Báo cáo khoa học: Functional characterization of front-end desaturases from trypanosomatids depicts the first polyunsaturated fatty acid biosynthetic pathway from a parasitic protozoan ppt

After phylogenetic and structural analysis of the deduced proteins, we predicted that the putative D6 desaturases could have D4 desaturase activity, based mainly on the conserved HX3HH m

trypanosomatids depicts the first polyunsaturated fatty acid biosynthetic pathway from a parasitic protozoan

Karina E J Tripodi, Laura V Buttigliero, Silvia G Altabe and Antonio D Uttaro

Instituto de Biologı´a Molecular y Celular de Rosario (IBR), CONICET, Departamento de Microbiologı´a, Facultad de Ciencias Bioquı´micas y Farmace´uticas, Universidad Nacional de Rosario, Santa Fe, Argentina

Trypanosoma brucei, T cruzi and Leishmania spp are

parasitic protozoa belonging to the order

Kinetoplast-ida They are causative agents of several highly

disab-ling and often fatal diseases, including African sleeping

sickness, Chagas disease and leishmaniasis The drugs

used in the treatment of trypanosomiasis and

leish-maniasis are toxic, and in some cases have low

effect-iveness, which makes imperative the development of

new chemotherapeutic compounds [1,2]

For a number of years it was believed that trypanosomatids are dependent on the host for their lipid content However, recently it was reported that they are able to synthesize de novo their own fatty acids (FA) through a prokaryotic or type II FA synthase [3] This pathway is inhibited in T brucei

by the antibiotic thiolactomycin, which kills the parasite, making it a promising chemotherapeutic target [3]

Keywords

front-end desaturases; Leishmania; lipids;

Trypanosoma; trypanosomatids

Correspondence

A D Uttaro, IBR-CONICET, Dpto

Microbiologı´a, Facultad de Ciencias

Bioquı´micas y Farmace´uticas, Universidad

Nacional de Rosario, Suipacha 531 (2000)

Rosario, Santa Fe, Argentina

Fax: +54 341 4390465

Tel: +54 341 4350661

E-mail: toniuttaro@yahoo.com.ar

(Received 30 September 2005, revised 28

October 2005, accepted 3 November 2005)

doi:10.1111/j.1742-4658.2005.05049.x

A survey of the three kinetoplastid genome projects revealed the presence

of three putative front-end desaturase genes in Leishmania major, one in Trypanosoma brucei and two highly identical ones (98%) in T cruzi The encoded gene products were tentatively annotated as D8, D5 and D6 desaturases for L major, and D6 desaturase for both trypanosomes After phylogenetic and structural analysis of the deduced proteins, we predicted that the putative D6 desaturases could have D4 desaturase activity, based mainly on the conserved HX3HH motif for the second histidine box, when compared with D4 desaturases from Thraustochy-trium, Euglena gracilis and the microalga, Pavlova lutheri, which are more than 30% identical to the trypanosomatid enzymes After cloning and expression in Saccharomyces cerevisiae, it was possible to functionally characterize each of the front-end desaturases present in L major and

T brucei Our prediction about the presence of D4 desaturase activity in the three kinetoplastids was corroborated In the same way, D5 desatu-rase activity was confirmed to be present in L major Interestingly, the putative D8 desaturase turned out to be a functional D6 desaturase, being 35% and 31% identical to Rhizopus oryzae and Pythium irregulare D6 desaturases, respectively Our results indicate that no conclusive pre-dictions can be made about the function of this class of enzymes merely

on the basis of sequence homology Moreover, they indicate that a com-plete pathway for very-long-chain polyunsaturated fatty acid biosynthesis

is functional in L major using D6, D5 and D4 desaturases In trypano-somes, only D4 desaturases are present The putative algal origin of the pathway in kinetoplastids is discussed

Abbreviations

BHT, 2,6-di-tert-butyl-p-cresol; FA, fatty acid; PUFA, polyunsaturated fatty acid.

Trypanosomatids contain the usual range of lipids

found in their eukaryote host (i.e triacylglicerols,

phospholipids, plasmalogens, sterols) but a higher

pro-portion of polyunsaturated fatty acids (PUFAs) [4,5]

