RESEARCH ARTICLE
EF-Hand Proteins and the Regulation of Actin-Myosin Interaction in the Eutardigrade Hypsibius klebelsbergi (Tardigrada) THIRUKETHEESWARAN PRASATH, HARTMUT GREVEN, AND JOCHEN D’HAESE∗ ¨ Institute of Cell Biology, Heinrich-Heine-University, Dusseldorf, Germany
ABSTRACT
Many tardigrade species resist harsh environmental conditions by entering anhydrobiosis or cryobiosis. Desiccation as well as freeze resistance probably leads to changes of the ionic balance that includes the intracellular calcium concentration. In order to search for protein modifications affecting the calcium homoeostasis, we studied the regulatory system controlling actin–myosin interaction of the eutardigrade Hypsibius klebelsbergi and identified full-length cDNA clones for troponin C (TnC, 824 bp), calmodulin (CaM, 1,407 bp), essential myosin light chain (eMLC, 1,015 bp), and regulatory myosin light chain (rMLC, 984 bp) from a cDNA library. All four proteins belong to the EF-hand superfamily typified by a calcium coordinating helix-loop-helix motif. Further, we cloned and obtained recombinant TnC and both MLCs. CaM and TnC revealed four and two potential calcium-binding domains, respectively. Gel mobility shift assays demonstrated calcium-induced conformational transition of TnC. From both MLCs, only the rMLC showed one potential N-terminal EF-hand domain. Additionally, sequence properties suggest phosphorylation of this myosin light chain. Based on our results, we suggest a dual-regulated system at least in somatic muscles for tardigrades with a calcium-dependent tropomyosin-troponin complex bound to the actin filaments and a phosphorylation of the rMLC turning on and off both actin and myosin. Our results indicate no special modifications of the molecular structure and function of the EF-hand proteins in tardigrades. Phylogenetic trees of 131 TnCs, 96 rMLCs, and 62 eMLCs indicate affinities to Ecdysozoa, but also to some other taxa suggesting that our results reflect the complex evolution of these proteins rather than phylogenetic relationships. J. Exp. Zool. 317:311–320, 2012. © 2012 Wiley Periodicals, Inc.
How to cite this article: Prasath T, Greven H, D’Haese J. 2012. EF-hand proteins and the regulation of actin-myosin interaction in the eutardigrade Hypsibius klebelsbergi (tardigrada). J. Exp. Zool. 317:311–320, 2012 J. Exp. Zool. 317:311–320.
Tardigrades (water bears) are minute metazoans now systematically placed within the monophyletic Ecdysozoa. Their interrelationship within Ecdysozoa is still uncertain. Either tardigrades are associated with the Cycloneuralia, particularly nematodes (Meusemann et al., 2010) or are Panarthropoda, which also include Onychophora and Arthropoda (Campbell et al., 2011). Biochemical experiments of tardigrades are hampered due to their small size (average approximately 500 μm) and the lack of a convenient culture system to obtain large amounts of animals for protein isolation. We recently constructed a cDNA
library of the eutardigrade Hypsibius klebelsbergi (Kiehl et al., 2007), a species that lives exclusively on glaciers in cryoconite Additional Supporting Information may be found in the online version of this article. ∗ Correspondence to: Jochen D’Haese, Institute of Cell Biology, Depart¨ ment Biology, Heinrich-Heine-University Dusseldorf, Universit¨atsstrasse 1, ¨ D-40225 Dusseldorf, Germany. E-mail: [email protected] Received 23 October 2011; Revised 29 January 2012; Accepted 16 February 2012 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jez.1724
© 2012 WILEY PERIODICALS, INC.
312 holes (Dastych et al., 2003) and obtained the complete nucleotide sequence of one actin gene and its deduced amino acid sequence (D’Haese et al., 2011). In continuation of these studies, we herein report data about four EF-hand proteins that trigger by calcium binding the interaction of actin–myosin. An increase in Ca2+ -concentration within the cytoplasm from 10−7 to 10−5 M induces the switching from the resting to the active state of muscles (Niggli, ’99). EF-hand proteins are characterized by a helix-loop-helix motif with a “canonical” consensus pattern of 12 amino acid residues spanning the loop and the beginning of the second helix. Within these 12 residues, Ca2+ is bound mainly via oxygen-containing amino acid side chains or alpha-carbonyl oxygen from the polypeptide backbone at positions 1[X], 3[Y], 5[Z], 7[−Y], 9[−X], and 12[−Z] (Bhattacharya et al., 2004). Muscle contraction is also controlled by EF-hand regula¨ tory proteins linked to actin and/or myosin (Szent-Gyorgyi, ’75; Lewit-Bentley and Rety, 2000). The proteins involved are calmodulin (CaM), troponin C (TnC), and the essential myosin light chain and regulatory myosin light chain (eMLC and rMLC). All these proteins are believed to have a common ancestor that contained four calcium-binding EF-hand domains. In the modern forms, typically one or more of the EF-hand domains of these proteins have lost the ability to bind Ca2+ (Kretsinger, ’80). The most widespread actomyosin control in animals is mediated by the actin-associated regulatory proteins troponin (Tn) and tropomyosin (Tm), which can sterically block myosin-binding sites on actin. Unblocking is initiated by calcium binding to the Tn subunit TnC (Gordon et al., 2000). Myosin-linked control is constituted by its associated myosin light chains (MLCs) that are wrapped around the lever arm of the myosin head by binding to an IQ (isoleucine, glutamine) sequence motif: IQxxxRGxxxR (Mooseker and Cheney, ’95). In molluscan- and annelid-muscle myosin is activated by direct calcium binding to eMLC and rMLC, ¨ respectively (Carlhoff and D’Haese, ’88; Szent-Gyorgyi et al., ’99; Himmel et al., 2009). In vertebrate smooth muscle and in nematode and arthropod-striated muscles as well as in nonmuscle cells myosin control occurs by phosphorylation of the rMLC (Harris et al., ’77; Takahashi et al., ’90; Seller, ’91). During desiccation or freezing considerable changes of ionic balance, including changes in intracellular calcium concentration, are expected. In the present study, we describe the nucleotide and amino acid sequences of four EF-hand proteins that potentially regulate muscle contraction in tardigrades and searched for specific modifications in view of harsh environmental conditions like anhydrobiosis or cryobiosis. Additionally, we obtained recombinant proteins of TnC, eMLC, and rMLC. We compare our findings with corresponding data of several Non-Ecdysozoa and Ecdysozoa to learn more about the evolution and/or phylogenetic significance of these proteins by constructing phylogenetic trees. J. Exp. Zool.
PRASATH ET AL.
