Proteins of the (hemo-)globin superfamily have been identified in many different animals but also occur in plants, fungi, and bacteria. Globins are renowned for their ability to store and to transport oxygen, but additional globin functions such as sensing, signaling, and detoxification have been proposed. Recently, we found that the zebrafish globin X protein is myristoylated and palmitoylated at its N-terminus. The addition of fatty acids results in an association with the cellular membranes, suggesting a previously unrecognized globin function. In this study, we show that N-terminal acylation likely occurs in globin proteins from a broad range of phyla. An N-terminal myristoylation site was identified in 90 nonredundant globins from Chlorophyta, Heterokontophyta, Cnidaria, Mollusca, Arthropoda, Nematoda, Echinodermata, Hemichordata, and Chordata (including Cephalochordata), of which 66 proteins carry an additional palmitoylation site. Bayesian phylogenetic analyses identified five major globin families, which may mirror the ancient globin diversity of the Metazoa. Globin X-like proteins form two related clades, which diverged before the radiation of the Eumetazoa. Vertebrate hemoglobin (Hb), myoglobin, cytoglobin, globin E, and globin Y form a strongly supported common clade, which is the sister group of a clade consisting of invertebrate Hbs and relatives. The N-terminally acylated globins do not form a single monophyletic group but are distributed to four distinct clades. This pattern may be either explained by multiple introduction of an N-terminal acylation site into distinct globin lineages or by the origin of animal respiratory globins from a membrane-bound ancestor. Similarly, respiratory globins were not monophyletic. This suggests that respiratory globins might have emerged independently several times and that the early metazoan globins might have been associated with a membrane and carried out a function that was related to lipid protection or signaling.
The family of vertebrate globins includes hemoglobin, myoglobin, and other O2-binding proteins of yet unclear functions. Among these, globin X is restricted to fish and amphibians. Zebrafish (Danio rerio) globin X is expressed at low levels in neurons of the central nervous system and appears to be associated with the sensory system. The protein harbors a unique N-terminal extension with putative N-myristoylation and S-palmitoylation sites, suggesting membrane-association. Intracellular localization and transport of globin X was studied in 3T3 cells employing green fluorescence protein fusion constructs. Both myristoylation and palmitoylation sites are required for correct targeting and membrane localization of globin X. To the best of our knowledge, this is the first time that a vertebrate globin has been identified as component of the cell membrane. Globin X has a hexacoordinate binding scheme and displays cooperative O2 binding with a variable affinity (P 50∼1.3–12.5 torr), depending on buffer conditions. A respiratory function of globin X is unlikely, but analogous to some prokaryotic membrane-globins it may either protect the lipids in cell membrane from oxidation or may act as a redox-sensing or signaling protein.
Studies of Marenzelleria species were often hampered by identification uncertainties when using morphological characters only. A newly developed PCR/RFLP protocol allows a more efficient discrimination of the three species Marenzelleria viridis, Marenzelleria neglecta and Marenzelleria arctia currently known for the Baltic Sea. The protocol is based on PCR amplification of two mitochondrial DNA gene segments (16S, COI) followed by digestion with restriction enzymes. As it is faster and cheaper than PCR/sequencing protocols used so far, the protocol is recommended for large-scale analyses. The markers allow an undoubted determination of species irrespective of life stage or condition of the worms in the samples. The protocol was validated on about 950 specimens sampled at more than 30 sites of the Baltic and the North Sea, and on specimens from populations of the North American east coast. Besides this test we used mitochondrial DNA sequences (16S, COI, Cytb) and starch gel electrophoresis to further investigate the distribution of the three Marenzelleria species in the Baltic Sea. The results show that M. viridis (formerly genetic type I or M. cf. wireni) occurred in the Ö resund area, in the south western as well as in the eastern Baltic Sea, where it is found sympatric with M. neglecta. Allozyme electrophoresis indicated an introduction by range expansion from the North Sea. The second species, M. arctia, was only found in the northern Baltic Sea, where it sometimes occurred sympatric with M. neglecta or M. viridis. For Baltic M. arctia, the most probable way of introduction is by ship ballast water from the European Arctic. There is an urgent need for a new genetic analysis of all Marenzelleria populations of the Baltic Sea to unravel the current distribution of the three species.
Since 1985, the nonindigenous polychaete species Marenzelleria neglecta has been found in the Baltic Sea. The species, which was introduced by ship ballast water, spreads rapidly and dominates in many habitats today. Using three gene segments of the mitochondrial DNA (16S rDNA, Cytochrom oxidase I, Cytochrom b), we investigated four populations of the western and northern Baltic Sea in a preliminary survey and compared them with four other populations from the North Sea, the Baltic Sea and from the Arctic. First, we could demonstrate the applicability of the markers to discriminate the species with certainty. Second, with M. viridis and M. arctia, we could detect two more species of the same genus, which have recently been introduced into the Baltic Sea. One of these, M. arctia, was hitherto known as an exclusive arctic member of the genus. The impact of these two recently invaded Marenzelleria species onto the autochthonous fauna needs to be evaluated in the future. The Baltic Sea as a 'natural aquarium' now offers the possibility to investigate sibling species simultaneously. However, correct identification and denomination of Marenzelleria species are indispensable prerequisites for all future studies. Molecular markers allow the exact identification of all Marenzelleria species and must be used whenever a classical taxonomic identification is uncertain.
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