Desaturases: Extreme cold adaptation and the biological significance in Antarctic organismsEntry ID: UNISA_DIFARMA_DESATURASES
Abstract: It has been shown that thermal stability of proteins is modified by very minor changes in the amino acid (aa) sequence. For ex., for malic dehydrogenase and other proteins, a single aa substitution results in alteration in thermal stability. Among other proteins, those involved in temperature adaptation, heat shock and desaturase proteins are particularly important for the comprehension, at a ... molecular level, of the mechanisms of temperature acclimation. This research project is aimed to further study the mechanisms adaptation and is a extension of previous studies on the mechanism of regulation of heat shock genes. We have shown that the expression of members of this gene family is under the control of membrane physical state (Vigh and Maresca, TIBS, 23: 369-74, 1998) and this in turn largely by the ratio saturated/unsaturated fatty acids. We have shown that desaturases that are responsible for the fine tuning of the SFA/UFA ratio and that can be used, by means of genetic manipulation, to reset the threshold of HS response and are involved in temperature adaptation. We will study under the tenure of this proposal, the biological significance of specific amino acid substitution in the first and second a-elix domain of a delta 9-desaturase in Antarctic fishes and in the krill, as a response to adaptation to extreme cold temperature and to the high fluid state of the membrane domain in which they resides. We have previously studied a delta 9-desaturase gene whose product introduces the first double bond in 16:0 and 18:0 fatty acids producing unsaturated fatty acids (UFA, 16:1 and 18:1) and is a key protein that modifies the phospholipid composition and physical state of the membranes all organisms. So far, analyses of the sequences of delta 9-desaturase proteins have focused primarily on the cytoplasmic regions of this protein. While these studies have shown a characteristic high conservancy in the amino acid sequence in all phyla analyzed, little attention has been given to the less conserved membrane spanning regions. This region is particularly important since the in addition the direct adaptation to the cold temperature, as the cytoplasmic portion of the protein, must also adapt to the membrane lipid bilayer which is a very specialized environment at this extreme and constant temperature. We have cloned and sequenced entirely two delta 9-desaturase genes of the Antarctic fishes Chionodraco hamatus and Trematomus bernacchii (acc. # AJ249579) and studied its physical arrangement.
To examine the level and size of the desaturase transcript, total cellular RNA was isolated from liver spleen and brain of C. hamatus and T. bernacchii. Northern blot analysis indicated that the size of the desaturase transcript is approximately 3.1 kb, moreover, a second transcript of 1.3 kb was detected in liver RNA. The levels of the desaturase transcript is higher in brain samples and lower in liver. Further, we have measured fatty acid content in the same tissues to compare gene activity to the actual UFA content. We reasoned that the delta 9-desaturase may have evolved a specific amino acid sequence in these membrane regions in response to extreme low temperature that could favor the interaction with the highly cold adapted membranes of these fishes. By RT-PCR we amplified the complete delta-9-desaturase ORF. Amplifications were performed on both C. hamatus and T. bernachii, using total RNA purified from liver, spleen and brain. All produced a single band of 1,375 bp. (coding for a polypeptide of 336 aa, only one ORF was obtained from all tissues. On the basis of the data relative to the general folding of membrane bound proteins, we analyzed the trans-membrane domains of delta 9-desaturase.
We identified in the protein distinctive aa residues characteristic of organisms living at low temperature. In particular, we identified in the gene of both fishes 2 Cys residues located in first and second trans-membrane domains. All other desaturases so far cloned lack these two cysteine residues, even in proteins of organisms living in cold but not in extreme and constant temperature. The 2 cyst are separated by 15 aa, a distance that is the canonical space in trans membrane domains that allows the formation of a cys-cys bridge. We have also analyzed the potential evolutionary significance of these 2 cysteine, amplifying and sequencing the corresponding regions of members of other closely related species of the Antarctic fish suborder of Notothenioidei: Pagotenia borchgrivinki, Pagotenia brachycephalum, Pagotenia coriiceps, Pagotenia charcoti and Pagotenia tessellata. All of them contains the cys pairing. In addition, we also sequenced the hortholog region of the gene in E. maclovinus which is a sub-Antarctic species, a member of the closest living sister group that separated from other Antarctic notothenioids before their adaptive radiation into the Antarctic waters. Significantly, E. maclovinus, now geographically limited to the Southern waters of the South American, contains only Cys83. Molecular dynamic calculations have shown that the pairing between trans-membrane helices deriving from Cys-Cys bridge, may affect the anchoring of the protein in the membrane and may influence the general folding and flexibility of the protein.