METHODS Library-Based Identification of cDNA Clones For the identification of cDNAs coding for CaM, TnC, and both MLCs, a series of primers (Supplementary Table S1) were designed based on conserved regions from pairwise and multiple alignments of hexapod, chelicerate, nematode, and crustacean sequences. The cDNA lambda phage library (Titer: 6 × 109 pfu/mL) of H. klebelsbergi was used as a template for the amplification of cDNA fragments by PCR (Kiehl et al., 2007). We used premixed PCR master mixes (Peqlab, Germany) adding 1 μL library and 0.5 μM of each primer with a total sample volume of 25 μL. Amplification was run with 15 min initial denaturation at 94◦ C followed by 35 cycles of 30 sec denaturation at 94◦ C, 1 min annealing at 50–55◦ C and 3 min elongation at 72◦ C. The program ended with a final incubation step for 10 min at 72◦ C. PCR products were separated in 1.2% agarose gels in TBE (90 mM Tris, 90 mM boric acid, 2 mM EDTA, 1 μg/mL ethidiumbro¨ mide) and custom sequenced by Seqlab (Gottingen, Germany). The resulting PCR fragments were cloned into pJET 1.2/blunt vector using CloneJET PCR Cloning kit (Fermentas, Germany) and transformed into TOP F10 cells (Invitrogen, Germany). Individual colonies were picked as templates for colony PCR reactions employing the supplemented primers pJet-forward (5 -CGACTCACTATAGGGAGAGCGGC-3 ) and pJet-reverse (5 AAGAACATCGATTTTCCATGGCAG-3 ). Products within the expected size range were sequenced and verified by comparison with expressed sequence tags (ESTs) of H. dujardini. By this strategy, partial cDNA sequences were identified and used to obtain complete cDNA sequences by combination of cDNA-specific and vector-based (Lambda TriplEx2) primers. All primers were synthesized by MWG-Biotech AG (Ebersberg, Germany). Cloning and Expression PCR products containing the open reading frame (ORF) of TnC and both MLCs were introduced into the PCR T7/NT TOPO TA expression system (Invitrogen, Germany). The expression construct was transformed into E. coli BL21 cells supplemented in the kit. A single colony was grown overnight in 10 mL LB medium containing 100 μg/mL ampicillin with shaking (250 rpm) at 37◦ C. 250 mL of LB medium were inoculated the next day with 5 mL of the overnight culture and incubated with shaking (250 rpm) at 37◦ C until an OD600 of 0.6 was obtained. Cultures were then induced with 1 mM isopropyl-β-d-thiogalactopyranoside and grown for another 4 hr. Cells were harvested by centrifugation at 6,000 × g for 10 min. The cell pellet was washed twice by suspending it in wash buffer 1 (50 mM Na2 HPO4 , 300 mM NaCl, pH 8.0) and centrifuged as before. Preparation and Protein Refolding of Inclusion Bodies Washed cell mass was collected by centrifuging at 15,000 × g, 4◦ C for 15 min. The pellet was suspended in wash buffer 1
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and lysed by sonication (3 × 20 sec pulses, with 1 min at 4◦ C between each sonication). After centrifugation at 12,000 × g for 15 min at 4◦ C, undissolved inclusion bodies were collected, washed twice with wash buffer 2 (PBS containing 0.5 [v/v] Triton X-100, 10 mM EDTA, and 50 mM NaCl, pH 7.4) and centrifuged at 12,000 × g for 5 min at 4◦ C. Inclusion bodies were then solubilized in 5 mM NaCl, 20 mM Tris-Maleate pH 7.9, 1 mM DTT, and 2 M guanidine hydrochloride. Protein refolding was achieved by dialyzing twice against 5 mM NaCl, 20 mM TrisMaleate pH 6.8, 5 mM 2-Mercaptoethanol. For analysis, samples were clarified by centrifugation at 12,000 × g for 30 min.
by sequencing and pairwise alignment with ESTs of H. dujardini, which showed 95–98% identity of their nucleotide and deduced amino acid sequences. All sequences contain a 5 and 3 untranslated region terminating in poly(A) tails. Sequences obtained have been deposited in GenBank (Accession Numbers: eMLC, HM628686; rMLC, HM628687; TnC, HM628688, and CaM, HM628689). For further analysis, TnC and the MLCs were obtained by recombinant protein expression and purified from inclusion bodies as described in Methods. The yield of purified recombinant protein was about 15% for TnC and 5% for both MLCs of the E. coli cell protein.
Gel Electrophoresis Expressions were analyzed by sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% gel according to Laemmli (’70). Calcium-induced mobility shift analysis was carried out by Urea-PAGE using a 7.5% gel containing 2 M urea after the method originally described by Creighton (’79). Probes were preincubated in 1 mM CaCl2 or 5 mM EGTA. Gels were stained with Coomassie Brilliant Blue G250 (Serva, Heidelberg, Germany). An unstained protein ladder (PageRulerTM , Fermentas, Germany) was used as molecular size marker.
Calmodulin The cDNA obtained for CaM was 1,407 nucleotides in length with an ORF of 447 bp (149 amino acids) and a calculated molecular weight of 16,836.64 Da. As typical for CaM, it is a highly acidic protein (pI of 4.11) due to a high content of aspartate and glutamate residues (24.8%). It lacks tryptophan and cysteine like most CaMs studied so far. Putative structure predictions (Fig. 1) reveal a dumbbell-shaped structure with Nand C-terminal globular regions linked by a long continuous α-helix. Each region contains two canonical helix-loop-helix EF-hand domains. All four Ca2+ -binding loops always show aspartate at both coordinates X and Y, while glutamate is to be found at coordinate Z. Furthermore, coordinates Z and −X are occupied by essential oxygen-containing side chain residues (Fig. 2 and Supplementary Table S2).
Sequence Analysis All protein sequences were retrieved from the UniProt database (http://www.uniprot.org). EST sequences of H. dujardini were retrieved from Tardibase (http://xyala.cap.ed.ac.uk/ tardigrades/tardibase.html) and from the Tardigrade workbench (http://waterbear.bioapps.biozentrum.uni-wuerzburg.de; see ¨ Forster et al., 2009). Blastn and tBlastx programs (http://www.ncbi.nlm.nih.gov) were used to search for similarity in NCBI databases and ClustalW (http://www.ebi.ac.uk/clustalw) was utilized to compare sequences (Altschul et al., ’97; Larkin et al., 2007).
RESULTS Searching for tardigrade EF-hand proteins only EST sequences from H. dujardini are documented in Tardibase and Tardigrade workbench (five TnC, 19 eMLC, and 13 rMLC sequences). No CaM data of tardigrades were found in any database. Sequence analysis of eMLC-ESTs revealed two different sequence clusters with about 90% nucleotide and deduced amino acid identity, whereas the TnC- and the rMLC-ESTs represented only one sequence each. All EST sequences shared 50–57% identity with TnC and both MLCs of various hexapods, chelicerates, nematodes, and crustaceans. In order to screen the H. klebelsbergi lambda phage library for Ca2+ -binding EF-hand proteins involved in regulation of actin–myosin interaction, conserved nucleotide regions were selected as PCR primer targets. We identified cDNA clones for all four EF-hand proteins by PCR-based screening. The authenticity of potential cDNA fragments amplified by PCR was confirmed
Troponin C The identified TnC clone had an 824 bp cDNA insert encoding a protein with an ORF of 498 bp (166 amino acids). It has a calculated molecular weight of 17,354.3 Da (apparent molecular mass of 19 kDa, Fig. 3B). The deduced amino acid sequence shows an excess of aspartate (11.8%) and glutamic acid (13.2%) that results in a pI of 4.1. The putative 3D model (Supplementary Fig. S1) displays similar to CaM a dumbbell shape with distinct N- and C-terminal regions linked by an α-helix with a short-loop segment (residues 81–85). Each region contains two EF-hand domains. EF-hand I differs from the canonical pattern at coordinate Z and −Z occupied by glutamine and at −X by histidine. EF-hand domain III shows glutamine at coordinate Y, where, however, an aspartate or asparagine is preferred (Supplementary Table S2). EF-hand domains II and IV display a canonical pattern (Fig. 3A). Gel mobility shift analysis of the recombinant protein showed that Ca2+ induced a considerable shift (Fig. 3B). This demonstrates calciuminduced conformational changes of the recombinant TnC of H. klebelsbergi. Protein BLAST search showed the highest similarity within the C-terminal region of arthropod and nematode TnCs with the highest score and 68% identity with the horseshoe crab Tachypleus tridentatus (GenBank Accession Number P15159). J. Exp. Zool.
