Effect of a T81A mutation at the subunit interface on catalytic properties of alkaline phosphatase from Escherichia coli.

Although alkaline phosphatase (APase) from Escherichia coli crystallizes as a symmetric dimer, it displays deviations from Michaelis-Menten kinetics supported by a model describing a dimeric enzyme with conformationally and kinetically non-equivalent subunits. The proposed model, explaining the mechanism of substrate hydrolysis, encompasses a conformational change mediated by subunit interactions [S. Orhanovic, M. Pavela-Vrancic, Eur. J. Biochem. 270 (2003) 4356-4364]. The significance of interactions at the subunit interface and the involvement of the beta-pleated sheet stretching from underneath the active site to the subunit surface, in the catalytic mechanism, has been probed by site-directed mutagenesis. The mutant APase, carrying alanine in place of Thr81, was analyzed in comparison to the wild-type protein. The T81A mutation, introduced at the subunit interface, significantly affected the protein kinetic properties, emphasizing the importance of subunit interactions in the catalytic process.

Bruton tyrosine kinase-like protein, BtkSD, is present in the marine sponge Suberites domuncula.

Sponges, the simplest and most ancient phylum of Metazoa, encode in their genome complex and highly sophisticated proteins that evolved together with multicellularity and are found only in metazoan animals. We report here the finding of a Bruton tyrosine kinase (BTK)-like protein in the marine sponge Suberites domuncula (Demospongiae). The nucleotide sequence of one sponge cDNA predicts a 700-aa-long protein, which contains all of the characteristic domains for the Tec family of protein tyrosine kinases (PTKs). The highest homology (38% identity, 55% overall similarity) was found with human BTK and TEC PTKs. Sponge PTK was therefore named BtkSD. Human BTK is involved in the maturation of B cells and mutations in the BTK gene cause X-linked agammaglobulinemia. Kinases from the Tec family are not present in Caenorhabditis elegans and, until now, they were found only in insects and higher animal taxa. Our finding implies that the BTK/TEC genes are of a very ancient origin.

Identification of highly conserved genes: SNZ and SNO in the marine sponge Suberites domuncula: their gene structure and promoter activity in mammalian cells(1).

Recently, we reported that cells from the sponge Suberites domuncula respond to ethylene with an increase in intracellular Ca(2+) level [Ca(2+)](i), and with an upregulation of the expression of (at least) two genes, a Ca(2+)/calmodulin-dependent protein kinase and the potential ethylene-responsive gene, termed SDSNZERR (A. Krasko, H.C. Schröder, S. Perovic, R. Steffen, M. Kruse, W. Reichert, I.M. Müller, W.E.G. Müller, J. Biol. Chem. 274 (1999)). Here, we describe for the first time that also mammalian (3T3) cells respond to ethylene, generated by ethephon, with an immediate and transient, strong increase in [Ca(2+)](i). Next, the promoter for the sponge SDSNZERR gene was isolated from S. domuncula. It was found that the SDSNZERR gene is positioned adjacent to the SNZ-related gene (SNZ-proximal open reading frame) (SDSNO) and linked, as in Saccharomyces cerevisiae, in a head-to-head manner. Until now, neither homologues nor orthologues of these two genes have been identified in higher metazoan phyla. The full-length genes share a bidirectional promoter. 3T3 cells were transfected with this promoter; the activity of the SDSNZERR promoter was strong and twice as high as that of the SV40 promoter, while the SDSNO promoter was less active. Surprisingly, the activity of the SDSNZERR promoter could not be modulated by ethylene or salicylic acid while it is strongly upregulated, by 4-fold, under serum-starved conditions. It is concluded that the modulation of the level of [Ca(2+)](i) by ethylene in mammalian cells is not correlated with an upregulation of the ethylene-responsive gene SDSNZERR. The data indicate that in mammalian cells, the activity of the SDSNZERR promoter is associated with the repression of serum-mediated growth arrest.

Modulation of intracellular calcium and proliferative activity of invertebrate and vertebrate cells by ethylene.

