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Bruton tyrosine kinase-like protein, BtkSD, is present in the marine sponge Suberites domuncula
Genomics 83 (2004) 743 – 745 www.elsevier.com/locate/ygeno
Short Communication
Bruton tyrosine kinase-like protein, BtkSD, is present in the marine sponge Suberites domuncula H. Cetkovic, a W.E.G. Mu¨ller, b and V. Gamulin a,* b
a Department of Molecular Genetics, Rudjer Boskovic Institute, Bijenicka cesta 54, 10000 Zagreb, Croatia Institut fu¨r Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Johannes Gutenberg Universita¨t, Mainz, Germany
Received 13 June 2003; accepted 10 September 2003
Abstract 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-aalong 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. D 2003 Elsevier Inc. All rights reserved. Keywords: Protein tyrosine kinase; Tec family; Porifera; AY299685
Sponges (Porifera) are excellent model organisms for molecular evolutionary studies. They represent the lowest metazoan phylum that existed prior to the Cambrian explosion [1] and can therefore be considered as living fossils. The analysis of phylogenetically conserved genes/proteins in sponges identified a high amount of very sophisticated proteins, believed to be important for the proper development and functioning of highly evolved and complex multicellular organisms [2 – 7]. Unusually high sequence conservation between sponge and mammalian homologous protein pairs was often observed and furthermore some sponge/human orthologues are absent from the genome of Caenorhabditis elegans [8], although sponges branched off first from the common ancestor of all Metazoa, at least 580 million years ago [1]. One large group of proteins that have evolved together with multicellularity and are found only in animals (Metazoa) is the family of protein tyrosine kinases (PTKs). PTKs have grown in step with the increasing complexity of multicellular organisms and over a hundred PTKs are
* Corresponding author. Fax: +385-1-4561-177. E-mail address: [email protected] (V. Gamulin). 0888-7543/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ygeno.2003.09.010
encoded in the human genome [9]. PTKs play a major role in intercellular communication and transduction of extracellular signals across the plasma membrane into the interior of the cells. They are generally divided into (a) receptor PTKs, transmembrane proteins that transmit signals in response to the binding of extracellular ligands, and (b) nonreceptor PTKs that are involved in transcytoplasmic signaling pathways. Members of both classes of PTKs are present in sponges [3– 7]. PTKs from the Tec family are nonreceptor, cytoplasmic kinases, and in mammals this family includes five members: Btk, Bmx, Itk, Tec, and Txk kinases. Kinases from the Tec family contain at their N-terminus the Pleckstrin homology (PH) domain, followed by the Tec homology (TH) domain, which includes a Zn2 +-binding Bruton tyrosine kinase (BTK) motif and a proline-rich region. Src homology 2 and 3 (SH2 and SH3) domains are in front of the tyrosine kinase (TK) catalytic domain [10,11]. Members of the Tec family are known only in insects (Drosophila and Anopheles), sea urchin, and several vertebrate species [11]. Tec PTKs are not encoded in the C. elegans genome. Extensive search for PTKs in the freshwater sponge Ephydatia fluviatilis [5] identified at least 10 homologues of vertebrate PTKs and not a single member of the Tec family. The detailed functions of Tec kinases are not well defined.