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PRASATH ET AL. Essential Myosin Light Chain The eMLC cDNA clone is 1,015 bp long and contains an ORF of 498 bp. It encodes an acidic protein of 166 amino acids (pI of 4.7) with a calculated molecular mass of 18,473.1 Da (apparent molecular mass of 20 kDa). Sequence analysis revealed two C-terminally located noncanonical EF-hand domains (Fig. 4 and Supplementary Table S2; for the putative 3D model see Supplementary Fig. S2). No calcium-dependent electrophoretic mobility shift could be seen (Fig. 4B). BLAST search showed the highest score and 50% identity with the tick Haemaphysalis qinghaiensis (GenBank Accession Number AAV41826).
Figure 1. Putative 3D structure of H. klebelsbergi calmodulin. All four EF-hand domains display a canonical Ca2+ -binding loop pattern (for details see text). Helices E (blue), F (green), and calciumbinding loops (red) are highlighted (EFh1–EFh4) in the predicted 3D model, based on template PDB ID: 2fp2 using SWISS-MODEL. N- and C-termini are indicated. Secondary structure predictions and 3D models were constructed using the SWISS-MODEL server (http://www.expasy.org/swissmod/SWISS-MODEL.html). Figures were drawn in Swiss-PDB viewer and rendered with POV-ray software (Guex and Peitsch, ’97; Arnold, 2006).
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Regulatory Myosin Light Chain The rMLC cDNA clone (984 bp) contains an ORF of 525 bp. The full-length translation product is predicted to be an acidic protein of 175 amino acids (pI of 4.87) with a calculated molecular mass of 18,924.3 Da. The apparent molecular mass of the recombinant protein calculated from SDS-PAGE was 18 kDa (Fig. 5B). The N-terminal region shows a phosphorylatable motif with threonine-serine at position 16 and 17. Both residues are flanked upstream by a consensus recognition sequence of positively charged amino acids including KKR xxR xxS xVF (position 9–20) essential for the catalytic activity of myosin light chain kinases. Each N- and C-terminus shows a single calcium-binding EF-hand domain. EF-hand I contains a canonical Ca2+ -binding loop pattern. EF-hand III shows at Ca2+ -binding loop coordinates Z and −Z amino acid substitutions with threonine and proline that lead to the loss of essential, negatively charged residues (Fig. 5A and Supplementary Table S2; for the putative 3D model, see Supplementary Fig. S3). A database search with BLAST revealed remarkable conservation with rMLCs of invertebrates with the highest score and 58% identity with the ants Camponotus floridanus (GenBank Accession Number EFN63580) and Harpegnathos saltator (GenBank Accession Number EFN80420). Notably, the rMLC of H. klebelsbergi exhibits an outstanding similarity to vertebrate smooth muscle (54% Homo sapiens). The highest conservation lies within the phosphorylatable threonine-serine region and the canonical EF-hand domain I (Supplementary Table S3). Electrophoretic mobility shift analysis shows only a slight mobility shift induced by Ca2+ of the recombinant rMLC of H. klebelsbergi (Fig. 5B).
DISCUSSION Calmodulin The identified CaM clone of H. klebelsbergi shares 99% amino acid identity with Drosophila melanogaster, Caenorhabditis elegans, the earthworm Lumbricus rubellus, the slug Aplysia californica, and remarkable 98% with Homo sapiens and Gallus
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Figure 2. EF-hand domains of H. klebelsbergi calmodulin. All four EF-hand domains (I–IV) display a canonical Ca2+ -binding loop pattern. Calcium coordinating key residues (X, Y, Z, −Y, −X, and −Z) that comply with the consensus pattern (for details see text) are boldunderlined. Helices E (dark gray), F (light gray), and calcium-binding loops (black) are shown as a block above the sequence. (The chosen sequence labeling for the EF-hand domains is likewise used for the troponin C, essential myosin light chain and regulatory myosin light chain figures.)
gallus. Differences are seen at position 99 and 143, where invertebrate CaMs contain phenylalanine, whereas H. sapiens, G. gallus, and other vertebrates show tyrosine (Klee et al., ’80). Our results confirm that CaM is highly conserved (Lewit-Bentley and Rety, 2000). Comparison of the four canonical EF-hand domains of CaM of H. klebelsbergi revealed the highest internal similarity between domains EF-hand I and EF-hand III (55%) and EF-hand II and EF-hand IV (59%). This characteristic is consistent with the concept that CaM evolved from an ancestral CaM gene with a single EF-hand by two tandem duplications (Kretsinger, ’80; Klee and Vanaman, ’82). CaM is involved in the regulation of numerous physiological pathways mediated by Ca2+ and interacts with a number of proteins by forming Ca2+ -dependent complexes (Means, ’88; James et al., ’95; Maria et al., ’99). Many unconventional myosins use several CaM molecules as light chains wrapped around the lever arm of the myosin head (Hasson and Mooseker, ’96). CaM of H. klebelsbergi is apparently a functional protein capable of binding four calcium ions and mediating the Ca2+ -signal similarly as in other organisms (Chin and Means, 2000) and reveals no special modifications in tardigrades.
bind Ca2+ during regulation while the C-terminal sites play a structural role in anchoring TnC to the troponin complex (Gordon et al., 2000). Due to substitutions of individual essential Ca2+ -coordinating residues, TnC of invertebrate muscles studied so far reveal the loss of two or even three Ca2+ -binding EF-hand domains: e.g., the crayfish Astacus leptodactylus (Kobayashi et al., ’89), the crustacean Balanus nubilis (Allhouse et al., ’99) or the nematode C. elegans (Ueda et al., 2001). These three are predicted to have a Ca2+ -specific binding site at EF-hand domain II and IV. Remarkably, the scallop Platinopecten yessoensis shows a functional temperature-sensitive TnC protein with only one Ca2+ -binding site at EF-hand domain IV (Ojima et al., 2000). The TnC cDNA of H. klebelsbergi shows two canonical calcium-binding EF-hand domains: one in the N-terminal region (EF-hand II) and one in the C-terminal region (EF-hand IV). Gel mobility shift analysis showed a calcium-induced conformational transition of the recombinant TnC of H. klebelsbergi. We expect TnC of H. klebelsbergi to regulate the “on” and “off” state of the actin filaments together with tropomyosin as do the TnCs from other organisms.
Troponin C A prerequisite of TnCs regulatory function is the reversible binding of Ca2+ in the narrow range of variations of the intracellular Ca2+ concentration. Vertebrate skeletal muscle TnC has four Ca2+ -binding EF-hand domains: two low affinity domains (I and II) in the N-terminal region that are Ca2+ specific and two high affinity domains (III and IV) in the C-terminal region that bind both Ca2+ and Mg2+ . N-terminal domains I and II reversibly
Essential Myosin Light Chain The cDNA of the eMLC of H. klebelsbergi reveals two noncanonical C-terminal EF-hand domains (III and IV). So far, only molluscan eMLCs (e.g., the scallop Argopecten irradians) have been shown to be capable of direct Ca2+ binding to trigger mus¨ cle contraction (Szent-Gyorgyi et al., ’99). Although the scallop eMLC contains one canonical EF-hand domain (EF-hand III), it has been shown that the region necessary for Ca2+ binding J. Exp. Zool.
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Figure 3. (A) The troponin C (TnC) of H. klebelsbergi shows two canonical calcium-binding EF-hand domains, EF-hand domain II, and EF-hand domain IV (for details see text). For sequence labeling see Fig. 2. (B) SDS-PAGE and Urea-PAGE mobility-shift analysis of the recombinant TnC of H. klebelsbergi with an apparent molecular mass of 19 kDa. Lane M: protein marker; lane 1: supernatant, soluble fraction; lane 2: pellet; lane 3: purified recombinant protein. Purified protein was incubated in 1 mM calcium [C] and in 5 mM EGTA [E].
and regulation exclusively resides in the noncanonical EF-hand ¨ domain I (Szent-Gyorgyi et al., ’99). However, a complex comprising the myosin lever arm and both light chains is necessary for regulation (Himmel et al., 2009).
