula
HPL
11 14
11-LOXT. rotula
14-LOXS. costatum
9
HPL HPL
20:5 ω3
2,4,7 decatrienal 2,4 heptadienal 2,4 octadienal
16:3 ω4
CO2H
HOO
OHC
Hydrolytic Actvity ?
Phospolipase A2 as key enzyme (Pohnert et al. Plant Physiology 2002 129:103)
![Page 14: Oxylipins in Marine Diatoms - MarBEF · Biosynthetic pahway of oxylipins in marine diatoms. ... Adrianna Ianora Antonio Miralto Giovanna Romano Ylenia Carotenuto University of Salerno](https://reader031.fdocuments.in/reader031/viewer/2022020319/5c6891fd09d3f263648baf8b/html5/thumbnails/14.jpg)
DistributionDistribution of of fattyfatty acidsacids in in complexcomplex lipidslipids of of T. rotulaT. rotula
GlycolipidsGlycolipids PhospholipidsPhospholipids NeutralNeutral lipidslipids
C16:3C16:3ωω44C16:4C16:4ωω11C18:4C18:4ωω33
17,5%17,5%3,6%3,6%10,4%10,4%
17,1%17,1%C16:0C16:0 2,8%2,8% 4,8%4,8% 11,2%11,2%
13,7%13,7%
2,5%2,5%0,73%0,73%8,6%8,6%
30,3%30,3% 18,22%18,22%C20:5C20:5ωω33
GlycolipidsGlycolipids PhospholipidsPhospholipids NeutralNeutral lipidslipids
C16:3C16:3ωω44
C16:4C16:4ωω11
C20:5C20:5ωω33
8,4%8,4%
21,5%21,5%16,9%16,9%
26,3%26,3%C16:0C16:0 1,5%1,5% 18,6%18,6% 90%90%
DistributionDistribution of of fattyfatty acidsacids in in complexcomplex lipidslipids of of S. S. costatumcostatum
![Page 15: Oxylipins in Marine Diatoms - MarBEF · Biosynthetic pahway of oxylipins in marine diatoms. ... Adrianna Ianora Antonio Miralto Giovanna Romano Ylenia Carotenuto University of Salerno](https://reader031.fdocuments.in/reader031/viewer/2022020319/5c6891fd09d3f263648baf8b/html5/thumbnails/15.jpg)
stdstdControl
TriglyceridesThalassiosira rotula
std
PhospholipidsOHC
decatrienal
std
GlycolipidsOHC
OHCoctadienaldecatrienal
heptadienal
octatrienal
![Page 16: Oxylipins in Marine Diatoms - MarBEF · Biosynthetic pahway of oxylipins in marine diatoms. ... Adrianna Ianora Antonio Miralto Giovanna Romano Ylenia Carotenuto University of Salerno](https://reader031.fdocuments.in/reader031/viewer/2022020319/5c6891fd09d3f263648baf8b/html5/thumbnails/16.jpg)
ExtractionPurification
3H-EPA-Glycolipids 3H-EPA-Phospholipids
Natural labelled complex lipids
24h incubationThe pelletted cells were traited with
boiled methanol
33HH1010--EPAEPA Labelling complex lipids
Skeletonema costatum
![Page 17: Oxylipins in Marine Diatoms - MarBEF · Biosynthetic pahway of oxylipins in marine diatoms. ... Adrianna Ianora Antonio Miralto Giovanna Romano Ylenia Carotenuto University of Salerno](https://reader031.fdocuments.in/reader031/viewer/2022020319/5c6891fd09d3f263648baf8b/html5/thumbnails/17.jpg)
S. costatumS. costatum: : incorporationincorporation of of labelledlabelled complexcomplex lipidslipids
33HH--EPAEPA--GlycolipidsGlycolipids181800 cpm181800 cpm
33HH--EPAEPA--PhospholipidsPhospholipids933200 cpm933200 cpm
Heptadienal2190 cpm
18 cpmC8:2
Heptaedienal480 cpm
22 cpmC8:2
S. costatumS. costatumS. costatum
S. costatumS. costatumS. costatum
HPLC
HPLC
d’Ippolito et al. BBA 2004, 1686, 100-107
Higher affinity for glycolipids rather than for phospolipids
![Page 18: Oxylipins in Marine Diatoms - MarBEF · Biosynthetic pahway of oxylipins in marine diatoms. ... Adrianna Ianora Antonio Miralto Giovanna Romano Ylenia Carotenuto University of Salerno](https://reader031.fdocuments.in/reader031/viewer/2022020319/5c6891fd09d3f263648baf8b/html5/thumbnails/18.jpg)
CH2
CH
O C
O
R'
OC
O
''R
CH2 O O OH
OH
OH
CH2R
1
2
3
sn-1sn-2
3 16.1
5 26.7
sn-1 sn-2
6 6.5
16:3ω416:4ω116:3ω416:3ω4
1 13.2
4 6.5
2 25.916:3ω4 16:4ω116:3ω420:5ω316:2ω420:5ω3
16:2ω420:5ω3
peak %
Glycolipids: main pool involved in the release of free fatty acids
LCMS/MS
1
2
3
4
5
6
![Page 19: Oxylipins in Marine Diatoms - MarBEF · Biosynthetic pahway of oxylipins in marine diatoms. ... Adrianna Ianora Antonio Miralto Giovanna Romano Ylenia Carotenuto University of Salerno](https://reader031.fdocuments.in/reader031/viewer/2022020319/5c6891fd09d3f263648baf8b/html5/thumbnails/19.jpg)
CO2H
HOO
CO2HCO2H
CO2H
HOO
OHC OHC
9-LOXS. costatumT. rotula
HPL
11
14
11-LOXT. rotula
14-LOXS.costatum
9
HPL HPL
20:5 ω3
2,4,7 decatrienal 2,4 heptadienal 2,4 octadienal
16:3 ω4
CO2H
HOO
OHC
Hydrolytic Actvity ?