BACKGROUND: Ethylene is a widely distributed alkene product which is formed enzymatically (e.g., in plants) or by photochemical reactions (e.g., in the upper oceanic layers from dissolved organic carbon). This gaseous compound was recently found to induce in cells from the marine sponge Suberites domuncula, an increase in intracellular Ca2+ level ([Ca2+]i) and an upregulation of the expression of two genes, the potential ethylene-responsive gene, SDERR, and a Ca2+/calmodulin-dependent protein kinase. RESULTS: Here we describe for the first time, that besides sponge cells, mammalian cell lines (mouse NIH-3T3 and human HeLa and SaOS-2 cells) respond to ethylene, generated by ethephon, with an immediate and strong, transient increase in [Ca2+]i level, as demonstrated using Fura-2 imaging method. A rise of [Ca2+]i level was also found following exposure to ethylene gas of cells kept under pressure (SaOS-2 cells). The upregulation of [Ca2+]i was associated with an increase in the level of the cell cycle-associated Ki-67 antigen. In addition, we show that the effect of ethephon addition to S. domuncula cells depends on the presence of calcium in the extracellular milieu. CONCLUSION: The results presented in this paper indicate that ethylene, previously known to act as a mediator (hormone) in plants only, deserves also attention as a potential signaling molecule in higher vertebrates. Further studies are necessary to clarify the specificity and physiological significance of the effects induced by ethylene in mammalian cells.

Construction and characterization of a Streptomyces rimosus recA mutant: the RecA-deficient strain remains viable.

Although previously reported attempts to construct recA null mutants in Streptomyces spp. have been unsuccessful, we have used the suicide plasmid pErmdeltaRecA to inactivate the recA gene in Streptomyces rimosus by gene disruption. pErmdeltaRecA carries the erythromycin resistance gene ermE and a 451-bp fragment of the S. rimosus recA gene (encoding amino acids 2-151). An erythromycin-resistant clone with single plasmid integration into the recA gene on the chromosome was analyzed in detail. This clone possesses one inactive copy of recA which lacks the entire promoter region and the ATG start codon, and a second, truncated gene that encodes only first 151 amino acids of the RecA protein. This S. rimiosus rec A mutant can therefore be considered a completely RecA-deficient strain. The mutant strain is highly sensitive to UV light. Introduction of a plasmid carrying the wild type S. rimosus recA gene completely restored the UV resistance of the recA mutant to wild-type levels. recA genes encoding RecA proteins with short deletions at the C-terminus (21 and 51 amino acids) could not fully rescue the UV sensitivity of the S. rimosus recA strain, when introduced in the same way.

The mitogen-activated protein kinase p38 pathway is conserved in metazoans: cloning and activation of p38 of the SAPK2 subfamily from the sponge Suberites domuncula.

Our recent data suggest that during auto- and allograft recognition in sponges (Porifera), cytokines are differentially expressed. Since the mitogen-activated protein kinase (MAPK) signal transduction modulates the synthesis and release of cytokines, we intended to identify one key molecule of this pathway. Therefore, a cDNA from the marine sponge Suberites domuncula encoding the MAPK was isolated and analyzed. Its encoded protein is 366 amino acids long (calculated Mr 42 209), has a TGY dual phosphorylation motif in protein kinase subdomain VIII and displays highest overall similarity to the mammalian p38 stress activated protein kinase (SAPK2), one subfamily of MAPKs. The sponge protein was therefore termed p38_SD. The overall homology (identity and similarity) between p38_SD and human p38alpha (CSBP2) kinase is 82%. One feature of the sponge kinase is the absence of threonine at position 106. In human p38alpha MAPK this residue is involved in the interaction with the specific pyridinyl-imidazole inhibitor; T106 is replaced in p38_SD by methionine. Inhibition studies with the respective inhibitor SB 203580 showed that it had no effect on the phosphorylation of the p38 substrate myelin basic protein. A stress responsive kinase Krs_SD similar to mammalian Ste20 kinases, upstream regulators of p38, had already previously been found in S. domuncula. The S. domuncula p38 MAPK is phosphorylated after treatment of the animal in hypertonic medium. In contrast, exposure of cells to hydrogen peroxide, heat shock and ultraviolet light does not cause any phosphorylation of p38. It is concluded that sponges, the oldest and most simple multicellular animals, utilize the conserved p38 MAPK signaling pathway, known to be involved in stress and immune (inflammatory) responses in higher animals.

Origin of insulin receptor-like tyrosine kinases in marine sponges.