Regulatory Myosin Light Chain Hypsibius klebelsbergi rMLC displays one canonical N-terminal calcium-binding EF-hand domain (EF-hand I). Although all the rMLCs we compared comply with the canonical calcium-binding loop pattern in their first EF-hand domain, the rMLC from obliquely striated body-wall muscle of the earthworm Lumbricus terrestris is the only known rMLC that regulates myosin J. Exp. Zool.
by direct binding of Ca2+ (Carlhoff and D’Haese, ’88). All but Pecten sp.-rMLCs exhibit a prolonged N-terminus of about 10– 15 basic residues, with L. terrestris having an outstanding high number of about 40. Comparing the rMLC-protein sequence of H. klebelsbergi with those of Mollusca, Vertebrata, Arthropoda, Annelida, and Nematoda, (Table S3) the highest sequence similarity is to the rMLCs of Arthropoda nonmuscle (49–54%) and notably Vertebrata smooth muscle (50–54%). The amino acid comparison exhibits two regions, of considerable conservation. The first region extending from position 11–58 of the H. klebelsbergi sequence, corresponds to the amino acids flanking the phosphorylatable threonine-serine motif and the canonical calciumbinding EF-hand domain I. The second region, extending from
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Figure 4. (A) The essential myosin light chain (eMLC) of H. klebelsbergi shows two noncanonical C-terminal calcium-binding EF-hand domains, EF-hand domain III, and EF-hand domain IV. Probable remnants of the ancestral EF-hand domains I and II are indicated. For sequence labeling see Fig. 2. (B) SDS-PAGE and Urea-PAGE mobility-shift analysis of the recombinant eMLC of H. klebelsbergi with an apparent molecular mass of 20 kDa. Lane M: protein marker; lane 1: supernatant, soluble fraction; lane 2: pellet; lane 3: purified recombinant protein. Purified protein was incubated in 1 mM calcium [C] and in 5 mM EGTA [E].
position 77–128, comprises the ancestral EF-hand domain II. Sequence properties suggest that the rMLC of H. klebelsbergi identified in this study is phosphorylatable. The sequence shows a phosphorylatable threonine-serine motif flanked by a consensus recognition sequence of positively charged amino acids including KKR xxR xxS xVF essential for the catalytic activity of myosin light chain kinase (Ikebe et al., ’94; Gao et al., ’95; Kamm and Stull, 2001). Phosphorylation of rMLC controls vertebrate smooth and nonmuscle myosin filament assembly as well as the interaction of myosin with actin (Sweeney, ’98). The rMLCs of skeletal and cardiac muscle are phosphorylated, yet this appears to play a minor modulatory
role (Szczesna et al., 2002). Studies on molluscan myosin reveal that Ca2+ -binding properties of both myosin light chains are al¨ tered by interaction with myosin heavy chains (Szent-Gyorgyi, ’99). This finding demonstrates that protein–protein interaction can influence Ca2+ -binding properties. The relative abundance of muscles in eutardigrades makes it probable that the regulatory proteins we describe can be assigned to muscle tissue especially to the somatic muscles (see Marcus, ’29; Schmidt-Rhaesa and Kulessa, 2007; Halberg et al., 2009). Somatic muscles of tardigrades represent an intermediate type between smooth muscles and obliquely striated muscles (Walz, ’74). But other muscle types (e.g., pharyngeal J. Exp. Zool.
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Figure 5. (A) The regulatory myosin light chain (rMLC) of H. klebelsbergi shows one canonical calcium-binding EF-hand domain (EF-hand domain I) and a consensus myosin-light-chain-kinase recognition motif (KKRxxRxxSxVF). Probable remnants of the ancestral EF-hand domains II and IV are indicated. For sequence labeling see Fig. 2. (B) SDS-PAGE and Urea-PAGE mobility-shift analysis of the recombinant rMLC of H. klebelsbergi with an apparent molecular mass of 18 kDa. Lane M: protein marker; lane 1: supernatant, soluble fraction; lane 2: pellet; lane 3: purified recombinant protein. Purified protein was incubated in 1 mM calcium [C] and in 5 mM EGTA [E].
bulb-, stylet-, and leg-muscle; see Halberg et al., 2009) and even nonmuscle cells present in tardigrades may also be the source of the four EF-hand proteins. Our data support that at least the somatic muscle is dual regulated by calcium binding to TnC and phosphorylation of the rMLC. The presence of a TnI-like protein has recently been demonstrated in the somatic muscle but it was absent in the pharyngeal muscle (Obinata et al., 2011). These findings suggest the possibility of diverse regulatory mechanisms in somatic and pharyngeal muscle. J. Exp. Zool.
Further study is required to localize both regulatory systems and determine their contribution to muscle and nonmuscle contractility in tardigrades.
CONCLUSIONS Studies of CaM, TnC, and both MLCs isoforms from invertebrates (for summary see Hooper and Thuma, 2005) suggest a diverse isoform pattern for H. klebelsbergi in nonmuscle and different muscle types. However, we assume that we have identified the major isoforms for all four EF-hand proteins expressed in the
EF-HAND PROTEINS OF TARDIGRADES somatic muscle of this tardigrade. We propose a dual-regulated system at least in somatic muscles for tardigrades. The CaM, TnC, and rMLC of H. klebelsbergi comply with sequence properties in which calcium triggers a Tn–Tm system and a CaM-mediated phosphorylation of the rMLC that turns on myosin as well as actin. Our results indicate no special modifications of EF-hand protein structure and function of tardigrades exposed to extreme environmental conditions. The phylogenetic trees (Supplementary Fig. S4) obtained from 131 TnC-, 96 rMLC-, and 62 eMLC-protein sequences appear to support some current views on tardigrade relationships. For example, the TnC tree indicates affinities to Ecdysozoa in general (nematodes and arthropods e.g., Garey et al., ’96; Giribet et al., ’96; Aguinaldo et al., ’97; Mallatt et al., 2004) and, within Ecdysozoa to nematodes (Philippe et al., 2005; Dunn et al., 2008; Meusemann et al., 2010). The rMLC tree suggests relationships with nematodes, whereas the eMLC tree suggests affinities with Annelida, i.e., “Articulata” and Mollusca. The protein-specific variations among these trees, however, suggest they reflect the complex evolution of muscle-regulatory proteins according to their various functional demands, as opposed to being useful for revealing phylogenetic relationships.
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PRASATH ET AL. Mooseker MS, Cheney RE. 1995. Unconventional myosin. Annu Rev Cell Dev Biol 11:633–675. Niggli E. 1999. Localized intracellular calcium signaling in muscle: calcium sparks and calcium quarks. Annu Rev Physiol 61:311–335. Obinata T, Ono K, Ono, S. 2011. Detection of a troponin I-like protein in non-striated muscle of the tardigrades (water bears). BioArchitecture 1:96–102. Ojima T, Koizumi N, Ueyama K, Inoue A, Nishita K. 2000. Functional role of Ca2+ -binding site IV of scallop troponin C. J Biochem 128:803–809. Philippe H, Lartillot N, Brinkmann H. 2005. Multigene analyses of bilaterian animals corroborate the monophyly of Ecdysozoa, Lophotrochozoa, and Protostomia. Mol Biol Evol 22:1246–1253. Schmidt-Rhaesa A, Kulessa J. 2007. Muscular architecture of Milnesium tardigradum and Hypsibius sp. (Eutardigrada, Tardigrada) with some data on Ramazzottius oberh¨auseri. Zoomorphology 126:265– 281. Sellers, JR. 1991. Regulation of cytoplasmic and smooth muscle myosin. Curr Opin Cell Biol 3:98—104. Sweeney HL. 1998. Regulation and tuning of smooth muscle myosin. Am J Respir Crit Care Med 158:S95–S99. Szczesna D, Zhao J, Jones M, Zhi G, Stull J, Potter JD. 2002. Phosphorylation of the regulatory light chains of myosin affects Ca2+ sensitivity of skeletal muscle contraction. J Appl Physiol 92:1661– 1670. Szent-Gy¨orgyi AG. 1975. Calcium regulation of muscle contraction. Biophys J 15:707–723. Szent-Gy¨orgyi AG, Kalabokis VN, Perreault-Micale CL. 1999. Regulation by molluscan myosins. Mol Cell Biochem 190:55–62. Takahashi S, Takano-Ohmuro H, Maruyama K. 1990. Regulation of Drosophila myosin ATPase activity by phosphorylation of myosin light chains-I. Wild-type fly. Comp Biochem Physiol B 95:179–181. Ueda T, Katsuzaki H, Terami H, Ohtsuka H, Kagawa H, Murase T, Kajiwara Y, Yoshioka O, Iio T. 2001. Calcium-bindings of wild type and mutant troponin Cs of Caenorhabditis elegans. Biochim Biophys Acta 1548:220–228. Walz B. 1974. The fine structure of somatic muscles of Tardigrada. Cell Tissue Res 149:81–89.
