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Oxylipins in Marine Diatoms
Istituto di Chimica Biomolecolare
Stazione Zoologica “A. Dohrn”
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
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
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
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
Oxylipin pathway in plants
Aldehydes formed through lipoxygenase/hydroperoxidelyase pathway
“Oxylipins” : oxygenated compoundsbiosynthesized from fatty acids byoxygenase-catalyzed oxygenation
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
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
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
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
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
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
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)
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
stdstdControl
TriglyceridesThalassiosira rotula
std
PhospholipidsOHC
decatrienal
std
GlycolipidsOHC
OHCoctadienaldecatrienal
heptadienal
octatrienal
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
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
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
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 ?
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)
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
Oxylipin pathway in plants
Oxylipin profile in three marine diatoms
LC-MS chromatogramm
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
CO2H CO2H
C16 fatty acids EPA
Glicolipids
Lipolytic acyl hydrolase (LAH)
Lipoxygenases
Hydroperoxides
Hydroperoxyde lyase
Aldehydesother oxylipins
Biosynthetic pahway of oxylipins in marine diatoms
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