One autapomorphic character restricted to all Metazoa including Porifera [sponges] is the existence of transmembrane receptor tyrosine kinases (RTKs). In this study we screened for molecules from one subfamily within the superfamily of the insulin receptors. The subfamily includes the insulin receptors (InsR), the insulin-like growth factor I receptors, and the InsR-related receptors--all found in vertebrates--as well as the InsR-homolog from Drosophila melanogaster. cDNAs encoding putative InsRs were isolated from the hexactinellid sponge Aphrocallistes vastus, the demosponge Suberites domuncula, and the calcareous sponge Sycon raphanus. Phylogenetic analyses of the catalytic domains of the putative RTKs showed that the sponge polypeptides must be grouped with the InsRs. The relationships revealed that all sponge sequences fall into one branch of this group, whereas related sequences from mammals (human, mouse, and rat), insects and molluscs, and polypeptides from one cephalochordate, fall together into a second branch. We have concluded that (i) the InsR-like molecules evolved in sponges prior to the "Cambrian Explosion" and contributed to the rapid appearance of the higher metazoan phyla; (ii) the sponges constitute a monophyletic taxon, and (iii) epidermal growth factor (EGF)-like domains are present in sponges, which allows the insertion of this domain into potential receptor and matrix molecules.

Characterization and phylogenetic analysis of a cDNA encoding the Fes/FER related, non-receptor protein-tyrosine kinase in the marine sponge sycon raphanus.

In search of ancient versions of phylogenetically conserved genes/proteins, which are typical for multicellular animals, we have decided to analyse marine sponges (Porifera), the most ancient and most primitive metazoan organisms. We report here the complete nucleotide sequence of Sycon raphanus cDNA coding for a 879 aa long protein, which displays high overall similarity in primary structure and organization of domains with non-receptor tyrosine kinases (TKs) from the Fes/FER family. The encoded protein, which we named Fes/FER_SR, has a highly conserved, 260 aa long tyrosine kinase domain at the C-terminus. Amino-terminal to the catalytic domain is an 85 aa long SH2 domain. The N-terminus is over 500 aa long and displays homology only with N-terminal domains of protein-tyrosine kinases (PTKs) from the Fes/FER family. Mammalian Fes/FER proteins show around 58% overall homology with Fes/FER_SR (identity and similarity) and lower homology was found with Drosophila melanogaster Fps (FER) protein (49%). Homologies in TK, SH2 and N-terminal domains are on average 78%, 65% and 49%, respectively. Fes/FER_SR shows next to best homology with the Abl family of non-receptor PTKs, while Src-related PTKs from the fresh-water sponge Spongilla lacustris are related only distantly to Fes/FER_SR. Phylogenetic analysis shows that the S. raphanus TK is indeed the most ancient known member of the Fes/FER family of non-receptor PTKs. The role of these PTKs in signal transduction in higher animals is still enigmatic; they are present in the nucleus as well as in the cytoplasm and FER is found in all cell types examined. The function of Fes/FER_SR in sponge, the most primitive multicellular animal which lacks specialized organ systems, remains to be elucidated.

The recA gene from Streptomyces rimosus R6: sequence and expression in Escherichia coli.

The recA gene from Streptomyces rimusus encodes a 376-amino acids polypeptide (M(r) 39,702) that is one of the largest bacterial RecA proteins observed. Detailed analyses of the Streptomyces RecA proteins showed that all possess an additional and unique C-terminal, rich in lysines and alanines, which can form an additional terminal alpha helix. Expression of the S. rimosus RecA protein in Escherichia coli FR333 (delta recA306) was demonstrated using antibodies raised against E. coli RecA protein; expression was possible only from the S. rimosus promoter. A Streptomyces-E. coli-like promoter sequence (TTGACA-18bp-TCTTAT) was found in the A+ T-rich region 135-165 base pairs upstream from the initiation codon and was related to Bacillus subtilis DNA damage-inducible promoters.

Experimental indication in favor of the introns-late theory: the receptor tyrosine kinase gene from the sponge Geodia cydonium.