This suggests a high membrane fluidity that may be

essential for the parasites in order to adapt themselves

to the dramatic changes in temperature and chemical

parameters experienced during their complex life

cycles

Bloodstream T brucei has an elevated amount of

linoleic acid (18:2) and 22:5 and 22:6 PUFAs, but

lower levels of oleate (18:1) and C16 FAs than the

human host Procyclic T brucei showed lower levels of

16:0 and 18:1, but higher ratios of 18:0 and 18:2 than

those present in the growth medium Remarkably,

they possess 22:5, a PUFA absent from culture media

[4,5] These and other [6,7] data indicate that

trypano-somatids are able to elongate and desaturate de novo

synthesized and exogenously acquired FAs

Leishmania has a high proportion of 18:3 [4],

although at present there is no conclusive evidence for

the isomeric nature of these molecules Meyer & Holz

[8] found 18:3 n-6 as the major FA in L tarentolae

grown on defined media, whose synthesis would

require a D6 desaturase, indicating the presence of the

‘animal pathway’ for PUFA biosynthesis Korn and

coworkers [9,10] found 18:3 n-3 as the main PUFA,

whereas the n-6 form was detected only in trace

amounts This latter finding could indicate the

pres-ence of a D15 or x3 desaturase, compatible with the

‘plant type’ pathway Currently, it is assumed that

both pathways could coexist in trypanosomatids, as

observed for Euglena, the organism most related to

kinetoplastids [4] In both cases, the presence of the

key enzyme D12 or oleate desaturase is imperative for

the de novo synthesis of these PUFAs Early work did

not show conclusive evidence for the presence of

enzymes involved in PUFA biosynthesis in

trypano-somatids, with the exception of oleate desaturase

Linoleate (18:2 n-6) synthesis from radioactive

satur-ated or monounsatursatur-ated FAs has been described for

T cruzi [6] and Leishmania spp [8,10] We have

recently confirmed the presence of this activity, by the

cloning and functional characterization of an oleate

desaturase from T brucei [11] Orthologous genes are

present in T cruzi and L major

Oleate desaturase is absent in mammals, so the

biosynthesis of PUFAs goes through the desaturation

and elongation of essential FAs taken from the diet,

linoleic and a-linolenic (18:3 n-3) acids, which allow

the production of n-6 and n-3 series of PUFAs,

respectively Essential FAs are first desaturated at

position 6 by a D6 desaturase, then elongated to

the corresponding 20:3 n-6 and 20:4 n-3 PUFAs

A D5 desaturase produces 20:4 and 20:5 that are finally converted to 22:5 n-6 and 22:6 n-3 PUFAs by the Sprecher pathway [12], which involves two elon-gation steps up to C24 FAs, a desaturation by the same D6 desaturase and one b-oxidation cycle in peroxisomes

At present, the Sprecher pathway has only been observed in mammals An alternative route was detec-ted in some protozoa, including Euglena gracilis and marine microalgae [13] Here, C20 FAs are elongated

to C22 and then desaturated by a D4 desaturase to the same final 22:5 n-6 and 22:6 n-3 PUFAs

Euglena exhibits another variation in the first steps

of the pathway C18 FAs are elongated to C20, and then a D8 desaturase produces the same kind of C20 FAs that will be the substrate of D5 desaturase Although D8 desaturase was cloned and functionally characterized only from E gracilis [14], the so-called

‘Euglena pathway’ was suggested to be present also

in the ciliate Tetrahymena [15], the marine microalga Isochrysis galbana [16] and the oyster protozoan para-site Perkinsus marinus [17]

The enzymes involved in PUFA biosynthesis (i.e D6, D8, D5 and D4 desaturases) are named ‘front-end’ desaturases, because they introduce a double bond between a pre-existent olefinic bond and the carboxyl end of the FA molecule On the other hand, methyl-end desaturases, such as D12 (x6) and x3 desaturases, introduce a double bond towards the methyl end of the aliphatic chain Front-end desaturases share struc-tural features that make them easy to recognize They contain a fused cytochrome b5 as an N-terminal domain and three histidine boxes in the desaturase (C-terminal) domain The third histidine box has the

QX2HH instead of the HX2HH motif present in other types of desaturases [18]

The genome projects of trypanosomatids revealed the presence of front-end desaturase genes, tentatively annotated as D8, D5 and D6 desaturases, on the basis

of sequence similarity However, functional predictions are never conclusive for desaturases, meaning that bio-chemical characterization is essential for the correct assignment of enzyme regioselectivities

Here, we describe the cloning and functional charac-terization of all the front-end desaturases detected in the T brucei and L major genomes This work allowed

us to assign the correct enzymatic activity to each desaturase and to depict, for the first time, the com-plete pathway for PUFA biosynthesis in a protozoon Moreover, it would help to resolve an old controversy related to the isomeric nature of the PUFAs synthes-ized by these organisms