744
H. Cetkovic et al. / Genomics 83 (2004) 743–745
However, it is well known that mutations in the BTK gene cause X-linked agammaglobulinemia (XLA) in human [12,13] and a similar defect is also seen in mice [14,15]. XLA is a severe hereditary immune disease; patients lack circulating B cells and have very low levels of immunoglobulins in the serum. We have used degenerate primers [16] designed to amplify parts of cDNAs encoding TK subdomains VIB –IX of PTKs to perform PCR on the total Suberites domuncula cDNA library prepared in a Lambda ZAP Express vector [6]. Amplified cDNA fragments (around 200 bp long) were cloned and sequenced. The majority of sequenced cDNA fragments indeed encoded parts of TK domains, which was proof that this sponge expresses a variety of PTK genes. In the next step, a S. domuncula cDNA library was plated at high density and
>2 106 plaques were replicated to solid membranes and hybridized with a PCR-amplified and digoxygenin-11 – dUTP-labeled mixture of 200-bp-long cDNA fragments encoding TK subdomains VIB – IX. Numerous positive signals were obtained. From the selected recombinant E phages, pBK-CMV phagemids with cDNA inserts were excised in vivo, amplified in Escherichia coli XLORL cells, and sequenced on the automated DNA sequencer AlfExpress. One cDNA (2610 bp, AY299685) coded for a 700-aminoacid (aa)-long protein with calculated Mr 78721 (Fig. 1). According to a BLAST search, this putative protein displays highest overall homology with human BTK and TEC PTKs. The protein was further analyzed in silico and all subdomains characteristic for Tec family PTKs were identified (Fig. 1): PH domain at the N-terminus, TH domain with BTK motif
Fig. 1. The alignment of BtkSD (AY299685) from the marine sponge Suberites domuncula with human Btk (Q06187) and Tec (P42680) proteins, produced by Clustal X. Amino acids (aa) conserved in all three proteins are shown in white on black and those conserved in two out of three proteins in black on gray. Arrows indicate borders of the five domains. Conserved aa in the PH domain and Btk motif are marked with a star, as well as two tyrosines (Y) whose phosphorylation is involved in the enzymatic activity of the protein. Btk motif and proline rich stretches are boxed.
H. Cetkovic et al. / Genomics 83 (2004) 743–745
and proline-rich regions, SH3 and SH2 domains, and TK domain at the C-terminus. Although human BTK (Q06187) and TEC (P42680) proteins share only 52% identity (70% similarity), the homology of the sponge kinase to the two human PTKs is the same: 38% identity and 55% overall similarity. Btk from Drosophila melanogaster, named Btk29A, exists in two splice forms [17]. However, the PH domain and BTK motif are present only in the longer, Btk29A type2 protein (17136510), which also shows an identical degree of homology (37% identity and 52% similarity) with human BTK and TEC proteins, as well as with the sponge protein. The sponge protein was therefore named BtkSD. Interestingly, BtkSD shares a higher overall homology with two human PTKs (55%) than with the Btk29A type 2 PTK from Drosophila (52%). All of the important and invariant amino acids characteristic of Tec family kinases are perfectly conserved in the sponge protein (Fig. 1). One notable difference is a single amino acid deletion in the BTK motif. In all other known kinases from the Tec family, the Zn2 +-binding BTK motif comprises a gapless stretch of 27 aa [11] and in BtkSD it is only 26 aa long. However, the invariant tyrosine, histidine, glycine, and cysteines are perfectly conserved. The TH domain in BtkSD is longer than in the two human proteins and, except for the BTK motif and two proline-rich stretches, this domain shows poor conservation compared with other TH domains. Our finding implies that the BTK/TEC genes/proteins are of very ancient origin. The gene(s) obviously already existed in the common metazoan ancestor (more than) 600 million years ago, i.e., before the separation of sponges from other animal lineages, and remained highly conserved during animal evolution. The biological role of BtkSD in the simple sponge organisms, lacking any organized