To study the correlation between protein structure and activity of delta-9-desaturase of C. hamatus in the adaptation to extreme temperatures we performed a complementation analysis of Saccharomyces cerevisiae Ole1- mutant (mat a, Ole1-/-::LEU2, ura3-52,his4) with a plasmid carrying the delta-9-desaturase of C. hamatus under the transcriptional control of S. cerevisiae Ole1 promoter. While the mutant strain grows in the absence of UFA when complemented with the C. hamatus sequence, it grows very slowly at temperatures between 14° and 30°C, and does not grow at 37°C. We could not test lower temperatures because of the chilling sensitivity of the yeast. These results support the model that trans-membrane regions play a crucial role for the functionality of delta 9-desaturase at extreme low temperature of adaptation (Porta et al., submitted). To analyze the roles of the cys-cys bridge of the a 2 helix domains of the delta 9-desaturase gene of Antarctic organisms in response to extreme cold adaptation we made a point mutations that reverted the G residue of the Cys83 codon (TGT) in the desCh coding sequences to the A residue to produce a tyrosine codon (Tyr TAT), and we complemented Ole1- mutant with the mutated gene of C. hamatus Cys83 - Tyr83. The transformant strain still complemented the Ole1- mutant, but grows slower then the yeast transformants with the delta-9-desaturase of C. hamatus.
Cloning and Sequencing of C.hamatus delta-9-desaturase cDNA. During an Italian Antarctic expedition (PNRA) brains, livers and spleens of sacrificed C.hamatus were collected. Organs were obtained from fish grown at the physiological temperature of -1.9°C. Total RNA of organs of C.hamatus have been extracted using Trireagen kit (GIBCO BRL), and then used for Reverse Transcriptase - PCR amplification of delta -9- desaturase cDNA. 5'-and 3'-end primers for RT-PCR will be deduced from available C.hamatus delta-9-desaturase gene (acc. # AJ249579). Specific 3'-end primer have been used to reverse transcribe ca. 5 mg of total RNA, using the SuperScript Preamplification System (GIBCO-BRL). First cDNA strand were amplified by PCR using both the 3'-end primer, used for RT reaction, and the primer specific for 5'-end of the gene. PCR amplified DNA fragment were-amplified by nested PCR using an internal primer to confirm specificity of RT-PCR amplication. PCR product will be cloned into the PCR2.1 vector using the Original TA Cloning Kit (Invtrogen). Cloned fragments were sequenced on both strands using, as seed sequencig points, universal primers present on both side of the cloning site of PCR2.1, and completed by walking on DNA insert. DNA region containing S.cerevisiae Ole1 promoter were amplified by PCR using specific primers and, as template, Yep352/OLE4.8 plasmid. PCR products were purified from agarose gel, using the Ultrafree-DA (Millipore), and subcloned into shuttle vector Yep352.
The resulting plasmid was used to subclone C.hamatus delta-9-desaturase cDNA, under the transcriptional control of S.cerevisiae Ole1 promoter. The harbouring construct was used to transform S. cerevisiae mutant Ole1- (L8-14C). Complementation of S.cerevisiae (Ole1-) mutant using C. hamatus delta-9-desaturase cDNA. S. cerevisiae containing a distrupted delta-9-desaturase (L8-14C) is unable to grow without UFA supplement in the medium. Complementation studies were performed using the S. cerevisiae mutant strain L8-14C (mata, Ole1-/-::LEU2, ura3-52,his4), grown in YPD rich medium [1% yeast extract (Difco), 1% bactopeptone (Difco), 2% glucosio] supplemented with 0.5 mM palmitoleic (16:1) and 0.5 mM oleic acids (18:1). To solubilize fatty acids, 1% tergitol was added to the culture medium. Plasmid, containing Ole1 promoter to drive the expression of C.hamatus delta-9-desaturase cDNA, was used to transform L8-14C strain. Complemented transformants were selected on minimal plates without UFA.
Delta-9-desaturase protein assays
Temperature stability of C.hamatus delta-9-desaturase protein was analyzed in S.cerevisiae Ole1- mutants. Yeasts was grown at 30°C, cells were homogenized and the fraction corresponding ER containing delta-9-desaturase protein, will be collected by differential centrifugation. Delta-9-desaturase enzymatic activity were assayed after incubation of ER fraction at temperatures ranging between 0° and 40°C. Protein activity was quantified by measuring formation of palmitoleic (16:1) and/or oleic (18:1) by separation with TLC on silver nitrate plate using palmitic and/or stearic acids as substrates. Moreover, S.cerevisiae strain, complemented with C.hamatus delta-9-desaturase cDNA was grown at temperatures ranging between 10°C and 40°C, to establish, in vivo, protein functionality at high temperatures.
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Dutra, G.F., G.R. Allen, T. Werner, and S. A. McKenna (Eds.). 2005. A Rapid Marine Biodiversity Assessment of the Abrolhos Bank, Bahia, Brazil. RAP Bulletin of Biological Assessment 38. Conservation International, Washington, DC, USA.
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