We have analyzed the gene that encodes receptor tyrosine kinase (RTK) from the marine sponge Geodia cydonium, which belongs to the most ancient and simple metazoan groups, the Porifera. RTKs are enzymes found only in metazoa. The sponge gene contains two introns in the extracellular part of the protein. However, the rest of the protein (transmembrane and intracellular part), including the tyrosine kinase (TK)-domain, is encoded by a single exon. In contrast, all TK genes, so far known only from higher animals (vertebrates), contain several introns especially in the TK-domain. The TK-domain of G. cydonium shows similarity with numerous members of receptor as well as nonreceptor TKs. Phylogenetic analysis of the sponge TK-domain indicates that this enzyme branched off first from the common tree of metazoan TK proteins. Consequently, we assume that introns, found in the TK-domains of genes from higher animals, were inserted into these genes after splitting off the sponge taxa from other metazoan organisms (over 600 million years ago). Our results support the view that ancient genes were not "in pieces."

Cathepsin, a major protease of the marine sponge Geodia cydonium: purification of the enzyme and molecular cloning of cDNA.

Sponges are suspension-feeders that are devoid of body cavities. Phagocytosis is the major route of nutrition in these animals. In an attempt to understand protein digestion, cathepsin was identified in crude extracts from the sponge Geodia cydonium. This enzyme was purified from lysosomes by a two-step procedure--pH precipitation and FPLC separation--to apparent homogeneity; it showed an M(r) of 26,000. Inhibitor as well as substrate studies showed that the sponge cathepsin belongs to the subfamily L of these cysteine proteases. The complete cDNA coding for cathepsin L was isolated and characterized. The deduced aa sequence contains 322 residues, has an M(r) of 36,085, and shows the characteristic signatures known from other cathepsins of the L subfamily: e.g., cleavage site for the proregion, the ERFNIN motif, and the conserved regions forming the catalytic triad of cysteine proteases. Phylogenetic analyses revealed that the sponge sequence groups with the cathepsin L subfamily and branches off first from the other metazoan members. The sponge sequence shows high homology to that isolated from Dictyostelium discoideum and only low similarity to the protozoan cathepsins L from Paramecium tetraurelia and Tetrahymena thermophila. From the data presented it is concluded that cathepsin L is the major digestive protease in sponges.

Regulation of motility of cells from marine sponges by calcium ions.

Sponges are known not to contain muscle and nerve cells. Since sponge cells are characterized by high motility we determined the effect of intracellular calcium ion concentration ([Ca2+]i) on their motility. Addition of the Ca2+ ionophore ionomycin to dissociated cells from the marine sponge Suberites domuncula caused in Ca(2+)-containing artificial seawater (ASW) an increase in motility from 0.2 micron/min (absence of the ionophore) to 3.7 microns/min (presence of ionomycin). When the experiments were performed in Ca(2+)-free medium, no effect of ionomycin could be observed. In parallel experiments the changes of [Ca2+]i using the dye Fura-2 were measured. The experiments revealed that ionomycin causes an influx of Ca2+ into the cytosol of cells suspended in Ca(2+)-containing artificial seawater. In contrast, if cells were suspended in Ca(2+)-free artificial seawater, no increase of [Ca2+]i occurred. Incubation of cells in the presence of inhibitors, specific for endoplasmatic Ca(2+)-ATPase in mammals such as thapsigargin, cyclopiazonic acid, or 2,5 di-t-butylhydrochinone, did not influence the [Ca2+]i if cells were suspended in Ca(2+)-free artificial seawater. From these data we conclude that the [Ca2+]i is primarily regulated through channels in the plasma membrane. In addition we summarize experimental evidence indicating that the [Ca2+]i is involved in the control of cell motility. From the marine sponge Geodia cydonium a partial sequence of the myosin cDNA has been cloned. The deduced amino acid sequence comprises highest homology to nonmuscle myosin type II found in higher invertebrates and vertebrates. Taken together, these data show that the [Ca2+]i level in sponge cells can be modulated by incubation with ionomycin. An increase of the Ca2+ level parallels with higher motility of cells, suggesting an activation of Ca(2+)-dependent protein kinases of myosin type II. Investigations on the ionomycin-activated influx of Ca2+ into the cytosol revealed that predominantly the Ca2+ channels in plasma membrane control the level of [Ca2+]i.

Molecular evolution of the metazoan protein kinase C multigene family.