Identification of putative front-end desaturase

genes in trypanosomatids, and a sequence-based

structural analysis of the encoded proteins

We performed, periodically, blast searches for

desatu-rase genes in the databases of the three

trypanosoma-tid genome projects, using, as queries, the sequences

of previously characterized desaturases present in the

public domain Three open reading frames encoding

amino acid sequences with high similarity to front-end

desaturases were detected in the L major genome

They have the GeneDB accession codes LmjF 36.6950,

LmjF 07.1090 and LmjF 14.1340, and were tentatively

annotated as D8, D5 and D6 desaturases, respectively

Entries for T brucei (Tb 10.6k15.3610) and T cruzi

(Tc00.1047053510181.20 and Tc00.1047053507609.40,

which are 98% identical) share 65–53% identity at the

protein sequence level with LmjF 14.1340 and were

also annotated as D6 desaturases

The protein encoded by LmjF 36.6950 showed

35.6% identity with Rhizopus oryzae D6 desaturase

(AAS93682) and 31% with E gracilis D8 desaturase

(AAQ19605) The LmjF 07.1090 protein is 43.1%

identical to Thraustochytrium sp D5 desaturase

(AAM09687), 40.2% to Ostreococcus tauri D6

rase (AAW70159), 31.3% to Homo sapiens D6

desatu-rase (AAH04901) and 30.2% to Danio rerio D5⁄ D6

bifunctional desaturase (Q9DEX7) The protein

enco-ded by LmjF 14.1340 and its trypanosome orthologue

are 31.5–40.1% identical to Pavlova lutheri D4

desatu-rase (AAQ98793), 36.3–37.6% identical to

Phaeodacty-lum tricornutum D5 desaturase (AAL92562) and 34–

34.2% identical to E gracilis D4 desaturase

(AAQ19605)

All structural features of front-end desaturases are

present in each of these trypanosomatid proteins,

including the three highly conserved histidine boxes

and the cytochrome b5 N-terminal domain (Fig 1)

Interestingly, the second histidine box of LmjF

14.1340, Tb 10.6k15.3610, Tc00.1047053510181.20 and

Tc00.1047053507609.40 has an additional amino acid,

with the motif HX3HH, as found in D4 desaturases

from E gracilis, Pav lutheri and Thraustochytrium sp

(AAN75710) In contrast, one other enzyme known

to have D4 desaturase activity, from I galbana

(AAV33631), possesses an HX2HH motif in its second

histidine box, characteristic of all front-end desaturases,

with the aforementioned exceptions In a phylogenetic

analysis [16], I galbana desaturase was located in a

cluster distinct from all other D4 desaturases,

indica-ting that it has evolved in an independent way, but

reached the same regioselectivity by a convergent evo-lutionary process Considering I galbana an exception, the HX3HH motif could be used to a priori character-ize LmjF 14.1340 and the Trypanosoma proteins as front-end D4 desaturases

Functional characterization of front-end desaturases

Functional characterization was carried out by deter-mining the FA profiles of Saccharomyces cerevisiae transformed either with vector p426GPD alone or with the vector containing an insert harbouring the putative

L major or T brucei front-end desaturases Yeast cul-tures were supplemented with the predicted substrates for each desaturase The absence of any endogenous PUFA makes yeast a suitable expression host for char-acterizing enzymes involved in PUFA biosynthesis The FA composition of the yeast transformed with p426GPD showed the four main FAs normally found

in S cerevisiae, namely 16:0, 16:1D9, 18:0 and 18:1D9 plus the supplemented FA that was incorporated from the culture medium Yeasts expressing the LmjF 14.1340 and Tb 10.6k15.3610 genes, and supplemented with the 22:4 n-6 substrate for D4 desaturase, cis-7,10,13,16 docosatetraenoic acid (22:4 n-6), showed an additional peak when compared with the FA profile of the control harbouring the empty vector (Fig 2) This

Fig 1 Sequence alignment of front-end desaturases by CLUSTALW

[32] Conserved motifs are highlighted in boxes I, II and III are the first, second and third histidine boxes, respectively; cytb5 is the heme-binding motif LmC36, Leishmania major LmjF36.6950 (CAJ09677); D8Eg, Euglena gracilis AAD45877; D6Hs, Homo sapiens AAD31282; D6Pi, Pythium irregulare AAL13310; LmC7, L major LmjF07.1090 (CAJ07076); D5Ts, Thraustochytrium

sp AAM09687; LmC14, L major LmjF14.1340 (CAJ03208); TbC10, Trypanosoma brucei Tb10.6k15.3610 (EAN78117); Tc20, T cruzi Tc00.1047053510181.20 (EAN90580); D4PL, Pavlova lutheri AAQ98793; D4Ts, Thraustochytrium sp AAN75710.