![Page 20: Oxylipins in Marine Diatoms - MarBEF · Biosynthetic pahway of oxylipins in marine diatoms. ... Adrianna Ianora Antonio Miralto Giovanna Romano Ylenia Carotenuto University of Salerno](https://reader031.fdocuments.in/reader031/viewer/2022020319/5c6891fd09d3f263648baf8b/html5/thumbnails/20.jpg)
CO2H
HOO
CO2HCO2H
CO2H
HOO
OHC OHC
9-LOXS. costatumT. rotula
HPL
11
14
11-LOXT. rotula
14-LOXS.costatum
9
HPL HPL
20:5 ω3
2,4,7 decatrienal 2,4 heptadienal 2,4 octadienal
16:3 ω4
CO2H
HOO
OHC
-hydrolysis of glycolipids and phospholipids-major affinity toward glycolipids-action on both sn positions-no activity on triglycerides
Lipolytic Acyl Hydrolase(LAH)
![Page 21: Oxylipins in Marine Diatoms - MarBEF · Biosynthetic pahway of oxylipins in marine diatoms. ... Adrianna Ianora Antonio Miralto Giovanna Romano Ylenia Carotenuto University of Salerno](https://reader031.fdocuments.in/reader031/viewer/2022020319/5c6891fd09d3f263648baf8b/html5/thumbnails/21.jpg)
Preliminary data on lipase activity
Aspecific(Comassie Blu)
Cutignano et al., ChemBioChem (submitted)
Specificstaining
SDS-PAGE Native molecular mass determination
215 kDa
The different mass values between denaturing and native conditions suggested:-an oligomeric structure -multi-molecular aggregate
![Page 22: Oxylipins in Marine Diatoms - MarBEF · Biosynthetic pahway of oxylipins in marine diatoms. ... Adrianna Ianora Antonio Miralto Giovanna Romano Ylenia Carotenuto University of Salerno](https://reader031.fdocuments.in/reader031/viewer/2022020319/5c6891fd09d3f263648baf8b/html5/thumbnails/22.jpg)
Oxylipin pathway in plants
![Page 23: Oxylipins in Marine Diatoms - MarBEF · Biosynthetic pahway of oxylipins in marine diatoms. ... Adrianna Ianora Antonio Miralto Giovanna Romano Ylenia Carotenuto University of Salerno](https://reader031.fdocuments.in/reader031/viewer/2022020319/5c6891fd09d3f263648baf8b/html5/thumbnails/23.jpg)
Oxylipin profile in three marine diatoms
LC-MS chromatogramm
![Page 24: Oxylipins in Marine Diatoms - MarBEF · Biosynthetic pahway of oxylipins in marine diatoms. ... Adrianna Ianora Antonio Miralto Giovanna Romano Ylenia Carotenuto University of Salerno](https://reader031.fdocuments.in/reader031/viewer/2022020319/5c6891fd09d3f263648baf8b/html5/thumbnails/24.jpg)
C16-fatty acids
6-HpHTrE 9(S)-HpHTrE
6-LOX
6-KHTrE
6-HHTrE
9(S)-HHTrE
CO2H
OOH
CO2H
OH
CO2H
O
CO2H
O
H
CO2HO
CO2HHO
Monounsaturated derivatives
T. rotula: unprecedented C16 oxylipin pathway
d’Ippolito et al., OBC, 2005 3:4065-4070
octadienal
CO2H
OOH
CO2H
OH
9S-LOX
![Page 25: Oxylipins in Marine Diatoms - MarBEF · Biosynthetic pahway of oxylipins in marine diatoms. ... Adrianna Ianora Antonio Miralto Giovanna Romano Ylenia Carotenuto University of Salerno](https://reader031.fdocuments.in/reader031/viewer/2022020319/5c6891fd09d3f263648baf8b/html5/thumbnails/25.jpg)
CO2H CO2H
C16 fatty acids EPA
Glicolipids
Lipolytic acyl hydrolase (LAH)
Lipoxygenases
Hydroperoxides
Hydroperoxyde lyase
Aldehydesother oxylipins
Biosynthetic pahway of oxylipins in marine diatoms
![Page 26: Oxylipins in Marine Diatoms - MarBEF · Biosynthetic pahway of oxylipins in marine diatoms. ... Adrianna Ianora Antonio Miralto Giovanna Romano Ylenia Carotenuto University of Salerno](https://reader031.fdocuments.in/reader031/viewer/2022020319/5c6891fd09d3f263648baf8b/html5/thumbnails/26.jpg)
Acknowledgements
ICB-CNRAngelo Fontana Guido CiminoAdele Cutignano
SZNAdrianna IanoraAntonio Miralto Giovanna RomanoYlenia Carotenuto
University of SalernoAldo Spinella
IBPE-CNRFerdinando Febbraio
University of BarcelonaPilar Diaz
University of TrentoGraziano Guella