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Characterization of Nme6-like gene/protein from marine sponge Suberites domuncula

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Abstract

Nucleoside diphosphate kinases (NDPKs) are evolutionarily conserved enzymes involved in many biological processes such as metastasis, proliferation, development, differentiation, ciliary functions, vesicle transport and apoptosis in vertebrates. Biochemical mechanisms of these processes are still largely unknown. Sponges (Porifera) are simple metazoans without tissues, closest to the common ancestor of all animals. They changed little during evolution and probably provide the best insight into the metazoan ancestors’ genomic features. The purpose of this study was to address structural and functional properties of group II Nme6 gene/protein ortholog from the marine sponge Suberites domuncula, Nme6, in order to elucidate its evolutionary history. Sponge Nme6 gene and promoter were sequenced and analysed with various bioinformatical tools. Nme6 and Nme6Δ31 proteins were produced in E. coli strain BL21 and NDPK activity was measured using a coupled pyruvate kinase-lactate dehydrogenase assay. Subcellular localization in human tumour cells was examined by confocal scanning microscopy. Our results show that the sponge Nme6 compared to human Nme6 does not possess NDPK activity, does not localize in mitochondria at least in human cells although it has a putative mitochondrial signal sequence, lacks two recent introns that comprise miRNAs and have different transcriptional binding sites in the promoter region. Therefore, we conclude that the structure of Nme6 gene has changed during metazoan evolution possibly in correlation with the function of the protein.

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Acknowledgements

This work was funded by Croatian MSES grants 098-0982913-2478 (H. Ćetković) and 098-09824642513 (M. Herak Bosnar).

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Authors and Affiliations

  1. Laboratory of Molecular Genetics, Division of Molecular Biology, Ruđer Bošković Institute, Bijenička cesta 54, P.P. 180, 10002, Zagreb, Croatia

    Drago Perina, Andreja Mikoč & Helena Ćetković

  2. Laboratory of Molecular Oncology, Division of Molecular Medicine, Ruđer Bošković Institute, Bijenička cesta 54, P.P. 180, 10002, Zagreb, Croatia

    Maja Herak Bosnar

  3. Institute for Physiological Chemistry and Pathobiochemistry, Johannes Gutenberg University, Mainz, Germany

    Werner E. G. Müller

Authors
  1. Drago Perina
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  2. Maja Herak Bosnar
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  4. Werner E. G. Müller
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  5. Helena Ćetković
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Correspondence to Maja Herak Bosnar or Helena Ćetković.

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Drago Perina and Maja Herak Bosnar contributed equally to this work

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Online resource 1

Sequences alignment of N-terminal predicted mitochondrial localization signal of Nme6 orthologs from human, rat, chicken, frog, fish, cnidarian, placozoan and sponge. PEM probability of export to mitochondria, CS cleavage site, np not predictable. Boxes indicate cleavage site (JPEG 223 kb)

Online resource 2

Kinase activity of Nme6Sd and Nme6SdΔ31 was determined by measuring the oxidation of NADH in a coupled pyruvate kinase-lactate dehydrogenase enzyme assay system. Reactions were initiated by the addition of 50 ng of enzyme in 50 mM Tris–HCl (pH 7.4), 75 mM KCl, 5 mM MgCl2, 1 mM phosphoenolpyruvate, 0.1 mg/mL NADH, 1 mM ATP, 0.2 mM dTDP, 2 U/mL of pyruvate kinase, 2 U/mL of lactate dehydrogenase and 1 mg/mL BSA. Activity was monitored measuring the decrease in absorbance at 340 nm for 10 min. Top line represents the control reaction (without protein). Nme1 exhibits NDPK activity. In contrast, Nme6Sd and Nme6SdΔ31 exhibit no detectible NDPK activity. All reactions were done in triplicate (JPEG 257 kb)

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Perina, D., Bosnar, M.H., Mikoč, A. et al. Characterization of Nme6-like gene/protein from marine sponge Suberites domuncula . Naunyn-Schmiedeberg's Arch Pharmacol 384, 451–460 (2011). https://doi.org/10.1007/s00210-011-0635-9

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