peak was identified as 22:5D4,7,10,13,16, and this was

confirmed by unequivocal determination of the double

bond positions through analysis of the mass spectrum

of the dimethyloxazoline derivative (Fig 2, inset) This

result was a confirmation of our predictions based on

primary structural analysis When the n-3 substrate,

cis-7,10,13,16,19 docosatetraenoic acid, was added to

the culture, it was converted to the corresponding 22:6 D4 product to a similar extent as the n-6 substrate (i.e 4.5–6%) (Table 1) No other FA was shown to be used

as a substrate, indicating that L major and T brucei D4 desaturases are highly specific for D7 PUFAs Using the same approach, the expressed desaturase from LmjF 07.1090 was, as predicted, able to use only FAs that represent the substrates characteristic for D5 desaturases, namely cis-8,11,14 eicosatrienoic acid (20:3 n-6) and cis-8,11,14,17 eicosatetraenoic acid (20:4 n-3), converting them to 20:4D5,8,11,14 (Fig 3) and 20:5D5,8,11,14,17, respectively (Table 1)

Fig 2 Gas chromatography analysis of fatty acid methyl esters

from yeasts expressing Leishmania major Lm14.1340 or

Trypano-soma brucei Tb10.6k15.3610 with exogenous substrate 22:4 n-6.

Cultures of yeast strain HH3 transformed with either the empty

vector (HH3p426) or the vector harbouring the desaturases

(HH3p426-Lm14, HH3p426-Tb10) were supplemented with

22:4D 7,10,13,16 The product of D4 desaturase activity (i.e 22:5

n-6) is highlighted in a box, and the spectrum of the

correspond-ing dimethyloxazoline derivative is shown The double bond in

position 4 is defined by the fingerprint ion at m ⁄ z ¼ 152* [38].

Each feeding experiment was repeated at least twice, and the

results of a representative experiment are shown Analysis of

fatty acid methyl esters was performed by using a SE-30

col-umn, as described in the Experimental procedures.

Table 1 Activity of desaturases on n-3 and n-6 substrates after expression in Saccharomyces cerevisiae strain HH3 The percentage of con-version was calculated taking into account the area resulting from the integration of substrate and product peaks in the respective chromato-gram after running fatty acid methyl ester samples in a PE-WAX column, as described in the Experimental procedures Data represent the mean and standard deviation of three independent determinations.

Substrate fatty acid

Activity (% of conversion)

a Conversion percentage less than 0.5% –, No activity detected.

Fig 3 Gas chromatography analysis of fatty acid methyl esters from yeast expressing Leishmania major Lm07.1090 with exogen-ous substrate 20:3 n-6 Cultures of yeast strain HH3 transformed with either the empty vector (HH3p426) or the vector harbouring the desaturase (HH3p426-Lm07) were supplemented with 20:3D8,11,14 The product of D5 desaturase activity (20:4, n-6), is in

a box and the spectrum of its corresponding dimethyloxazoline derivative is shown Analysis was performed as described in Fig 2 The double bond in position 5 is confirmed by the fingerprint ion at

m ⁄ z ¼ 153* [38], while the remainder are located by the gaps of

12 atomic mass units, as indicated.

Finally, the protein encoded on LmjF 36.6950, and

tentatively annotated as D8 desaturase, was assayed

using the substrates typical for this class of

regioselec-tivity: 20:2D11,14 and 20:3D11,14,17 [14] No activity at

all was evident with these FAs, but a small peak of

16:2D6,9was detected on the chromatogram, suggestive

for the action of a D6 desaturase on the endogenous

16:1D9FA (Fig 4) Curiously, although 18:2D9is a

rel-atively abundant endogenous substrate, we were able

to detect its D6desaturation product only in very small

amounts (less than 0.1%) The addition of linoleic

(18:2D9,12) or a-linolenic (18:3D9,12,15) acids resulted in

the formation of the corresponding 18:3D6,9,12 and

18:4D6,9,12,15FAs, indicating that the real

regioselectiv-ity of the protein encoded by LmjF 36.6950

corres-ponds to that of a D6 desaturase (Fig 4 and Table 1)

Evolutionary relationships of front-end

desaturases from trypanosomatids

In order to gain information about the evolution of

front-end desaturases in trypanosomatids, we

com-pared them with desaturases from a variety of

organ-isms (i.e algae, fungi, marine protists, nematodes,

vertebrates, plants and mosses) A similar detailed

ana-lysis has been performed by Sperling et al [18], but we

included a number of recently described sequences, plus the trypanosomatid sequences, and obtained the phylogenetic tree depicted in Fig 5 D4 desaturases from L major, T brucei and T cruzi group together with D4 and D5 desaturases from fungi, algae and mosses, but form a subgroup with algae desaturases (i.e D4 from Pav lutheri and D5 desaturases from Phaeod tricornutum and Thalassiosira pseudonana) Interestingly, E gracilis D4 desaturase is located in the other subgroup, comprising also the highly related D4 desaturases from the alga Thal pseudonana and Thrau-stochytriumsp., and the D5 desaturases from fungi As previously reported, the D6 desaturase from cyanobac-teria is also present in this group [18] In contrast,