EF-Hand Proteins and the Regulation of Actin-Myosin Interaction in the Eutardigrade Hypsibius klebelsbergi (Tardigrada) THIRUKETHEESWARAN PRASATH, HARTMUT GREVEN, AND JOCHEN D’HAESE∗ ¨ Institute of Cell Biology, Heinrich-Heine-University, Dusseldorf, Germany
ABSTRACT
Many tardigrade species resist harsh environmental conditions by entering anhydrobiosis or cryobiosis. Desiccation as well as freeze resistance probably leads to changes of the ionic balance that includes the intracellular calcium concentration. In order to search for protein modifications affecting the calcium homoeostasis, we studied the regulatory system controlling actin–myosin interaction of the eutardigrade Hypsibius klebelsbergi and identified full-length cDNA clones for troponin C (TnC, 824 bp), calmodulin (CaM, 1,407 bp), essential myosin light chain (eMLC, 1,015 bp), and regulatory myosin light chain (rMLC, 984 bp) from a cDNA library. All four proteins belong to the EF-hand superfamily typified by a calcium coordinating helix-loop-helix motif. Further, we cloned and obtained recombinant TnC and both MLCs. CaM and TnC revealed four and two potential calcium-binding domains, respectively. Gel mobility shift assays demonstrated calcium-induced conformational transition of TnC. From both MLCs, only the rMLC showed one potential N-terminal EF-hand domain. Additionally, sequence properties suggest phosphorylation of this myosin light chain. Based on our results, we suggest a dual-regulated system at least in somatic muscles for tardigrades with a calcium-dependent tropomyosin-troponin complex bound to the actin filaments and a phosphorylation of the rMLC turning on and off both actin and myosin. Our results indicate no special modifications of the molecular structure and function of the EF-hand proteins in tardigrades. Phylogenetic trees of 131 TnCs, 96 rMLCs, and 62 eMLCs indicate affinities to Ecdysozoa, but also to some other taxa suggesting that our results reflect the complex evolution of these proteins rather than phylogenetic relationships. J. Exp. Zool. 317:311–320, 2012. © 2012 Wiley Periodicals, Inc.
How to cite this article: Prasath T, Greven H, D’Haese J. 2012. EF-hand proteins and the regulation of actin-myosin interaction in the eutardigrade Hypsibius klebelsbergi (tardigrada). J. Exp. Zool. 317:311–320, 2012 J. Exp. Zool. 317:311–320.
Tardigrades (water bears) are minute metazoans now systematically placed within the monophyletic Ecdysozoa. Their interrelationship within Ecdysozoa is still uncertain. Either tardigrades are associated with the Cycloneuralia, particularly nematodes (Meusemann et al., 2010) or are Panarthropoda, which also include Onychophora and Arthropoda (Campbell et al., 2011). Biochemical experiments of tardigrades are hampered due to their small size (average approximately 500 μm) and the lack of a convenient culture system to obtain large amounts of animals for protein isolation. We recently constructed a cDNA
library of the eutardigrade Hypsibius klebelsbergi (Kiehl et al., 2007), a species that lives exclusively on glaciers in cryoconite Additional Supporting Information may be found in the online version of this article. ∗ Correspondence to: Jochen D’Haese, Institute of Cell Biology, Depart¨ ment Biology, Heinrich-Heine-University Dusseldorf, Universit¨atsstrasse 1, ¨ D-40225 Dusseldorf, Germany. E-mail: [email protected] Received 23 October 2011; Revised 29 January 2012; Accepted 16 February 2012 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jez.1724
© 2012 WILEY PERIODICALS, INC.
312 holes (Dastych et al., 2003) and obtained the complete nucleotide sequence of one actin gene and its deduced amino acid sequence (D’Haese et al., 2011). In continuation of these studies, we herein report data about four EF-hand proteins that trigger by calcium binding the interaction of actin–myosin. An increase in Ca2+ -concentration within the cytoplasm from 10−7 to 10−5 M induces the switching from the resting to the active state of muscles (Niggli, ’99). EF-hand proteins are characterized by a helix-loop-helix motif with a “canonical” consensus pattern of 12 amino acid residues spanning the loop and the beginning of the second helix. Within these 12 residues, Ca2+ is bound mainly via oxygen-containing amino acid side chains or alpha-carbonyl oxygen from the polypeptide backbone at positions 1[X], 3[Y], 5[Z], 7[−Y], 9[−X], and 12[−Z] (Bhattacharya et al., 2004). Muscle contraction is also controlled by EF-hand regula¨ tory proteins linked to actin and/or myosin (Szent-Gyorgyi, ’75; Lewit-Bentley and Rety, 2000). The proteins involved are calmodulin (CaM), troponin C (TnC), and the essential myosin light chain and regulatory myosin light chain (eMLC and rMLC). All these proteins are believed to have a common ancestor that contained four calcium-binding EF-hand domains. In the modern forms, typically one or more of the EF-hand domains of these proteins have lost the ability to bind Ca2+ (Kretsinger, ’80). The most widespread actomyosin control in animals is mediated by the actin-associated regulatory proteins troponin (Tn) and tropomyosin (Tm), which can sterically block myosin-binding sites on actin. Unblocking is initiated by calcium binding to the Tn subunit TnC (Gordon et al., 2000). Myosin-linked control is constituted by its associated myosin light chains (MLCs) that are wrapped around the lever arm of the myosin head by binding to an IQ (isoleucine, glutamine) sequence motif: IQxxxRGxxxR (Mooseker and Cheney, ’95). In molluscan- and annelid-muscle myosin is activated by direct calcium binding to eMLC and rMLC, ¨ respectively (Carlhoff and D’Haese, ’88; Szent-Gyorgyi et al., ’99; Himmel et al., 2009). In vertebrate smooth muscle and in nematode and arthropod-striated muscles as well as in nonmuscle cells myosin control occurs by phosphorylation of the rMLC (Harris et al., ’77; Takahashi et al., ’90; Seller, ’91). During desiccation or freezing considerable changes of ionic balance, including changes in intracellular calcium concentration, are expected. In the present study, we describe the nucleotide and amino acid sequences of four EF-hand proteins that potentially regulate muscle contraction in tardigrades and searched for specific modifications in view of harsh environmental conditions like anhydrobiosis or cryobiosis. Additionally, we obtained recombinant proteins of TnC, eMLC, and rMLC. We compare our findings with corresponding data of several Non-Ecdysozoa and Ecdysozoa to learn more about the evolution and/or phylogenetic significance of these proteins by constructing phylogenetic trees. J. Exp. Zool.
