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