L major D5 desaturase is separated from this cluster;

it is found in a heterogeneous group that includes D5 desaturase from Thraustochytrium sp., D4 desaturase from I galbana and the O tauri D6 acyl-CoA desatu-rase [19]

Lastly, D6 desaturase from L major was found in another major branch, mainly containing D6 desatu-rases from lower and higher eukaryotes and the D8 desaturase from E gracilis It is reasonably clear from the analysis of this branch that D5 desaturases in nem-atodes and vertebrates may have diverged from an ancestral D6 desaturase in each of these lineages after gene duplication L major D6 desaturase is located in

a subgroup containing D6 desaturases from lower euk-aryotes and shows most relationship with E gracilis D8 desaturase and the nematode desaturases The remaining desaturases in this branch form two other groups; one containing vertebrate desaturases and the other formed by desaturases from higher plants and from the alga Thal pseudonana

Discussion

We have functionally characterized all front-end desaturases detected in the genome databases of the three trypanosomatids The completion of these genome projects [20] allowed us to unravel the biosynthetic pathway for PUFAs in these parasitic protozoa In

L major, the pathway involves the consecutive action of D6, D5 and D4 desaturases on C18, C20 and C22 FAs, respectively The n-6 and n-3 isomers were used equally well by the enzymes Recent evidence from our laborat-ory showed that D12 and x3 desaturases are present

in L major (A Alloati and A D Uttaro, unpublished results), indicating that the biosynthesis of 18:2 n-6 and 18:3 n-3 FAs is functional in Leishmania (Fig 6A)

It was shown previously that Leishmania sp mainly accumulates 18:3 FAs, but their isomeric nature remained controversial [4] Our results confirm that

Fig 4 Gas chromatography analysis of fatty acid methyl esters

from yeast expressing Leishmania major Lm36.6950 Cultures

of yeast strain HH3 transformed with the desaturase insert

(HH3p426-Lm36) were fed (+) or not (–) with 18:3D 9,12,15 (A) and

18:2D9,12 (B) The products of D6 desaturase activity are boxed:

18:4 n-3 (A); 18:3 n-6 (B); and 16:2 (A and B) In each case,

dimethyl-oxazoline derivatives were obtained and the double bond positions

in products were ascertained (data not shown) Analysis was

per-formed as described in Fig 2.

both 18:3D6,9,12(n-6) and 18:3D9,12,15(n-3) can be

syn-thesized by L major, but we cannot anticipate the

proportion of each isomer from our heterologous

expression experiments This ratio will depend on the

relative expression of the D6 and x3 desaturases in

different Leishmania species and on the growth condi-tions Interestingly, the groups of Holz and Korn, although both working with the same organism,

L tarentolae, reached opposing conclusions and sugges-ted the presence of the ‘animal’ or ‘plant’ pathway, respectively [8,9] Now, these conclusions have to be considered as an oversimplification, as new evidence showed that alternative pathways are present in lower eukaryotes [13,14,16,18,21] Algae synthesize PUFAs by two different ways, using the combination of D6, D5 and D4 desaturases plus D6 and D5 elongases, or by D8, D5 and D4 desaturases plus D9 and D5 elongases The only complete set of desaturases characterized to date from

an alga is that for Thal pseudonana [22], which has a pathway similar to the one described here for L major

A complete characterization of all the desaturases involved in PUFA biosynthesis in other algae or proto-zoa is still lacking The pathway involving D8

desaturas-es was proposed to be prdesaturas-esent in some algae, such as Isochrysis sp., and in some marine protists [16,17], but only conclusively proved in the protozoan E gracilis [14], which is the organism most related to Kinetoplast-ids, both grouped in the Euglenozoa Interestingly,

L major D6 desaturase showed high similarity to

E gracilis D8 desaturase (Fig 5) On the other hand, the phylogenetic analysis locates L major D4 desaturase

in a subgroup with the same enzymes from trypano-somes and the microalga Pav lutheri, and separated from E gracilis D4 desaturase Unfortunately, D4 desaturase is the only known Pavlova desaturase [23],

Fig 5 Phylogenetic analysis of front-end desaturases The

phylo-genetic tree was created using the neighbour-joining method, with

10 000 replicates, in MEGA -3 [33] For the analysis we used

sequences of desaturases from the trypanosomatids characterized

here, D4 Leishmania major (D4Lm14: CAJ03208), D5 L major

(D5Lm07: CAJ07076); D6 L major (D6Lm36: CAJ09677), D4

Try-panosoma brucei (EAN78117) along with the following sequences:

D4 desaturases from T cruzi (EAN90580), Euglena gracilis

(AAQ19605), Isochrysis galbana (AAV33631), Pavlova lutheri

(AAQ98793), Thraustochytrium sp (AAN75710), Thalassiosira

pseu-donana (AAX14506); D5 desaturases from Caenorhabditis elegans

(AAC95143), Mortierella alpina (AAC72755), Phaeodactylum

tri-cornutum (AAL92562), Pythium irregulare (AAL13311),

Thraustochy-trium sp (AAM09687), Phytophthora megasperma (CAD53323),

Mus musculus (AAH26848), Thal pseudonana (AAX14502),

Physc-omitrella patens (CAH05235), Homo sapiens (AAF29378); D8

de-saturases from E gracilis (AAD45877); D6 dede-saturases from

C elegans (AAC15586), Rhyzopus oryzae (AAS93682), Thal

pseu-donana (AAX14504), P tricornutum (AAL92563), P irregulare

(AAL13310), P patens (CAA11033), Ceratodon purpureus

(CAB94993), H sapiens (AAD31282), M musculus (AAD20017),

Ostreococcus tauri (AAW70159), Borago officinalis (AAD01410),

Primula vialii (AAP23036), Synechocystis sp (Q08871) and D5 ⁄ D6

bifunctional desaturase from Danio rerio (AAG25710) Numbers

represent bootstrap values The bar represents the percentage of

substitutions.

Fig 6 Hypothetical routes followed by Leishmania (A) and trypano-somes (B) for polyunsaturated fatty acid (PUFA) biosynthesis FAD, fatty acid desaturase; Elo, elongase; Elo5, D5 elongase; Elo6, D6 elongase; AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid Light grey ovals are front-end desaturases identified in the present work Grey ovals represent desaturases already characterized by our group White ovals correspond to activ-ities that await characterization.

making it impossible to establish the relationship

between L major D6 and D5 desaturases and related

enzymes from this alga L major D5 desaturase is also

phylogenetically related to algal enzymes such as the

O tauriD6 [19] desaturase and the D5 desaturase from

the marine protist Thraustochytrium sp [24]

Hannaert et al have recently proposed that the high

number of plant-like genes detected in the

trypano-somatid genomes could be explained by an early event

of endosymbiosis into the Euglenozoa, where an alga

acted as the secondary endosymbiont [25] After the

divergence, euglenids have retained a chloroplast as

the only remnant of the alga, whereas kinetoplastid

ancestors have lost the chloroplast, and retained only

nuclear and chloroplast genes that were transferred to

the nucleus We have shown here the phylogenetic

relationship of the trypanosomatid front-end

desatu-rases with algal and Euglena enzymes However, it is

expected that all trypanosomatid front-end desaturases

would be related more to the Euglena counterpart than

to any other organism in order to support that

hypo-thesis, which is not the case for D4 desaturases It may

indicate that more than one event of secondary

endo-symbiont acquisition could have occurred, as suggested

previously [26]

The only front-end desaturase detected in T brucei,

characterized as a D4 desaturase, is able to use n-3

and n-6 FA isomers Two highly identical protein

sequences that share 66% of identity with the T brucei

enzyme were detected in the T cruzi database, most

probably representing the products from orthologous

genes In addition, we have previously characterized an

oleate (D12) desaturase from T brucei [11], and also

detected the T cruzi counterpart No other methyl-end

desaturases could be identified in the trypanosome

genomes This indicates that these parasites are only

able to synthesize 18:2D9,12 (n-6), which is the main

FA in both species Other PUFAs detected in total

lipid extracts from T brucei are 22:5 and 22:6 They

are present in trace amounts, but in a higher

propor-tion with respect to the levels found in the host or in

culture media [4,5] This last observation is in

agree-ment with the existence of a D4 desaturase acting on

exogenous n-6 and n-3 substrates The pathways shown

in Fig 6 involve the presence of putative D6 and D5

elongases in L major, whereas in trypanosomes,

poss-ibly a D5 elongase or no elongase activity would be

present As a consequence, the nature of the exogenous

substrates for trypanosome D4 desaturases can only be

speculated A probable candidate is the relatively

abundant PUFA in the mammalian host, arachidonic

acid (20:4; n-6), which would imply the presence of a

D5 elongase (Fig 6B)

We have shown here that the prediction of front-end desaturase regioselectivity cannot be based merely on sequence similarity or phylogenetic analysis (Fig 5) However, we can gain some structural insights, as new desaturases are functionally characterized It is the case for D4 desaturases, with four new enzymes conclusively characterized, including that recently reported from Thal pseudonana [22] and the three trypanosomatid enzymes described here As discussed previously, Isochrysis sp D4 desaturase appears to have evolved independently, probably from a D5 desaturase, conser-ving the consensus motifs for the three histidine boxes characteristic of D6 and D5 desaturases, which includes the motif HX2HH in the second box The other seven D4 desaturases present a variation in this box with the motif HX3HH This structural characteristic may thus

be taken as a diagnostic feature to identify D4 desatu-rases, although the presence of the canonical HX2HH cannot rule out this kind of regioselectivity