PRASATH ET AL.
METHODS Library-Based Identification of cDNA Clones For the identification of cDNAs coding for CaM, TnC, and both MLCs, a series of primers (Supplementary Table S1) were designed based on conserved regions from pairwise and multiple alignments of hexapod, chelicerate, nematode, and crustacean sequences. The cDNA lambda phage library (Titer: 6 × 109 pfu/mL) of H. klebelsbergi was used as a template for the amplification of cDNA fragments by PCR (Kiehl et al., 2007). We used premixed PCR master mixes (Peqlab, Germany) adding 1 μL library and 0.5 μM of each primer with a total sample volume of 25 μL. Amplification was run with 15 min initial denaturation at 94◦ C followed by 35 cycles of 30 sec denaturation at 94◦ C, 1 min annealing at 50–55◦ C and 3 min elongation at 72◦ C. The program ended with a final incubation step for 10 min at 72◦ C. PCR products were separated in 1.2% agarose gels in TBE (90 mM Tris, 90 mM boric acid, 2 mM EDTA, 1 μg/mL ethidiumbro¨ mide) and custom sequenced by Seqlab (Gottingen, Germany). The resulting PCR fragments were cloned into pJET 1.2/blunt vector using CloneJET PCR Cloning kit (Fermentas, Germany) and transformed into TOP F10 cells (Invitrogen, Germany). Individual colonies were picked as templates for colony PCR reactions employing the supplemented primers pJet-forward (5 -CGACTCACTATAGGGAGAGCGGC-3 ) and pJet-reverse (5 AAGAACATCGATTTTCCATGGCAG-3 ). Products within the expected size range were sequenced and verified by comparison with expressed sequence tags (ESTs) of H. dujardini. By this strategy, partial cDNA sequences were identified and used to obtain complete cDNA sequences by combination of cDNA-specific and vector-based (Lambda TriplEx2) primers. All primers were synthesized by MWG-Biotech AG (Ebersberg, Germany). Cloning and Expression PCR products containing the open reading frame (ORF) of TnC and both MLCs were introduced into the PCR T7/NT TOPO TA expression system (Invitrogen, Germany). The expression construct was transformed into E. coli BL21 cells supplemented in the kit. A single colony was grown overnight in 10 mL LB medium containing 100 μg/mL ampicillin with shaking (250 rpm) at 37◦ C. 250 mL of LB medium were inoculated the next day with 5 mL of the overnight culture and incubated with shaking (250 rpm) at 37◦ C until an OD600 of 0.6 was obtained. Cultures were then induced with 1 mM isopropyl-β-d-thiogalactopyranoside and grown for another 4 hr. Cells were harvested by centrifugation at 6,000 × g for 10 min. The cell pellet was washed twice by suspending it in wash buffer 1 (50 mM Na2 HPO4 , 300 mM NaCl, pH 8.0) and centrifuged as before. Preparation and Protein Refolding of Inclusion Bodies Washed cell mass was collected by centrifuging at 15,000 × g, 4◦ C for 15 min. The pellet was suspended in wash buffer 1
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and lysed by sonication (3 × 20 sec pulses, with 1 min at 4◦ C between each sonication). After centrifugation at 12,000 × g for 15 min at 4◦ C, undissolved inclusion bodies were collected, washed twice with wash buffer 2 (PBS containing 0.5 [v/v] Triton X-100, 10 mM EDTA, and 50 mM NaCl, pH 7.4) and centrifuged at 12,000 × g for 5 min at 4◦ C. Inclusion bodies were then solubilized in 5 mM NaCl, 20 mM Tris-Maleate pH 7.9, 1 mM DTT, and 2 M guanidine hydrochloride. Protein refolding was achieved by dialyzing twice against 5 mM NaCl, 20 mM TrisMaleate pH 6.8, 5 mM 2-Mercaptoethanol. For analysis, samples were clarified by centrifugation at 12,000 × g for 30 min.
by sequencing and pairwise alignment with ESTs of H. dujardini, which showed 95–98% identity of their nucleotide and deduced amino acid sequences. All sequences contain a 5 and 3 untranslated region terminating in poly(A) tails. Sequences obtained have been deposited in GenBank (Accession Numbers: eMLC, HM628686; rMLC, HM628687; TnC, HM628688, and CaM, HM628689). For further analysis, TnC and the MLCs were obtained by recombinant protein expression and purified from inclusion bodies as described in Methods. The yield of purified recombinant protein was about 15% for TnC and 5% for both MLCs of the E. coli cell protein.
Gel Electrophoresis Expressions were analyzed by sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% gel according to Laemmli (’70). Calcium-induced mobility shift analysis was carried out by Urea-PAGE using a 7.5% gel containing 2 M urea after the method originally described by Creighton (’79). Probes were preincubated in 1 mM CaCl2 or 5 mM EGTA. Gels were stained with Coomassie Brilliant Blue G250 (Serva, Heidelberg, Germany). An unstained protein ladder (PageRulerTM , Fermentas, Germany) was used as molecular size marker.
Calmodulin The cDNA obtained for CaM was 1,407 nucleotides in length with an ORF of 447 bp (149 amino acids) and a calculated molecular weight of 16,836.64 Da. As typical for CaM, it is a highly acidic protein (pI of 4.11) due to a high content of aspartate and glutamate residues (24.8%). It lacks tryptophan and cysteine like most CaMs studied so far. Putative structure predictions (Fig. 1) reveal a dumbbell-shaped structure with Nand C-terminal globular regions linked by a long continuous α-helix. Each region contains two canonical helix-loop-helix EF-hand domains. All four Ca2+ -binding loops always show aspartate at both coordinates X and Y, while glutamate is to be found at coordinate Z. Furthermore, coordinates Z and −X are occupied by essential oxygen-containing side chain residues (Fig. 2 and Supplementary Table S2).
Sequence Analysis All protein sequences were retrieved from the UniProt database (http://www.uniprot.org). EST sequences of H. dujardini were retrieved from Tardibase (http://xyala.cap.ed.ac.uk/ tardigrades/tardibase.html) and from the Tardigrade workbench (http://waterbear.bioapps.biozentrum.uni-wuerzburg.de; see ¨ Forster et al., 2009). Blastn and tBlastx programs (http://www.ncbi.nlm.nih.gov) were used to search for similarity in NCBI databases and ClustalW (http://www.ebi.ac.uk/clustalw) was utilized to compare sequences (Altschul et al., ’97; Larkin et al., 2007).
RESULTS Searching for tardigrade EF-hand proteins only EST sequences from H. dujardini are documented in Tardibase and Tardigrade workbench (five TnC, 19 eMLC, and 13 rMLC sequences). No CaM data of tardigrades were found in any database. Sequence analysis of eMLC-ESTs revealed two different sequence clusters with about 90% nucleotide and deduced amino acid identity, whereas the TnC- and the rMLC-ESTs represented only one sequence each. All EST sequences shared 50–57% identity with TnC and both MLCs of various hexapods, chelicerates, nematodes, and crustaceans. In order to screen the H. klebelsbergi lambda phage library for Ca2+ -binding EF-hand proteins involved in regulation of actin–myosin interaction, conserved nucleotide regions were selected as PCR primer targets. We identified cDNA clones for all four EF-hand proteins by PCR-based screening. The authenticity of potential cDNA fragments amplified by PCR was confirmed
Troponin C The identified TnC clone had an 824 bp cDNA insert encoding a protein with an ORF of 498 bp (166 amino acids). It has a calculated molecular weight of 17,354.3 Da (apparent molecular mass of 19 kDa, Fig. 3B). The deduced amino acid sequence shows an excess of aspartate (11.8%) and glutamic acid (13.2%) that results in a pI of 4.1. The putative 3D model (Supplementary Fig. S1) displays similar to CaM a dumbbell shape with distinct N- and C-terminal regions linked by an α-helix with a short-loop segment (residues 81–85). Each region contains two EF-hand domains. EF-hand I differs from the canonical pattern at coordinate Z and −Z occupied by glutamine and at −X by histidine. EF-hand domain III shows glutamine at coordinate Y, where, however, an aspartate or asparagine is preferred (Supplementary Table S2). EF-hand domains II and IV display a canonical pattern (Fig. 3A). Gel mobility shift analysis of the recombinant protein showed that Ca2+ induced a considerable shift (Fig. 3B). This demonstrates calciuminduced conformational changes of the recombinant TnC of H. klebelsbergi. Protein BLAST search showed the highest similarity within the C-terminal region of arthropod and nematode TnCs with the highest score and 68% identity with the horseshoe crab Tachypleus tridentatus (GenBank Accession Number P15159). J. Exp. Zool.