tissue, is yet to be elucidated. However, recent studies with human mononuclear cells have shown that PTKs of the Tec family, in particular BTK, are key elements of lipopolysaccharide (LPS)-induced tumor necrosis factor a production [18] and therefore BTK plays an important role in the cellular response to bacterial infections. Molecular response to bacterial infection, or LPS exposure, is very efficient in S. domuncula [19,20] and involves several highly conserved proteins. It is very likely that the basic role of BTK in sponges (as well in mammals) is in antimicrobial defense. References [1] C.W. Li, J.Y. Chen, T.E. Hua, Precambrian sponges with cellular structures, Science 279 (1998) 879 – 882. [2] K. Pfeifer, et al., S-type lectins occur also in invertebrates: unusual
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
745
subunit composition and high conservation of the carbohydrate recognition domain in the lectin genes from the marine sponge Geodia cydonium, Glycobiology 3 (1993) 179 – 184. H. Scha¨cke, et al., Molecular cloning of a tyrosine kinase gene from the marine sponge Geodia cydonium: a new member belonging to the receptor tyrosine kinase class II family, Mol. Membr. Biol. 11 (1994) 101 – 107. H. Cetkovic, I.M. Mu¨ller, W.E.G. Mu¨ller, V. Gamulin, Characterization and phylogenetic analysis of a cDNA encoding the Fes/FER related, non-receptor protein-tyrosine kinase in the marine sponge Sycon raphanus, Gene 216 (1998) 77 – 84. H. Suga, K. Katoh, T. Miyata, Sponge homologs of vertebrate protein tyrosine kinases and frequent domain shufflings in the early evolution of animals before the parazoan – eumetazoan split, Gene 280 (2001) 195 – 201. M. Kruse, I.M. Mu¨ller, W.E.G. Mu¨ller, Early evolution of metazoan serine/threonine and tyrosine kinases: identification of selected kinases in marine sponges, Mol. Biol. Evol. 12 (1997) 1326 – 1334. S. Ottilie, F. Raulf, A. Barnekow, G. Hannig, M. Schartl, Multiple srcrelated kinase genes, srk 1-4, in the fresh water sponge Spongilla lacustris, Oncogene 7 (1992) 1625 – 1630. V. Gamulin, W.E.G. Mu¨ller, I.M. Mu¨ller, Sponge proteins are more similar to those of Homo sapiens than to Caenorhabditis elegans, Biol. J. Linn. Soc. London 71 (2000) 821 – 828. G. Manning, D.B. Whyte, R. Martinez, T. Hunter, S. Sudarsanam, The protein kinase complement of the human genome, Science 298 (2002) 1912 – 1934. A. Takesono, L.D. Finkelstein, P.L. Schwartzberg, Beyond calcium: new signaling pathways for Tec family kinases, J. Cell Sci. 115 (2002) 3039 – 3048. C.I. Smith, et al., The Tec family of cytoplasmic tyrosine kinases: mammalian Btk, Bmx, Itk, Tec, Txk and homologs in other species, Bioessays 23 (2001) 436 – 446. S. Tsukada, et al., Deficient expression of a B cell cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia, Cell 72 (1993) 279 – 290. D. Vetrie, et al., The gene involved in X-linked agammaglobulinaemia is a member of the src family of protein-tyrosine kinases, Nature 361 (1993) 226 – 233. D.J. Rawlings, et al., Mutation of unique region of Bruton’s tyrosine kinase in immunodeficient XID mice, Science 261 (1993) 358 – 361. J.D. Thomas, et al., Colocalization of X-linked agammaglobulinaemia and X-linked immunodeficiency genes, Science 261 (1993) 355 – 358. A. Iwama, K. Okano, T. Sudo, Y. Matsuda, T. Suda, Molecular cloning of a novel receptor tyrosine kinase gene, STK, derived from enriched hematopoietic stem cells, Blood 83 (1994) 3160 – 3169. K. Baba, et al., The Drosophila Bruton’s tyrosine kinase (Btk) homolog is required for adult survival and male genital formation, Mol. Cell Biol. 19 (1999) 4405 – 4413. N.J. Horwood, et al., Bruton’s tyrosine kinase is required for lipopolysaccharide-induced tumor necrosis factor alpha production, J. Exp. Med. 197 (2003) 1603 – 1611. V.A. Grebenjuk, et al., Induction of (2V– 5V) oligoadenylate synthetase in the marine sponges Suberites domuncula and Geodia cydonium by the bacterial endotoxin lipopolysaccharide, Eur. J. Biochem. 269 (2002) 1382 – 1392. M. Bo¨hm, et al., Molecular response of the sponge Suberites domuncula to bacterial infection, Mar. Biol. 139 (2001) 1037 – 1045.