Unsaturated FAs have a key role in maintaining the correct membrane fluidity in poikilothermic organisms, which is necessary for the mobility and function of embedded proteins and in giving shape to membrane curvatures, which in turn are required for the forma-tion of organelles, the vesicular system and the nuclear envelope They represent more than 70% of total FAs

in trypanosomatids [27] This fact could indicate that the unsaturated FA biosynthetic pathway is essential

in these parasitic protozoa This is not unexpected con-sidering the complexity of their life cycles, where they have to adapt to dramatic changes of temperature and morphology It may be very interesting to evaluate the possibility of using this pathway as a putative chemo-therapeutic target The high proportion of C18 mono- and polyunsaturated FAs can be ascribed to the physiological processes described above, but the role of C20 and C22 PUFAs is more difficult to explain In mammals, PUFAs are converted to eicosa-noids, like prostaglandins and leukotrienes They are potent mediators involved in numerous homeostatic biological functions and inflammation Two recent reports highlighted the significance of prostaglandins

in trypanosomatid life cycles [28,29] Prostaglandins are produced from arachidonic acid, both in T brucei bloodstream forms and in L major promastigotes, and

a role of prostaglandins in the pathogenesis of the dis-eases was suggested An additional function for C22 FAs could be assigned as trypanosomes have con-served a D4 desaturase This function cannot be antici-pated, although it is known that some PUFAs act as second messengers in other organisms Recent experi-ments on T brucei and L major showed that free arachidonic acid might induce the mobilization of

Ca2+ and other nonspecific effects This role could

also be shared by linoleic acid and linolenic acid [30]

Experimental procedures

a-Linolenic (18:3, D9,12,15); linoleic (18:2, D9,12); cis-11,

14-eicosadienoic; cis-11,14,17-eicosatrienoic;

cis-8,11,14-eico-satrienoic and cis-7,10,13,16-docosatetraenoic acids (all

more than 99% pure); Tergitol (type Nonidet P-40);

sodium methoxide; ampicillin, yeast nitrogen base; glucose;

amino acids; 2-amino-2-methyl-1-propanol (95%) and

2,6-di-tert-butyl-p-cresol (BHT) were obtained from Sigma

(Sigma-Aldrich, St Louis, MI, USA)

Cis-7,10,13,16,19-do-cosapentaenoic acid and cis-8,11,14,17-eicosatetraenoic acid

were from Cayman Chemical Company (Ann Arbor, MA,

USA) All organic solvents were purchased from Merck

(Whitehouse Station, NJ, USA)

Cloning, sequencing and sequence analysis

Front-end desaturase gene sequences were retrieved from the

databases of the trypanosomatid genome projects and

analysed using tools available online (http://www.genedb.org

and http://www.ncbi.nlm.nih.gov/BLAST)

Promastigote L major (Friedlin strain) and procyclic

T brucei(strain 427) cells were grown in SDM-79 medium

supplemented with 10% fetal bovine serum and hemin [31]

Genomic DNA from both sources was prepared by

stand-ard methods Four sets of primers were designed to amplify

three types of front-end desaturases: Lm14 forward, 5¢-CG

for Lm14.1340; Lm07 forward, 5¢-CGGGATCCATGGCC

CTCGACAATGTCC-3¢ and Lm07 reverse; 5¢-CCAAGCT

TAGTTCCCAGCAACGATGAA-3¢ for Lm07.1090; Lm36

5¢-CGGGATCCATGGTCTTCGAGCTCACTC-3¢ and Lm36 reverse, 5¢-CCAAGCTTCTACTTCCCGCTC

TTGGCCTC3¢ for Lm36.6950; and Tb10 forward, 5¢-CG

Tb10 reverse, 5¢-CCAAGCTTCACAACCGTTTGTCTTC

TAT-3¢ for Tb 10.6k15.3610 The underlined sequences

rep-resent BamHI sites for forward primers and HindIII sites

for reverse primers, with the exception of Lm14 reverse,

where EcoRI was introduced; oligonucleotides also include

the natural initiation and stop codons Amplifications were

carried out in a 50 lL volume under the following

condi-tions: initial incubation at 94
C for 4 min, followed by 30

cycles of denaturation at 94
C for 1 min, annealing at

60
C (for Lm14.1340, Lm07.1090 and Tb10.6k15.3610

desaturases) or 58
C (for Lm36.6950 desaturase) for 30 s,

and extension at 72
C for 2 min Amplified fragments were

cloned using pGEM-T Easy vector (Promega, Madison,

WI, USA), according to the manufacturer’s procedure, and

transformed into competent Escherichia coli (XL1-Blue)