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PRASATH ET AL. Essential Myosin Light Chain The eMLC cDNA clone is 1,015 bp long and contains an ORF of 498 bp. It encodes an acidic protein of 166 amino acids (pI of 4.7) with a calculated molecular mass of 18,473.1 Da (apparent molecular mass of 20 kDa). Sequence analysis revealed two C-terminally located noncanonical EF-hand domains (Fig. 4 and Supplementary Table S2; for the putative 3D model see Supplementary Fig. S2). No calcium-dependent electrophoretic mobility shift could be seen (Fig. 4B). BLAST search showed the highest score and 50% identity with the tick Haemaphysalis qinghaiensis (GenBank Accession Number AAV41826).
Figure 1. Putative 3D structure of H. klebelsbergi calmodulin. All four EF-hand domains display a canonical Ca2+ -binding loop pattern (for details see text). Helices E (blue), F (green), and calciumbinding loops (red) are highlighted (EFh1–EFh4) in the predicted 3D model, based on template PDB ID: 2fp2 using SWISS-MODEL. N- and C-termini are indicated. Secondary structure predictions and 3D models were constructed using the SWISS-MODEL server (http://www.expasy.org/swissmod/SWISS-MODEL.html). Figures were drawn in Swiss-PDB viewer and rendered with POV-ray software (Guex and Peitsch, ’97; Arnold, 2006).
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Regulatory Myosin Light Chain The rMLC cDNA clone (984 bp) contains an ORF of 525 bp. The full-length translation product is predicted to be an acidic protein of 175 amino acids (pI of 4.87) with a calculated molecular mass of 18,924.3 Da. The apparent molecular mass of the recombinant protein calculated from SDS-PAGE was 18 kDa (Fig. 5B). The N-terminal region shows a phosphorylatable motif with threonine-serine at position 16 and 17. Both residues are flanked upstream by a consensus recognition sequence of positively charged amino acids including KKR xxR xxS xVF (position 9–20) essential for the catalytic activity of myosin light chain kinases. Each N- and C-terminus shows a single calcium-binding EF-hand domain. EF-hand I contains a canonical Ca2+ -binding loop pattern. EF-hand III shows at Ca2+ -binding loop coordinates Z and −Z amino acid substitutions with threonine and proline that lead to the loss of essential, negatively charged residues (Fig. 5A and Supplementary Table S2; for the putative 3D model, see Supplementary Fig. S3). A database search with BLAST revealed remarkable conservation with rMLCs of invertebrates with the highest score and 58% identity with the ants Camponotus floridanus (GenBank Accession Number EFN63580) and Harpegnathos saltator (GenBank Accession Number EFN80420). Notably, the rMLC of H. klebelsbergi exhibits an outstanding similarity to vertebrate smooth muscle (54% Homo sapiens). The highest conservation lies within the phosphorylatable threonine-serine region and the canonical EF-hand domain I (Supplementary Table S3). Electrophoretic mobility shift analysis shows only a slight mobility shift induced by Ca2+ of the recombinant rMLC of H. klebelsbergi (Fig. 5B).
DISCUSSION Calmodulin The identified CaM clone of H. klebelsbergi shares 99% amino acid identity with Drosophila melanogaster, Caenorhabditis elegans, the earthworm Lumbricus rubellus, the slug Aplysia californica, and remarkable 98% with Homo sapiens and Gallus
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Figure 2. EF-hand domains of H. klebelsbergi calmodulin. All four EF-hand domains (I–IV) display a canonical Ca2+ -binding loop pattern. Calcium coordinating key residues (X, Y, Z, −Y, −X, and −Z) that comply with the consensus pattern (for details see text) are boldunderlined. Helices E (dark gray), F (light gray), and calcium-binding loops (black) are shown as a block above the sequence. (The chosen sequence labeling for the EF-hand domains is likewise used for the troponin C, essential myosin light chain and regulatory myosin light chain figures.)
gallus. Differences are seen at position 99 and 143, where invertebrate CaMs contain phenylalanine, whereas H. sapiens, G. gallus, and other vertebrates show tyrosine (Klee et al., ’80). Our results confirm that CaM is highly conserved (Lewit-Bentley and Rety, 2000). Comparison of the four canonical EF-hand domains of CaM of H. klebelsbergi revealed the highest internal similarity between domains EF-hand I and EF-hand III (55%) and EF-hand II and EF-hand IV (59%). This characteristic is consistent with the concept that CaM evolved from an ancestral CaM gene with a single EF-hand by two tandem duplications (Kretsinger, ’80; Klee and Vanaman, ’82). CaM is involved in the regulation of numerous physiological pathways mediated by Ca2+ and interacts with a number of proteins by forming Ca2+ -dependent complexes (Means, ’88; James et al., ’95; Maria et al., ’99). Many unconventional myosins use several CaM molecules as light chains wrapped around the lever arm of the myosin head (Hasson and Mooseker, ’96). CaM of H. klebelsbergi is apparently a functional protein capable of binding four calcium ions and mediating the Ca2+ -signal similarly as in other organisms (Chin and Means, 2000) and reveals no special modifications in tardigrades.
bind Ca2+ during regulation while the C-terminal sites play a structural role in anchoring TnC to the troponin complex (Gordon et al., 2000). Due to substitutions of individual essential Ca2+ -coordinating residues, TnC of invertebrate muscles studied so far reveal the loss of two or even three Ca2+ -binding EF-hand domains: e.g., the crayfish Astacus leptodactylus (Kobayashi et al., ’89), the crustacean Balanus nubilis (Allhouse et al., ’99) or the nematode C. elegans (Ueda et al., 2001). These three are predicted to have a Ca2+ -specific binding site at EF-hand domain II and IV. Remarkably, the scallop Platinopecten yessoensis shows a functional temperature-sensitive TnC protein with only one Ca2+ -binding site at EF-hand domain IV (Ojima et al., 2000). The TnC cDNA of H. klebelsbergi shows two canonical calcium-binding EF-hand domains: one in the N-terminal region (EF-hand II) and one in the C-terminal region (EF-hand IV). Gel mobility shift analysis showed a calcium-induced conformational transition of the recombinant TnC of H. klebelsbergi. We expect TnC of H. klebelsbergi to regulate the “on” and “off” state of the actin filaments together with tropomyosin as do the TnCs from other organisms.