Short Communication
Bruton tyrosine kinase-like protein, BtkSD, is present in the marine sponge Suberites domuncula H. Cetkovic, a W.E.G. Mu¨ller, b and V. Gamulin a,* b
a Department of Molecular Genetics, Rudjer Boskovic Institute, Bijenicka cesta 54, 10000 Zagreb, Croatia Institut fu¨r Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Johannes Gutenberg Universita¨t, Mainz, Germany
Received 13 June 2003; accepted 10 September 2003
Abstract 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-aalong 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. D 2003 Elsevier Inc. All rights reserved. Keywords: Protein tyrosine kinase; Tec family; Porifera; AY299685
Sponges (Porifera) are excellent model organisms for molecular evolutionary studies. They represent the lowest metazoan phylum that existed prior to the Cambrian explosion [1] and can therefore be considered as living fossils. The analysis of phylogenetically conserved genes/proteins in sponges identified a high amount of very sophisticated proteins, believed to be important for the proper development and functioning of highly evolved and complex multicellular organisms [2 – 7]. Unusually high sequence conservation between sponge and mammalian homologous protein pairs was often observed and furthermore some sponge/human orthologues are absent from the genome of Caenorhabditis elegans [8], although sponges branched off first from the common ancestor of all Metazoa, at least 580 million years ago [1]. One large group of proteins that have evolved together with multicellularity and are found only in animals (Metazoa) is the family of protein tyrosine kinases (PTKs). PTKs have grown in step with the increasing complexity of multicellular organisms and over a hundred PTKs are
* Corresponding author. Fax: +385-1-4561-177. E-mail address: [email protected] (V. Gamulin). 0888-7543/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ygeno.2003.09.010
encoded in the human genome [9]. PTKs play a major role in intercellular communication and transduction of extracellular signals across the plasma membrane into the interior of the cells. They are generally divided into (a) receptor PTKs, transmembrane proteins that transmit signals in response to the binding of extracellular ligands, and (b) nonreceptor PTKs that are involved in transcytoplasmic signaling pathways. Members of both classes of PTKs are present in sponges [3– 7]. PTKs from the Tec family are nonreceptor, cytoplasmic kinases, and in mammals this family includes five members: Btk, Bmx, Itk, Tec, and Txk kinases. Kinases from the Tec family contain at their N-terminus the Pleckstrin homology (PH) domain, followed by the Tec homology (TH) domain, which includes a Zn2 +-binding Bruton tyrosine kinase (BTK) motif and a proline-rich region. Src homology 2 and 3 (SH2 and SH3) domains are in front of the tyrosine kinase (TK) catalytic domain [10,11]. Members of the Tec family are known only in insects (Drosophila and Anopheles), sea urchin, and several vertebrate species [11]. Tec PTKs are not encoded in the C. elegans genome. Extensive search for PTKs in the freshwater sponge Ephydatia fluviatilis [5] identified at least 10 homologues of vertebrate PTKs and not a single member of the Tec family. The detailed functions of Tec kinases are not well defined.