Protein kinases C (PKCs) comprise closely related Ser/Thr kinases, ubiquitously present in animal tissues; they respond to second messengers, e.g., Ca2+ and/or diacylglycerol, to express their activities. Two PKCs have been sequenced from Geodia cydonium, a member of the lowest multicellular animals, the sponges (Porifera). One sponge G. cydonium PKC, GCPKC1, belongs to the "novel" (Ca2+-independent) PKC (nPKC) subfamily while the second one, GCPKC2, has the hallmarks of the "conventional" (Ca2+-dependent) PKC (cPKC) subfamily. The alignment of the Ser/Thr catalytic kinase domains, of the predicted aa sequences for these cDNAs with respective segments from previously reported sequences, revealed highest homology to PKCs from animals but also distant relationships to Ser/Thr kinases from protozoa, plants, and bacteria. However, a comparison of the complete structures of the sponge PKCs, which are-already-identical to those of nPKCs and cPKCs from higher metazoa, with the structures of protozoan, plant, and bacterial Ser/Thr kinases indicates that the metazoan PKCs have to be distinguished from the nonmetazoan enzymes. These data indicate that metazoan PKCs have a universal common ancestor which they share with the nonmetazoan Ser/Thr kinases with respect to the kinase domain, but they differ from them in overall structural composition.

Immunoglobulin-like domain is present in the extracellular part of the receptor tyrosine kinase from the marine sponge Geodia cydonium.

We have isolated and characterized two cDNAs from the marine sponge Geodia cydonium coding for a new member of a receptor tyrosine kinase of class II. The deduced amino acid sequence shows two characteristic domains: (i) the tyrosine kinase domain; and (ii) an immunoglobulin-like domain. The latter part shows high homology to the vertebrate C2 type immunoglobulin domain. This result demonstrates that immunoglobulin domains are not recent achievements of higher animals but exist also in those animals which have diverged from other organisms about 800 million years ago.

Molecular evidence for the presence of a developmental gene in the lowest animals: identification of a homeobox-like gene in the marine sponge Geodia cydonium.

During the development of higher animals, morphogenetic programs are switched on which are frequently controlled by homeotic genes. Until now these genes have not been identified in the lowest animals, the marine sponges. Since sponges show (i) an antero-posterior and/or dorso-ventral axis during embryogenesis and (ii) a complex differentiation pattern during spicula formation, we hypothesized that in sponges homeotic genes--if present--are also involved in the control of these processes. Therefore, we searched for homeobox or homeobox-like sequences in the marine sponge Geodia cydonium. Here we describe a homeobox-like sequence from these animals; it was isolated from a cDNA library of an adult specimen. The deduced amino acid sequence of the complete homeodomain shares over 70% similarity with other homeodomain sequences, including those from hydra, insects and vertebrates. These data indicate that the sponge homeodomain-like sequence is similar with respect to structure to those of other animals and may suggest that the sponge homeodomain-like sequence(s) might function during developmental processes and/or during spiculogenesis in a similar manner to that known for higher animals.

Phylogenetic relationship of ubiquitin repeats in the polyubiquitin gene from the marine sponge Geodia cydonium.

Ubiquitin is a 76-residue protein which is highly conserved among eukaryotes. Sponge (Porifera) ubiquitin, isolated from Geodia cydonium, is encoded by a gene (termed GCUBI) with six repeats, GCUBI-1 to GCUBI-6. All repeat units encode the same protein (with one exception: GCUBI-4 encodes ubiquitin with a change of Leu to Val at position 71). On the nt level the sequences of the six repeats differ considerably. All changes (except in GCUBI-4) are silent substitutions, which do not affect the protein structure. However, there is one major difference between the repeats: Codons from both codon families (TCN and AGPy) are simultaneously used for the serine at position 65. Using this characteristic the repeats were divided into two groups: Group I: GCUBI-1,3 (TCT codon) and GCUBI-5 (TCC); Group II: GCUBI-2,4,6 (AGC codon). Mutational analysis suggests that the sponge polyubiquitin gene evolved from an ancestral monoubiquitin gene by gene duplication and successive tandem duplications. The ancestral monoubiquitin gene most probably coded for threonine (ACC) at position 65. The first event, duplication of the monoubiquitin gene, happened some 110 million years ago. Since sponges from the genus Geodia are known from the Cretaceous (145 million) to recent time, it is very likely that all events in the evolution of polyubiquitin gene occurred in the same genus. Alignment data of the "consensus" ubiquitin nt sequences of different animals (man to protozoa) reflect very well the established phylogenetic relationships of the chosen organisms and show that the sponge ubiquitin gene branched off first from the multicellular organisms.