Troponin C A prerequisite of TnCs regulatory function is the reversible binding of Ca2+ in the narrow range of variations of the intracellular Ca2+ concentration. Vertebrate skeletal muscle TnC has four Ca2+ -binding EF-hand domains: two low affinity domains (I and II) in the N-terminal region that are Ca2+ specific and two high affinity domains (III and IV) in the C-terminal region that bind both Ca2+ and Mg2+ . N-terminal domains I and II reversibly
Essential Myosin Light Chain The cDNA of the eMLC of H. klebelsbergi reveals two noncanonical C-terminal EF-hand domains (III and IV). So far, only molluscan eMLCs (e.g., the scallop Argopecten irradians) have been shown to be capable of direct Ca2+ binding to trigger mus¨ cle contraction (Szent-Gyorgyi et al., ’99). Although the scallop eMLC contains one canonical EF-hand domain (EF-hand III), it has been shown that the region necessary for Ca2+ binding J. Exp. Zool.
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Figure 3. (A) The troponin C (TnC) of H. klebelsbergi shows two canonical calcium-binding EF-hand domains, EF-hand domain II, and EF-hand domain IV (for details see text). For sequence labeling see Fig. 2. (B) SDS-PAGE and Urea-PAGE mobility-shift analysis of the recombinant TnC of H. klebelsbergi with an apparent molecular mass of 19 kDa. Lane M: protein marker; lane 1: supernatant, soluble fraction; lane 2: pellet; lane 3: purified recombinant protein. Purified protein was incubated in 1 mM calcium [C] and in 5 mM EGTA [E].
and regulation exclusively resides in the noncanonical EF-hand ¨ domain I (Szent-Gyorgyi et al., ’99). However, a complex comprising the myosin lever arm and both light chains is necessary for regulation (Himmel et al., 2009).
Regulatory Myosin Light Chain Hypsibius klebelsbergi rMLC displays one canonical N-terminal calcium-binding EF-hand domain (EF-hand I). Although all the rMLCs we compared comply with the canonical calcium-binding loop pattern in their first EF-hand domain, the rMLC from obliquely striated body-wall muscle of the earthworm Lumbricus terrestris is the only known rMLC that regulates myosin J. Exp. Zool.
by direct binding of Ca2+ (Carlhoff and D’Haese, ’88). All but Pecten sp.-rMLCs exhibit a prolonged N-terminus of about 10– 15 basic residues, with L. terrestris having an outstanding high number of about 40. Comparing the rMLC-protein sequence of H. klebelsbergi with those of Mollusca, Vertebrata, Arthropoda, Annelida, and Nematoda, (Table S3) the highest sequence similarity is to the rMLCs of Arthropoda nonmuscle (49–54%) and notably Vertebrata smooth muscle (50–54%). The amino acid comparison exhibits two regions, of considerable conservation. The first region extending from position 11–58 of the H. klebelsbergi sequence, corresponds to the amino acids flanking the phosphorylatable threonine-serine motif and the canonical calciumbinding EF-hand domain I. The second region, extending from
EF-HAND PROTEINS OF TARDIGRADES
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Figure 4. (A) The essential myosin light chain (eMLC) of H. klebelsbergi shows two noncanonical C-terminal calcium-binding EF-hand domains, EF-hand domain III, and EF-hand domain IV. Probable remnants of the ancestral EF-hand domains I and II are indicated. For sequence labeling see Fig. 2. (B) SDS-PAGE and Urea-PAGE mobility-shift analysis of the recombinant eMLC of H. klebelsbergi with an apparent molecular mass of 20 kDa. Lane M: protein marker; lane 1: supernatant, soluble fraction; lane 2: pellet; lane 3: purified recombinant protein. Purified protein was incubated in 1 mM calcium [C] and in 5 mM EGTA [E].
position 77–128, comprises the ancestral EF-hand domain II. Sequence properties suggest that the rMLC of H. klebelsbergi identified in this study is phosphorylatable. The sequence shows a phosphorylatable threonine-serine motif flanked by a consensus recognition sequence of positively charged amino acids including KKR xxR xxS xVF essential for the catalytic activity of myosin light chain kinase (Ikebe et al., ’94; Gao et al., ’95; Kamm and Stull, 2001). Phosphorylation of rMLC controls vertebrate smooth and nonmuscle myosin filament assembly as well as the interaction of myosin with actin (Sweeney, ’98). The rMLCs of skeletal and cardiac muscle are phosphorylated, yet this appears to play a minor modulatory
role (Szczesna et al., 2002). Studies on molluscan myosin reveal that Ca2+ -binding properties of both myosin light chains are al¨ tered by interaction with myosin heavy chains (Szent-Gyorgyi, ’99). This finding demonstrates that protein–protein interaction can influence Ca2+ -binding properties. The relative abundance of muscles in eutardigrades makes it probable that the regulatory proteins we describe can be assigned to muscle tissue especially to the somatic muscles (see Marcus, ’29; Schmidt-Rhaesa and Kulessa, 2007; Halberg et al., 2009). Somatic muscles of tardigrades represent an intermediate type between smooth muscles and obliquely striated muscles (Walz, ’74). But other muscle types (e.g., pharyngeal J. Exp. Zool.
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Figure 5. (A) The regulatory myosin light chain (rMLC) of H. klebelsbergi shows one canonical calcium-binding EF-hand domain (EF-hand domain I) and a consensus myosin-light-chain-kinase recognition motif (KKRxxRxxSxVF). Probable remnants of the ancestral EF-hand domains II and IV are indicated. For sequence labeling see Fig. 2. (B) SDS-PAGE and Urea-PAGE mobility-shift analysis of the recombinant rMLC of H. klebelsbergi with an apparent molecular mass of 18 kDa. Lane M: protein marker; lane 1: supernatant, soluble fraction; lane 2: pellet; lane 3: purified recombinant protein. Purified protein was incubated in 1 mM calcium [C] and in 5 mM EGTA [E].
bulb-, stylet-, and leg-muscle; see Halberg et al., 2009) and even nonmuscle cells present in tardigrades may also be the source of the four EF-hand proteins. Our data support that at least the somatic muscle is dual regulated by calcium binding to TnC and phosphorylation of the rMLC. The presence of a TnI-like protein has recently been demonstrated in the somatic muscle but it was absent in the pharyngeal muscle (Obinata et al., 2011). These findings suggest the possibility of diverse regulatory mechanisms in somatic and pharyngeal muscle. J. Exp. Zool.
Further study is required to localize both regulatory systems and determine their contribution to muscle and nonmuscle contractility in tardigrades.
CONCLUSIONS Studies of CaM, TnC, and both MLCs isoforms from invertebrates (for summary see Hooper and Thuma, 2005) suggest a diverse isoform pattern for H. klebelsbergi in nonmuscle and different muscle types. However, we assume that we have identified the major isoforms for all four EF-hand proteins expressed in the
EF-HAND PROTEINS OF TARDIGRADES somatic muscle of this tardigrade. We propose a dual-regulated system at least in somatic muscles for tardigrades. The CaM, TnC, and rMLC of H. klebelsbergi comply with sequence properties in which calcium triggers a Tn–Tm system and a CaM-mediated phosphorylation of the rMLC that turns on myosin as well as actin. Our results indicate no special modifications of EF-hand protein structure and function of tardigrades exposed to extreme environmental conditions. The phylogenetic trees (Supplementary Fig. S4) obtained from 131 TnC-, 96 rMLC-, and 62 eMLC-protein sequences appear to support some current views on tardigrade relationships. For example, the TnC tree indicates affinities to Ecdysozoa in general (nematodes and arthropods e.g., Garey et al., ’96; Giribet et al., ’96; Aguinaldo et al., ’97; Mallatt et al., 2004) and, within Ecdysozoa to nematodes (Philippe et al., 2005; Dunn et al., 2008; Meusemann et al., 2010). The rMLC tree suggests relationships with nematodes, whereas the eMLC tree suggests affinities with Annelida, i.e., “Articulata” and Mollusca. The protein-specific variations among these trees, however, suggest they reflect the complex evolution of muscle-regulatory proteins according to their various functional demands, as opposed to being useful for revealing phylogenetic relationships.
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