744
H. Cetkovic et al. / Genomics 83 (2004) 743–745
However, it is well known that mutations in the BTK gene cause X-linked agammaglobulinemia (XLA) in human [12,13] and a similar defect is also seen in mice [14,15]. XLA is a severe hereditary immune disease; patients lack circulating B cells and have very low levels of immunoglobulins in the serum. We have used degenerate primers [16] designed to amplify parts of cDNAs encoding TK subdomains VIB –IX of PTKs to perform PCR on the total Suberites domuncula cDNA library prepared in a Lambda ZAP Express vector [6]. Amplified cDNA fragments (around 200 bp long) were cloned and sequenced. The majority of sequenced cDNA fragments indeed encoded parts of TK domains, which was proof that this sponge expresses a variety of PTK genes. In the next step, a S. domuncula cDNA library was plated at high density and
>2 106 plaques were replicated to solid membranes and hybridized with a PCR-amplified and digoxygenin-11 – dUTP-labeled mixture of 200-bp-long cDNA fragments encoding TK subdomains VIB – IX. Numerous positive signals were obtained. From the selected recombinant E phages, pBK-CMV phagemids with cDNA inserts were excised in vivo, amplified in Escherichia coli XLORL cells, and sequenced on the automated DNA sequencer AlfExpress. One cDNA (2610 bp, AY299685) coded for a 700-aminoacid (aa)-long protein with calculated Mr 78721 (Fig. 1). According to a BLAST search, this putative protein displays highest overall homology with human BTK and TEC PTKs. The protein was further analyzed in silico and all subdomains characteristic for Tec family PTKs were identified (Fig. 1): PH domain at the N-terminus, TH domain with BTK motif
Fig. 1. The alignment of BtkSD (AY299685) from the marine sponge Suberites domuncula with human Btk (Q06187) and Tec (P42680) proteins, produced by Clustal X. Amino acids (aa) conserved in all three proteins are shown in white on black and those conserved in two out of three proteins in black on gray. Arrows indicate borders of the five domains. Conserved aa in the PH domain and Btk motif are marked with a star, as well as two tyrosines (Y) whose phosphorylation is involved in the enzymatic activity of the protein. Btk motif and proline rich stretches are boxed.
H. Cetkovic et al. / Genomics 83 (2004) 743–745
and proline-rich regions, SH3 and SH2 domains, and TK domain at the C-terminus. Although human BTK (Q06187) and TEC (P42680) proteins share only 52% identity (70% similarity), the homology of the sponge kinase to the two human PTKs is the same: 38% identity and 55% overall similarity. Btk from Drosophila melanogaster, named Btk29A, exists in two splice forms [17]. However, the PH domain and BTK motif are present only in the longer, Btk29A type2 protein (17136510), which also shows an identical degree of homology (37% identity and 52% similarity) with human BTK and TEC proteins, as well as with the sponge protein. The sponge protein was therefore named BtkSD. Interestingly, BtkSD shares a higher overall homology with two human PTKs (55%) than with the Btk29A type 2 PTK from Drosophila (52%). All of the important and invariant amino acids characteristic of Tec family kinases are perfectly conserved in the sponge protein (Fig. 1). One notable difference is a single amino acid deletion in the BTK motif. In all other known kinases from the Tec family, the Zn2 +-binding BTK motif comprises a gapless stretch of 27 aa [11] and in BtkSD it is only 26 aa long. However, the invariant tyrosine, histidine, glycine, and cysteines are perfectly conserved. The TH domain in BtkSD is longer than in the two human proteins and, except for the BTK motif and two proline-rich stretches, this domain shows poor conservation compared with other TH domains. Our finding implies that the BTK/TEC genes/proteins are of very ancient origin. The gene(s) obviously already existed in the common metazoan ancestor (more than) 600 million years ago, i.e., before the separation of sponges from other animal lineages, and remained highly conserved during animal evolution. The biological role of BtkSD in the simple sponge organisms, lacking any organized