Plasmids purified from positive clones were sequenced completely

Phylogenetic analyses

Available front-end desaturase protein sequences were aligned using clustalw [32] Positions with gaps were removed Phylogenetic analyses were carried out by the neighbour-joining method using the program mega3, ver-sion 3.0 [33] with 10 000 bootstrap samplings or by mini-mum evolution with 5000 bootstrap replicates Both methods gave very similar tree topologies

Expression of front-end desaturase genes

Cloned sequences were ligated into the BamHI and HindIII (or EcoRI) sites of p426GPD, a yeast expression vector containing the glyceraldehyde-3-phosphate dehydrogenase constitutive promoter [34] A selectable marker gene in this vector confers uracil prototrophy to the host Either the vector alone, or the vector harbouring different desaturases,

was used to transform S cerevisiae strain HH3 (MATa,

trp1-1, ura3-52, ade2-101, his3-200, lys2-801, leu2-1 [35]) by electroporation Transformed clones were selected on min-imal agar plates lacking uracil [36]

In order to determine enzyme activities, transformed yeasts were cultured for 2 days at 30
C in 0.67% (w ⁄ v) yeast nitrogen base, 2% (w⁄ v) glucose and leucine, and tryptophan, lysine, adenine and histidine (all at

20 mgÆL)1), and inoculated Cultures were diluted to an absorbance at 600 nm of 0.2, and grown for 72 h at

20
C with constant agitation FAs were prepared in eth-anol containing BHT at a stock concentration of 2% (w⁄ v) and added to a final concentration of 0.002% (w ⁄ v) into 20 mL cultures containing 0.2% (v⁄ v) Tergitol (type Nonidet P-40)

Fatty acid analysis

Twenty millilitre cultures were centrifuged at 5000 g for

5 min, and pelleted cells were washed twice with an equal volume of distilled water Afterward, lipids were extracted

as described by Bligh & Dyer [37] The organic phase was dried under N2, and fatty acid methyl esters were obtained

by incubation with 1 mL of 0.5 m sodium methoxide in methanol for 20 min at room temperature Following neut-ralization with 6 m HCl and extraction with 2 mL of hex-ane, the solvent was evaporated to dryness under a N2 atmosphere Alternatively, dimethyloxazoline derivatives were prepared by adding 0.25 g of 2-amino-2-methyl-1-pro-panol to up to 2 mg of lipid sample, as described by Chris-tie [38] In both cases, after evaporation of the solvent, the product was dissolved in isohexane containing BHT (50 p.p.m.) for GC-MS analysis

The composition of fatty acid methyl esters was analysed

by running samples through a polyethylene glycol column

(PE-WAX; 30 m· 0.25 mm inside diameter; Perkin Elmer,

Norwalk, CT, USA) isothermically at 180
C, or through an

SE-30 column (25 m· 0.22 mm inside diameter; Scientific

Glass Engineering, Ringwood, Australia) for 4 min

isother-mically at 164
C, followed by a temperature increase, at a

rate of 4
CÆmin)1, to 215
C Gas chromatographic analysis

was performed with a Perkin Elmer AutoSystem XL gas

chromatograph, and GC-MS was carried out in a Perkin

El-mer mass detector (model TurboMass) at a ionization

volt-age of 70 eV with a scan range of 20–500 Da Retention

times and mass spectra of peaks obtained were compared

with those for standards (Sigma) or with those available on

NBS75K (National Bureau of Standards database, Perkin

Elmer) Dimethyloxazoline derivatives were submitted to

GC through an SE-30 column After holding the

tempera-ture at 150
C for 3 min, the column was

temperature-pro-grammed to increase to 320
C at a rate of 4
CÆmin)1

Helium was the carrier gas, at a constant flow rate of 1 mLÆ

min)1or 1.2 mLÆmin)1for the SE-30 and the PE-WAX

col-umn, respectively MS was used in the electron impact mode

at 70 eV with a scan range of 40–500 Da

Acknowledgements

We wish to thank Mo´nica Hourcade and Daniel Elı´as

for technical assistance, and Paul A M Michels for

comments and suggestions on the manuscript We also

acknowledge the Trypanosoma brucei, T cruzi and

Leishmania major genome projects, The Institute of

Genomic Research (TIGR) and The Sanger Institute,

for the availability of sequence data S.G.A and

A.D.U are members of Carrera del Investigador

Cien-tı´fico, CONICET, Argentina K.E.J.T has a

postdoc-toral fellowship from ANTORCHAS and CONICET,

Argentina This work was supported by Fondo

Nac-ional de Ciencia y Tecnologı´a, SECyT, Argentina, by

grants PICT 99 No.1-7160 and PICT 03

No.1-13842

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