tissue, is yet to be elucidated. However, recent studies with human mononuclear cells have shown that PTKs of the Tec family, in particular BTK, are key elements of lipopolysaccharide (LPS)-induced tumor necrosis factor a production [18] and therefore BTK plays an important role in the cellular response to bacterial infections. Molecular response to bacterial infection, or LPS exposure, is very efficient in S. domuncula [19,20] and involves several highly conserved proteins. It is very likely that the basic role of BTK in sponges (as well in mammals) is in antimicrobial defense. References [1] C.W. Li, J.Y. Chen, T.E. Hua, Precambrian sponges with cellular structures, Science 279 (1998) 879 – 882. [2] K. Pfeifer, et al., S-type lectins occur also in invertebrates: unusual
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
745
subunit composition and high conservation of the carbohydrate recognition domain in the lectin genes from the marine sponge Geodia cydonium, Glycobiology 3 (1993) 179 – 184. H. Scha¨cke, et al., Molecular cloning of a tyrosine kinase gene from the marine sponge Geodia cydonium: a new member belonging to the receptor tyrosine kinase class II family, Mol. Membr. Biol. 11 (1994) 101 – 107. H. Cetkovic, I.M. Mu¨ller, W.E.G. Mu¨ller, V. Gamulin, Characterization and phylogenetic analysis of a cDNA encoding the Fes/FER related, non-receptor protein-tyrosine kinase in the marine sponge Sycon raphanus, Gene 216 (1998) 77 – 84. H. Suga, K. Katoh, T. Miyata, Sponge homologs of vertebrate protein tyrosine kinases and frequent domain shufflings in the early evolution of animals before the parazoan – eumetazoan split, Gene 280 (2001) 195 – 201. M. Kruse, I.M. Mu¨ller, W.E.G. Mu¨ller, Early evolution of metazoan serine/threonine and tyrosine kinases: identification of selected kinases in marine sponges, Mol. Biol. Evol. 12 (1997) 1326 – 1334. S. Ottilie, F. Raulf, A. Barnekow, G. Hannig, M. Schartl, Multiple srcrelated kinase genes, srk 1-4, in the fresh water sponge Spongilla lacustris, Oncogene 7 (1992) 1625 – 1630. V. Gamulin, W.E.G. Mu¨ller, I.M. Mu¨ller, Sponge proteins are more similar to those of Homo sapiens than to Caenorhabditis elegans, Biol. J. Linn. Soc. London 71 (2000) 821 – 828. G. Manning, D.B. Whyte, R. Martinez, T. Hunter, S. Sudarsanam, The protein kinase complement of the human genome, Science 298 (2002) 1912 – 1934. A. Takesono, L.D. Finkelstein, P.L. Schwartzberg, Beyond calcium: new signaling pathways for Tec family kinases, J. Cell Sci. 115 (2002) 3039 – 3048. C.I. Smith, et al., The Tec family of cytoplasmic tyrosine kinases: mammalian Btk, Bmx, Itk, Tec, Txk and homologs in other species, Bioessays 23 (2001) 436 – 446. S. Tsukada, et al., Deficient expression of a B cell cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia, Cell 72 (1993) 279 – 290. D. Vetrie, et al., The gene involved in X-linked agammaglobulinaemia is a member of the src family of protein-tyrosine kinases, Nature 361 (1993) 226 – 233. D.J. Rawlings, et al., Mutation of unique region of Bruton’s tyrosine kinase in immunodeficient XID mice, Science 261 (1993) 358 – 361. J.D. Thomas, et al., Colocalization of X-linked agammaglobulinaemia and X-linked immunodeficiency genes, Science 261 (1993) 355 – 358. A. Iwama, K. Okano, T. Sudo, Y. Matsuda, T. Suda, Molecular cloning of a novel receptor tyrosine kinase gene, STK, derived from enriched hematopoietic stem cells, Blood 83 (1994) 3160 – 3169. K. Baba, et al., The Drosophila Bruton’s tyrosine kinase (Btk) homolog is required for adult survival and male genital formation, Mol. Cell Biol. 19 (1999) 4405 – 4413. N.J. Horwood, et al., Bruton’s tyrosine kinase is required for lipopolysaccharide-induced tumor necrosis factor alpha production, J. Exp. Med. 197 (2003) 1603 – 1611. V.A. Grebenjuk, et al., Induction of (2V– 5V) oligoadenylate synthetase in the marine sponges Suberites domuncula and Geodia cydonium by the bacterial endotoxin lipopolysaccharide, Eur. J. Biochem. 269 (2002) 1382 – 1392. M. Bo¨hm, et al., Molecular response of the sponge Suberites domuncula to bacterial infection, Mar. Biol. 139 (2001) 1037 – 1045.